Supplements - Haematologica

haematologica 

journal ofh 

hematology 

volume 85 

supplement 

to no. 12 

december 2000 

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ISSN 0390-6078

haematologica 

journal ofh 

hematology 

volume 85 

supplement to no. 12 

december 2000 

ISSN 0390-6078 

editor-in-chief 

edoardo ascari 

published by the 

Ferrata Storti Foundation 

established in 1920

Haematologica – Vol. 85, supplement to n. 12, December 2000

haematologica 

journal of 

hematology 

hOfficial Organ of 

the Italian Society of Hematology, 

the Italian Society of Experimental Hematology, 

the Spanish Association of Hematology and Hemotherapy, 

the Italian Society of Hemostasis and Thrombosis, 

and 

the Italian Society of Pediatric Hematology/Oncology 

Editor-in-Chief 

Edoardo Ascari (Pavia) 

Executive Editor 

Mario Cazzola (Pavia) 

Editorial Committee 

Tiziano Barbui (President of the Italian Society of Hematology, Bergamo); Ciril 

Rozman (Representative of the Spanish Association of Hematology and 

Hemotherapy, Barcelona); Pier Mannuccio Mannucci (Italian Society of 

Hemostasis and Thrombosis, Milan) 

Associate Editors 

Carlo Brugnara (Boston), Red Cells & Iron. Federico Caligaris Cappio (Torino), 

Lymphocytes & Immunology. Carmelo Carlo-Stella (Milano), Hematopoiesis & 

Growth Factors. Paolo G. Gobbi (Pavia), Lymphoid Neoplasia. Franco Locatelli 

(Pavia), Pediatric Hematology. Francesco Lo Coco (Roma), Translational Research. 

Alberto Mantovani (Milano), Leukocytes & Inflammation. Giuseppe Masera 

(Monza), Geographic Hematology. Cristina Mecucci (Perugia), Cytogenetics. 

Pier Giuseppe Pelicci (Milano, Italian Society of Experimental Hematology), 

Molecular Hematology. Paolo Rebulla (Milano), Transfusion Medicine. Miguel 

Angel Sanz (Valencia), Myeloid Neoplasia. Salvatore Siena (Milano), Medical 

Oncology. Jorge Sierra (Barcelona), Transplantation & Cell Therapy. Vicente 

Vicente (Murcia), Hemostasis & Thrombosis 

Editorial Board 

Adriano Aguzzi (Zürich), Adrian Alegre Amor (Madrid), Claudio Anasetti (Seattle), 

Jeanne E. Anderson (San Antonio), Nancy C. Andrews (Boston), William Arcese 

(Roma), Andrea Bacigalupo (Genova), Carlo Balduini (Pavia), Luz Barbolla (Madrid), 

Giovanni Barosi (Pavia), Javier Batlle Fourodona (La Coruña), Yves Beguin (Liège), 

Marie Christine Béné (Nancy), Yves Beuzard (Paris), Andrea Biondi (Monza), Mario 

Boccadoro (Torino), Niels Borregaard (Copenhagen), David T. Bowen (Dundee), 

Ronald Brand (Leiden), Salut Brunet (Barcelona), Ercole Brusamolino (Pavia), Clara 

Camaschella (Torino), Dario Campana (Memphis), Maria Domenica Cappellini 

(Milano), Angelo Michele Carella (Genova), Gian Carlo Castaman (Vicenza), Marco 

Cattaneo (Milano), Zhu Chen (Shanghai), Alan Cohen (Philadelphia), Eulogio Conde 

Garcia (Santander), Antonio Cuneo (Ferrara), Björn Dahlbäck (Malmö), Riccardo 

Dalla Favera (New York), Armando D’Angelo (Milano), Elisabetta Dejana (Milano), 

Jean Delaunay (Le Kremlin-Bicêtre), Consuelo Del Cañizo (Salamanca), Valerio De 

Stefano (Roma), Joaquin Diaz Mediavilla (Madrid), Francesco Di Raimondo 

(Catania), Charles Esmon (Oklahoma City), Elihu H. Estey (Houston), Renato Fanin 

(Udine), José-María Fernández Rañada (Madrid), Evarist Feliu Frasnedo (Barcelona), 

Jordi Fontcuberta Boj (Barcelona), Francesco Frassoni (Genova), Renzo Galanello 

(Cagliari), Arnold Ganser (Hannover), Alessandro M. Gianni (Milano), Norbert 

C. Gorin (Paris), Alberto Grañena (Barcelona), Eva Hellström-Lindberg (Huddinge), 

Martino Introna (Milano), Rosangela Invernizzi (Pavia), Achille Iolascon (Bari), 

Sakari Knuutila (Helsinki), Myriam Labopin (Paris), Catherine Lacombe (Paris), 

Francesco Lauria (Siena), Mario Lazzarino (Pavia), Roberto M. Lemoli (Bologna), 

Giuseppe Leone (Roma), A. Patrick MacPhail (Johannesburg), Ignazio Majolino 

(Palermo), Patrice Mannoni (Marseille), Guglielmo Mariani (Palermo), Estella 

Matutes (London), Alison May (Cardiff), Roberto Mazzara (Barcelona), Giampaolo 

Merlini (Pavia), José Maria Moraleda (Murcia), Enrica Morra (Milano), Kazuma 

Ohyashiki (Tokyo), Alberto Orfao (Salamanca), Anders Österborg (Stockholm), 

Pier Paolo Pandolfi (New York), Ricardo Pasquini (Curitiba), Andrea Pession 

(Bologna), Franco Piovella (Pavia), Giovanni Pizzolo (Verona), Domenico Prisco 

(Firenze), Susana Raimondi (Memphis), Alessandro Rambaldi (Bergamo), Fernando 

Ramos Ortega (León), José Maria Ribera (Barcelona), Damiano Rondelli (Bologna), 

Giovanni Rosti (Ravenna), Bruno Rotoli (Napoli), Domenico Russo (Udine), Stefano 

Sacchi (Modena), Giuseppe Saglio (Torino), Jesus F. San Miguel (Salamanca), 

Guillermo F. Sanz (Valencia), Hubert Schrezenmeier (Berlin), Mario Sessarego 

(Genova), Pieter Sonneveld (Rotterdam), Yoshiaki Sonoda (Kyoto), Yoichi Takaue 

(Tokyo), José Francisco Tomás (Madrid), Giuseppe Torelli (Modena), Antonio Torres 

(Cordoba), Pinuccia Valagussa (Milano), Andrea Velardi (Perugia), Ana Villegas 

(Madrid), Françoise Wendling (Villejuif), Pier Luigi Zinzani (Bologna) 

Publication Policy Committee 

Edoardo Storti (Chair, Pavia), John W. Adamson (Milwaukee), Carlo Bernasconi 

(Pavia), Gianni Bonadonna (Milano), Gianluigi Castoldi (Ferrara), Albert de la 

Chapelle (Columbus), Peter L. Greenberg (Stanford), Fausto Grignani (Perugia), 

Lucio Luzzatto (New York), Franco Mandelli (Roma), Massimo F. Martelli (Perugia), 

Emilio Montserrat (Barcelona), David G. Nathan (Boston), Alessandro Pileri 

(Torino), Vittorio Rizzoli (Parma), Eduardo Rocha (Pamplona), Pierluigi Rossi Ferrini 

(Firenze), George Stamatoyannopoulos (Seattle), Sante Tura (Bologna), Herman 

van den Berghe (Leuven), Zhen-Yi Wang (Shanghai), David Weatherall (Oxford), 

Soledad Woessner (Barcelona) 

Editorial Office 

Igor Ebuli Poletti, Paolo Marchetto, Michele Moscato, Lorella Ripari, 

Rachel Stenner

h 

journal of 

hematology 

haematologica 

Associated with USPI, Unione Stampa Periodica 

Italiana. Premiato per l’alto valore culturale dal 

Ministero dei Beni Culturali ed Ambientali 

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haematologica 

hInstructions to Authors 

For additional information, the scientific 

staff of Haematologica can be reached 

through: 

mailing address: Haematologica, Strada 

Nuova 134, I-27100 Pavia, Italy. 

Tel. +39.0382.531182. 

Fax +39.0382.27721. 

e-mail: office@haematologica.it 

web: http://www.haematologica.it 

Haematologica publishes monthly Editorials, Original Papers, Reviews and Scientific Correspondence 

on subjects regarding experimental, laboratory and clinical hematology. 

Editorials and Reviews are normally solicited by the Editor, but suitable papers of this type 

may be submitted for consideration. Appropriate papers are published under the headings 

Decision Making and Problem Solving and Molecular Basis of Disease. 

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observations and ideas in hematology. As such, our objective is to review a paper in 4 

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However, it must be noted that Haematologica strongly encourages authors to send their 

papers via Internet. Haematologica believes that this is a more reliable way of speeding 

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Submit papers to: 

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Manuscript preparation. Manuscripts must be written in English. Manuscripts with 

inconsistent spelling will be unified by the English Editor. Manuscripts should be prepared 

according to the Uniform Requirements for Manuscripts Submitted to Biomedical Journals, 

N Engl J Med 1997; 336:309-15; the most recent version of the Uniform Requirements can 

be found on the following web site: 

http://www.ama-assn.org/public/peer/wame/uniform.htm 

With respect to traditional mail submission, manuscripts, including tables and figures, 

should be sent in triplicate to facilitate rapid reference. In order to accelerate processing, 

author(s) should also enclose a 3.5” diskette (MS-DOS or Macintosh) containing the manuscript 

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Title Page. The first page of the manuscript must contain: (a) title, name and surname of 

the authors; (b) names of the institution(s) where the research was carried out; (c) a running 

title of no more than 50 letters; (d) acknowledgments; (e) the name and full postal 

address of the author to whom correspondence regarding the manuscript as well as 

requests for abstracts should be sent. To accelerate communication, phone, fax number 

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Abstract. The second page should carry an informative abstract of no more than 250 words 

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Editorials should be concise. No particular format is required for these articles, which 

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Original Papers should normally be divided into abstract, introduction, design and methods, 

results, discussion and references. 

The section Decision Making and Problem Solving presents papers on health decision science 

specifically regarding hematologic problems. Suitable papers will include those 

dealing with public health, computer science and cognitive science. This section may also 

include guidelines for diagnosis and treatment of hematologic disorders and position 

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Reviews provide a comprehensive overview of issues of current interest. No particular format 

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Scientific Correspondence should be no longer than 500 words (a word count should be 

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Correspondence, i.e. comments on articles published in the Journal will only appear in

haematologica 

hInstructions to Authors 

For additional information, the scientific 

staff of Haematologica can be reached 

through: 

mailing address: Haematologica, Strada 

Nuova 134, I-27100 Pavia, Italy. 

Tel. +39.0382.531182. 

Fax +39.0382.27721. 

e-mail: office@haematologica.it 

web: http://www.haematologica.it 

our Internet edition. Pictures of particular interest will be published in appropriate spaces 

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Tables and Illustrations. Tables and illustrations must be constructed in consideration of 

the size of the Journal and without repetitions. They should be sent in triplicate with each 

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In preparing composite illustrations, each section should be marked with a small letter 

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page. Authors are also encouraged to submit illustrations as electronic files together 

with the manuscript text (please, provide what kind of computer and software 

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Units of measurement. All hematologic and clinical chemistry measurements should be 

reported in the metric system according to the International System of Units (SI) (Ann 

Intern Med 1987; 106:114-29). Alternative non-SI units may be given in addition. Authors 

are required to use the standardized format for abbreviations and units of the International 

Committee for Standardization in Hematology when expressing blood count results 

(Haematologica 1991; 76:166). 

References should be prepared according to the Vancouver style (for details see: 

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Journal abbreviations are those of the List of the Journals Indexed, printed annually 

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on Internet through the US National Library of Medicine website, at the following worldwide-web 

address: http://www.nlm.nih.gov/tsd/serials/lji.html). 

List all authors when six or fewer; when seven or more, list only the first three and add et 

al. Examples of correct forms of references follow (please note that the last page must be 

indicated with the minimum number of digits): 

Journals (standard journal article, 1,2 corporate author, 3 no author given, 4 journal supplement 

5 ): 

1. Najfeld V, Zucker-Franklin D, Adamson J, Singer J, Troy K, Fialkow PJ. Evidence for clonal 

development and stem cell origin of M7 megakaryocytic leukemia. Leukemia 1988; 

2:351-7. 

2. Burgess AW, Begley CG, Johnson GR, et al. Purification and properties of bacterially 

synthesized human granulocyte-macrophage colony stimulating factor. Blood 1987; 

69:43-51. 

3. The Royal Marsden Hospital Bone-Marrow Transplantation Team. Failure of syngeneic 

bone-marrow graft without preconditioning in post-hepatitis marrow aplasia. Lancet 

1977; 2:242-4. 

4. Anonymous. Red cell aplasia [editorial]. Lancet 1982; 1:546-7. 

5. Karlsson S, Humphries RK, Gluzman Y, Nienhuis AW. Transfer of genes into hemopoietic 

cells using recombinant DNA viruses [abstract]. Blood 1984; 64(Suppl 1):58a. 

Books and other monographs (personal authors, 6,7 chapter in a book, 8 published proceeding 

paper, 9 abstract book, 10 monograph in a series, 11 agency publication 12 ): 

6. Ferrata A, Storti E. Le malattie del sangue. 2nd ed. Milano: Vallardi; 1958. 

7. Hillman RS, Finch CA. Red cell manual. 5th ed. Philadelphia: FA Davis; 1985. 

8. Bottomley SS. Sideroblastic anaemia. In: Jacobs A, Worwood M, eds. Iron in biochemistry 

and medicine, II. London: Academic Press; 1980. p. 363-92. 

9. DuPont B. Bone marrow transplantation in severe combined immunodeficiency with an 

unrelated MLC compatible donor. In: White HJ, Smith R, eds. Proceedings of the third 

annual meeting of the International Society for Experimental Hematology. Houston: 

International Society for Experimental Hematology; 1974. p. 44-6. 

10.Bieber MM, Kaplan HS. T-cell inhibitor in the sera of untreated patients with Hodgkin’s 

disease [abstract]. Paper presented at the International Conference on Malignant 

Lymphoma Current Status and Prospects, Lugano, 1981:15. 

11.Worwood M. Serum ferritin. In: Cook JD, ed. Iron. New York: Churchill Livingstone; 1980. 

p. 59-89. (Chanarin I, Beutler E, Brown EB, Jacobs A, eds. Methods in hematology; vol 

1). 

12.Ranofsky AL. Surgical operation in short-stay hospitals: United States-1975. Hyattsville, 

Maryland: National Center for Health Statistics; 1978. DHEW publication no. (PHS) 

78-1785, (Vital and health statistics; series 13; no. 34). 

References to Personal Communications and Unpublished Data should be incorporated in 

the text and not placed under the numbered References. Please type the references exactly 

as indicated above and avoid useless punctuation (e.g. periods after the initials of 

authors’ names or journal abbreviations). 

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Transfer of Copyright and Permission to Reproduce Parts of Published Papers. Authors 

will grant copyright of their articles to the Ferrata Storti Foundation. No formal permission 

will be required to reproduce parts (tables or illustrations) of published papers, provided 

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haematologica 

hTable of Contents 

2000; vol. 85; supplement to no. 12 

(indexed by Current 

Contents/Life Sciences and in Faxon 

Finder and Faxon XPRESS, also available 

on diskette with abstracts) 

Papers 

CD34-positive cells: biology and clinical relevance 

Carmelo Carlo Stella, Mario Cazzola, Paolo De Fabritiis, 

Armando De Vincentiis, Alessandro Massimo Gianni, 

Francesco Lanza, Francesco Lauria, Roberto M. Lemoli, 

Corrado Tarella, Paola Zanon, Sante Tura .........................................1 

Peripheral blood stem cells in acute myeloid leukemia: 

biology and clinical applications 

Massimo Aglietta, Armando De Vincentiis, Luigi Lanata, 

Francesco Lanza, Roberto Massimo Lemoli, Giacomo 

Menichella, Agostino Tafuri, Paola Zanon, Sante Tura ....19 

Peripheral blood stem cell transplantation for 

the treatment of multiple myeloma: biological 

and clinical implications 

Federico Caligaris Cappio, Michele Cavo, 

Armando De Vincentiis, Luigi Lanata, Roberto Massimo 

Lemoli, Ignazio Majolino, Corrado Tarella, Paola Zanon, 

Sante Tura ......................................................................................................................32 

Allogeneic hematopoietic stem cells from sources other 

than bone marrow: biological and technical aspects 

Francesco Bertolini, Armando De Vincentiis, Luigi Lanata, 

Roberto Massimo Lemoli, Rita Maccario, Ignazio Majolino, 

Luisa Ponchio, Damiano Rondelli, Antonio Tabilio, 

Paola Zanon, Sante Tura..................................................................................49 

Clinical use of allogeneic hematopoietic stem cells from 

sources other than bone marrow 

William Arcese, Franco Aversa, Giuseppe Bandini, 

Armando De Vincentiis, Michele Falda, Luigi Lanata, 

Roberto Massimo Lemoli, Franco Locatelli, 

Ignazio Majolino, Paola Zanon, Sante Tura................................69 

Ex vivo expansion of hematopoietic cells 

and their clinical use 

Massimo Aglietta, Francesco Bertolini, Carmelo 

Carlo-Stella, Armando De Vincentiis, Luigi Lanata, Roberto 

Massimo Lemoli, Attilio Olivieri, Salvatore Siena, Paola 

Zanon, Sante Tura ..................................................................................................92 

Cell therapy: achievements and perspectives 

Claudio Bordignon, Carmelo Carlo-Stella, Mario Paolo 

Colombo, Armando De Vincentiis, Luigi Lanata, Roberto 

Massimo Lemoli, Franco Locatelli, Attilio Olivieri, 

Damiano Rondelli, Paola Zanon, Sante Tura ...........................117 

Antitumor vaccination: where we stand 

Monica Bocchia, Vincenzo Bronte, Mario Paolo Colombo, 

Armando De Vincentiis, Massimo Di Nicola, Guido Forni, 

Luigi Lanata, Roberto Massimo Lemoli, Massimo Massaia, 

Damiano Rondelli, Paola Zanon, Sante Tura ...........................156

eview 

CD34-positive cells: 

biology and clinical relevance 

haematologica 2000; 85(supplement to no. 12):1-18 

Correspondence: Prof. Sante Tura, Istituto di Ematologia L. e A. Seràgnoli, 

Policlinico S. Orsola, via Massarenti 9, 40138 Bologna, Italy. 

Acknowledgments: the preparation of this manuscript was supported 

by grants from Dompé Biotec SpA, Milano, and Amgen S.p.A., Milano, 

Italy. Received October 18, 1994; accepted May 2, 1995. 

CARMELO CARLO STELLA, MARIO CAZZOLA,* 

PAOLO DE FABRITIIS,° ARMANDO DE VINCENTIIS, # 

ALESSANDRO MASSIMO GIANNI, @ FRANCESCO LANZA,^ 

FRANCESCO LAURIA,** ROBERTO M. LEMOLI,°° 

CORRADO TARELLA, ## PAOLA ZANON, @@ SANTE TURA°° 

Cattedra di Ematologia, Università di Parma, Parma; *Dipartimento 

di Medicina Interna e Terapia Medica, Sezione di Medicina 

Interna e Oncologia Medica, Università di Pavia e IRCCS 

Policlinico S. Matteo, Pavia; °Dipartimento di Biopatologia 

Umana, Sezione di Ematologia, Università “La Sapienza”, 

Roma; # Dompé Biotec SpA, Milano; @ Centro Trapianto Midollo 

Osseo “Cristina Gandini”, Divisione di Oncologia Medica C, 

Istituto Nazionale Tumori, Milano; ^Istituto di Ematologia e 

Fisiopatologia dell’Emostasi, Università degli Studi di Ferrara, 

Ferrara; **Istituto di Scienze Mediche, Università degli Studi 

di Milano, Milano; °°Istituto di Ematologia “Lorenzo e Ariosto 

Seragnoli”, Università degli Studi di Bologna, Bologna; ## Cattedra 

di Ematologia, Università di Torino, Torino; @@ Amgen 

S.p.A., Milano, Italy 

The CD34-positive cell: definition and 

morphology 

Cellular expression of the CD34 antigen identifies a 

morphologically and immunologically heterogeneous 

cell population that is functionally characterized by the 

in vitro capability to generate clonal aggregates derived 

from early and late progenitors and the in vivo capacity 

to reconstitute the myelo-lymphopoietic system in 

a supralethally irradiated host. 1-3 

Immunohistochemical studies have demonstrated 

that the CD34 antigen is stage but not lineage specific. 

In fact, independently of the differentiative lineage, 

it is expressed only by ontogenetically early cells. 4 For 

years a major obstacle to the morphological identification 

of putative hematopoietic stem cell has been the 

difficulty in separating them from their direct progeny. 

The use of CD34 and other suitable cell surface markers 

(i.e. CD33, CD38, HLA-DR antigens) in fluorescenceactivated 

cell-sorting techniques or other cell separation 

methods has allowed considerable progress in this 

field. 

Positively selected, lineage committed CD34 + cells 

and more immature, lineage negative CD34 + CD33 – 

HLA-DR – cells are shown in Figure 1 and Figure 2, 

respectively. On May-Grünwald-Giemsa stained preparations, 

CD34 + cells are medium sized cells having large 

nuclei, eccentrically surrounded by narrow rims of deep 

blue cytoplasm occasionally containing cytoplasmic 

granules. Some normal CD34 + cell nuclei show one or 

more pale blue nucleoli. Taken together, these findings 

reflect the heterogeneous proliferative status and protein 

synthesis of this cell population. Conversely, earlier 

hematopoietic progenitors, identified as CD34 + 

CD33– HLA-DR–, seem to be more homogeneous in size 

(small lymphocyte-like cells) and lack cytoplasmic granules 

and prominent nucleoli. Again, the morphology of 

this cellular population appears to reflect the functional 

characteristics of these cells (e.g. low protein synthesis, 

very low proliferative activity with predominantly 

G0 phase). 

Several monoclonal antibodies (MY10, 12.8, B1-3C5, 

115.2, ICH3, TUK3, etc.) raised against the leukemic cell 

lines KG1 or KG1a and an anti-endothelial cell antibody 

(QBEND10) assigned to the CD34 cluster have 

been shown to identify a transmembrane glycoproteic 

antigen of 105-120 kD expressed on 1-3% of normal 

bone marrow cells, 0.01-0.1% peripheral blood cells 

and 0.1-0.4% cord blood cells. 5 Different antibodies 

recognize distinct epitopes of the same antigen. CD34 

antigen expression is associated with concomitant 

expression of several other markers that can be classified 

as lineage non-specific markers (Thy1, CD38, HLA- 

DR, CD45RA, CD71) and lineage specific markers, 

including T-lymphoid (TdT, CD10, CD7, CD5, CD2), B- 

lymphoid (TdT, CD10, CD19), myeloid (CD33, CD13) and 

megakaryocytic (CD61, CD41, CD42b) markers. 5 The 

expression of lineage non-specific markers allows the 

heterogeneous CD34 + population to be divided into two 

distinct subpopulations characterized, respectively, by 

low or high expression of Thy1, CD38, HLA-DR, CD45RA, 

CD71. These two cell subpopulations contain early and 

late hematopoietic progenitor cells, respectively. 6-8 

In addition to conventional immunological markers 

classified on the basis of their assignation to specific 

clusters of differentiation, CD34 cells express receptors 

for a number of growth factors. Two distinct families of 

haematologica vol. 85(suppl. to n. 12):December 2000

2 

C. Carlo Stella et al. 

Figure 1. May-Grünwald-Giemsa (MGG) staining of cytospin 

preparation of enriched CD34+ cells, highly purified by 

avidin-biotin immunoaffinity. 

Figure 2. MGG staining of cytospin preparation of enriched 

CD34+ CD33– HLA-DR– cells. The CD34+ cell fraction was 

further depleted of CD33+ HLA-DR+ cells by immunomagnetic 

separation. 

related receptors have been identified: (i) tyrosine kinase 

receptors, including the stem cell factor receptor (SCF- 

R, CD117) and the macrophage colony-stimulating factor 

receptor (M-CSF-R, CD115); (ii) hematopoietic 

receptors not containing a tyrosine kinase domain, such 

as the granulocyte-macrophage colony-stimulating factor 

receptor (GM-CSF-R, CDw116). 5,9 

The identification of new markers selectively 

expressed on primitive lymphohematopoietic cells 

(CD34 + CD38 – ) represents a stimulating research field. In 

this context, stem cell tyrosine kinase receptors (STK), 

such as STK-1, a human homologue of the murine Flk- 

2/Flt-3, are of particular relevance. 10-12 The ligands for 

these receptors might represent new factors able to 

selectively control stem cell self-renewal, proliferation 

and differentiation. 13-15 

Clonogenic and biologic activity 

The structural and functional integrity of the 

hematopoietic system is maintained by a relatively small 

population of stem cells, located mainly in the bone 

marrow, that can (i) undergo self-renewal to produce 

more stem cells or (ii) differentiate to produce progeny 

which are progressively unable to self-renew, irreversibly 

committed to one or another of the various hematopoietic 

lineages, and able to generate clones of up to 10 5 

lineage-restricted cells that mature into specialized 

cells. 16-18 

The decision of a stem cell to either self-renew or differentiate 

and the selection of a specific differentiation 

lineage by a multipotent progenitor during commitment 

are intrinsic properties of stem cell progenitor cells and 

are regulated by stochastic mechanisms. 19 Survival and 

amplification of hematopoietic progenitors are controlled 

by a number of regulatory molecules (hematopoietic 

growth factors) interacting according to complex 

modalities (synergism, recruitment, antagonism). 19 A further 

level of hematopoietic control is exerted by nuclear 

transcription factors that activate lineage-specific genes 

regulating growth factor responsiveness and/or the proliferative 

capacity of hematopoietic cells. 20 

Detection of the most primitive hematopoietic cell 

types is now possible due to the technique of long-term 

bone marrow culture. In the case of human bone marrow, 

a 5- to 8-week time period between initiating cultures 

and assessing clonogenic progenitor numbers 

allows quantification of a very primitive cell in the starting 

population, the so-called long-term culture-initiating 

cell (LTC-IC). 21 Committed progenitors of the various 

hematopoietic cell classes can be quantitated by a number 

of short-term culture clonogenic assays. 19 CD34 antigen 

expression associated with low CD38 and CD45RA 

expression and variable HLA-DR expression is a typical 

feature of LTC-IC, CFU-Blast, CFU-T, CFU-B. In contrast, 

CD34 antigen expression associated with CD38 and HLA- 

DR expression is a typical feature of multipotent (CFU- 

GEMM) and lineage-restricted (CFU-GM, CFU-G, CFU-M, 

BFU-E, CFU-E, BFU-Meg, CFU-Meg) hematopoietic progenitor 

cells 5 (Figure 3). Recently reported data have 

shown that low or absent expression of the Thy1 or SCF 

receptor can be efficiently used to enrich primitive 

hematopoietic progenitors from the heterogeneous 

CD34 + cell population. 7,9 Although the CD34 antigen is 

virtually expressed by all progenitor cells, the percentage 

of CD34 + cells with assayable in vitro clonogenic activity 

ranges from 10 to 30%. The problem of non-clonogenic 

CD34 + cells is still open and not adequately 

explained by the presence of lymphoid progenitors which 

are not assayable with current in vitro systems. Nonproliferating 

CD34 + cells might represent a subpopulation 

that is not responsive to conventional myeloid 

hematopoietic growth factors. The non-proliferating 

CD34+ subset might require the presence of co-factors, 

such as the ligand of STK-1 or the hepatocyte growth 

factor, able to activate stem cell-specific genes whose 

expression is a prerequisite for acquiring responsiveness 

to conventional growth factors. 14,22,23 

In lethally irradiated non-human primates, both autologous 

and allogeneic CD34 + cells have been shown to 

have the capacity to reconstitute the myelo-lymphopoietic 

system, thus suggesting that the stem cell responsible 

for hematopoietic reconstitution is CD34 + . 24,25 It 


CD34 + cells: biology and clinical relevance 3 

has also been shown that human CD34 + HLA-DR – cells 

transplanted in utero in the fetus of sheep initiate and 

sustain a chimeric hematopoiesis producing human 

progenitor cells of all differentiative lineages. 26 Autologous 

CD34 + cells enriched by avidin-biotin columns have 

been shown to be able to reconstitute myelo-lymphopoiesis 

in patients receiving high-dose chemoradiotherapy. 

27 The results of studies using CD34 + bone marrow 

cells in the allogeneic setting in patients receiving 

both related as well as unrelated allogeneic marrow 

transplants will soon be available. 27 In addition, trials are 

planned that will use allogeneic peripheral blood CD34 + 

cells either alone or with marrow. 27 

Figure 3. Cellular organization of the hematopoietic system. 

Characterization and function of the CD34 

cell surface molecule 

The CD34 cell surface molecule has been biochemically 

characterized and both the human cDNA and gene 

have been cloned and sequenced in the last few 

years. 28,29 

CD34 is a one-pass type I transmembrane glycoprotein 

with a molecular weight of 105-120 kDs in either 

the reduced or unreduced form 28 (Figure 4). CD34 protein 

is not homologous to any other known protein. The 

minimum size of the CD34 protein is 354-amino acids 

and contains nine sites for N-glycosylation and a several 

for O-glycosylation that are essential constituents 

of the three epitopes of the molecule; this molecule is 

also rich in sialic acid. Its biochemical composition suggests 

a mucin-like structure and in some respects 

resembles leucosialin (CD43). Sequence comparisons 

between human and mouse CD34 show a very low level 

of identity in the glycosylated region, 70% identity in 

the globular domain, and 92% in the transmembrane 

and cytoplasmic regions. 

Using a KG1 cell line library, it has been shown that 

the human CD34 gene is located on chromosome 1, and 

recent studies with in situ hybridization have assigned 

its localization to band 1q32, in close proximity to other 

genes that encode growth factors or function molecules 

such as CD1, CD45, TGF2, laminin, LAM/GMP, 

etc. 28 

Seven CD34 monoclonal antibodies (MoAbs) were 

clustered at the 4th Workshop on Leukocyte Differentiation 

Antigens (Vienna, 1988), 30,31 and another 15 

MoAbs were verified as recognizing the CD34 molecule 

during the 5th International Workshop on Leukocyte 

Differentiation Antigens (Boston, 1983), the most direct 

evidence being reactivity with cells transfected with 

CD34 cDNA and binding to CD34 protein. 32,33 The epitope 

specificity of the CD34 antibodies was classified 

into three distinct groups according to the sensitivity of 

the epitopes to enzymatic cleavage (which was performed 

using neuraminidase, chymopapain and glycoprotease 

from Pasteurella haemolytica), reactivity with 

fibroblasts and high endothelial venules, and cross 

blocking experiments (Table 1). 34,35 We know, in fact, 

that glycoprotease from Pasteurella haemolytica specifically 

cleaves only proteins containing sialylated O- 

linked glycans. 34 Based on these data, it can be further 

postulated that class III epitopes are more proximal to 

the extracellular side of the cell membrane than class I 

and class III epitopes. 

Furthermore, for most CD34 MoAbs (with few exceptions) 

cross blocking experiments are in agreement with 

the classification based on enzymatic cleavage of the 

CD34 protein. In other words, using a cocktail of CD34 

MoAbs, CD34 reactivity is blocked only in the case that 

MoAbs belonging to the same CD34 epitope are simultaneously 

employed. On the contrary, the combined use 

of MoAbs recognizing class II and III or class I and II or 

class III epitopes does not affect cell reactivity. Moreover, 

CD34 MoAbs defining class III epitopes are unable 

to react with CD34 glycoprotein in Western blots 

because this epitope is sensitive to denaturation. 

The pattern of expression of CD34 antibodies exhibited 

by CD34 + acute leukemias is partially in accordance 

with that derived from epitope mapping based on the 

differential sensitivity of CD34 to enzymatic treatment. 

However, about one third of CD34 MoAbs do not seem 

to belong to any of these subgroups and for this peculiar 

pattern of expression are referred to as atypical 

CD34 reagents. The widest variation in CD34 MoAb 

reactivity has been demonstrated in acute myeloid 

leukemia (AML) samples, allowing us to postulate the 

occurrence of aberrant antigens or of distinct epitopes 

in subgroups of leukemias. Alternatively, it can be 

hypothesized that the expression of different antibod- 


4 


Lipid bilayer 

GPI anchor 

N-glycosylation site 

O-glycosylation site 

Figure 4. Schematic representation of the CD34 cell surface 

molecule. 

Fig. 4 

ies could reflect the degree of maturation of leukemic 

cells. The presence of new, distinct non overlapping epitopes 

could be proposed on the basis of the data published 

so far in the literature. As far as the expression of 

CD34 in normal and leukemic cells is concerned, it has 

been calculated by flow cytometry that the number of 

molecular equivalents of soluble fluorochrome (MESF) 

expressed by leukemic and normal progenitors ranges 

from 18,200 to 322,000 and from 8,000 to 124,000, 

respectively. 

Recent data collected by Lanza et al. 36 seem to indicate 

that class I-type MoAbs are more sensitive to freezing 

procedures than class II and III MoAbs, since the 

epitope is not identifiable following a frozen/thawed 

methodology. 

The function of CD34 surface glycoprotein in hematopoietic 

stem and progenitor cells is still the object of 

debate. In light of recent findings, it would seem to play 

a relevant role in modulating cell adhesion. 36 Furthermore, 

it has been demonstrated that CD34 probably acts 

as an adhesive ligand for L-selectin. It has been further 

postulated that the CD34 molecule could play a protective 

role against proteolytic enzyme-mediated damage 

due to its high number of O-glycosylation sites. The cytoplasmic 

domain contains two sites for protein kinase C 

phosphorylation and one for tyrosine phosphorylation. 28 

Given the discordant reactivity of these molecules, 

the choice of the CD34 MoAb to employ may be important 

when analyzing cell positivity for the CD34 molecule 

in both leukemic and normal samples, and may be 

responsible for the differences reported by various 

authors in the literature concerning the prognostic role 

played by this antigen in acute myeloid leukemias. 37 The 

type of CD34 MoAb used to enumerate progenitor cells 

is probably relevant in the peripheral blood stem cell 

autograft setting as well, since both early and late 

engraftment following transplantation are, to some 

extent, related to the number of hemopoietic stem cells 

collected at the time of blood or bone marrow harvests, 

and to the degree of progenitor cell maturation related 

to the expression of lineage markers such as HLA-DR, 

CD71, CD38, CD33, and myeloperoxidase. 

Techniques for CD34 + cell separation 

A number of different techniques have been proposed 

for separating CD34 + cells. The common aim is to produce 

a cell population with optimal purity and viability 

by means of a low cost, rapid and simple separation 

technique. The first separation techniques exploited 

parameters such as size and cell density and were represented 

by Ficoll-Hypaque and Percoll density gradi- 

Epitope class Clones CD34 reactivity 

paraffin frozen western 

section section blotting 

I. Sensitive to neuraminidase (from Vibrio 

cholerae), chymopapain, glycoprotease (from 

Pasteurella haemolytica) 

II. Resistant to neuraminidase. Glycoprotease, 

and chymopapain sensitive 

14G3, BI3C5, My10,* 12.8,* 

ICH3,* 

Immu-133, Immu-409 

43A1, MD34.3, 

MD34.1, MD34.2, QBend10, 

4A1, 9044, 9049 

positive negative positive 

positive positive positive 

III. Resistant to neuraminidase, chymopapain 

and glycoprotease 

CD34 9F2,HPCA2, 

581, 553.563, Tuk 3, 115.2 

negative positive negative 

Table 1. Epitope specificity of 

CD34 MoAbs as assessed by 

their differential sensitivity. 

*Incomplete digestion by neuroaminidase. 



ents. In the last two decades, the development of monoclonal 

antibodies has allowed a more specific and careful 

cell selection by identifying surface antigens used as 

targets for cell separation (Table 2). 

FACS (Fluorescence Activated Cell Sorter) 

Flow cytometry is able to physically separate different 

populations after incubation of cells with fluorochrome-conjugated 

monoclonal antibodies. This cell 

sorting technique can yield a highly purified (> 98%) 

cell population. In addition, the use of electronic gates 

allows selection and recovery of several subpopulations 

according to antigenic expression and different characteristics 

such as size and cytoplasmic granularity. This 

technique has been very useful for studying CD34 + subpopulations 

but cannot be employed to select large 

numbers of cells due to its complexity and low recovery. 

The recent development of high-speed cell sorting, 

however, might allow clinical utilization of this technique. 

Panning 

Anti-CD34 monoclonal antibodies bound to one of 

the surfaces of cell culture flasks were recently used to 

select CD34 + cells. When a cell suspension is introduced 

into the flask, the positive population is blocked on the 

plastic surface, while CD34 negative cells remain in suspension 

and can be easily eliminated. Adherent cells 

should contain the CD34 + population with a viability 

> 90%. 38 

Immunomagnetic systems 

Immunomagnetic beads are uniform, super-paramagnetic, 

polystyrene beads with affinity purified antimouse 

Ig covalently bound to the surface. They are 

equally suited for negative and positive cell separation; 

the rosetted target cell can easily be isolated by applying 

a magnet on the outer wall of the test tube for 1-2 

minutes. Immunomagnetic beads coupled with CD34 

monoclonal antibodies can be utilized for positive selection 

of CD34 + cells to obtain a population with > 80% 

viable CD34 + cells. 39 Similarly, immunomagnetic beads 

can be employed for negative depletion with monoclonal 

antibodies binding lineage-specific antigens. 

High-affinity chromatography based on 

biotin-avidin interaction 

This method is based on an immunoadsorption technique 

that relies on the high affinity interaction between 

the protein avidin and the vitamin biotin. Avidin-biotin 

immunoadsorption has been employed for both positive 

selection and depletion of specific cell populations. 40 

The instrument includes a set of non-reusable products 

including biotinylated anti CD34 antibody, plastic bags, 

filters and a column of avidin-biotin beads. An automated 

version controlled by a computer which guarantees 

reproducibility of operation and reduces risks of 

operator errors has been developed for clinical use. Its 

capacity has recently been increased so that a single 

column can process more than 50×10 10 bone marrow or 

peripheral blood cells in 1-2 hours and sustain bone 

marrow engraftment in patients submitted to autograft. 

41 

CD34-positive subpopulations: 

phenotypic and functional analysis 

The normal CD34 + cell population likely contains 

progenitors of all human lympho-hematopoietic lineages, 

including stem cells capable of hematopoietic 

reconstitution after bone marrow transplantation. 1 Levels 

of CD34 expression decline with differentiation; consequently, 

the earliest clonogenic cells (CFU-blast, LTC- 

IC, etc.) express the highest levels of CD34, while the 

most differentiated (CFU-G, CFU-M, CFU-E, CFU-Meg) 

express only low levels of CD34 (Figure 5). The CD34 

antigen has been used to identify, enumerate and isolate 

cells from different lympho-hematopoietic lineages, 

as well as develop in vitro tests for indirect evaluation 

of cells with different functional and clonogenic capacity. 

42 

Pre-clinical studies 

Several animal-human systems have been created to 

utilize chimeras for the study of lympho-hematopoiesis 

in vivo. The first experiments demonstrated the feasibility 

of transplanting human fetal stem cells to sheep 

fetuses; the postnatal presence of human cells in the 

sheep was documented at several points in time. 43 Furthermore, 

some early CD34 + subpopulations were able 

to repopulate sheep bone marrow; animals were transplanted 

in utero with CD34 + /DR – cells and the presence 

of a chimeric population with the functional characteristics 

of hemopoietic progenitors was demonstrated in 

the marrow and peripheral blood in a percentage of cases. 

44 Berenson et al. 45 also showed how hematopoietic 

progenitors (positive for the Ia antigen and subsequently 

for the CD34 antigen) could reconstitute the marrow of 

lethally irradiated dogs. Of the seven animals treated, all 

showed complete marrow engraftment after reinfusion 

of Ia-positive cells; only one dog died from infection. 45 

Similarly, marrow cells from 5 primates (baboons) were 

treated in vitro with a biotinylated anti-CD34 antibody 

and then passed through a column of avidin; after autograft, 

all the animals showed marrow engraftment followed 

by hematologic reconstitution comparable to that 

of control animals. 46 Furthermore, the demonstration 

that allogeneic CD34 + cells can reconstitute the hematopoietic 

system in lethally irradiated baboons confirmed 

that this cell population includes pluripotent 

stem cells. 25 

Lymphoid precursor cells 

The CD34 + cell compartment contains all the cells 

expressing terminal deoxynucleotidyltransferase (TdT), 

which is an intranuclear enzyme expressed in early lymphoid 

cells undergoing immunoglobulin or T-cell receptor 

gene rearrangement. Flow cytometry has shown that 

the great majority of TdT + cells in the marrow coexpress 

CD34, CD19 and CD10 (B-cell precursors), as well as T- 

cell differentiation antigens such as CD7, CD5 and CD2. 


6 


A small proportion of CD34 + /TdT + cells coexpress CD10, 

which might represent a common lymphoid progenitor 

for both B and T lineages. 47 Recently, Miller et al. reported 

that CD34 + cells may also generate NK cells in vitro. 48 

Granulocyte-macrophage precursors 

Marrow erythroid progenitors lack specific markers 

and therefore are difficult to identify. Glycophorin A- 

directed monoclonal antibodies recognize all hemoglobinized 

cells, but this molecule is expressed in only a 

small proportion of CD34 + cells and is absent in clonogenic 

cells. 49 

High levels of CD45 are present on BFU-E, but this 

antigen is lost by the CFU-E stage; 50 however, the 

CD45RO isoform is well represented on earlier erythroid 

progenitors, while the CD45RA isoform is present on 

committed myeloid progenitors. The expression of CD71 

(transferrin receptor) is currently considered to be the 

specific antigen for the CD34 + erythroid population. 

CD71 is present at high levels on erythroid progenitor 

cells and at very low levels in all the other progenitor 

cells. 51 Expression of CD71 increases from stem cells to 

BFU-E, then declines during erythroid maturation. In 

addition, marrow CD34 + erythroid cells might express IL- 

3, GM-CSF (CD116) and erythropoietin receptors, based 

on the action of these growth factors on CFU-erythroid 

cells. 52 

Myeloid precursor clonogenic cells (CFU-GM, CFU-G, 

CFU-M, CFU-MK) coexpress CD34, HLA-DR, CD117 (ckit), 

CD45RA, CD33 and CD13; CD15 is present at low 

levels on CFU-G, while CFU-M specifically express 

CD115 (M-CSF receptor). CFU-MK are the only CD34 + 

cells which express the platelet glycoproteins identified 

by the CD61, CD42 and CD41 monoclonal antibodies. 5 

Dendritic cells also originate from bone marrow, but the 

conditions that direct their growth and differentiation 

are still poorly characterized. GM-CSF stimulates the 

growth of dendritic cells from mouse peripheral blood; 

however, it was recently reported that CD34 + cells may 

give rise to dendritic/Langherans cells after stimulation 

with GM-CSF and tumor necrosis factor-α. 53 

Multilineage progenitors and stem cells 

CFU-GEMM contain precursor clonogenic cells of both 

myeloid and erythroid lineages and express CD34, HLA- 

DR, CD38, CD117 and CD45RA. They also express low 

amounts of CD33, but not CD13. In a hypothetical differentiation 

scheme involving pluripotent stem cells, the 

lympho-hemopoietic compartment is the next cell type 

and can be identified in vitro with CFU-Blast and LTC-IC. 

The lack of expression of CD38 is the most important 

characteristic of these early progeny, which represent 

1% of CD34 and less than 0.01% of mononuclear cells. 

The lack of CD38 allows separation of committed progenitors 

(CD34 + /CD38 + ) from earlier compartments 

(CD34 + /CD38 – ) by a single marker combination. 54 

Furthermore, the earliest CD34 + cells coexpress low 

levels of CD45RO6 and are negative for staining with the 

fluorescent dye rhodamine 123. The role of HLA-DR in 

defining earlier cell types is still controversial; a series 

Table 2. Recovery of CD34-positive cells after different separation techniques. 

Enrichment Recovery Large-scale 

separation 

(% CD34+ 

in the recovered 

(% CD34+ 

of the original 

population) sample) 

Negative depletion 

by immunomagnetic 

beads 

20-60 30-60 no 

Positive selection by 

immunomagnetic 

beads 

60-80 30-60 yes 

Fluorescence activated 

cell sorter (FACS) > 95 30 - 50 time consuming 

Panning 50 - 80 30 - 60 yes 

Ceprate SC® 50 - 80 40 - 70 yes 

of evidence indicates that the stem cell compartment 

has the CD34 + /CD38 – /HLA-DR + phenotype. 8,54 Recent 

works, however, have not found HLA-DR in the earliest 

cells. 55 These data were confirmed by the possibility that 

HLA-DR expression may discriminate the Ph-positive 

leukemic compartment (HLA-DR + ) from normal residual 

cells (HLA-DR – ) in chronic myeloid leukemia. 56 

Adhesion molecules and cytokine 

receptors 

A number of molecules within the integrin family have 

been shown to mediate interactions between early 

CD34-positive cells and bone marrow stromal cells. 

These include VLA-4/VCAM-1, VLA-5/fibronectin, LFA-1 

or ICAM, and several others. Each adhesion molecule 

appears to mediate a specific cell interaction. 

Hematopoietic growth factors may be active in a soluble 

form or in a membrane-anchored form; adhesion 

molecules may be crucial for allowing anchored growth 

factors to bind the target cell. Table 3 lists the main 

adhesion molecule and growth factor receptors 

expressed on CD34 + cells. 

CD34 expression on normal and neoplastic 

cells 

It has been known that CD34 monoclonal antibodies 

bind specifically to vascular endothelium ever since a 

110 kd protein extracted from freshly isolated umbilical 

vessel endothelium was identified with CD34 antibodies 

in Western blots and in Northern blot analysis. 57,58 

CD34 molecules have a striking ultrastructural localization 

on endothelial cells: they are concentrated primarily 

on the luminal side, in particular on membrane 

processes, many of which interdigitate between adjacent 

endothelial cells. 57,58 Since this region is an important 

site for leukocyte adhesion and transendothelial 



traffic, in contrast to previous experiences, 33 it has been 

hypothesized that CD34 may be antagonistic or 

inhibitory to the adhesive functions of vascular endothelium. 

This was supported by the demonstration that 

CD34 gene expression at the mRNA level is reciprocally 

down-regulated when adhesion molecules ICAM-1 

and ELAM-1 are up-regulated by IL-1 during inflammatory 

skin lesions associated with leukocyte infiltration. 

33,59 Furthermore, additional studies conducted on 

both paraffin embedded and cryopreserved sections 

demonstrated that fibroblasts also react with anti-CD34 

MoAbs. 60 However, it should be noted that while CD34 

MoAbs reacted with all classes of epitopes on cryopreserved 

sections, class III epitopes were not recognized by 

specific anti-CD34 MoAbs on paraffin embedded sections. 

Levels of CD34 expression, highest in immature 

hematopoietic precursor cells, decrease progressively 

with cell maturation. 

Regarding hematologic malignancies, CD34 is 

expressed in a large percentage of acute leukemias. 61 

The fluorescence intensity of CD34 expression is variable 

and higher in acute lymphoblastic (ALL) than in acute 

myeloblastic leukemia (AML). In these latter patients, 

the CD34 antigen is found on 40-60% of leukemic blasts 

and is most frequently associated with M0, M1 and M4 

FAB cytotype, secondary leukemias, karyotypic abnormalities 

involving chromosome 5 or 7, P170 expression 

and poor prognosis. 61-64 Thus, CD34 expression may be 

considered the most predictive negative factor in AML 

patients strictly correlated with intensity of expression. 

65,66 In ALL, CD34 is expressed in 70% of patients, 

particularly in those with a B-lineage phenotype. In 

these patients, unlike AML cases, the clinical relevance 

of CD34 expression is controversial; however, according 

to the findings of a Pediatric Oncology Group, 67 its 

expression in B-lineage cases was associated with 

hyperdiploidy, lower frequency of initial central nervous 

system (CNS) leukemia and a favorable prognosis. In T- 

cell ALL cases, on the other hand, CD34 expression 

showed a positive correlation with initial CNS leukemia 

and CD10 negativity, but not with any presenting favorable-risk 

characteristics. 67,68 

Lastly, CD34 and HLA-DR expression may be very useful 

in discriminating between the very few benign primitive 

hematopoietic progenitors and their malignant 

counterparts in patients with chronic myeloid leukemia 

(CML). In fact, it has been demonstrated that normal 

progenitor cells are CD34 + and HLA-DR – , while malignant 

progenitor cells, which exhibit Ph and bcr/abl 

rearrangement, express HLA-DR antigens. 56 

Positive and negative regulators of 

hematopoietic progenitor cells 

The hematopoietic stem cell is defined by its extensive 

self-renewal capacity, multilineage differentiation 

potential and capacity for of long-term reconstitution 

of normal marrow function in lethally irradiated animals. 

68 Transplantation of retrovirally marked murine 

Figure 5. Functional differentiation and antigenic expression of hematopoietic cells. 


8 


Table 3. Adhesion molecule and growth factor receptors 

expressed on CD34-positive cells. 

Antigen name CD Ligand 

GlyCAM-L/selectin none adhesion molecule 

ICAM1,2,3 CD11a/CD18 adhesion molecule 

H-CAM CD44 adhesion molecule 

VLA-4 CD49d/CD29 adhesion molecule 

VLA-5 CD49d/CD29 adhesion molecule 

FGF-R none growth factor 

M-CSF CD115 growth factor 

GM-CSF-R CD116 growth factor 

SCF (c-kit) CD117 growth factor 

Interferon γ-R CD119 growth factor 

IL 7-R CD127 growth factor 

stem cells has shown that only a few multilineage progenitor 

cells induce the repopulation of engrafted 

hematopoietic tissue, 69 suggesting that hematopoiesis 

may be supported by a succession of short-lived clones. 

Moreover, experimental evidence indicates that the 

processes of self-renewal, differentiation and selection 

of lineage potentials are intrinsic properties of the stem 

cells and occur according to a stochastic (random) model. 

70 In the steady state, most of the stem cells are quiescent 

(G0 phase) and begin active cycling randomly. 71 

Conversely, survival and proliferation of hematopoietic 

progenitors are regulated by cytokines, which also act 

in preventing apoptosis. 72 According to this model, the 

induction of differentiation by a cytokine may be considered 

as the consequence of the proliferation of a specific 

population stimulated by that factor. The broad 

term cytokines includes growth factors such as fibroblast 

growth factor, colony stimulating factors (CSFs) (e.g. 

granulocyte-CSF, granulocyte-macrophage-CSF), and 

interleukins (ILs). Depending on their target cells and 

different proliferative potential, cytokines can be divided 

into three categories: 71 1) late-acting, lineage-specific 

factors; 2) intermediate-acting, lineage-nonspecific 

factors; 3) early-acting growth factors inducing the 

recruitment of dormant early progenitors in the cell 

cycle (Figure 6). 71 

The majority of late-acting factors support the proliferation 

and maturation of lineage-committed progenitors 

and the functional properties of terminally differentiated 

cells. Erythropoietin (Epo) is the physiologic 

regulator of erythropoiesis 73 and thrombopoietin has the 

same role in thrombopoiesis, 74 while M-CSF and IL-5 

are considered specific for macrophages and eosinophils, 

respectively. G-CSF exerts its activity on committed 

neutrophil precursors, although it has been shown to 

be a synergistic factor for primitive hematopoietic 

cells. 75 

Intermediate-acting, lineage-nonspecific factors 

include IL-3, IL-4 and GM-CSF. Their activity is mainly 

directed toward progenitor cells in the intermediate 

stages of hematopoietic development. However, they 

interact with later-acting growth factors for the production 

of more mature cells, 76 as well as with the 

cytokines capable of triggering stem cell cycling. On 

their own, they act as survival factors for stem cells and 

appear to stimulate early hematopoietic precursors only 

after their exit from G0. 77 

Several cytokines have recently been identified for 

their capacity to stimulate the proliferation of the earliest 

hematopoietic cells. Mapping studies of normal 

blast cell colony formation from single progenitors have 

shown that IL-6, G-CSF, IL-11, stem cell factor (SCF), IL- 

12 and leukemia inhibitory factor (LIF) recruit dormant 

stem cells into the cell cycle that are then able to 

respond to additional growth factors. 77 Whereas the permissive 

factors retain limited proliferative potential by 

themselves, they strongly enhance the stimulatory activity 

of IL-3, GM-CSF, G-CSF and EPO on CD34 + and more 

immature CD34 + lineage-progenitors. 78 In addition to 

positive interaction with intermediate-and late-acting 

growth factors, SCF synergistically or additively augments 

the colony-forming ability of other early-acting 

growth factors, such as IL-11, IL-12 and IL-6, on myeloid 

and bilineage (i.e. lymphomyeloid) primitive cells. 79 

Recently, the ligands for the STK-1 or FLT3 receptor, and 

the hepatocyte growth factor were shown to be able to 

stimulate very primitive hematopoietic progenitor cells. 

However, their biological activity is still under investigation. 

The main sources of both positive and negative regulatory 

proteins are accessory myeloid cells and the 

stromal component of the bone marrow. In general, 

microenvironment cells do not constitutively produce 

cytokines, rather transcription and/or translation 

processes are rapidly induced by cytokines such as IL-1, 

TNF. The extracellular matrix also participates in the 

regulation of hematopoiesis by binding growth factors 

and presenting them in a biologically active form to 

bone marrow progenitor cells. 80 

Most data suggest that stem cells express low levels 

of growth factor receptors and require multiple proliferative 

stimuli to enter into the cell cycle, while committed 

progenitor cells can be effectively stimulated by 

individual cytokines. 23 Combinations of two or more 

growth factors 81 can stimulate hematopoietic cells 

either by amplifying the progeny cell production of single 

precursors (synergy) or by inducing additional clonogenic 

cells to proliferate (recruitment). Examples of 

these two types of enhancement are given in Figures 7 

and 8, where CD34 + CD33 – DR – cells are simultaneously 

stimulated by two or three growth factors. The molecular 

basis regulating the complex interplay between 

cytokines is still largely unknown. However, one proposed 

mechanism for growth factor synergism is the 



induction of CSF receptors on early hematopoietic progenitor 

cells. 82 This appears to be a coordinate cascade 

transactivation via up-modulation of growth factor 

receptors that leads to proliferation and differentiation 

of human marrow cells. 83 Conversely, the structural 

homology between some growth factors, 84 the presence 

of shared receptor subunits on the cell membrane 85 and 

common signal-transduction proteins 86 provide a potential 

explanation for their functional similarities and the 

apparent redundancy of their activity. 

The growth of hematopoietic progenitor cells is also 

regulated by soluble negative regulators such as 

macrophage inflammatory protein-1a (MIP-1a), tumor 

necrosis factor-α (TNF-α), interferons (IFNs), prostaglandins 

and transforming growth factor-β (TGF-β). The 

TGF-β family of proteins includes at least five isoforms 

(TGF-β1-5) which are encoded by different genes 87 and 

produced by stromal cells, platelets and bone cells. 

Moreover, a subset of very primitive murine hematopoietic 

cells has been shown to secrete active TGF-β1 by an 

autocrine mechanism. 88 TGF-β1 and 2 isoforms are 

bimodal regulators of murine and human hematopoietic 

progenitor cells, and their activity is based upon the 

differentiation state of the target cells and the presence 

of growth factors. 89 In the human system, for 

instance, CFU-GEMM derived from purified CD34 + 

CD33 – cells are inhibited by TGF-β1, whereas more committed 

progenitors such as CFU-G or CFU-GM are not 

affected. In addition, high proliferative potential-CFC 

(HPP-CFC) responsive to a combination of CSFs are 

markedly inhibited by TGF-β1, while more mature CFU- 

GM are actually enhanced when GM-CSF is used as 

colony forming factor. 78 TGF-β1 and 2-induced myelosuppression 

is partially counteracted by early-acting 

growth factors such as G-CSF, IL-6 and fibroblast 

growth factor. 90 On the other hand, TGF-β3 has been 

shown to be a more potent suppressor of human BM 

precursors and its activity on hematopoiesis is only 

inhibitory, 89 although the synergistic growth factors IL- 

11 and IL-9 seem to be able to oppose the negative regulation 

of TGF-β3 on human CD34 + cells. 91 Several 

potential modes of action of the TGF-β family have been 

suggested, including down-modulation of cytokine 

receptors, 92 interaction with the underphosphorylated 

form of the retinoblastoma gene product in late G1 

phase, 93 and alteration of gene expression. 94 Early studies 

have shown that TGF-β1 and 3 exert their activity on 

normal and leukemic cells by lengthening or arresting 

the G1 phase of the cell cycle, 95 and this effect is functional 

to protect normal CD34 positive cells from the 

toxicity of alkylating agents in vitro. 96 More recent 

investigations have demonstrated that TGF-β regulates 

the responsiveness of mice hematopoietic cells to SCF, 

which is known to be the main synergistic factor for 

both murine and human stem cells, through a decrease 

in c-kit mRNA stability that leads to decreased cell-surface 

expression. 97 

Similarly to TGF-β, TNF-α has been reported to have 

both inhibitory and stimulatory effects on hematopoietic 

progenitor cells. TNF-α potentiated the IL-3 and 

GM-CSF-mediated growth of human CD34 + cells in 

short-term liquid culture assay. However, it inhibited 

STEM CELLS 

SCF 

 

IL-6 

 

 

IL-1 

 

IL-3 

IL-9 

IL-11 

IL-12 

G-CSF 

GM-CSF 

PROGENITOR 

CELLS 

IL-4 

G-CSF 

 

IL-3 

 

SCF 

M-CSF 

IL-5 

 

IL-3 

EPO 

IL-9 

SCF 

 

SCF 

IL-3 

 

IL-6 

IL-11 

 

GM-CSF 

GM-CSF 

GM-CSF 

MATURING 

CELLS 

 

 

 

IL-5 

 

 

EPO 

 

 

 

IL-6 

 

EPO 

 

G-CSF 

GM-CSF 

M-CSF 

MEG-CSF 

 

Neutrophils 

 

 

 

 

Red cells 

Platelets 

Figure 6. Cytokines exert their activity at different levels of the hematopoietic differentiation pathway. Each progenitor cell is 

concurrently affected by multiple regulators. 


10 


the growth promoting activity of G-CSF. 98 Two TNF 

receptors with molecular weights of 55 and 75 kd, 

respectively, have recently been identified. 99 Whereas 

the p55 receptor mediates TNF-α effects on committed 

progenitor cells, the p75 receptor is involved in signaling 

the inhibition of murine primitive cells. 

Furthermore, it was recently shown that TNF-α is 

capable of inhibiting the multi-growth factor (GM-CSF, 

IL-3, G-CSF, SCF, IL-1)-dependent growth of human 

HPP-CFC derived from CD34 + cells through interaction 

with both p55 and p75 receptors, while the p55 receptor 

exclusively mediates the bifunctional activity of TNFα 

on more mature BM precursors responsive to single 

cytokines. 100 

MIP-1a is a peptide of 69-amino acids with a molecular 

weight of 7.8 kd produced by activated macrophages, 

T-cells and fibroblasts. 101 It belongs to a large 

family of putative cytokines that includes MIP-1β, MIP- 

2 and IL-8 (chemokines). Biologic characterization has 

shown that MIP-1α enhances the M-CSF- and GM-CSFdependent 

growth of CFU-GM, while it inhibits the 

colony-forming ability of hematopoietic precursors present 

in a cell population enriched for CD34 ++ DR + cells 

stimulated with erythropoietin, IL-3 and GM-CSF. 102 

Figure 7. Human CD34 + CD33- DR- cells were stimulated by 

IL-9 (A) or IL-9 and SCF (B) in the presence of EPO. The addition 

of SCF induced the growth of large multicentered BFU- 

E colonies containing > 10,000 cells. The different size of 

the colonies indicates amplification of stem cell progeny 

(synergy). 

A 

B 

Taken together, these results indicate that a complex 

interplay between positive and negative regulatory proteins 

determines the proliferation or inhibition of early 

hematopoietic progenitor cells (Figure 9). In general, the 

activity of inhibitors of hemopoiesis appears to be 

reversible, lineage-nonspecific and directed at the early 

stages of differentiation. In addition, TGF-β has shown 

some degree of differential activity between normal and 

neoplastic lymphoid cells. 96 Thus, negative regulators 

may be clinically relevant to the protection of the hematopoietic 

stem cell compartment from the dose-limiting 

toxicity of neoplastic disease therapy. 96,103,104 

Collection of CD34 + cells 

Bone marrow and peripheral blood are the only 

sources of immature hemopoietic precursors identified 

as CD34 + cells. A diagnostic marrow sample contains 

only a few CD34 + cells, while even fewer of them are 

present in peripheral blood samples taken under steady 

state conditions. Large quantities of CD34 + cells can be 

collected with massive marrow harvests, such as for 

transplantation purposes. Nevertheless, marrow CD34 + 

cells are somewhat elusive due to their scattering 

among the predominant CD34 + hematopoietic population. 

105 Recently developed cell separation procedures 

allow collection of highly enriched CD34 + cell populations. 

However, this is generally accomplished through 

aspecific and often unacceptable cell loss. 106 So far the 

limited number of immature precursors commonly 

obtained from both bone marrow and peripheral blood 

has been the major obstacle to a simple identification 

and analysis of marrow CD34 + cells. Indeed, the growing 

interest in CD34 + cells is primarily the result of the 

development of new therapeutic modalities that allow 

easy access to large quantities of hemopoietic precursors 

through the peripheral blood. The key role in these 

innovative approaches is represented by the introduction 

of hemopoietic growth factors for clinical use. 107 

At present GM-CSF and G-CSF are the most extensively 

employed and studied hemopoietic growth factors 

in the clinical setting. From the very beginning, it was 

observed that GM-CSF or G-CSF administration was 

associated with an increase in circulating hemopoietic 

progenitors. 108,109 

Later on, it was demonstrated that this phenomenon 

could be extensively and reproducibly amplified by combining 

growth factor administration with high-dose 

chemotherapy. 110-112 

Indeed hemopoietic progenitors are massively, though 

transiently, mobilized into peripheral blood during 

hemopoietic recovery following high-dose chemotherapy 

given with growth factor support. Such an abundance 

of immature hemopoietic cells makes them easily 

recognizable by cell sorting techniques that employ 

anti-CD34 MoAbs. 113 Under optimal conditions, the proportion 

of CD34 + cells may reach values as high as 20- 

30% of the total leukocyte count. In steady state conditions 

CD34 + cells do not exceed 4% of the total marrow 

population, while they are undetectable by cell sort- 



Figure 8. The addition of SCF to IL-11, in the presence of 

EPO, results in a much higher number of colony-forming 

cells derived from CD34+ CD33– DR– progenitors (right), as 

compared to the IL-11 and EPO combination (left) (recruitment). 

ing techniques in the peripheral blood. Chemotherapy 

and growth factors do not merely induce a relative 

increase of immature hemopoietic cells; their absolute 

number is amplified several times over basal conditions. 

This allows collection of sufficient amounts of hemopoietic 

progenitors for autografting purposes by means 

of a few leukapheresis procedures. 110,113,114 

Values of 10-20×10 4 CFU-GM/kg represent the minimal 

required dose for marrow engraftment with peripheral 

blood progenitors. 115-117 In fact, much higher quantities 

of circulating progenitors can be collected using 

appropriate mobilization procedures. Under optimal 

conditions, circulating CD34 + cell peak values may range 

between 150 and 700/µL on days of maximal mobilization. 

As a rule, at least 8×10 6 CD34 + cells/kg or more can 

thus be collected with 1 to 3 leukapheresis procedures. 

114 These huge quantities, approximately corresponding 

to 50×10 4 CFU-GM/kg, must be considered 

the ideal threshold dose of peripheral blood progenitors 

for autografting purposes. Indeed values of 8×10 6 CD34 + 

cells/kg or more guarantee a rapid and durable hemopoietic 

recovery when circulating progenitors are used 

as the sole source for marrow reconstitution following 

submyeloablative treatment. 118-121 

Thus far, massive CD34 + cell mobilization has been 

most commonly observed when growth factor is administered 

following high-dose cyclophosphamide, given at 

7 g/m 2 . Indeed chemotherapy that induces profound 

leukocytopenia seems to be crucial for optimal mobilization; 

for instance, cyclophosphamide at doses lower 

than 7 g/m 2 produces a reduced mobilizing stimulus. 

111,122 Several other chemotherapy schedules have 

also been successfully employed for mobilization purposes. 

The principal chemotherapy protocols reported 

to be highly effective in inducing CD34 + cell mobilization 

are summarized in Table 4. 

110, 111, 115, 117, 122-127 

As stressed earlier, hemopoietic growth factors play a 

key role in mobilization. This has been clearly documented 

with cyclophosphamide. A median peak value of 

75 CD34 + cells/µL has been recorded following highdose 

cyclophosphamide alone, whereas 420 and 500/µL 

are the median values recorded when GM-CSF or G- 

CSF, respectively, are added to cyclophosphamide. 

112,113,128,129 Extensive growth factor-induced mobilization 

is further substantiated by the possibility of collecting 

sufficient CD34 + cells using growth factor 

alone. 130,131 In this setting the most promising experiences 

have been produced with G-CSF. The results 

reported indicate new opportunities for the utilization 

of mobilized progenitors in allogeneic transplantation 

procedures. 132,133 Combined chemotherapy and growth 

Self renewal 

Differentiation 

Negative Positive Survival 

IFN , , TGF- 1-2 

TGF- 3 TNF- 

MIP-1 

SCF 

IL-11 

IL-12 

IL-6 

G-CSF 

IL-1 

IL-9(?) 

IL-11 

GM-CSF 

Figure 9. Interplay between positive 

and negative regulatory proteins 

acting on hematopoietic 

stem cells. 


12 


factors remain the most efficient mobilization procedure 

at this time. However, ongoing studies are directed 

at improving mobilization efficiency with growth factors 

alone. For example, G-CSF at doses higher than 5 

µg/kg/day up to 20 µg/kg/day may have higher mobilization 

capacity. 134 Further improvement might derive 

from the clinical availability of new cytokines, such as 

IL-3, SCF and others, to be employed alone or in combination 

with G- or GM-CSF. 135-137 However, the potentially 

high mobilizing activity of such new cytokine combinations 

must be accompanied by no or very few side 

effects in order to be considered for a wide clinical 

applicability. 

Mobilizing protocols generally include daily delivery 

of growth factors, starting 1 to 3 days after chemotherapy 

administration and continuing until harvesting procedures 

are completed. Growth factor is usually administered 

for a total of 7-12 consecutive days. The most 

convenient route of delivery is subcutaneous, with 1 to 

2 doses per day. Progenitor cell harvests are performed 

during hemopoietic recovery, provided that progenitor 

cell mobilization is documented. Indeed various parameters 

have been considered as an indirect indication of 

progenitor mobilization, including an increase in WBC or, 

alternatively, in monocytes, basophils or platelets. 138-140 

However, CD34 + cell evaluation remains mandatory for 

an accurate definition of the extent of progenitor mobilization. 

141,142 

CD34 + cells should be monitored daily from the early 

Table 4. Main chemotherapy protocols employed in hematopoietic progenitor 

mobilization. 

Authors Protocol Drug(s) Dosage 

characteristics employed 

Gianni et al.110 single agent cyclophosphamide high 

Tarella et al.123 single agent etoposide high 

Gianni et al.124 

Kotasek et al.121 single agent cyclophosphamide intermediate 

Tarella et al.122 

Dreyfus et al.125 single agent cytarabine high 

Schimazaki et al.126 multiple drugs etoposide high 

cytarabine 

Kawano et al.115 multiple drugs daunorubicin intermediate 

cyclophosphamide 

etoposide 

Pettengell et al.127 multiple drugs doxorubicin intermediate 

cyclophosphamide 

etoposide 

Hass et al.117 multiple drugs cytarabine high 

mitoxantrone 

stages of hemopoietic recovery. Detection of circulating 

CD34 + cells is not sufficient reason for starting leukapheresis 

procedures; an adequate number of CD34 + cells 

(>20-50/µL) and WBC > 1.0×10 9 /L and platelet count 

>30×10 9 /L are required for safe and effective progenitor 

cell harvesting. 114,142 Leukaphereses are performed 

using continuous-flow blood cell separators. A complete 

leukapheresis procedure generally takes 2-3 hours and 

the total blood volume processed ranges from 6 to 10 

liters. 114,140,143,144 The harvested cells are resuspended in 

freezing medium and then cryopreserved for subsequent 

transplantation. One to 3 leukaphereses repeated on 

consecutive days usually provide large amounts of progenitor 

cells capable of rapid engraftment after submyeloablative 

treatments. 

Factors and procedures favoring CD34 + cell mobilization 

have been well established. However, other conditions 

adversely influencing the mobilization phenomenon 

should be considered. A major limitation is represented 

by impaired marrow function, as can occur in 

previously treated patients. 111 In fact, patients at first 

relapse following a single treatment protocol maintain 

an adequate mobilization capacity; 145 however, few if 

any mobilized CD34 + cells can be obtained from heavily 

treated patients previously exposed to multiple 

chemotherapy courses. Mobilization is also profoundly 

reduced and often totally abolished in patients previously 

exposed to radiotherapy, especially if it was delivered 

to the pelvis or to extended vertebral areas. 117,123 

Lastly, marrow invasion by tumor cells may negatively 

affect mobilization capacity. This is typically reported in 

myeloma patients in whom the extent of CD34 + cells 

often correlates with the degree of marrow involvement 

by tumoral plasma cells. 122 In conclusion, several new 

findings have dramatically improved the procedures for 

collection of large amounts of CD34 + cells. However, 

optimal marrow function is a prerequisite for exploiting 

fully the activity of all those factors known for their 

mobilization-inducing capacity. 

CD34 + positive cells in umbilical cord blood 

Significant numbers of human hematopoietic stem 

cells can be found in umbilical cord blood and can be 

used for allogeneic bone marrow transplantation. 

Mayani et al. 146 found that 1-2% of the total number of 

cord blood-derived low-density cells express high levels 

of the CD34 antigen and low or undetectable levels of 

the antigens CD45RA and CD71. These populations were 

highly enriched in clonogenic cells (34%), in particular 

in multipotent progenitors (12%). By culturing these 

cells at low concentration for 8-10 days in highly 

defined serum-free liquid cultures supplemented with 

various hematopoietic cytokines, it was possible to 

achieve a significant expansion (about 100-fold) of the 

CD34 + cell population. 

These last results were recently confirmed by Traycott 

et al. 147 in an elegant study showing that umbilical cord 

blood CD34 + cells rapidly exit G0-G1 phases and start to 

cycle in response to stem cell factor. This feature would 



make cord blood CD34 + cells more suitable candidates 

than bone marrow cells for ex vivo expansion. 

It is clear that in vitro expansion and maturation of 

hematopoietic progenitor cells might be of particular 

relevance for transplantation of cord blood hematopoietic 

cells; however, information about the effects of 

such an expansion on the cells required for long-term 

hematopoietic reconstitution is highly desirable. 

The dual role of peripheral blood 

hematopoietic progenitor cells in oncohematology 

CD34 + progenitor cells circulating in the peripheral 

blood represent an enriched and easily accessible source 

of two distinct cell populations: committed progenitor 

cells and hematopoietic stem cells. 

Although circulating progenitors are commonly called 

stem cells, the presence of circulating stem cells has 

been formally proved only in mice. 148 In humans the 

presence of hematopoietic stem cells in the peripheral 

blood is almost certain (see below), but to call reinfusion 

of circulating progenitors transplantation of 

peripheral blood stem cells (PBSC or similar acronyms) 

is hardly appropriate. This terminology overlooks the fact 

that the tremendous interest in peripheral blood autografting 

is not (at least so far) a consequence of its being 

a simple surrogate for autologous bone marrow transplantation 

(as the term PBSC would suggest), but rather 

derives from the unique property of this procedure to 

reduce the duration of the severe pancytopenia that follows 

submyeloablative treatments from two-three 

weeks (when bone marrow cells are used) to a few days 

only. 110,149 The reason for the rapid recovery which occurs 

after circulating progenitor autotransfusion (CPAT) has 

not been formally proved, but it is most likely a consequence 

of the much larger (10- to 100-fold higher) 

amount of committed progenitors reinfused when a 

patient is autografted with blood-derived (as opposed to 

marrow-derived) cells. The most likely hypothesis is that 

these late progenitors (post-progenitors) of granulocytes 

and platelets are capable of giving rise to mature 

progeny within a few days, thus allowing submyeloablated 

patients to survive the initial aplastic phase. 

The fundamental role of committed progenitor cells 

was elegantly proved in mice by Jones et al. 150 None of 

the lethally irradiated animals transplanted with a pure 

population of stem cells free of more mature progenitors 

(CFU-GM, CFU-S) survived the initial aplasia. A 

more recent paper 151 challenged this ‘conventional wisdom’ 

model, and maintained that peripheral blood stem 

cells per se are capable of rapidly maturing in vivo. 

Other authors, using a very similar approach, reached 

an opposing conclusion. 152 In humans the most convincing, 

albeit indirect, evidence in favor of the role of 

committed progenitors in accelerating post-transplant 

recovery was provided by Robertson et al., 153 who documented 

a significantly prolonged hematopoietic recovery 

for patients undergoing autologous bone marrow 

transplantation purged ex vivo with anti-CD33 monoclonal 

antibodies. This result, which occurred after a 

treatment that selectively kills the most mature progenitor 

cells (expressing the CD33 surface antigen), represents 

convincing indirect proof of the role of committed 

progenitors in early engraftment. 

The clinical role of committed progenitors in reducing 

the morbidity and mortality of high-dose therapy 

has expanded since hemopoietic growth factors have 

become clinically available. In fact, infusion of rhGM- 

CSF or rhG-CSF, in particular after administration of 

myelotoxic doses of certain stem cell-sparing agents, 

allows easy collection of an amount of CFU-GM/kg body 

weight 10 to 100 times higher than that contained in a 

bone marrow harvest. 110 

As already documented by a large number of papers, 

the use of circulating progenitors has brought about a 

dramatic change in the perspectives of high-dose submyeloablative 

regimens. In fact, these once specialized, 

expensive and highly toxic treatments are now well tolerated, 

easy to administer, and clinically useful (cost 

effective). As an example, it is worth mentioning the 

initial Milan Cancer Institute experience in a group of 

over 50 poor-risk breast cancer patients who received 

high-dose melphalan plus an optimal amount of circulating 

progenitors (≥ 5×10 4 CFU-GM/kg body weight). 

These patients required a median of 10.5, 11 and 12.5 

days to score > 0.5, 1.0 and 2.0×10 9 /L neutrophils/µL, 

respectively. Moreover, more than half of them did not 

require platelet transfusions, while the remaining ones 

needed only one or two transfusions during the first 

week after autografting. Such mild to moderate toxicity 

was never described before the clinical availability of 

committed progenitor cells. 

In conclusion, born as a compassionate surrogate of 

bone marrow autografting, today CPAT is rapidly replacing 

ABMT. In fact, today it is the latter that should be 

considered a compassionate need procedure, useful in 

those few patients unable to mobilize a sufficient number 

of circulating progenitor cells. 

The second, distinct role of circulating progenitors is 

related to the presence of stem cells, i.e. totipotent precursors 

responsible for durable reconstitution of all lympho-hematopoietic 

lineages following marrow ablative 

therapy. Since virtually no high-dose treatment that can 

be safely administered to humans is genuinely myeloablative, 

formal proof of the existence of circulating 

stem cells must await either stable transduction of DNA 

markers into autografted cells or the use of this cell 

population for allografting. 

These experiments are presently underway in several 

laboratories, 154,155 and preliminary data do confirm the 

presence of stem cells in the peripheral blood of humans. 

Their utilization is expected to revolutionize fields like 

allogeneic bone marrow transplantation and somatic 

gene therapy, whenever the target of genetic manipulations 

is the hematopoietic cell. 156 


14 


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eview 

Peripheral blood stem cells in acute 

myeloid leukemia: biology and clinical 

applications 


MASSIMO AGLIETTA, ARMANDO DE VINCENTIIS, LUIGI LANATA, 

FRANCESCO LANZA, ROBERTO M. LEMOLI, GIACOMO MENICHELLA, 

AGOSTINO TAFURI, PAOLA ZANON, SANTE TURA 

Clinica Medica, Università di Torino, Novara; Dompé Biotec 

SpA, Milano; Amgen Italia SpA; Cattedra di Ematologia, Università 

di Ferrara, Ferrara; Istituto di Ematologia Seràgnoli, 

Università di Bologna, Bologna; Servizio di Ematologia ed 

Emotrasfusione, Università Cattolica del Sacro Cuore, Roma; 

and Cattedra di Ematologia, Università La Sapienza, Roma 

Correspondence: Prof. Sante Tura, Istituto di Ematologia L. e A. 

Seràgnoli, Policlinico S. Orsola, via Massarenti 9, 40138 Bologna, 

Italy. 

Received September 22, 1995; accepted November 27, 1995. 

Ackowledgments: preparation of this manuscript was supported by a 

grant from Dompé Biotec SpA and Amgen Italia SpA, Milan, Italy. 

Clinical application of circulating stem cells for 

autologous transplantation is steadily expanding. 1 

It has become increasingly clear that mobilized 

peripheral blood progenitor cells (PBSC) induce faster 

hematopoietic recovery, fewer febrile days, lower transfusion 

requirement and shorter hospitalization than 

bone marrow (BM)-derived cells. 2,3 More recently, rapid 

and sustained engraftment has also been reported using 

granulocyte colony-stimulating factor (G-CSF)-mobilized 

allogeneic PBSC following myeloablative therapy. 4 

In contrast to solid tumors and many hematological 

malignancies, PBSC transplantation is not widely used 

for acute myeloblastic leukemia (AML) patients. In this 

setting there are still unanswered questions such as the 

role of autologous stem cell transplantation in postremission 

therapy, as well as major issues concerning 

PBSC mobilization and collection: the expression of 

CD34 antigen on leukemic stem cells as compared to 

their normal counterparts, the biologic significance of 

CD34 + AML, the response of leukemic cells to CSFs used 

to optimize PBSC harvest, the potential contamination 

of PBSC grafts by residual AML cells and the role of exvivo 

purging of leukemic cells. 

This review analyzes the most recent advances in this 

field, addressing clinical and biological issues relevant 

to the use of autologous PBSC for AML patients. 

Growth factor receptor expression and 

response of leukemic cells to human CSFs 

The CD34 antigen is a 105-120 KD glycoprotein 

expressed on the cell surface of hematopoietic progenitors 

and stem cells, but it is not expressed on late 

hematopoietic cells or on many tumor cells. 1 CD34 + cells 

are responsible for the self-renewal and the expansion 

of the large majority of AML. It has recently been shown 

that most of the clonogenic cells in AML derive from the 

CD34 + cell fraction as opposed to CD34 – cells. 5,6 Moreover, 

CD34 + cells co-expressing differentiation markers 

(CD33, CD38) have a reduced proliferative potential 

since in vitro they give rise to small colonies unable to 

originate secondary clones. This phenomenon is likely 

the expression of a limited self-renewal potential. 6 Lapidot 

et al. provided the most convincing evidence of the 

stem cell role of CD34 + cells in AML by showing that 

only the CD34 + /CD38 – cell fraction was capable of generating 

acute leukemia when transplanted into SCID 

mice. 7 

These observations indicate the relevance of defining 

the growth and receptor expression pattern of leukemic 

CD34 + cells, their response to CSFs as well as their 

kinetic status compared to their normal counterparts. 

Among the different cytokines involved in the regulation 

of hemopoiesis, a key role in the pathogenesis of 

the leukemic growth is probably played by stem cell 

factor (SCF), interleukin 3 (IL-3), granulocyte-macrophage 

CSF (GM-CSF) and G-CSF. 8-12 

SCF receptor (c-kit) is expressed by the vast majority 

of AML. 8,9 Both high and low affinity receptors have 

been demonstrated (Table 1). c-kit shares structural 

similarities with the receptors for M-CSF and PDGF. A 

linear correlation between the percentage of CD34 + 

cells and c-kit expression has been documented, thus 

indicating that CD34 + AML express high levels of c-kit. 

In adult patients, the presence of a high number of 

CD34 + cells has been shown to correlate with a bad 

prognosis. 

c-kit activation plays a foundamental role in the regulation 

of the early phases of CD34 + cell stimulation. 

The interaction of SCF with its ligand exerts a modest 

proliferative stimulus on immature quiescent cells and 

up-regulates the expression of receptors for other 

growth factors. While in normal hematopoiesis this triggers 

myeloid differentiation, in AML it may activate 

self-renewal and expansion of the leukemic population. 

11-14 

High affinity receptors for GM-CSF and IL-3 (Table 1) 

are expressed by nearly all AML, irrespective of the FAB 

subtype. 15,16 IL-3, GM-CSF (and IL-5) receptors consist of 

an α subunit (ligand specific) and a shared β subunit. 

While the α subunit has a low affinity for the ligand and 

alone is incapable of transducing the signal, the association 

of the two subunits gives rise to a functioning 


20 

M. Aglietta et al. 

high affinity receptor which is a type I receptor devoid 

of endogenous tyrosine-kinase activity. β chain activation 

induces several tyrosine-kinases like Fyn, Lyn, Fps, 

Jaks, which transduce a signal common to IL-3 and GM- 

CSF, 17 whereas a subunit activation induces ligand specific 

pathways. In the majority of AML cases IL-3 and 

GM-CSF induce the proliferation of CD34 + , although to 

variable extents. Correlations have been observed 

between responses to different factors but no significant 

additive effects have been noted. 

Exposure of leukemic cells to GM-CSF or IL-3 in vitro 

can give rise to a generation of mature cells. However, 

the persistence of blast cells capable of secondary 

leukemic colony formation indicates that the differentiation 

potential of IL-3 and GM-CSF is negligible and 

that they are unable to abolish the self-renewal of the 

leukemic population. 18-20 SCF synergizes with IL-3 and 

GM-CSF in inducing large clones primarily composed of 

undifferentiated cells. 

G-CSF receptor is expressed by nearly all AML (Table 

1). 16,21 However, M2 and M3 AML appear to express the 

highest number of receptors. In vitro growth stimulation 

is not consistent except for M2 and M3 AML; G-CSF 

action is additive or synergic with that of IL-3, SCF and, 

to a lesser extent, with that of GM-CSF. G-CSF also 

induces some degree of differentiation of leukemic 

CD34 + cells, and the presence of the growth factor 

affects the formation of secondary colonies. In addition, 

CSF treatment seems to prevent cell death in AML. 22 

The in vivo use of growth factors in AML patients 

derives from contrasting hypotheses: 

a) use of growth factors before and during cytostatic 

treatment to induce the proliferation of quiescent 

leukemic progenitors. The increased proliferative rate 

and, possibly, the intracellular accumulation of some 

cytotoxic drugs (i.e. Ara-CTP) should increase the fraction 

of cells killed. 23,24 This approach has never been tested 

in randomized trials specifically addressing this issue. 

It seems, however, to be of modest value with G-CSF or 

GM-CSF. 25,26 It remains to be seen if this approach would 

be more useful with molecules such as SCF that are particularly 

active on leukemic CD34 cells; 

Table 1. High affinity receptors for hematopoietic growth factors in AML. 

Affinity, kd 

(range) 

No. of receptors 

per cell 

Reference 

SCF 16-158 pmol/L 200-8000 14 

G-CSF 36-130 pmol/L 55-1200 16 

GM-CSF 64-404 pmol/L 40-1263 15,16 

IL-3 26-467 pmol/L 21-145 15,16 

b) use of growth factors as differentiating agents with 

the aim of exhausting the self-renewal potential of the 

leukemic progenitors. On the basis of in vitro and preliminary 

(although still to be confirmed) in vivo data, G- 

CSF seems the most promising molecule; 27 

c) use of growth factors for accelerating the recovery 

of residual normal progenitors after induction chemotherapy. 

This approach has been pursued with G-CSF 

and GM-CSF in AML patients > 60 years of age, for 

whom the pancytopenia following cytotoxic treatment 

is particularly profound and long-lasting and carries a 

relevant risk of life-threatening infections. In this setting, 

both G-CSF and GM-CSF given after induction 

chemotherapy reduce the duration of neutropenia without 

affecting the rate of severe infections. 28,29 Moreover, 

G-CSF, but not GM-CSF, appears to increase the complete 

remission rate. Both cytokines, however, have no 

impact on the survival rate. Of interest, no evidence of 

accelerated growth of residual leukemic cells has been 

observed. 

All these data demonstrate how controversial the use 

of hemopoietic growth factors in the treatment of AML 

is, although the most recent results suggest the safety 

of G-CSF administration following induction-consolidation 

treatment. 29 

Stem cell kinetics in AML 

The hematopoietic cell renewal process is supported 

by a small population of bone marrow cells termed 

hematopoietic stem cells. They are defined as cells capable 

of long-term hematopoietic reconstitution and differentiation 

into multiple hematopoietic lineages. It is 

generally held that, in the steady state, the majority of 

normal stem cells are dormant in the cell cycle and only 

a few of them supply all the hematopoietic cells at a given 

time. More than thirty years ago, stem cell kinetic 

studies 30 proposed the concept of a true resting state 

and coined the term G0 as the state from which stem 

cells randomly move to the active cell cycle. 

Subsequent studies 31 confirmed Lajtha’s observations 

by showing that brief in vitro exposure of bone marrow 

cells to highly specific radioactive thymidine does not 

reduce the number of multipotential progenitors. As 

shown in Figure 1, most normal bone marrow CD34 + 

progenitor cells are indeed quiescent in G0. Culture of 

enriched human progenitors documented that they 

remain as single cells for as long as 2 weeks in culture 

and begin proliferation upon stimulation with combinations 

of cytokines. 32 Based on mathematical studies, 

stem cell function was seen as a model in which the 

decision to self-renew and differentiate followed a stochastic 

process. 33 By replating individual blast cell 

colonies, Till and coworkers showed that the production 

of secondary blast cell colonies is a self-renewal process 

and that the generation of secondary multilineage 

colonies is differentiation. Thus the self-renewal process 

is associated with renewed dormancy in the cell cycle 

while the differentiation process is characterized by 

continuous cell doubling. 


Peripheral blood stem cells in acute myeloid leukemia 

21 

Similar work was performed on leukemic stem cells by 

several authors and two fundamental antithetical models 

were proposed, based on the presence of quiescent 

progenitor cells in human leukemia. 

It was postulated that leukemic progenitors were predominantly 

involved in rapid cell cycling as judged by 

measuring the proportion of S-phase cells using H3- 

TdR or hydroxyurea. 34 However, this was in contrast with 

previous observations obtained in vivo by continuous 

infusions of 3H-thymidine for 8-10 days. 35 In those 

experiments, 88 to 93% of the leukemic cells were 

labeled at the end of infusion, whereas almost all the 

smallest leukemic cells were not, suggesting that they 

were in an extended G0. Using in vivo pulse labeling with 

tritiated thymidine in AML patients, it was further 

shown that blasts with a high proliferative rate do not 

behave as a pool of normal self-maintaining cells, but 

rather as a normal multiplication-maturation compartment. 

36 Data obtained with different techniques (labeling 

and cell culture methods) and the heterogeneity of 

study cell populations may represent the reason for 

these discordant results. The hypothesis that AML progenitors 

are characterized by a substantial number of 

nonproliferating or very slowly proliferating blast cells 

(lower RNA content) 37,38 was the rationale for different 

approaches to AML treatment. For instance, the combined 

use of cytokines and chemotherapy to recruit quiescent 

cells into the cell cycle, enhancing the cytotoxicity 

of cycle-specific agents. 39,40 

Raymakers et al. 41 have studied the proliferative 

capacity of the bone marrow fraction double stained 

for CD34 and CD33 in AML patients. The cloning efficacy 

was highly variable in different AML samples, with 

predominant cluster growth. Cluster and colony growth 

was similar between CD34 – /CD33 + and CD34 + /CD33 + , 

in contrast to what is observed in normal bone marrow. 

The most primitive CD34 + /CD33 – fraction was found in 

highly proliferative colony growth. When this analysis 

was extended to AML with a more mature phenotype 

(small fraction of CD34 + /33 – ), the highly proliferative 

colonies deriving from the CD34 + /33 – fraction were 

found to be disomic by in situ hybridization in all 

patients who were characterized by chromosomal 

abnormalities. Nevertheless, the authors could not 

exclude the presence of leukemic stem cells kinetically 

characterized by low or no proliferation under their 

experimental culture conditions. 

A further study 42 aimed at evaluating the specific 

activity of SCF on enriched CD34 + in suspension culture 

by measuring Ki67 expression and flow cytometric DNA 

content showed no difference in cell cycle distribution 

among progenitors obtained from normal bone marrow, 

umbilical cord blood and chronic myeloid leukemia 

CD34 + peripheral blood stem cells. 

Further investigations on the role of a family of proteins 

recently identified as cell cycle regulators, such as 

cyclin A, B, D, E and of their catalytic subunits, the cyclindependent 

kinases cdk2, cdk4, cdk6 and cdc2, may help 

to identify kinetic features and fine differences between 

normal and leukemic hemopoietic stem cells, as well as 

events involved in neoplastic transformation. 43 

In conclusion, the kinetic characteristics of leukemic 

stem cells have still not been defined, mainly because 

different experimental conditions allow evaluation of 

progenitors with different degrees of maturation and 

therefore with different proliferative characteristics. The 

heterogeneity among different leukemia subtypes 

should also be taken into account. 

Expression of CD34 antigen in AML and 

CD34 + leukemias: clinical and biological 

significance 

Based on current information, there is no doubt that 

a substantial number of acute leukemias express the 

CD34 antigen on the cell membrane of blast cells. However, 

the incidence of such expression in AML has been 

found to be highly variable (25-64% of the patients 

examined), depending on a number of factors, as shown 

in Table 2. 

The variability in the reported incidence of CD34 + AML 

has also influenced the prognostic relevance of CD34 

expression in AML. Most authors found a clear association 

between CD34 + AML and a lower incidence of 

complete remission following induction therapy. In 

addition, the relapse rate was higher in AML showing 

positivity for the CD34 antigen compared to that of the 

CD34 – group. 44-53 However, other authors did not confirm 

these results and found no significant difference in 

the complete remission rate or overall survival of CD34 + 

and CD34 – AML patients. 54-60 

Expression of the CD34 antigen in AML and its association 

with different survival rates could be due to a 

Figure 1. DNA/RNA cellular content (acridine orange) of 

normal enriched CD34+ cells. Cell cycle measurements confirms 

that the majority of progenitors are quiescent (G0) 

with only few going into cycle (G1). 


22 


number of factors. First of all, the cut-off point for CD34 

positivity that should be used to decided whether an 

AML sample is carrying this antigen. Since the proportion 

of CD34 + cells is around 1% of normal bone marrow 

mononuclear cells and 0.01-0.1% of peripheral 

blood leukocytes, many authors have considered 5% as 

the optimal cutoff level for classifying CD34 + AML. 

Nowadays, most authors agree that the cutoff point for 

CD34 should be 20% in order to avoid misinterpretation 

of the data coming from surface marker analysis. However, 

there is no scientific basis for considering a sample 

with 20% positive cells as positive, while another 

specimen with 19% positivity as negative, since the level 

of CD34 expression in a substantial number of 

patients is characterized by a continuous spectrum.53 

Furthermore, it must be kept in mind that the choice of 

a cutoff level of 5% could give rise to erroneous results, 

since it can be influenced by the methods used to detect 

antigen expression, which are characterized by different 

levels of sensitivity and specificity. Indirect immunofluorescence 

staining is more sensitive, although less specific, 

while the opposite is true for the direct technique. 

As far as the instrumentation is concerned, it must be 

underlined that modern flow cytometers are highly sensitive 

in detecting surface marker positivity with respect 

to microscope analysis and immunoenzymatic techniques 

such as APAAP, PAP, etc. In addition, whenever 

possible immunophenotype analysis should be preferentially 

performed on fresh, not cryopreserved bone 

marrow samples, and if this is not possible the number 

Table 2. Possible explanations for the differences reported in the literature 

concerning the incidence of CD34 + AML. 

1. Cut-off levels for the discrimination of positive and negative cases 

2. Detection systems employed (flow cytometry, type of flow cytometer, 

immunoenzymatic technique-APAAP, immunogold, PAP-immunofluorescence 

microscope) 

3. Specimen analyzed (bone marrow, peripheral blood) 

4. Percentage of leukemic cells present in the sample examined 

5. Use of cryopreserved rather than fresh cells 

6. Use of different CD34 antibodies recognizing distinct 

CD34 epitopes 

7. Percentage value and level of intensity for CD34 

8. Light scattering properties of CD34+ cells 

9. Patients analyzed (de novo AML or secondary AML) 

10. Biologic characteristics of AML cells (chromosome aberrations, 

gene abnormalities, immunophenotypic profile of CD34+ AML blasts) 

11. Type of chemotherapy regimen employed 

of blasts present in the specimen analyzed should be 

carefully evaluated. 61-65 

A recent report showed that CD34 antigen expression 

in AML samples having a marked heterogeneity in cell 

size was found preferentially on small leukemic cells 

with little or no side scatter. This feature was also associated 

with shorter remission duration and survival, suggesting 

that this morphological heterogeneity could 

reflect a peculiar biological behavior of AML. 66,67 

Moreover, discrimination of blast cells from residual 

normal nucleated cells is less likely to be obtained in 

AML cells by looking at light scattering properties (forward 

and side scatter) and expression of the CD45 antigen. 

For this reason, a multiparametric approach using 

two-three-colour analysis is strongly recommended in 

order to define the predominant leukemic population as 

well as minor pathological clones or subclones. In addition, 

CD34 positivity has to be evaluated solely on the 

blast population in order to avoid misinterpretation of 

the data. In fact, the percentage of blasts could vary 

from 30% to 99% in the bone marrow, and from 1 to 

99% in the peripheral blood. 

Another point which deserves careful discussion is 

represented by the level of expression for CD34 in AML. 

In normal hemopoiesis, the CD34 antigen is expressed 

on virtually all colony forming cells (CFU) and lymphocyte 

progenitors of either T or B lineage. However, within 

the progenitor cell compartment the degree of positivity 

for CD34 decreases with cell differentiation (maximum 

for multipotent cells and minimum for unipotent 

cells), and disappears in morphologically identifiable 

bone marrow precursors. Studies performed at the V 

International Workshop on Leukocyte Differentiation 

Antigens (Boston, 1993) recognized three main subsets 

of CD34 + normal bone marrow cells with CD34 antigen 

densities: low (2,000-5,000 binding sites per cell-ABC), 

medium (10,000-20,000 ABC), high intensities (25,000- 

40,000 ABC). This heterogeneity in CD34 antigen expression 

in normal progenitors makes it difficult to use this 

molecule for the monitoring of minimal residual disease 

(MRD) in AML patients treated with chemotherapy 

and/or bone marrow transplantation. 68 Flow cytometry 

allows the recognition of a subset of CD34 + AML characterized 

by bright expression for CD34 (> 50,000 ABC), 

which could therefore be easily recognized even when 

present in a very low percentage (< 0.1% of nucleated 

cells). This subset represents about 20-30% of CD34 + 

AML, so the remaining AML patients should be checked 

for MRD by using alternative ways (strategical double or 

triple staining: CD34/CD56; CD34/CD65/TdT; cytogenetics, 

molecular biology, etc.). 68 

Another source of variability in detecting CD34 + AML 

is represented by the type of CD34 monoclonal antibody 

used for immunophenotypic analysis. It has been 

demonstrated that at least three distinct CD34 epitopes 

exist, based on their differential sensitivity to enzymatic 

cleavage (using neuroaminidase, chymopapain and 

glycoprotease), Western blotting analysis, cell reactivity 

studies, and cross blocking experiments. 1,69-75 So far 



23 

at least twenty-two CD34 monoclonal antibodies 

(McAbs) have been shown to recognize the CD34 molecule, 

the most direct evidence being reactivity with 

cells transfected with CD34 cDNA and binding to CD34 

glycoprotein. 72 Recently, it has been reported that a 

number of AML cases are positive for some CD34 McAbs 

and negative for others (especially if they belong to a 

different epitope class), confirming the necessity of 

using the same CD34 McAbs in order to achieve comparable 

results between different centers. 71,72,74 

Another point which needs to be considered when 

evaluating the incidence of CD34 + AML is represented by 

patient characteristics at diagnosis. The number of CD34 + 

cases is higher in secondary than in newly diagnosed 

AML. If we consider that both the biology and the clinical 

pattern of secondary AML are quite different from 

those of de novo AML, one may argue that including of 

both types of leukemias in a clinical setting could interfere 

significantly with the prognostic relevance of CD34 

expression on AML blast cells. In this context, the type of 

treatment utilized by various authors (chemotherapy regimen, 

allogeneic and autologous bone marrow transplantation), 

which can influence the outcome of the disease, 

is also of some relevance. 

CD34 + AML are characterized by a higher incidence of 

chromosomes abnormalities involving chromosomes 5 

(–5, 5q–), 7 (–7.7q–), and to a lesser extent chromosomes 

16 (16q), 17 (17p), 11 (trisomy 11), or multiple 

chromosomes at the same time, generating the socalled 

major karyotypic abnormalities. 44,46,48,52,57,76 Recent 

studies have found a close relationship between CD34 

expression in AML and previous exposure to chemotherapy, 

radiotherapy and/or pesticides. 47 CD34 + AML are 

also associated with trilineage myelodysplasia, dysgranulopoiesis, 

and/or abnormalities of the p53 tumor 

suppressor gene. 77 

The correlation between CD34 + AML and FAB subtypes 

is illustrated in Table 3. On the other hand, most 

biphenotypic acute leukemias (BAL) show positivity for 

the CD34 antigen. 

Finally, the antigenic profile of CD34 + AML is rather 

heterogeneous, depending essentially on the morphological 

subtype and to a lesser extent on the differentiation 

stage of the leukemic clone. The large majority of 

CD34 + AML coexpress a number of antigens which are 

not associated with cell commitment, such as HLA-DR, 

CD38, CD45RO, CD45RA, CD71, and IL3 receptor. In 

addition, some surface and cytoplasmic glycoproteins 

expressed by committed myeloid cells were found to be 

positive in CD34 + AML, i.e. CD33, CD13, CD117 (stem 

cell factor receptor), CDw116 (GM-CSF receptor), G-CSF 

receptor, myeloperoxidase, lysozyme 78-82 (Figures 2 and 

3). The stem cell- associated antigen Thy-1 (CD90) is 

negative in this AML subtype, while nuclear TdT is sometimes 

positive. CD34 + cells also express high levels of P- 

glycoprotein, which is the product of the multiple drug 

resistance (MDR) gene. 

Post-remission therapy of acute myeloid 

leukemia and potential role of autologous 

stem cell transplantation 

About two thirds of previously untreated adults with 

primary acute myeloid leukemia enter complete remission 

(CR) after induction therapy based on cytarabine 

and an anthracycline. 83 However, long-term disease-free 

survival occurs in a minority of cases since most subjects 

relapse from proliferation of occult residual leukemic 

cells. Following conventional consolidation treatment 

less than 25% of patients remain in complete remission 

at four years. 84,85 

In order to eradicate residual AML cells and improve 

disease-free survival, three approaches have been 

employed in the last ten years: (a) intensive postremission 

chemotherapy; (b) allogeneic bone marrow transplantation 

(BMT), and (c) myeloablative conditioning 

regimens followed by autologous BMT as supportive 

therapy. 

Intensive postremission chemotherapy is essentially 

based on the use of high-dose cytarabine (1 to 3 g/m 2 

3 6 to 12 doses), either alone or in combination with 

other agents. Results of uncontrolled studies performed 

in the late ’80s and early ’90s (reviewed by Cassileth et 

al. 85 ) indicated that intensive post-remission therapy 

achieves long-term disease-free survival in 25-30% of 

patients in first CR. Mayer et al. 83 recently reported a 

prospective study aimed at evaluating the effect of the 

intensity of postremission chemotherapy on survival of 

leukemic patients. Acute leukemia individuals in first CR 

were randomly treated with four courses of cytarabine 

at one of three doses: 100 mg/m 2 per day for 5 days by 

continuous infusion; 400 mg/m 2 per day for 5 days by 

continuous infusion, or 3 g/m 2 in a 3-hour infusion every 

Table 3. Clinical and biological characteristics of CD34 + AML. 

Incidence: 30-50% (de novoAML); 50-70% (secondary AML) 

History: previous exposure to chemo-radiotherapy or pesticides 

Correlation with FAB subtypes: M0,M1,M5 (70-90%); M2,M4 (20-60%); M3: 

1-5%; M6, M7: 20-50% 

Prognosis: poor (mean survival rates less than 12-18 months) 

Cellular density for CD34: variable from case to case (range: 3,000- 100,000 

per blast cell) 

Immunophenotypic profile: in most cases CD45+, HLA-DR+, CD38+, CD33+, 

CD117+, Thy1– 

Chromosome abnormalities: -5, -7, 5q-, 7q-, 16q, 17p, major karyotype 

aberrations 

Therapy: to be defined (more aggressive chemotherapy regimens?) 


24 


Figure 2. Dual color fluorescence analysis with a flow cytometer 

(CD34/FITC; c-kit-CD117-PE) in a patient with AML FAB M4. Bone marrow 

sample shows a blast percentage of 84%. 

A) Light scattering properties of blast cells (forward scatter= cell volume; 

side scatter= cell granularity). 

B)Contour plot diagram showing 83% of CD34 + cells, 71% of c-kit + 

cells, and 68.5% of cells co-expressing CD34 and c-kit. 

C)Histogram distribution of cells stained for CD34 monoclonal antibody. 

D)Histogram distribution of cells labelled with CD117 monoclonal antibody. 

Figure 3. Discordant reactivity between different epitope class antibodies 

is shown in a patient with AML M1 (% blasts = 90%); 36.5% of 

blasts are positive for class I, 0.1% for class II, and 47% for class III. 

The intensity of fluorescence varied significantly from antibody to antibody 

even within the same epitope class. 

12 hours on days 1, 3 and 5. In patients 60 years of age 

or younger the probability of remaining disease-free 

after four years correlated with the postremission 

cytarabine dose: 24% for the 100-mg group, 29% for 

the 400-mg group, and 44% for the 3-g group (p = 

0.002). In patients older than 60 the probability of 

remaining disease-free after four years was 16% or less 

in each of the three postremission cytarabine groups, 

with no significant difference between groups. It should 

be noted that a significant proportion of AML patients 

achieving CR cannot proceed to intensive chemotherapy 

due to persistent bone marrow aplasia. 

Allogeneic bone marrow transplantation offers many 

advantages, including the graft-versus-leukemia effect; 

however, the availability of a histocompatible sibling 

donor is restricted to approximately 25% of potential 

candidates. Published studies report disease-free survival 

rates at four years ranging from 45 to 58%. 86-88 

These figures should be considered with caution since 

they are biased by the exclusion of patients who 

relapsed before allogeneic BMT. The new approach 

recently described by Aversa et al., 89 i.e. a strong 

immunosuppressive and myeloablative conditioning 

regimen followed by transplantation of a large number 

of haploidentical stem cells depleted of T lymphocytes, 

may open new perspectives for allogeneic bone marrow 

transplantation in AML patients. In this setting, the 

mobilization and collection of allogeneic PBSC is crucial 

to overcoming HLA-disparity. 89 

Pilot studies on the use of autologous BMT as postremission 

therapy 90,91 indicate that disease-free survival at 

four years is on the order of 50% (i.e. comparable to that 

of allogeneic BMT). Autologous BMT has the advantage 

of lower procedure-related mortality than allogeneic BMT 

(approximately 10-15%), but involves a high risk of 

leukemic relapse (about one half treatment failures). A 

recent trial by Zittoun et al. 92 showed that both autologous 

and allogeneic BMT performed in first CR resulted 

in a significantly better disease-free survival than intensive 

consolidation chemotherapy. The projected rate at 

four years was 55% for allogeneic BMT, 48% for autologous 

BMT and 30% for intensive chemotherapy with no 

differences between allogeneic and autologous BMT. 

Taken together, these results demonstrate the potential 

benefit of autologous stem cell transplantation for 

leukemic patients. However, the high incidence of 

leukemic relapse and delayed hematological recovery 

after ABMT have prompted several authors to investigate 

the use of mobilized PBSC. 

CD34 + mobilized hematopoietic cells for 

support of intensive postremission 

chemotherapy of AML 

As stated above, autologous transplantation of mobilized 

hematopoietic progenitor cells has been shown to 

reconstitute hematopoiesis more efficiently than BMderived 

grafts. 2 Moreover, early studies have failed to 

detect neoplastic cells in the peripheral blood of AML 

patients during the early recovery phase following 



25 

Table 4. Reported studies on autologous stem cell transplantation in AML 

patients. PBSC indicates peripheral stem cell transplantation, while ABMT 

refers to autologous bone marrow transplantation. 

PBSC/ABMT PBSC/ABMT PBSC/ABMT 

Reference # 99 102 100 

Pts 28/683 38/13° 20/23* 

CFU-GM reinfused 

(x104/Kg) 

NR 86.6/12.1** 2.3/0.1 

(0.2-4.1/0-1) 

Median time to: 

> 0.5 x 109PMN/L 

(9-60/9-389) 

15.5/27 

(9-17/12-35) 

11/22 14/42 

NR 

> 50 x 109PLT/L 

(11-713/10-700) 

58.5/50 

(9-NA/21-NA) 

13.5/32 30/46 

NR 

PLT transfusion NR 5.4/8.8** NR 

Days on antibiotics NR 9.3/14.3** NR 

Hospitalization 

(days) 

NR 27.5/35.1** 45/73 

Relapse rate 57%/48% NR NR 

DFS 39%/42% NR 35%/51% 

°AML pts = 19 in PBSC group and 1 in ABMT group. 

* Comparison was made with 23 pts receiving purged marrow. ** Results expressed as 

the mean. Abbreviations: NR, not reported; NA, not achieved; DFS, disease free survival; 

PMN, neutrophil; PLT, platelet. 

remission induction/consolidation chemotherapy. 93,94 

Based on these reports, several investigators have 

addressed the question of whether the use of PBSC 

might result in a more rapid engraftment and a lower 

risk of relapse in AML patients. Relevant issues include 

the level of malignant cell contamination, the threshold 

dose of hematopoietic precursors (i.e. CFU-GM and 

CD34 + cells), the optimal timing for stem cell collection 

and the potential benefit derived from the use of selected 

cytokines to improve PBSC harvest. 

To et al. 93 studied leukemia-associated cytogenetic 

abnormalities in myeloid colonies derived from early 

remission PBSC. At a sensitivity level of 2:100 cells, they 

were not able to detect the t(8;21) in 293 samples 

examined. Recently, the more sensitive nested reverse 

transcriptase polymerase chain reaction (RT-PCR) was 

used to monitor minimal residual disease in the BM and 

peripheral blood of leukemic patients considered in 

remission by morphologic analysis. 95 In that study, the 

authors found no differences between PBSC collections 

and simultaneous BM harvests. However, the degree of 

leukemic contamination may have been different 

among the PCR-positive groups because the two-step 

PCR is highly sensitive but not quantitative. 

The issue of leukemia-free autograft was recently 

underscored by gene-marking studies showing that 

residual contaminating AML cells contribute to relapse 

when reinfused into patients. 96 In this regard, leukemic 

recurrence remains the most frequent cause of treatment 

failure in AML patients 97,98 and preliminary nonrandomized 

clinical studies have not reported any 

advantage for ABMT over PBSC 99-101 in terms of diseasefree 

survival and overall survival rate (Table 4). Moreover, 

it could be argued that the interval between complete 

remission and myeloablative therapy may be 

shorter for PBSC patients, since the exclusion rate is 

higher for ABMT patients. 101 Thus, randomized studies 

are warranted to rule out selection bias. Reinfusion of 

PBSC markedly shortened the period of marrow aplasia 

compared to purged and unpurged ABMT 99-102 and 

reduced morbidity and resource utilization (Table 4). In 

particular, To et al. 102 demonstrated an advantage of 11 

and 19 days in the median time to achieve 0.5×10 9 neutrophils/L 

and 50×10 9 platelets/L, respectively, in favor 

of PBSC, whereas two other studies 99,100 showed a more 

rapid neutrophil engraftment (28 days and 12 days, 

respectively) but not a highly significant faster platelet 

recovery. Most likely the acceleration of hematopoietic 

reconstitution derives from the reinfusion of a higher 

number of early pluripotent precursors and large 

amounts of committed progenitor cells which require 

less time to reach maturation. 103 

Early studies in acute leukemia indicated that an optimal 

CFU-GM dose of 50×10 4 /kg body weight is required 

for complete and sustained reconstitution of BM function. 

104 Other reports 99-101 and our own preliminary experience 

(Tables 4 and 5) have demonstrated similar 

results with lower CFU-GM numbers. These differences 

are probably related to different assay methods, whereas 

the more reproducible evaluation of progenitor cells 

by flow cytometry has suggested a threshold dose of 

2×10 6 CD34 + cells/kg. 105 Interestingly, the number of 

progenitor cells infused is only indirectly related to longterm 

engraftment, suggesting that additional measurements 

of CD34 + cell fraction subsets may be helpful in 

predicting sustained recovery of hematopoiesis. Moreover, 

a transient secondary fall in neutrophil and 

platelet count has been described 104 during the 3 rd -8 th 

weeks after transplantation, indicating a time lag 

between exhaustion of the committed progenitor cell 

pool, which is responsible for early engraftment, and 

expansion of the more immature pluripotent stem cell 

compartment. 

Lastly, the timing of BM harvest or PBSC collection in 

the autologous setting plays an important role in the 

quality of the autograft. It has been clearly established 

that the amount and the length of previous therapy 

affects the number of circulating CD34 + cells; 106 however, 

reinfusion of PBSC collected in the recovery phase 

of induction therapy has resulted in a higher relapse 

rate 101,107 indicating that returning a larger quantity of 

cells to patients without careful analysis of minimal 

residual disease may increase the probability of transplantation 

of leukemic cells. Thus, at least one consolidation 

cycle of treatment should be performed in the 


26 


case of stem cell mobilization to take advantage of in 

vivo purging, coupled with a low number of apheresis 

procedures. To this end, it has been shown 108 that 

administration of G-CSF during the recovery phase of 

consolidation chemotherapy in acute leukemia patients 

increased the peak level of CFU-GM and CFU-MIX by 5.8 

and 4.3 times, respectively, compared to cycles were G- 

CSF was not used, and significantly prolonged the period 

of mobilization of stem cells. Although the role of 

cytokine treatment in AML patients is still under evaluation, 

preliminary data from a large cohort of leukemic 

patients suggest that G-CSF administration does not 

affect either the remission or relapse rate, 109,110 whereas 

a protective effect regarding relapse was shown in a 

randomized study. 29 

In practice, leukapheresis sessions should be started 

after a careful evaluation of CD34 + cells in the peripheral 

blood by flow cytometry, at time of hematopoietic 

recovery from transient myelosuppression. 

When adequate mobilization of progenitors (CD34 + 

cells > 10-15/µL) occurs, daily leukaphereses should be 

performed until the collection of a minimum number of 

2×10 6 /kg CD34 + cells. The leukapheresis products should 

be evaluated for the presence of residual contaminating 

leukemic cells by immunophenotyping, karyotypic 

analysis and RT-PCR-based molecular analysis in those 

samples deriving from patients who had shown a specific 

phenotypic and/or molecular marker at diagnosis. 

Moreover, because the content of circulating progenitors 

is generally low (< 1% of the mononuclear cell 

fraction), blood cell separator efficiency must be optimized. 

Collection efficiency (CE) is the percentage of 

cells entering the system that are eventually collected: 

CE (%) = 

No. harvested cells 

× processed blood volume 

No. of cells in preapheresis 

blood unit volume 

Acceptable CE should not be lower than 50%. CE is a 

useful parameter for evaluating blood cell separator 

effectiveness in harvesting PBSC, independently of the 

patient’s clinical condition. 

Purging in AML 

Considering the possibility of relapse from minimal 

residual disease (MRD) derived from autologous graft, 

several investigators addressed the issue of ex vivo purging 

of leukemic cells prior to stem cell reinfusion. Using 

the Brown Norway myelocytic leukemia rat model, it 

has been shown that injection of 25 leukemic cells 

induces leukemia in 50% of recipients. 111 By applying 

the same mathematical model to humans, it has been 

suggested that reinfusion of 10,000 residual leukemic 

cells may result in a relapse rate as high as 50%. 111 More 

recently, the role of residual tumor cells in clinical 

relapse after autograft was indicated by a clinical study 

Table 5. Experience of the Institute of Hematology “Seragnoli”, Bologna 

on autologous stem cell transplantation in AML patients. The results are 

expressed as median (range) and refer to AML (n=7) and RAEB-T (n=2) 

patients in I CR. PBSC collections were carried out following consolidation 

treatment. PMN and PLT recovery was recorded as such when the PB 

count was > 0.5 and 20 x 10 9 /L, respectively. 

Apheresis products 

Pts PBSC MNC CFU-GM CD34+ 

collections (108/Kg) (104/Kg) (106/Kg) 

9 3 (2-3) 7 (2.8-11.6) 11.8 (2.8-78.2) 7 (3.1-17.5) 

Hematological reconstitution 

Pts day to PMN day to PLT PLT RBC 

Hospital 

recovery recovery transfusion transfusion 

discharge 

8 14 (11-34) 18 (10-NR) 2.5 (0->10) 4 (1-9) 18 (14-38) 

Abbreviations: MNC, mononuclear cells; RBC, red blood cells. 

involving 114 B-cell lymphoma patients with t(14;18) 

who received autologous marrow treated with a combination 

of monoclonal antibodies directed against B- 

cell associated antigens plus complement. 112 Following 

purging, no lymphoma cells could be detected by PCR 

amplification of the bcl-2 gene in the marrow of 57 

patients. Disease-free survival was increased in these 

individuals with respect to that of patients whose marrow 

contained detectable tumor cells. Moreover, the 

ability to remove lymphoma cells was the most important 

prognostic factor for predicting relapse (39% versus 

5% of purged patients after a median follow-up of 

23 months). 112 Lastly, genetic marking of marrow cells 

with the neomycin-resistance gene has provided the 

formal proof that reinfusion of residual leukemic cells in 

AML patients contributes to a recurrence of the disease. 

113 Taken together, these findings demonstrate the 

need for effective ex vivo treatments to improve the 

outcome of autologous transplantation. 

Among the many purging methods proposed for the 

elimination of MRD, 114 cyclophosphamide (Cy) derivatives 

are the most widely used agents in AML patients, 

since preclinical models demonstrated that these compounds 

were able to eliminate residual BM neoplastic 

cells in the Brown Norway rat system. 115 The main mechanism 

of action of Cy active metabolites is based upon 

marked inhibition of leukemic progenitor cell (CFU-L) 

growth 115 while sparing normal primitive hematopoietic 

cells. 116 Furthermore, these alkylating agents seem to 

induce apoptotic death of leukemic cells 117 as well as the 

activation of immune mechanisms capable of controlling 

leukemic cell proliferation. 118 Combinations of 4- 

hydroperoxycyclophosphamide (4-HC) or nitrogen mustard 

and VP-16 have also been proposed to increase the 

selective toxicity of pharmacologic purging towards 

neoplastic cells. 119 Several monoclonal antibodies that 



27 

recognize tumor-associated or cell-differentiation antigens 

not expressed by primitive cells responsible for 

hematopoietic engraftment have been selected for clinical 

trials after in vitro studies demonstrated a high 

purging efficacy with the use of complement, toxins and 

radioactive molecules. However, the heterogeneity of 

antigen expression on neoplastic cells and the lack of 

tumor-specific determinants may greatly affect the efficiency 

of antibody-based strategies for depletion of 

leukemic cells. In this context, the combination of two 

purging techniques has been explored with promising 

results. 120 Other approaches include the use of photoactive 

compounds which sensitize leukemic cells and 

specifically damage their cell membranes upon exposure 

to light, 121 and biological methods based on the different 

proliferative patterns of leukemic cells and their normal 

counterparts when cultured ex vivo for several days 

in the presence of stromal cells. 122 

Clinical trials 

Clinical retrospective data supporting the beneficial 

effect of purging have been progressively accumulating. 

The Baltimore team provided indirect evidence in favor 

of purging by correlating effective CFU-GM colony elimination 

with a significant decrease in relapse. 123 Furthermore, 

the same authors associated the sensitivity to 

4-HC of CFU-L grown in remission with the posttransplant 

outcome. 124 The 3 most recent surveys of the 

Leukemia Working Party of the EBMT group have consistently 

reported lower relapse rates following reinfusion 

of BM purged with mafosfamide, 91,125,126 especially 

in patients transplanted within 6 months of CR and in 

slow remitters (> 40 days to achievement of CR), two 

patient populations considered at high risk of disease 

recurrence. In fact, the proportion of patients relapsing 

in the purged and unpurged groups was 29±5% vs 

50±4%, respectively, following a conditioning regimen 

that included total body irradiation (p < 0.0001). More 

striking differences were found when considering only 

those patients autografted early after CR (16±6% vs 

60±6%) and patients with an interval from diagnosis to 

CR greater than 40 days (20±8% vs 61±6%). Gulati et 

al. and Laporte et al. 127, 128 reported disease free survival 

which approximated 60% in AML patients in I CR reinfused 

with autologous marrow treated with 4-HC and 

VP-16 and mafosfamide. In the same paper by the Paris 

group, 151 it was suggested that the higher the initial 

content of BM CFU-GM, the lower the risk of transplant 

related mortality and the higher the chance of curing 

the disease. 

Despite these results in favor of ex vivo elimination of 

contaminating leukemic cells, this procedure is not routinely 

performed in the majority of transplant centers. 

The main reasons might be: 1) the delay in hematological 

recovery after reinfusion of purged autografts; 2) 

the increase in the cost of ABMT; 3) the need for technical 

training and, most of all, 4) the lack of results 

derived from prospective clinical studies demonstrating 

the effects of purging. The feasibility of such trials is 

rather limited due to the high number of patients needed 

to obtain adequate statistical power. 

So far, no data are available on purging protocols for 

PBSC collections; however, there are several reasons for 

proposing purging strategies for autograft of circulating 

autologous stem cells. Unlike solid tumors and 

malignant lymphomas, acute leukemias easily involve 

PB. Moreover, the number of hematopoietic progenitor 

cells (and possibly leukemic precursors) in PB autotransplants 

is usually at least 10 times higher than that 

of ABMT. Finally, as discussed above, the interval 

between CR and autotransplant may be shorter for PBSC 

patients, who may be thus considered high risk patients 

for relapse. Critical issues for designing experimental 

studies in this setting would include the proper assessment 

of MRD before and after purging, the establishment 

of reproducible technical protocols (cell concentration, 

RBC contamination, etc.), and careful evaluation 

of the toxicity of purging agents on PB progenitors, since 

the kinetic status of circulating stem cells following 

mobilization protocols (especially if CSFs are used) may 

be different from BM stem cells. 129 

Conclusions 

Autologous BMT has been widely used as consolidation 

therapy in AML patients in first or second remission; 

however, delayed hematopoietic engraftment occurs in 

a substantial proportion of patients resulting in significant 

morbidity and mortality. This is mainly due to the 

adverse effects of prior intensive chemotherapy on BM 

harvest, a decrease in the normal stem cell pool in 

leukemic patients and, perhaps, toxic damage to the 

marrow microenvironment. Thus, several groups have 

investigated the use of circulating progenitor cells with 

the twofold aim of reducing transplant-related toxicity 

and widening the number of potential candidates for 

myeloablative therapy with the support of autologous 

stem cells. 

As for hematopoietic reconstitution, previous studies 

have provided evidence that PBSC transplantation may 

offer some advantages over BM autografting. However, 

crucial issues such as asynchronous mobilization of normal 

vs leukemic cells and potential contamination of 

PBSC collections, timing of PBSC harvest, detection of 

minimal residual disease, and the role of growth factors 

to accelerate hematological recovery and optimize stem 

cell collection have not been fully addressed. 

In the present paper, the latest advances in this field 

have been reviewed with special focus on the biology of 

putative leukemic stem cells; operative guidelines have 

also been provided for those investigators who wish to 

design proper clinical trials on PBSC autotransplantation 

in acute leukemia. 

Definitive answers regarding the role of PBSC will be 

coming from a large European randomized trial which 

is currently comparing peripheral blood stem cell and 

BM-derived graft. 


28 


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eview 

Peripheral blood stem cell 

transplantation for the treatment 

of multiple myeloma: 

biological and clinical implications 


FEDERICO CALIGARIS CAPPIO, MICHELE CAVO, 

ARMANDO DE VINCENTIIS, LUIGI LANATA, ROBERTO MASSIMO 

LEMOLI, IGNAZIO MAJOLINO, CORRADO TARELLA, PAOLA ZANON, 

SANTE TURA 

Cattedra di Immunologia Clinica, Dipartimento di Scienze 

Biomediche e Oncologia Umana, Università di Torino, Turin; 

Istituto di Ematologia ed Oncologia Medica“Lorenzo e Ariosto 

Seràgnoli”, Università di Bologna, Bologna; Dompé Biotec 

SpA, Milan; Dipartimento di Ematologia, Unità Trapianti di 

Midollo Osseo, Ospedale Cervello, Palermo; Divisione Universitaria 

di Ematologia, Università di Torino, Azienda 

Ospedaliera S. Giovanni, Turin; Amgen Italia SpA, Milan; Italy 

Correspondence: Prof. Sante Tura, Istituto di Ematologia ed Oncologia 

Medica “Seràgnoli”, Policlinico S. Orsola, via Massarenti 9, 

40138 Bologna, Italy. 

Received January 11, 1996; accepted June 4, 1996. 

Acknowledgments: preparation of this manuscript was supported by 

grants from Dompé Biotec SpA and Amgen Italia SpA, Milan, Italy. 

The aim of this review is to define the role of 

peripheral blood stem cell transplantation for the 

treatment of multiple myeloma. Therefore, we 

first review our present knowledge of this disease and 

then analyze the clinical trials based on the use of 

autologous bone marrow or peripheral stem cell 

transplantation. Optimal methods for peripheral 

blood stem cell transplantation will also be discussed. 

Myelomagenesis 

Multiple myeloma (MM), the prototype plasma cell 

malignancy, is characterized by the uncontrolled 

accumulation of plasma cells that replace normal 

bone marrow (BM) and by the overproduction of 

monoclonal immunoglobulins (Ig) and cytokines. A 

number of observations provided both by basic sciences 

and by clinical investigation allow us to place 

the disease and its unusual features in a more coherent 

perspective and to discuss new therapeutic 

options properly. 

Epidemiology 

The reported incidence of MM is available for the 

years up to 1982 and varies substantially in different 

countries. 1 A striking increase in the incidence of MM 

has been noticed in the last thirty years and is only 

partially 2 explained by amelioration of diagnostic 

capabilities. 3 Between 1973 and 1990 an increase of 

40% among people over 65 and of almost 15% 

among people under 65 has been recorded in US Cancer 

Death rates. 4 Ethnic differences are apparent: the 

incidence is twice as high and the mortality rate has 

quadrupled in blacks, while doubling in whites. 4 By 

contrast, rates among Asians are lower than those of 

whites living in the same geographic area. 5 

Both genetic and environmental factors can be 

invoked to explain these ethnic differences. A significant 

increase has been detected in first-degree relatives 

of patients. 5 Moreover, an increased risk has 

been observed to be associated with occupational 

and environmental elements that include farming 

exposure to pesticides, exposure to ionizing radiations, 

petroleum and rubber processing, as well as 

persistent (viral) infections. 3 The main conclusion that 

can be drawn from a large body of observations is 

the necessity of discriminating the genetic roots from 

the environmental links of the disease. As a corollary, 

it may be asked which elements (genetic vs. environmental) 

are associated with the development of monoclonal 

gammopathy of undetermined significance 

(MGUS) and how they relate to the progression of 

MGUS to overt MM. 

Cytogenetics and molecular biology 

Two major pieces of information have emerged 

from cytogenetic studies. The first is that no consistent 

(yet not random) chromosome abnormalities 

have been detected in MM. 6 The second is that 

numeric chromosome abnormalities are shared by 

MGUS and MM. 7,8 Both facts lead us to ask what the 

prerequisite is and what the additional events are in 

the development of plasma cell malignancies. We still 

do not know the prerequisite events that lead to 

MGUS, to MM or to the evolution of MGUS into MM, 

or how they differ from collateral elements that simply 

favor the malignant process. Along the same vein, 

it is interesting that no known specific oncogene has 

yet been related to the development of MM or to the 

transition from MGUS to overt MM. The genes most 

commonly implicated in MM, like N-RAS, P53 and 

retinoblastoma gene (RB), are all involved in the late 

stages of the disease. 9 

If the same cytogenetic abnormalities are shared by 

two clinical situations as different as MGUS and overt 

MM, a patrolling role for the immune system can be 

envisaged in the natural history of plasma cell disorders. 

It is not unreasonable to suspect that if the 

immune system is able to keep a malignant clone 


PBSC transplantation in multiple myeloma 

33 

under control, a benign MGUS is the resulting disease; 

the breakdown of this control would lead to MM. Little 

direct, but much indirect evidence is available in murine 

models to suggest the immunomodulation of myeloma 

cell growth by host effector cells. 10 

Immunochemistry and B cell differentiation 

studies 

Three major findings have been obtained through 

immunochemistry and by a more proper understanding 

of the differentiation processes of B lineage cells. First, 

MM paraproteins may be directed against a wide variety 

of infectious agents, suggesting that MM development 

and antigen (Ag) stimulation may be causally related. 

11-13 Second, the Ig isotype of MM plasma cells is generally 

IgG or IgA, demonstrating that the predominant 

phenotype of MM tumor cells is post-switch. 9 Third, 

clonal proliferation involves a cell population that has 

already passed through the stage of Ig genes somatic 

hypermutation. 14,15 Since this process occurs in the germinal 

centers (GC) of secondary follicles, 16 its presence 

is a clear marker of the differentiative and functional 

level reached by the cell population being analyzed. 

By and large, the observation that MM is a neoplasm 

of plasma cells that have a post-switch phenotype, 

show somatic mutations and may produce monoclonal 

Ig with targeted antibody (Ab) activity leads to the conclusion 

that MM is an Ag-driven process, even if the 

specific causal Ag is generally unknown. This assumption 

has to be confronted with the simple, though basic, 

lesson from clinical medicine that MM is a BM disorder. 

In contrast with the distribution of normal plasma cells, 

MM plasma cells localize uniquely within the BM. 9 

Although the lamina propria of the intestine contains 

more Ig-producing cells than all other tissues in the 

body, it is never a site where MM develops, not even 

IgA1- and IgA2-producing MM. 17 Likewise, involvement 

of the spleen and/or lymph nodes, though typical of 

Waldenström’s macroglobulinemia, is very unusual in 

MM. 17 The exclusive BM localization of MM plasma cells 

appears to conflict with the extensive somatic hypermutations 

of the Ig they produce, which indicate a 

peripheral origin of malignant cells. However, while the 

steps of Ag processing and presentation that lead to 

the generation of somatically mutated IgG and IgA plasma 

cells occur only in secondary lymphoid follicles, the 

BM is a major site of IgG and IgA production in T-celldependent 

secondary immune responses. 18-20 Plasma cell 

precursors with specific traffic commitments originate 

from secondary lymphoid organs and migrate to the BM 

a few days after the Ag challenge (Figure 1). 20,21 

The issue whether MM plasma cell precursors are early 

BM stem cells or late peripheral B cells is misleading. 

The cell whose original transformation has ultimately 

generated the malignant plasma cell progeny that we 

see in MM cannot be equated with the B cell population 

that disseminates the disease throughout the axial 

skeleton. 21 The identity of the hypothetical MM stem 

cell is unknown, i.e. we do not know either the cellular 

target of the primary transforming event or where, 

when and how the unknown cellular target was hit by 

the transforming event. By contrast, the information 

available on the B cell population that feeds the downstream 

compartment of plasma cells and disseminates 

the disease indicates that this population has been generated 

in peripheral lymphoid organs during secondary 

T-cell-dependent Ab response, is programmed to home 

to the BM, and is committed to differentiate in close 

association with the BM microenvironment (Figure 

2). 21,22 On the basis of existing data, the most likely candidate 

for the physiological B lymphocyte equivalent of 

the MM plasma cell precursor is either a B memory cell 

or a plasma blast (Figure 1). 14,15,23,24 

Microenvironment and cytokines 

It is assumed that BM-seeking plasma cell precursors 

receive a differentiation signal after contact with the 

BM stromal microenvironment (Figure 2). 25,26 Microenvironmental 

stromal cells play an essential role in the 

growth of plasma cell tumors both in mice 27 and in 

humans. 28 MM BM stromal cells are well equipped with 

a large series of adhesion and extracellular matrix molecules 

that mediate homotypic and heterotypic interactions 

and provide anchorage sites to cells selectively 

exposed to locally released growth factors. 22,29,30 MM 

BM stromal cells produce cytokines like IL-6 known to 

play a crucial role in the evolution of the disease both 

in experimental systems, including IL-6 transgenic mice, 

extracellular matrix proteins 

B memory cell 

Stromal cell 

plasma blast 

centroblast 

stromal cell released cytokines (IL-6) 

Plasma cell 

BONE MARROW 

B memory cells 

centrocyte 

LYMPHOID FOLLICLES 

Figure 1. Plasma cell precursors generated in peripheral 

lymphoid organs differentiate in contact with bone marrow 

stromal cells. 


34 

F. Caligaris Cappio et al. 

Precursors 

Macrophages 

Fibroblasts 

T lymphocytes 

Bone Marrow 

IL-1, TNF-, M-CSF 

IL-1, IL-3, GM-CSF, 

TNF 

IL-6, IL-7, 

stem cell factor 

Microenvironment cells 

Osteoclasts 

Plasma cells 

Reciprocal 

Activation 

Figure 2. Model of multiple myeloma growth and progression 

based upon a series of Fig. mutual 2 interactions between the 

B-cell clone and the bone marrow microenvironment. 

and in vivo. 31-34 High levels of IL-6 are observed in the 

sera of patients with aggressive or progressive MM, 35 

and infusion of anti-IL-6 antibodies in patients with 

plasma cell leukemia or MM refractory to therapy has 

decreased the size of the plasma cell pool and hampered 

the proliferative activity of plasma cells. 36 

Malignant MM plasma cells are not inert vehicles of 

monoclonal Ig. They also produce a number of cytokines, 

including interleukin (IL)-1b, tumor necrosis factor 

(TNF)- and monocyte-macrophage colony stimulating 

factor (M-CSF), that activate stromal and accessory 

cells, aa well as having significant osteoclast activating 

factor (OAF) activity. 22,37 A minority of human MM cell 

lines autonomously produce small amounts of IL-6, but 

it is unclear whether fresh MM plasma cells can also 

produce IL-6. 34 IL-6, besides promoting B cell proliferation 

and differentiation, has recently been shown to 

have important OAF activity. 32,33 

These experimental findings linked to clinical observations 

lead to the attractive hypothesis (Figure 2) that 

a self-maintaining series of mutual interactions between 

the malignant B cell clone and the BM microenvironment 

may explain the progression of MM 22 through the 

production of ever-increasing amounts of cytokines 

capable of recruiting and activating several microenvironmental 

cells, including osteoclasts. 

The role of autologous transplantation 

in the treatment of multiple myeloma 

Investigations into the use of myeloablative therapy 

for the management of MM were pionereed in the mid- 

1980s and were stimulated by a persistent lack of 

progress in prognosis with conventional chemotherapy. 

38,39 As is the case with any experimental approach, 

initial trials were restricted to the treatment of patients 

with advanced refractory or relapsing disease and were 

focused mainly on defining the feasibility and toxicity of 

the procedure. These preliminary experiences were performed 

without the support of hemopoietic stem cells 

and demonstrated that high-dose melphalan (HDM), given 

intravenously (i.v.) at doses ranging between 100 and 

140 mg/m 2 , yielded an increase in the complete remission 

(CR) rate, albeit at the expense of prolonged marrow 

aplasia and an unacceptably high early mortality 

rate. 40-42 On the basis of these observations later studies 

with chemotherapeutic agents administered at myeloablative 

doses, and possibly added total body irradiation 

(TBI), were carried out with the support of autologous 

BM and/or peripheral blood hemopoietic stem cells 

(PBSC). 43 Demonstration of the safety and relative efficacy 

of autotransplants in refractory MM 41,44-46 encouraged 

subsequent application of this procedure in earlier 

phases of the disease 45,46 and, more recently, in newly 

diagnosed patients as well. 47,48 Over the past decade 

interest in this new treatment strategy has progressively 

grown, and the number of reported patients receiving 

autologous hemopoietic stem cell-supported myeloablative 

therapy is now approximately one thousand 

worldwide. 

What lessons have we learned from this collective 

experience? It is difficult to draw firm conclusions from 

published trials since none of them were controlled and 

patient populations were different, as were the preparative 

treatments and the criteria used for evaluating 

tumor response. In addition, the bias introduced by 

patient selection and, in most of the cases, the lack of 

an adequate follow-up also helped complicate correct 

interpretation of the data. As a consequence, the exact 

role of autotransplantation in the management of MM 

still remains poorly defined and could be properly 

addressed only in controlled clinical studies comparing 

autografting and conventional chemotherapy. There are 

at least several such trials in progress at the moment in 

Europe and the United States. Data reported at the last 

ASH meeting in Seattle (1995) by the Intergroupe 

Français du Myelome are promising and suggest an 

advantage for autografted patients in terms of increased 

CR rate and extended survival duration. 49 

Obviously, these results warrant confirmation in larger 

independent series. For this reason, similar investigations 

are currently being conducted in the United 

States under the auspices of the National Cancer Institute. 

While the conclusions of these studies are being 

awaited, analyses of available transplant data have provided 

the following important information. 



35 

Group No. % Source % % Median mos. 

pts. sens. % BM % PB ED CR PFS Surv. 

EBMT 130 68 63 25 6 48 17 27 

Univ. Arkansas (USA) 287 60 unknown

36 


Remission duration 

As previously emphasized, myeloablative therapy 

requiring autologous hemopoietic stem cell support provides 

substantial antitumor response, especially in 

patients with good prognosis (see below). However, even 

in this favorable condition, a considerable relapse rate, 

approaching 60% at 3 years, is reported after autotransplant 

and no plateau is yet apparent on relapse-free 

survival curves. 50-52 These results contrast with the 30% 

probability of long-term unmaintained remissions (and 

possible cures) reported by several groups for patients 

receiving allogeneic transplantation. 60 It has been suggested 

that the lack of an immunological effect by the 

donor’s marrow T lymphocytes on the residual myeloma 

cells (i.e. graft-versus myeloma) 61,62 and/or possible 

tumor reseeding may account for the apparently less 

durable duration of disease control following autologous 

as opposed to allogeneic transplantation. For this 

reason, important issues currently under clinical investigation 

in the autografting setting include further 

increases in the cytotoxic dose intensity level and depletion 

of tumor cells from the graft (see below). 

Prognostic variables 

Several important variables affecting the outcome of 

autologous transplantation have been identified (Table 

3), including 2 -microglobulin ( 2 -M) levels, 45,47,50-52,56 

pre-transplant disease status, 45,51,52 age, 45,51,52 performance 

status, 45 Ig isotype 45,51,52 and response to myeloablative 

therapy (e.g. attainment or non-attainment of 

CR). 47,48 In particular, at multivariate regression analysis 

early mortality was reported to be highest among resistant 

relapsing patients, who also had the poorest 

response to myeloablative therapy and the shortest 

relapse-free survival duration. 45 In contrast, low serum 

2 -M levels, both at diagnosis and before autografting, 

and prior responsiveness to conventional chemotherapy 

conferred the highest CR rate, as well as prolonged 

relapse-free and overall survival durations. 45,47,50-52,56 In 

addition, the timing of autotransplant also emerged as an 

important and independent prognostic parameter. 56,64 

This observation, on the one hand, was related to the 

generally reported improved outcome of patients transplanted 

earlier and, on the other hand, reflected the 

acquisition of multiple biological abnormalities in 

advanced phases of the disease 63 that ultimately led to 

refractoriness even to high-dose therapy. 64 Conversely, 

retaining sensitivity to high-dose therapy in earlier phases 

of MM assured better results, even in patients with 

primary refractory disease. 45,47,65 

New perspectives under clinical investigation 

Based on the assumption that the failure of the conditioning 

regimen to eradicate the myeloma clone contributes 

most to post-transplant relapse, attempts to 

increase the intensity, and possibly the efficacy, of 

treatment by means of repeated courses of myeloablative 

therapy have recently been undertaken. 46,53 The 

more rapid recovery of hemopoiesis assured by the 

combined use of PBSC and post-transplant administration 

of hemopoietic growth factors 59 made the double 

transplant strategy feasible for approximately 60% 

of patients within one year. 53 Results of pilot trials in 

primary refractory MM indicated that such an approach 

provided superior antitumor effect with improved 

event-free and overall survival durations with respect 

to a single transplant. 53 

A controlled clinical study comparing in a randomized 

fashion single vs. double autografting in newly 

diagnosed patients is currently underway in France. A 

similar trial is already in the early accrual stage in Italy. 

These studies will clarify in the next several years 

whether double transplant is associated with better 

prognosis. Alternatively, efforts to improve the clinical 

impact of autotransplant have been carried out by several 

groups and have included depletion of tumor cells 

from autografts by both negative selection of myeloma 

cells and positive selection of CD34 + hemopoietic 

stem cells, 66,67 as well as post-transplant immunomodulation 

with interferon- (IFN-). 47,49,68 

In summary, hemopoietic stem cell-supported myeloablative 

therapy holds the promise of being a safe and 

effective treatment modality for MM. It yields better 

overall response and CR rates than conventional 

chemotherapy and may prolong the duration of survival. 

49 

These conclusions, while encouraging, have been 

drawn mainly from uncontrolled studies carried out in 

select groups of patients and obviously warrant confirmation 

in controlled clinical trials which are currently 

under way. Therefore the next several years will clarify 

whether newly diagnosed patients with symptomatic 

MM can be routinely offered a single or double autotransplant 

as first-line or early salvage therapy for their 

disease. 

Table 3. Variables affecting the outcome of autotransplants 

for multiple myeloma. @ 

Disease status 

Variable Refractory Refractory + Responsive 

CR RFS Surv. CR RFS Surv. 

Low 2 M – +* +* + +* +* 

Early transplant + + + + +* +* 

CR achievement +* +* 

Double transplant +* +* 

CT responsiveness + + + 

Younger age – + + + + + 

Non IgA isotype – + + – + + 

*In multivariate analyses. Abbreviations: CT, conventional chemotherapy; 

RFS, relapse-free survival. @ Ref.: 45,47,48,50,51,52,56,63,64,65. 



37 

While the results of these studies are being awaited, 

wider application of myeloablative therapy should probably 

be encouraged. Less heavily pretreated patients 

who did not respond to prior conventional chemotherapy 

are more likely to benefit primarily from autotransplant. 

In addition, data available from the literature do 

suggest that a superior outcome of this procedure can 

be anticipated in patients with chemosensitive disease 

and low tumor burden at diagnosis. Hence, ongoing 

clinical trials aimed at comparing conventional versus 

myeloablative therapy will also address the important 

issue of the role of autotransplant as early consolidation 

therapy in patients with intrinsically good prognosis. 

However, even in this favorable situation, recurrence 

of the underlying malignant disease remains a major 

problem and is the most common cause of treatment 

failure. For this reason, attempts to improve the clinical 

impact of autografting are under active clinical 

investigation. 

In addition, many other problems regarding autologous 

transplantation for MM are still unresolved and 

should be formally addressed in future clinical trials. 

The most important of these issues include the choice 

of the best conditioning regimen, the optimal source of 

hemopoietic stem cells, the nature of relapse after autografting, 

the benefit from purging techniques and, finally, 

the likelihood of long-term disease control, especially 

for patients with molecularly defined CR. 69 

Advantages offered by the use of 

PBSCs in the treatment of multiple 

myeloma 

The use of PBSC in support of high-dose chemoradiotherapy 

(peripheral blood stem cell transplantation) 

(PBSCT) is a valid alternative to autologous bone 

marrow transplantation (ABMT) in the treatment of 

both hematologic and non-hematologic neoplastic disorders. 

70-72 The growing interest in this procedure can be 

explained by: i) the possibility of mobilizing and collecting 

large amounts of hemopoietic progenitors, 73,74 

and ii) the rapid hemopoietic recovery observed following 

PBSCT. 70,71,74-78 

Progenitor collection represents the critical step in the 

procedure. Daily monitoring of circulating CD34 + cells is 

an essential assay in predicting the number and timing 

of leukaphereses. 79,80 Under proper conditions, only a few 

leukapheresis procedures are required to collect enough 

progenitor cells for marrow reconstitution after myeloablative 

treatments. Indeed, when circulating CD34 + 

cells rise to >50/µL, 1-2 leukaphereses may yield more 

than 5010 4 /CFU-GM/kg or 810 6 /CD34 + cells/kg, 

which are considered the ideal values for optimal 

engraftment. 80-82 In addition, it has been shown that 

large quantities of very immature elements, identified as 

long-term culture-initiating cells (LTC-IC), are mobilized 

as well. 83-85 

Inclusion in the harvested material of very immature 

elements is responsible for the stable and durable marrow 

reconstitution observed in patients autografted with 

circulating progenitors. 77,83 Thus the term PBSC, now 

commonly employed to identify mobilized hemopoietic 

progenitors, relies on both biological and clinical observations. 

As previously emphasized, the rapidity of 

engraftment is the major advantage offered by PBSC. 

Nevertheless, some authors argued that BM cells stimulated 

by growth factor administration might be at least 

as efficient as mobilized progenitors in ensuring rapid 

engraftment following myeloablative treatment. 87-89 

However, it has recently been shown that both committed 

and early progenitors are by far more frequent 

in PB than in BM during maximal mobilization. 90 This 

conclusive observation points toward the preferential 

use of PBSC as the hemopoietic cell source for grafting 

purposes. 

Since its introduction into clinical practice, PBSCT 

has been considered a promising approach for MM 

patients. 91,92 Several studies have been designed in the 

last few years. 

Reported results have shown a significant decrease in 

hemopoietic toxicity following this procedure as compared 

to ABMT, with recovery of granulocytes >0.510 9 /L 

and plateles >25-3010 9 /L within approximately 2 weeks 

after autograft (Table 4) 42,44-48,52,54,56,93-98 This was paralleled 

by rather good tolerability with rare early fatal 

events. 52,56,97,98 

In addition, hemopoietic reconstitution by PBSC 

seems to be long lasting. MM patients may require 

repeated exposure to high-dose cytotoxic therapy. 

Reducing hemopoietic toxicity might be critical for the 

ultimate treatment outcome. Therefore, also for its 

long-term effect, PBSCT may have a positive impact on 

the life expectancy of those patients who are suitable 

for intensified chemo-radiotherapy treatments. 99 

PBSC mobilization and collection in 

multiple myeloma 

PBSC mobilization in myeloma patients 

PBSC collection presents specific problems in patients 

with MM, where a decrease of progenitors in the bone 

marrow is due in part to a defect of the monocyte/macrophage 

activation pathway. In fact, CD34 + cells 

from MM patients grow normal numbers of colonies 

when stimulated by normal monocytes, while normal 

CD34 + cells have a reduced growth rate with MM monocytes. 

100 Another aspect is prior treatment. Repeated 

courses of chemo-radiotherapy are able to exhaust the 

pool of pluripotent stem cells, 101 resulting in insufficient 

progenitor cell harvests. 59,102-105 Studies specifically 

addressed at MM patients show that melphalan 106 and 

treatment-free interval prior to PBSC mobilization 107 also 

have an influence on the release of progenitors into the 

peripheral blood, while the value of BM plasmacytosis as 

an independent factor is more questionable. 108,109 As a 

consequence of these and other unknown factors, progenitor 

yields in MM are often unpredictable and lower 

than those observed in other malignant disorders. 110 


38 


Time to recovery from° 

Intensified treatments* leukopenia thrombocytopenia Treatment-related References 

(days) (days) deaths (%) 

Without autograft 28 # 27 17 42,46,54,93-95 

With BMT 20 26 7 44,45,47,48,96 

With PBSCT 14 18 3.7 52,56,97,98 

Table 4. Toxicity of intensified 

treatments with or without 

autologous stem cell support in 

multiple myeloma patients. 

*intensified treatments consisted of HDM (60-200 mg/sqm) in most studies; the association of TBI/HDM was also used in some programs with autograft; 

°time to recovery from leukopenia and thrombocytopenia was reported as days to reach >0.5x10 9 ANC/µL and > 25x10 9 platelets/L, respectively, 

in nearly all studies; # table data have been calculated as medians from median values of hemopoietic recovery and from percentages of treatmentrelated 

deaths reported in each quoted study. 

Nonetheless, cell harvests sufficient for one or two subsequent 

autografts are usually obtained, 59,97,108,111-113 even 

in patients with markedly infiltrated marrow or primary 

resistant disease. 109 To avoid the adverse influence of premobilization 

treatment, PBSC collection in MM patients 

should be planned as early as possible in the course of 

disease, and alkylating drugs should be omitted in the 

primary treatment. It should also be kept in mind that 

heavily pre-treated patients require more leukaphereses 

and show slower platelet recovery after autograft. 109 The 

key issues in the apheretic harvest of PBSCs in MM are 

presented in Table 5. 

PBSC also may be collected from patients with malignancies 

in steady state conditions; 114 however, multiple 

aphereses are required with this method. Mobilization 

of progenitors with cytotoxic chemotherapy, hemopoietic 

growth factors, or a combination of the two is 

therefore generally preferred. The hematopoietic recovery 

that occurs after cytotoxic chemotherapy is accompanied 

by a PBSC rise that is proportional to the intensity 

of myelosuppression. 

102, 108 

In MM, chemotherapy alone with either HDM, 115 or 

CHOP-like regimens 112,113,116 or intermediate- to highdose 

cyclophosphamide (Cy) 97,117,118 has been used to 

mobilize PBSC. However, the failure rate, defined as the 

percentage of patients with a low progenitor cell peak 

in the blood or poor collections at the end of the 

apheresis program, was relatively high, ranging from 20 

to 30%. Moreover, when using high-dose therapy protocols 

without growth factor support, one should consider 

that this implies an undue risk of severe toxicity. 118 

G-CSF 73,119-121 and GM-CSF, 75,112 as well as other 

cytokines are able to promote a dramatic rise of progenitors 

in the circulation. In a study of MM patients, 

administration of G-CSF at 10 µg/kg alone for six days 

induced a considerable increase in CFU-GM and CD34 + 

cells, 111 with rapid recovery of counts after autograft. 

However, the use of growth factors alone in patients 

with neoplastic disorders produces little enthusiasm 

among hematologists. In fact, the spike of progenitor 

cells can be further amplified by combining growth factors 

with chemotherapy. 71 Together with the demonstration 

that tumor cells are also mobilized by growth 

factors, 123 this fact makes the combination of 

chemotherapy with G-CSF or GM-CSF the most reliable 

approach. 86,109,112,113 

In MM as in other diseases, 74,77 the use of growth factors 

following cytotoxic treatment proves to be superior 

to chemotherapy alone in terms of progenitor cell 

yield, 108,112 and significantly contributes to minimizing 

treatment toxicity. 112,124 High progenitor peak levels are 

reported 108 with high-dose chemotherapy, namely Cy at 

7 g/m 2 or etoposide (VP16) at 2 g/m 2 followed by G-CSF 

or GM-CSF, and results seem to compare favorably with 

intermediate-dose Cy with or without G-CSF or GM- 

CSF. In conclusion, the optimal schedule for PBSC mobilization 

in MM has not yet been defined, though the 

most experience is with Cy at 7 g/m 2 followed by G-CSF 

or GM-CSF. A review of the mobilization schedules 

reported so far in MM patients is presented in Table 6. 

Target of collections and cell monitoring 

CD34 + cell number and CFU-GM dose are both reliable 

predictors of engraftment time. 125-129 The amount of 

PBSC necessary for engraftment is not clearly defined, 

but values of 10 to 2010 4 /kg CFU-GM represent a reasonable 

minimal dose. 110,120 Irrespective of disease, rapid 

neutrophil engraftment has been reported with 

2010 4 /kg CFU-GM or 210 6 /kg CD34 + cells. 125,130,131 

However, a higher dose may be necessary for rapid and 

full platelet engraftment. 105,132 In a recent study of MM 

autografts, Tricot et al. 59 found that platelet engraftment 

is influenced by previous history and cell dose. In 

patients with more than 24 months of chemotherapy 

before the autograft, they found a dose ≥510 6 /kg to 

be required for rapid and full platelet recovery post 

graft. This number of CD34 + cells may be obtained with 

1 or 2 apheretic runs, and only a minority of patients, 

namely those with prolonged pre-mobilization treatment, 

need a higher number of apheretic procedures. 

The number of cells needed is obviously greater when a 

double autograft is planned. When this is the case, since 

recovery after a second autograft is influenced by the 

same factors as the first, 59 the number of CD34 + cells to 

be collected simply has to be doubled. 

CD34 + cell monitoring in blood and collection products 

is undoubtely the most reliable and rapid method 

for apheresis planning, 131,133-135 though the assay 



39 

Issue 

Dysregulated or suppressed hematopoiesis 

Methods for mobilization 

Toxicity of mobilization therapy 

Kinetics of recovery after mobilization 

Prediction of harvest 

Progenitor cell assays 

Target of collections 

Apheresis method 

Tumor contamination of harvest 

Related aspects 

Decreased rate of progenitors,* defective monocyte activation,* prior 

chemotherapy 

Type (and doses) of chemotherapy, use of growth factors 

Fever, allergy, infections, thrombosis 

Timed and asynchronous use of WBC, monocytes and platelets 

Prior chemotherapy, G-CSF test 

CD34 + cells, CFU-C 

Need for >510 6 /kg CD34 + cells in heavily pre-treated patients* 

Cell separator, volume processed, schedule of aphereses 

Purging technique 

Table 5. Key issues in mobilization 

and collection of 

PBSCs. 

Note. Most aspects are 

shared with other malignant 

disorders, and only a few 

may specifically affect multiple 

myeloma. These latter 

are marked with an *. 

requires skillful personnel and carries a substantial cost. 

The issue has been reviewed extensively by Rowley. 136 

Siena et al. 133 initially suggested starting the collection 

program as soon as CD34 + cells were detectable in the 

peripheral blood. However, in terms of efficiency, the 

best collections are performed when CD34 + cells are at 

their peak. In practice, aphereses should be started as 

soon as the CD34 + cells in the blood exceed a given level. 

We suggest a value of 20 CD34 + cells/µL combined 

with a WBC level >1.010 9 /L and a platelet count 

>3010 9 /L before starting collections. 82,133 Mononuclear 

cells (MNC) in DNA synthesis also predict a good 

yield when their level in the blood is >5% (or 

>250/µL). 137 

Few studies report detailed data on apheretic PBSC 

collection in MM. Dimopoulos et al. 109 began the 

aphereses when the MNC count went above 0.310 9 /L, 

having as target the collection of > 210 6 /kg CD34 + 

cells. They were able to collect >3.010 6 /kg CD34 + cells 

daily in patients with ≤ 4 months of prior chemotherapy, 

but the mean daily yield was uniformly lower 

(< 110 6 CD34 + cells/kg) in patients with more than 12 

months of chemo-radiotherapy. Tricot et al. 59 initiated 

collections upon recovery of a WBC count > 0.510 9 /L, 

and assumed a target of > 63,108/kg MNC to support 

two autografts. In a recent study 113 aphereses were 

started as soon as the WBC count exceeded 510 9 /L 

after a CHOP-like regimen followed by G-CSF, and 

>610 6 /kg CD34 + cells were collected from all patients 

in 1 to 3 aphereses. 

A predictive test with G-CSF, a single dose of 10 

mcg/kg, followed by CD34 + cell monitoring on days 4 

and 5 has been proposed. 138 The study included patients 

with MM, but the sample was too small to draw any 

conclusions. Steady-state CD34 + cell counts seem to 

predict the yield of PBSCs after mobilization with 

chemotherapy and G-CSF, 139 but not after G-CSF 

alone. 140 Table 7 shows the first apheresis day reported 

with different mobilization methods. 98,108,111,112,117,141 It 

is clear that the CD34 + cell peak occurs very early 

(approximately day 5 or 6) during mobilization with 

growth factors alone. When chemotherapy is included 

in the mobilization schedule, the CD34 + cell peak day 

occurs later (approximately day 20), but the subsequent 

use of growth factors will shorten it by a week or so. 

To conclude, we suggest (Table 8) mobilizing PBSC 

with the combination of chemotherapy and growth factors 

(G-CSF or GM-CSF), and performing serial determinations 

of CD34 + cells in the blood. Aphereses should 

be started as soon as the level of CD34 + cells exceeds 

20/µL, and collections should be performed daily with 

twice the blood volume processed each time. Continuous-flow 

separators are to be preferred. As target for 

collections, the figure of 210 6 /kg CD34 + cells per single 

autograft should be adopted for patients with < 24 

months of prior chemotherapy, while a greater number 

(> 510 6 /kg) should be collected in patients with a 

longer treatment history. 

Assessment of myeloma cells in the 

peripheral blood and role of ex vivo 

purging 

PBSC collections are generally believed to have lower 

tumor cell contamination than BM harvests in cancer 

patients eligible for autografting. Moreover, the use 

of circulating progenitor cells has shown more rapid 

hematopoietic reconstitution than reinfusion of BMderived 

cells, thus reducing the incidence of serious 

infections and virtually eliminating mortality. 142 Consequently, 

PBSCT is widely used after myeloablative therapy 

for the treatment of myeloma patients. 53,56,59 However, 

myeloma-related B-cells bearing the same idiotypic 

determinant as the neoplastic plasma cells have 

been identified in the blood of MM patients under 

steady-state conditions, 143-149 and they may play a crucial 

role in the pathogenesis of the disease. 144,147 Therefore 

in this chapter we will review the published data 

concerning: i) the presence of MM elements in PB and 

their kinetics in response to mobilization protocols; ii) 

methods for myeloma cell assessment; iii) methods for 

ex vivo removal of contaminating tumor cells and the 

role of purging with respect to disease relapse. 


40 


Table 6. PBSC mobilization schedules in multiple myeloma. 

Authors No. pts Treatment Growth factor Day of Peaked Peaked Notes 

progenitor peak CD34 + /µL CFU-GM/mL 

Reiffers 117 15 Cy 7 g/m 2 no nr nr nr 5/13 failures 

Jagannath 97 36 Cy 6 g/m 2 no nr nr nr better with GM-CSF 

39 Cy 6 g/m 2 GM-CSF 17 nr nr 

Tarella 108 11 Cy 7 g/m 2 or VP16 2 g/m 2 GM-CSF 15 (13-16) 126 6432 

4 Cy 21.2 g/m 2 no 16 (16-18) 31 462 

4 Cy 21.2 g/m 2 GM-CSF 14 (14-15) 77 2588 

Ossenkoppele 111 6 no G-CSF6 gg 6 845 

Majolino 112 7 VCAD no 20 (17-30) 622 

7 VCAD G-CSF 13 (9-17) 22 893 

Vasta 113 6 VCED G-CSF 13 (12-15) 70 2391 

Legend. Cy: cyclophosphamide; VCAD: vincristine 1 mg, cyclophosphamide 4x500 mg/m 2 , adriamycin 2x50 mg/m 2 , dexamethasone 4x40 mg. VCED was identical to VCAD except that epirubicin 2x60 to 80 

mg/m 2 was substituted for adriamycin. nr: not reported. 

Identification of circulating myeloma cells 

Circulating B-cells belonging to the malignant clone 

were originally thought to be pre-B-cells on the basis of 

the surface expression of the CD10 (CALLA) Ag, 150 an 

endopeptidase present on all fetal pre-B and B-cells, on 

adult pre-B-cells and their neoplastic counterparts. 151 

However, the CD10 Ag has also been found on activated 

B-cells 151 and does not seem to be restricted to the early 

stages of B-lineage differentiation. Moreover, PB 

abnormal B-lymphocytes express plasma cell markers 

such as PCA-1 and PC-1 and the CD45RO Ag isoform, 

which is typical of late B-cells. 145 Thus phenotypic analysis 

of circulating CD19 + cells indicates a heterogeneous, 

continuously differentiating B-lineage. 145 By physical 

parameters, CD19 + cells include a small and a large subset 

that are mainly late B-cells (pre-plasma cells) coexpressing 

CD20, CD10, PCA-1, CD45RO and CD24 Ag. 148 

The majority of large B-cells also express the CD56 Ag 

and high density CD38, whereas small lymphocytes show 

only minor expression of these 2 antigenic determinants. 

This phenotypic profile (i.e. CD19 + CD20 + CD38 ++ CD56 + ) 

is not found in normal resting B-cells. Interestingly, 

malignant cells were detected at diagnosis, irrespective 

of tumor burden and stage of disease, 148 and treatment 

had no detectable effect on the large B-cell subset. Conversely, 

a significant decrease in the number of small B- 

lymphocytes followed chemotherapy, although these 

cells returned to baseline value once the therapy was 

discontinued. In this regard, it was previously shown that 

circulating CD19 + cells in MM express the functional 

multidrug transporter p-glycoprotein, 147,169 thus suggesting 

that blood B-cells include a highly drug-resistant 

subset capable of inducing disease recurrence in myeloma 

patients. However, it should be noted that mature 

plasma cells do not always express the CD19 Ag, whereas 

the presence of the CD56 Ag discriminates clonal plasma 

cells from normal ones. 152 In addition, the recently 

described monoclonal antibody B-B4 152 seems to be highly 

specific for BM and circulating terminal plasma cells. 

More recently, the issue of myeloma cell contamination 

in leukapheresis products and the kinetics of circulating 

tumor cells in response to mobilization protocols have 

been addressed. 67,69,153-155 These studies have consistently 

shown that the majority of PBSC collections, if not all, 

are contaminated by myeloma cells, which represent up 

to 10% of PB mononuclear cells by immunophenotyping 

and molecular analysis using polymerase chain reaction 

(PCR) with consensus oligonucleotides to the Ig 

heavy chain complementary determining region III (CDR 

III) (see below). 155 The same pattern of contamination 

has been shown following high-dose Cy and either G- or 

GM-CSF, 67,69,155 as well as after G-CSF alone, 154 suggesting 

that growth factors for stem cell mobilization, 

regardless of the use of chemotherapy, may influence 

the expression of adhesion molecules associated with 

the myeloma cell membrane. Notably, kinetic analysis 

has demonstrated that following high-dose Cy and G- 

CSF, the concomitant mobilization of plasma cells and 

hematopoietic progenitor cells in the PB takes place with 

the maximum peak of neoplastic elements occurring 

within the optimal time period for collection of circulating 

CD34 + cells. 67 Conversely, GM-CSF seems to 

reduce asynchronous mobilization of neoplastic elements 

and hematopoietic stem cells into PB, so that the 

contamination of actively proliferating myeloma cells is 

minimal in the first two days of apheresis. 156 

Methods for assessment of minimal residual 

disease 

A number of methods have been proposed to detect 

malignant cells in the blood of myeloma patients, including 

immunologic assessment by monoclonal antibodies, 

flow cytometry analysis of DNA and cytoplasmic Ig, 

studies on gene rearrangement. Each of these techniques 

has limitations in sensitivity and, in some cases, specificity. 

For instance, analysis of the hypervariable region 

of the Ig heavy chain (IgH) gene using a set of familyspecific 

primers (IgH fingerprinting) requires 0.1% mon- 


PBSC transplantation in multiple myeloma 41 

Regimen Day of Day of No. apheresis References 

progenitor peak first apheresis 

G-CSF 10 mcg/kg/day 6 d 6 6 phlebotomy 2 112 

Cy 7 g/m 2 nr 20 6 117 

Cy 7 g/m 2 + GM-CSF 15 nr 4 98-108 

Cy 7 g/m 2 + G-CSF 15 14 2-3 141 

VCAD 20 14 6 112 

VCAD + G-CSF 13 12 2-3 112 

Table 7. Day of cell peak and 

of first apheresis after PBSC 

mobilization in patients with 

MM. The addition of G-CSF 

or GM-CSF shortens the 

time to progenitor peak and 

consequently the time to 

apheresis. Mean number of 

apheretic procedures was 

lower when growth factors 

were employed. 

Legend: nr: not reported. 

oclonal cells 157 and may produce false positive results. 

Conversely, dual-parameter flow cytometric analysis 

(e.g. CD19/monoclonal light chain) and evaluation of 

intracytoplasmic monoclonal heavy or light chain are 

highly specific and allow detection as low as 0.1% 67 (Figure 

3); however, they only assess mature Ig + B-cells. 

Recently, several laboratories have described applications 

of PCR techniques to increase significantly the sensitivity 

and specificity of detection of minimal residual 

disease (MRD). Both consensus oligonucleotides (ODN) 146 

and family-specific primers 69 have been used to amplify 

the CDRIII of rearranged heavy chain alleles (Figure 4) 

from myeloma samples. From the sequence of the amplified 

products, allele-specific (tumor-specific) oligonucleotides 

(ASO) were synthesized and used directly in 

PCR amplification reactions (ASO-PCR) for each patient 

sample to detect the malignant clone. The sensitivity of 

this method is 1:10 5 normal cells and a quantitative 

analysis can be performed by generating titrations 

curves of tumor cells. Alternatively, direct fingerprinting 

of CDRIII IgH gene rearrangement may be used, although 

the sensitivity is 1:10 4 normal cells. 67 

The biological and prognostic significance of cancer 

cells present in autologous grafts is still unknown and 

circulating myeloma cells may only reflect advanced 

stages of the disease; therefore relapse may be caused 

by regrowth of residual clonogenic cells in vivo. However, 

considering that MM is a disease intrinsic to BM 

and recent studies clearly show that reseeding of reinfused 

malignant cells contributes to relapse, 158 several 

attempts have been made to remove myeloma cells 

from BM or PBSC autografts using different strategies. 

Ex vivo purging of myeloma cells 

Of the purging methods proposed for the elimination 

of MRD, the cyclophosphamide derivative 4-hydroperoxycyclophosphamide 

(4-HC) was the first used, 159 on the 

basis of in vitro models demonstrating that this compound 

was able to eliminate BM-infiltrating MM cell 

lines. 160 The main mechanism of action of 4-HC is based 

on a marked inhibition of myeloma cell growth, whereas 

it spares normal primitive hematopoietic cells. 161 Moreover, 

this alkylating agent seems to induce the apoptotic 

death of tumor cells 162 as well as activate immune 

mechanisms capable of controlling malignant cell proliferation. 

163 Because 4-HC does not affect surface antigen 

expression of myeloma cells, it is also a potential candidate 

for combined treatment with monoclonal antibodies 

(MoAbs), and preliminary in vitro data confirm the 

additive effect of these two purging techniques. 160 Several 

MoAbs directed against tumor-associated or cell differentiation 

antigens not expressed by primitive cells 

responsible for hematopoietic engraftment have been 

selected for clinical trials after in vitro studies demonstrated 

high purging efficacy with the use of complement, 

164,165 toxins 166,167 or immunoaffinity columns. 168 

Gobbi et al. developed a series of MoAbs that recognize 

mature plasma cells as well as B-cell precursors. One of 

them (8A) was conjugated with the ribosome-inactivat- 

Table 8. Recommendations for PBSC mobilization and their 

apheretic harvest in patients with multiple myeloma. 

• Mobilization with chemotherapy + growth factors (G-CSF or GM-CSF) 

• Serial CD34 + determinations according to institutional protocol 

• Start apheresis when CD34 + cells in blood >2010 6 /L 

• Continuous flow separator, volume processed 2 blood volume per run 

• Collect at least 210 6 /kg CD34 + cells in patients with < 24 months prior 

chemothrapy, at least 510 6 /kg CD34 + cells in patients with > 24 months 

prior chemotherapy 

Figure 3. Circulating monoclonal B-lymphocytes and plasma 

cells assessed by double fluorescence immunostaining: 

intracytoplasmic Ig (green)/nuclear BRDU (red). Bromodeoxyuridine 

(BRDU) is incorporated in actively proliferating 

cells. 


42 


ing toxin momordin and clinically tested in 8 advanced 

stage MM patients to eliminate, ex vivo, contaminating 

myeloma cells prior to ABMT. 166 Although a marked tumor 

reduction was observed in all evaluable patients, none of 

them achieved CR and hematopoietic reconstitution following 

the myeloablative conditioning therapy was significantly 

delayed in 3 patients. These preliminary results 

showed the feasibility of this purging approach despite 

the poor selection of patients. 

The same MoAbs were also employed in vitro to remove 

myeloma cells through the avidin-biotin immunoabsorption 

technique, and the result was a greater than 3 log 

reduction in tumor cells with acceptable recovery of BM 

progenitors. 168 More recently, Goldmacher et al. 167 reported 

the development of an anti-CD38 immunotoxin capable 

of killing 4-6 logs of human myeloma and lymphoma 

cell lines. The immunotoxin was composed of an anti- 

CD38 antibody conjugated to a chemically modified ricin 

molecule (blocked ricin). However, the CD38 Ag may not 

be the proper target for purging because it is strongly 

expressed on myeloma plasma cells (see above) and on 

committed hematopoietic progenitor cells, 169 which are 

thought to be essential for rapid BM reconstitution. More 

specific antibodies directed either toward B-cells (anti- 

CD10 and CD20) or mature plasma cells (PCA-1) and complement 

were used to deplete tumor cells from the graft 

before ABMT by Anderson et al. 165 Following a TBI-containing 

conditioning regimen, a neutrophil count greater 

than 0.510 9 /L and an unsupported platelet count 

greater than 2010 9 /L were reached at a median of 21 

days (range 12-46) and 23 days (range 12-53), respectively. 

Similarly, immunologic reconstitution was not different 

from that commonly observed in cancer patients 

receiving unmanipulated autograft. This study documented 

that high-dose chemo-radiotherapy can produce 

a high response rate in pretreated patients with sensitive 

disease, and MoAb-based purging methods do not prevent 

rapid and sustained engraftment. However, the occurrence 

of relapses post-ABMT and partial responses will 

not define the need, if any, for marrow purging until more 

effective ablative strategies are developed. Taken together, 

these data demonstrate that the heterogeneity of Ag 

expression on neoplastic cells and the lack of true tumorspecific 

determinants may greatly influence the efficacy 

of antibody-based strategies for the depletion of myeloma 

cells. Alternatively, long-term Dexter-type marrow 

cultures have been used to select normal myeloid progenitors 

from heavily infiltrated myeloma BM, on the basis 

of the selective growth advantage of benign cells over 

malignant cells in this system. 170 

Enrichment of hematopoietic CD34 + cells has lately 

been shown to be an alternative approach to myeloma 

cell removal with a limited loss of normal stem cells. The 

CD34 Ag is a 110-120 kD glycoprotein that is mainly 

CDRIII junctional region 

VH N DH N JH 

FRI CDRI FRII CDRII FRIII 

VH family 

consensus oligo 

VH consensus oligo 

CDRIII 

VH N DH N JH 

clonal CDR-III PCR product at diagnosis 

JH consensus oligo 

Sequencing 

ASO probe construction 

(allele specific oligonucleotide probe) 

ASO 

hybridization negative 

ASO 

hybridization positive 

VH N DH N JH 

non clonal CDRIII PCR product 

= molecular remission 

VH N DH N JH 

clonal CDRIII PCR product 

= molecular persistance of minimal residual disease 

Figure 4. Schematic representation of the genomic region of rearranged CDRIII of IgH gene and further utilization of the PCR 

product for detection of MRD. For further details see text. 



43 

expressed on the earliest identifiable precursor cells and 

committed myeloid progenitors. 169 In normal individuals, 

CD34 + cells represent 1% to 4% of the mononuclear cell 

fraction in the BM, whereas they are barely detectable in 

the PB. 169 In addition, the CD34 Ag is not expressed on 

the surface of mature plasma cells in MM, although the 

possibility that this glycoprotein may be present on clonally 

less differentiated B-lymphocytes is still a matter of 

debate. As reported above, recent data support the 

hypothesis that MM originates in the later stages of B- 

cell differentiation when B lymphocytes have lost the 

CD34 Ag, 171 whereas other studies have found CD34 + 

cells to be part of the neoplastic clone. 172,173 It should be 

underlined, however, that reverse transcription-PCR, 

which was used to detect MRD in those studies, is an 

extremely sensitive technique, and the potential contamination 

of the CD34 + cell fraction by unwanted cells 

should be carefully avoided. 

In this respect, Vescio et al. 171 did not find IgH gene 

clonal rearrangement in collections of 99.99% pure 

CD34 + cells obtained after using a combination of two 

methods of stem cell purification. Schiller et al. 66 and 

Lemoli et al. 67 reported the first studies on purified, CD34- 

selected PBSCT conducted in patients with advanced MM. 

A median of 4.65 and 410 6 CD34 + cells/kg were reinfused 

in the two trials with a median purity of 77% and 

88.5%, respectively. The median time to neutrophil and 

platelet recovery was 12 days and 10 and 11 days, respectively, 

with no difference with respect to a group of 

patients receiving unmanipulated PBSCs. 67 

Both reports utilized rigorously quantitative immunofluorescence 

and/or IgH gene rearrangement analysis, 

and tumor cell depletion ranging from 2.5 to 4.5 logs was 

achieved. However, the persistence of myeloma cells in 

the CD34 + cell fraction was documented by sensitive PCR 

assay in all cases heavily contaminated before positive 

selection of CD34 + cells. Thus an additional purging step 

may be necessary to achieve a virtually tumor-free autograft. 

In this regard, studies aimed at optimizing myeloma 

cell depletion by positive selection of primitive CD34 + 

Lin – Thy + cells have already been performed 155 and clinical 

trials are currently in progress. 

In summary, all these studies show the capacity of 

purging techniques to eliminate a substantial proportion 

of the myeloma cells from autologous grafts without 

affecting their engraftment potential. The clinical impact 

of purging on disease relapse remains to be determined 

in future randomized trials. 

Post-transplant (immuno)therapy 

In MM as well as in other hematologic malignancies, 

the primary objective of high-dose therapy with hemopoietic 

stem cell support is to prolong survival and possibly 

to cure an otherwise incurable disease. The aim of 

post-transplant therapy is to prevent recurrence of the 

disease while assuring good quality of life. From this 

latter point of view, there is no room for additional 

chemotherapy as a preventive means. In addition, highdose 

chemotherapy itself involves a risk of secondary 

myelodysplastic syndrome or acute myeloid leukemia. 

This risk is apparently related to prolonged alkylating 

agent therapy prior to transplantation and would 

undoubtedly increase with additional post-transplant 

chemotherapy. 

In the past few years interferon- (IFN-) has been 

extensively evaluated in the management of MM, either 

as part of the induction program or as maintenance therapy. 

173 Although controversial findings were frequently 

reported, several clinical trials showed a prolongation of 

the remission phase, and even of the survival duration, 

for patients receiving IFN- after a favorable response 

to conventional chemotherapy. 174,175 These results suggested 

that IFN- might be particularly useful in patients 

with low tumor burden or minimal residual disease, and 

led to clinical investigations of this agent in the autograft 

setting. 

The European Group for Blood and Marrow Transplantation 

(EBMT) has recently presented a retrospective study 

of a large series of MM patients treated with autologous 

stem cell transplantation. 50 Interestingly, post-transplant 

treatment with IFN- was independently associated with 

extended survival of responding patients, i.e. those achieving 

either CR or partial remission. Moreover, Powels et 

al. 176 designed a randomized clinical trial aimed at comparing 

maintenance IFN- therapy with no maintenance 

after HDM and ABMT. 175 The authors found that IFN- 

prolonged remission and improved the survival after autotransplant, 

and that this effect was particularly marked in 

the group of patients achieving CR. 

Maintenance IFN- is usually started three months 

after transplant and is given sc at a dosage of 310 6 

U/m 2 , 3 times weekly. This dose usually induces mild 

hematological and non-hematological toxicity, thus 

allowing good quality of life. Available data indicate that 

about 50% of the MM patients who achieve CR and are 

then treated with IFN- remain in remission four years 

after transplantation. 

Alternatively, maintenance treatments aimed at prolonging 

the duration of disease control after transplantation 

may also include the administration of interleukin 

2 (as nonspecific immunotherapy) 177 or humanized antiidiotype 

monoclonal antibodies, which could allow selective 

killing of myeloma cells and might be particularly 

useful for controlling minimal residual disease. 

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eview 

Allogeneic hematopoietic stem cells 

from sources other than bone marrow: 

biological and technical aspects 



Medica “Seràgnoli”, Policlinico S. Orsola, via Massarenti 9, 40138 

Bologna, Italy. Received August 22, 1996; accepted January 20, 

1997. 

FRANCESCO BERTOLINI, ARMANDO DE VINCENTIIS, LUIGI LANATA, 

ROBERTO M. LEMOLI, RITA MACCARIO, IGNAZIO MAJOLINO, 

LUISA PONCHIO, DAMIANO RONDELLI, ANTONIO TABILIO, 

PAOLA ZANON, SANTE TURA 

Division of Oncology, IRCCS Fondazione Maugeri, Pavia; 

Dompé Biotec SpA, Milan; Amgen Italia SpA, Milan; Istituto 

di Ematologia ed Oncologia Medica “Seràgnoli”, University of 

Bologna, Bologna; Department of Pediatrics, IRCCS Policlinico 

S. Matteo di Pavia, Pavia; Department of Hematology and 

BMT Unit, Ospedale “V. Cervello”, Palermo; Department of 

Internal Medicine, University of Pavia and IRCCS Policlinico S. 

Matteo, Pavia; Institute of Hematology, University of Perugia, 

Italy 

Background and Objectives. Identification and characterization 

of hematopoietic stem cells in peripheral 

blood (PB) and cord blood (CB) have suggested feasible 

alternatives to conventional allogeneic bone marrow 

(BM) transplantation. The growing interest in this 

use of allogeneic stem cells has prompted the Working 

Group on CD34-positive Hematopoietic Cells to review 

this subject by analyzing its biological and technical 

aspects. 

Evidence and Information Sources. The method used 

for preparing this review was informal consensus development. 

Members of the Working Group met three 

times, and the participants at these meetings examined 

a list of problems previously prepared by the 

chairman. They discussed the individual points in order 

to reach an agreement on the various concepts, and 

eventually approved the final manuscript. Some of the 

authors of the present review have been working in the 

field of hematopoietic stem cell biology and processing, 

and have contributed original papers published in 

peer-reviewed journals. In addition, the material examined 

in the present review includes articles and 

abstracts published in journals covered by the Science 

Citation Index® and Medline®. 

State of Art. Several studies have now shown that 

hematopoietic stem cells collected from peripheral 

blood after the administration of G-CSF, or from cord 

blood upon delivery, are capable of supporting rapid 

and complete reconstitution of BM function in allogeneic 

recipients. Perhaps more importantly, reinfusion 

of large numbers of HLA-matched T-cells from PB collections 

or T-cells with various degrees of HLA disparity 

from CB did not result in a higher incidence or greater 

severity of acute graft-versus-host disease than expected 

with BM. Based on the data reviewed, operative 

guidelines for mobilization, collection and graft processing 

are provided. 

Perspectives. It should be remembered that despite 

the growing interest, these procedures must be still 

considered as advanced clinical research and should 

be included in formal clinical trials aimed at demonstrating 

their definitive role in stem cell transplantation. 

In this regard, a large European randomized study is 

currently comparing PB and BM allografts. However, 

the possibility of collecting large quantities of 

hematopoietic progenitor-stem cells, perhaps with 

reduced allo-reactivity, offers an exciting perspective 

for widening the number of potential stem cell donors 

and greater leeway for graft manipulation than is possible 

with BM. 

©1997, Ferrata Storti Foundation 

Key words: hematopoietic stem cells, bone marrow, cord blood, 

peripheral blood, allogeneic transplantation, graft-versus-host disease 

Allogeneic bone marrow transplantation has progressed 

from a highly experimental procedure to 

being accepted as the preferred form of treatment 

for a wide variety of diseases. 1 There have been 

impressive improvements in this therapeutic procedure 

in the last two decades, but the most important 

advances probably took place in the last few years and 

concern the source of hematopoietic stem cells itself. 

Whereas this had always been by definition the bone 

marrow since the very beginning, identification of stem 

cells in peripheral and cord blood has now provided 

useful alternatives. 

In 1994 the growing interest in the use of peripheral 

blood stem cells (PBSC) in the setting of allogeneic 

bone marrow transplantation induced the GITMO 

(Gruppo Italiano Trapianto di Midollo Osseo) to promote 

a Study Committee for evaluating the key aspects of 

allogeneic PBSC collection and transplantation. This 

Committee produced a list of recommendations that 

were published as a position paper in this Journal at the 

beginning of 1995. 2 In summary, the authors strongly 

recommended the use of allogeneic PBSC in experienced 

centers, in well-defined clinical settings, and possibly 

– for the time being – in patients with advanced 

disease. 

As the use of PBSC expanded both in the autologous 

and the allogeneic setting, expression of the CD34 antigen 

became increasingly important for their character- 


50 

F. Bertolini et al. 

ization. An ad hoc working group reviewed the biology 

and clinical relevance of CD34-positive cells in this journal 

in 1995. 3 In particular, techniques for CD34-positive 

cell separation and procedures for their collection from 

peripheral blood were analyzed. A brief chapter was also 

devoted to CD34-positive cells in cord blood. 3 

The working group on CD34-positive hematopoietic 

cells subsequently reviewed the use of PBSC in acute 

myeloid leukemia 4 and multiple myeloma. 5 The growing 

interest in the use of PBSC and cord blood stem cells in 

the setting of allogeneic transplantation has now 

prompted the working group to review this subject by 

analyzing its biological and technical aspects. 

PBSC mobilization and collection in 

normal donors 

Until recently, the collection of hematopoietic cells for 

allogeneic transplantation has required general or spinal 

anesthesia and multiple punctures of iliac bones. However, 

marrow harvesting is not completely devoid of 

complications, side effects or patient discomfort. In a 

report on 1270 harvest procedures in Seattle, 6 6 donors 

suffered life-threatening complications and 10 showed 

significant operative site morbidity. As many as 10 percent 

of donations were associated with fever, and 

increasing donor age was significantly linked to poor 

cell harvest. In a different survey, 10 percent of donors 

recovered completely from marrow donation only more 

than 30 days after the procedure. 7 

PBSC transplantation represents an alternative 

approach. In autologous transplantation peripheral 

blood is now replacing bone marrow as a source of progenitor 

cells. 8 The advantage is quicker hematopoietic 

recovery 9,10 with consequently fewer complications and 

shorter hospital stay. 

In the autologous setting, PBSC can be collected after 

mobilization with chemotherapy, 11,12 growth factors, 13 

or a combination of the two. 14 In a randomized study, 

leukaphereses created less anxiety and pain than bone 

marrow harvest. 15 

Table 1. Relationship between CD34 + cell yield and G-CSF 

dose in allogeneic PBSC donors. Only clinical experiences 

are reported. 

Authors (ref.#) 

Donors 

No. 

G-CSF, 

dose/kg and 

days of 

administration 

CD34+ 

collected 

x 10 6 

Apheresis 

No. 

Weaver (31) 4 16 µg, 4 d 9.6/kg 2 

Korbling (16) 9 12 µg, 7 d 13.1/kg 3 

Bensinger (18) 8 16 µg, 6 d 13.1/kg 2 

Schmitz (17) 8 5-10 µg, 5-6 d 6.7/kg 1-3 

Russell (34) 9 6-8 µg, 2-4 d 4.7/kg 1-2 

Majolino (27) 5 

6 

10 µg, 5 d 

16 µg, 4 d 

754 

789 

Tabilio (1996) 39 12 µg, 4-7 d 132.6 2-4 

2 

2 

In allogeneic transplantation, the use of PBSC has 

been somewhat delayed by a possible increase in graftversus-host 

disease (GVHD) as a consequence of the 

much higher number of lymphocytes in the graft inoculum, 

and by the need for a mobilization treatment for 

healthy individuals in order to obtain a good cell yield. 

However, the clinical experience of the last two years 

suggests that the incidence of acute GVHD is not 

increased with PBSC as compared to marrow, and that 

in healthy donors a sufficient cell number can be 

obtained by using growth factors alone, in particular G- 

CSF. 16-20 As a consequence, the number of allogeneic 

PBSC transplants is increasing rapidly. The European 

Blood and Marrow Transplant Group (EBMT) registered 

only 12 PBSC allografts in 1993, but their number 

increased to 180 in 1994 and to 537 in 1995 (Gratwohl, 

personal communication). 

Collection of PBSC in normal donors 

On biological grounds, there are several means of 

mobilizing progenitor cells into the peripheral blood, but 

their ultimate modality of action is always detachment 

of the CD34 + progenitor cell from marrow stroma and 

endothelium, to which it is normally bound by interactions 

with different integrin-adhesion molecules. 3,21 We 

may induce detachment either by an inhibition of the 

link between CD34 + cells and stroma, or by inducing a 

stress to the hematopoietic system capable of favoring 

the egress of progenitor cells from marrow to circulation. 

The former is obtained by means of monoclonal 

antibodies directed against adhesion molecules, 22 while 

the latter is based on the use of a drug or a combination 

of drugs. Richman et al. 23 demonstrated for the first 

time in man that chemotherapy-induced cytopenia is 

followed by a substantial increase of CFU-C in blood. 

In normal donors, however, the use of chemotherapy 

is ethically unacceptable, and only growth factors must 

be employed. Though a number of other cytokines are 

able to induce an increase of PBSC, only G-CSF and GM- 

CSF have been utilized in clinical practice. G-CSF in particular 

has an excellent mobilizing effect when used 

alone. 13,24-29 

The pilot experience with stem cell mobilization in 

normal donors is the one reported by the Seattle group. 

They administered G-CSF 300 µg/day or 6 µg/kg/day to 

increase WBC levels in apheresis collections from granulocyte 

donors. 30 A number of different schedules were 

later applied to mobilize PBSC for allogeneic transplantation. 

The results in terms of CD34 + cell collection are 

reported in Table 1. In most of the studies the G-CSF 

dose ranged from 10 to 16 µg/kg/day. With 16 µg/kg/day 

for 5 days, Weaver et al. 31 collected 1.6 to 12.6 (median 

9.6)×10 6 /kg CD34 + cells with two aphereses. All transplants 

were syngeneic, and recovery of 0.5×10 9 /L granulocytes 

and 20×10 9 /L platelets occurred on day 13 and 

10, respectively. With the same dose administered for 4 

days, Majolino et al. 27 were able to mobilize (median) 

147×10 6 /L CD34 + cells on day 4, a 65-fold increase over 

the baseline level. The median collection was 754×10 6 


Non bone marrow allogeneic hemopoietic stem cells 

51 

CD34 + cells and 270×10 8 CD3 + cells with 2 aphereses. 

With 12 µg/kg for 6 days, Körbling et al. 16 collected a 

mean of 13.1×10 6 /kg CD34 + cells with 3 aphereses, and 

their patients recovered >0.510 9 /L granulocytes on day 

10 and >20×10 9 /L platelets on day 14. However, their 

short recovery times were also influenced by the absence 

of methotrexate from GVHD prophylaxis. 

The relationship between G-CSF dose and CD34 + cell 

mobilization is supported in part by the study by Dreger 

et al., 26 who compared 5 µg/kg/day and 10 µg/kg/day G- 

CSF in normal volunteers. They found 10 µg/kg to be 

superior in terms of progenitor cell yield. With higher 

doses the advantage seems to decline, and no statistical 

difference was found between 10 µg/kg for 5 days 

and 16 µg/kg for 4 days in a retrospective non-randomized 

study (Figure 1). 32 Dührsen 33 has suggested that 

the maximal effect in terms of progenitor cell increase 

is that obtained at a dose level of 10 µg/kg/day. This is 

also the dose recommended by the GITMO in its recently 

published guidelines. 2 

The number of apheretic procedures necessary for a 

good collection is critical for the donor, and may vary 

with the dose and schedule of G-CSF as well as with the 

volume processed. 

Bensinger et al. 28 routinely employ a schedule of 16 

µg/kg/day for 5-6 days in an effort to minimize the 

number of apheretic procedures. With this dose, a median 

of approximately 7×10 6 /kg CD34 + cells are obtained 

with a single apheresis performed on day 5. At the M.D. 

Anderson Cancer Center in Houston 29 a schedule of 12 

µg/kg/day for 4-6 days is used. With a single large volume 

apheresis the target CD34 + cell dose of > 4×10 6 /kg 

is reached in nearly 80% of the donors. Russell et al. 34 

mobilized their donors with 6-8 µg/kg/day for 2-4 days. 

By daily monitoring of CD34 + levels, the target of 

2.5×10 6 /kg CD34 + cells was achieved with a single 2-4 

hour harvest in 12 out of 14 donors. With 24 µg/kg/day 

Figure 1. Schematic representation of timing in PBSC mobilization 

in healthy donors. G-CSF is administered at a daily 

dose of 10 µg/kg, apheretic harvest is performed on day 5 

(and 6). Day 5 collection cells are stored at 4°C till the following 

day, when they are infused together with day 6 cells. 

Parentheses indicate that G-CSF is given and aphereses 

performed only if the target number of CD34 + cells is not 

reached with the day 5 apheretic run. 

G-CSF for 4 days Waller et al. 35 were able to collect 

13×10 6 /kg CD34 + with a single apheresis; however, one 

donor suffered severe side effects and the G-CSF dose 

had to be halved. 

The mobilization kinetics of PBSC under low daily doses 

of G-CSF has also been investigated. With 2.5 

µg/kg/day G-CSF on days 1 to 6 followed by 5.0 

µg/kg/day on days 7 to 10, a CFU-GM peak was obtained 

on day 6, but continuing G-CSF administration at 5 

µg/kg/day did not increase the level of circulating CFU- 

GM. 36 

With a single G-CSF dose of 15 µg/kg a significant rise 

in CD34 + cells, CFU-GM and BFU-E was obtained, 37 but 

the reported counts of 250/mL, 3.2×10 3 /mL and 

1.75×10 3 /mL, respectively, are not comparable with 

those obtained with prolonged administration schedules. 

Bishop et al. 38 reported their experience with G-CSF 

at 5 µg/kg/day. Aphereses began on day 4 of G-CSF 

administration. However, the target cell CD34 + dose of 

>1×10 6 /kg required 3 to 4 aphereses. With this method, 

median time to ANC > 0.5×10 9 /L was 12 days but all 

patients received G-CSF after the allograft. 

With G-CSF doses ranging from 10 to 16 µg/kg/day, 

the progenitor cell peak occurs on day 4 or 5. 2,20,27,28,32,39 

Since the CD34 + cell level rapidly declines after growth 

factor withdrawal, it is highly recommended that its 

administration be continued till the end of apheretic 

harvests. 

In both the Seattle and the GITMO experiences, the 

WBC peak occurred approximately the same day as the 

CD34 + cell peak. In the GITMO study, 39 the level of CD34 + 

cells reached a peak of (mean) 135.5×10 6 /L CD34 + cells, 

a 19-fold increase over the mean baseline level. 

Lymphocytes also increased, doubling their counts on 

day 5. A number of CD34 + cells > 4×10 6 /kg was collected 

in 51% of donors with a single apheresis, in 85% 

with two. Optimal collections are obtained on days 4 

and 5 of G-CSF administration. 28,39 It is likely that starting 

PBSC collection on day 4 is best when using 16 

µg/kg/day, whereas day 5 is better when lower doses are 

employed (Figure 2). 

In normal volunteers GM-CSF has found application 

less frequently than G-CSF. Lane et al. 40 studied G- and 

GM-CSF alone and a combination of the two. The total 

number of CD34 + cells collected from the G-CSF group 

with a single apheresis was 119×10 6 , and was not significantly 

different from that collected from the group 

treated with G- and GM-CSF (101×10 6 CD34 + cells), but 

both were greater than that from the group treated with 

GM-CSF (12.6×10 6 ). However, a higher fraction of an 

early CD34 + /HLA-DR – /CD38 – cell population was found 

among the CD34 + cells after GM-CSF administration. 

Whether this early fraction is associated with more rapid 

engraftment is presently unknown. 

Predictive factors for progenitor cell yield have not 

been studied in normal volunteers. Though there is anecdotal 

experience of donors failing to respond, only age 

was reported to influence the quality of collections in a 

single study. 26 


52 


The number of PBSC necessary for rapid and stable 

engraftment is unknown. In the autologous setting a 

dose of >2×10 6 /kg CD34 + cells has been suggested, 41 

but the requirement might be higher in allogeneic transplantations 

as a consequence of the immunological 

mechanisms involved. In Seattle 28 4 out of the 53 normal 

donors yielded only 0.6, 1.49, 1.55 and 1.74×10 6 

CD34 + cells/kg. Despite the low cell numbers, successful 

engraftment was achieved in all cases. We suggest 

that collection of >4×10 6 /kg CD34 + cells is the target for 

a safe allogeneic transplantation. Lower doses, however, 

may be sufficient. A lower limit of >210 6 /kg CD34 + 

cells would be reasonable for those patients whose 

donors respond poorly to cell mobilization. 

There are currently no contraindications to cryopreservation 

of cells for later use after thawing. Though 

most centers currently infuse freshly collected apheresis 

products, one may consider the advantage of separating 

the mobilization/collection phase from transplantation 

in terms of logistics and patient safety. 

Side effects and toxicity of the procedure 

Early toxic effects of G-CSF in healthy donors are now 

well known. The GITMO survey 39 on 76 healthy subjects 

aged 6 to 67 years receiving G-CSF for PBSC mobilization 

reveals that the side effects of G-CSF administration 

are acceptable, the only problem being moderate to 

severe bone pain in 13% of donors (Table 2). 

Twenty-three percent of donors also said the 

apheretic procedures were demanding. Comparable side 

effects are reported in other studies. 34,28 Additional problems 

could include pneumothorax due to jugular vein 

cannulation and paresthesia. 34 Nonetheless, donors who 

had previously given marrow mostly agreed that they 

preferred blood cell mobilization and collection to marrow 

harvest. 34-39 A good policy would be to avoid the use 

of venous catheters. In autologous PBSC harvesting 

where mobilization treatment often includes chemotherapy, 

central venous catheter (CVC) occlusion necessitating 

thrombolytic therapy was the most commonly 

Figure 2. Variations of blood cell counts in normal donors 

during G-CSF treatment and apheretic collection of mononuclear 

cells. The curves represent mean values. Data are 

those of 76 normal donors. 39 

Table 2. Incidence and grading of side effects reported during 

G-CSF administration in 76 healthy donors from the GIT- 

MO. 39 

% donors 

Absent Mild Moderate Severe 

Bone pain 27.4 59.6 9.6 3.2 

Arthralgias 54.8 32.2 11.2 1.6 

Headache 70.9 20.9 8 0 

Fatigue 74.5 22 3.3 0 

Fever 91.5 6.7 1.6 0 

observed complication, occurring in 15.9% of CVC-aided 

collections. 42 

Variations in blood counts mainly consist of a pronounced 

WBC increase, a moderate thrombocytopenia 

and a slight decrease of hematocrit values. In the Italian 

survey, thrombocytopenia from mild (< 70×10 9 /L) to 

moderate (< 50×10 9 /L) followed PBSC harvests in 40% 

and 10% of cases, respectively. WBC counts exceeded 

50×10 9 /L in 40% of cases, and 70×10 9 /L in 8%. Bensinger 

et al. 28 report their experience with 124 donors treated 

with G-CSF at various doses and scheduling. Forty-one 

were granulocyte donors, while 13 were PBSC syngeneic 

and 63 allogeneic donors. One donor had a myocardial 

infarction after the first apheresis, but he had a previous 

history of infarction. Thrombocytopenia was in part 

related to G-CSF dosage, in part to the volume of blood 

processed. A count


53 

The studies on leukemia development after G-CSF 

treatment must be considered with caution. A Japanese 

group 46 reported data on 170 children with aplastic anemia. 

Eleven out of the 108 receiving G-CSF had a transformation 

to MDS or leukemia, while this evolution 

occurred in none of the 62 patients not receiving G- 

CSF. Another study 47 reports the evolution to 

MDS/leukemia in 13 patients with congenital neutropenia 

treated with G-CSF, with the occurrence of 

monosomy 7 in 10 of them. However, as suggested by 

Smith et al. 48 in a study on the leukemic evolution of 

Kostmann’s disease, the fact that congenital neutropenia 

may evolve into MDS and AML under G-CSF treatment 

has no implication for normal donors, since it is 

the underlying hemopoietic defect that represents a preleukemic 

condition. In fact, in Kostmann’s disease not all 

the chromosomal aberrations involve chromosome 7, 

and when other abnormalities are detected leukemia 

does not develop. 

The use of G-CSF for mobilization of PBSC in children 

should be considered with more attention. Though there 

may be a specific advantage in collecting PBSC from 

children in the case of considerable disparity in body 

weight with the recipient, the GITMO stated that such 

a practice should be discouraged in standard allogeneic 

transplants. 2 This is also the opinion of the Italian 

Association of Pediatric Hemato-Oncology (AIEOP). 

PBSC have also been employed for allogeneic engraftment 

in MUD transplants. A small series was presented 

by Ringdén et al. 49 in Geneva. Six patients with high risk 

hematological malignancies received PBSC from unrelated 

donors, 4 of them as primary treatment and 2 for 

treatment of graft failure. For PBSC mobilization the 

donors received G-CSF 5 to 12 µg/kg and leukaphereses 

were performed using continuous flow devices. One 

donor complained of rib pain and one of nausea, dizziness 

and anxiety. 

One advantage of using PBSC for MUD transplants 

could derive from the higher number of progenitor cells, 

with better engraftment and reduction of failures. For 

the donor, the chance of obtaining stem cells for unrelated 

transplants without the need for general anesthesia 

is certainly appealing, and would probably encourage 

more volunteers to donate stem cells. It would also be 

easier to expand especially the number of donors belonging 

to ethnic minorities. Apheresis-derived mononuclear 

cells might be stored in liquid nitrogen and shipped when 

needed. Age limit for donors could be expanded. However, 

because of the limited experience with G-CSF 

mobilization in normal donors, National Marrow Donor 

Registries have not approved the use of PBSC as first 

choice. We expect this will remain the case in the foreseeable 

future. 

In general, the use of growth factors for any purpose 

in healthy subjects should be considered experimental. 

The donor should be informed of the potential short and 

long-term risks of growth factors and leukapheresis, as 

well as of anesthesia, and he should be given the possibility 

of choosing. Donor consent should be asked on 

the basis of a protocol previously approved by an official 

ethical committee. 2 

Characterization of CD34 + hematopoietic 

progenitor cells mobilized into peripheral 

blood of normal donors by rHG-CSF 

The preliminary results of clinical trials on allogeneic 

PBSC transplantation have demonstrated the capacity of 

G-CSF to mobilize true stem cells capable of long-term 

reconstitution of marrow function. Moreover, similarly 

to autografting, the most striking finding of PBSC transplantation 

has been the faster recovery of hematopoiesis 

after myeloablative conditioning regimens as compared 

to transplantation of BM-derived stem cells. Thus, clinical 

investigators asked the question of whether circulating 

progenitor cells may differ from their BM counterparts 

with respect to kinetic status, immunophenotype, 

frequency of both committed and primitive precursors, 

and their proliferative response to colony stimulating 

factors (CSFs). 

One early report 50 has shown a high expression of 

myeloid antigens on PB CD34 + cells (i.e. CD33, CD13) at 

the expense of B-lineage-associated antigens (i.e. CD10, 

CD19, CD20), coupled with a high colony-forming 

capacity of G-CSF-stimulated apheresis products. Moreover, 

Roberts and Metcalf 51 have clearly shown in an 

animal model and in humans that only a small minority 

of mobilized PBSC undergo active DNA synthesis, 

whereas BM cells contain more than 30% of S-phase 

clonogenic progenitors. This finding, coupled with the 

lack of expression of CD71 antigen (transferrin receptor) 

and the Rhodamine 123 dull status 52 observed in CD34 + 

cells from cancer patients mobilized with G-CSF, has 

suggested that PB progenitors may be functionally inactive 

since they are in deep G0-phase of the cell cycle. 

However, these results are somewhat in contrast with 

clinical data indicating rapid BM recovery after autologous 

and allogeneic transplantation and the experimental 

evidence that circulating CD34 + cells represent 

an optimal target for efficient retroviral infection requiring 

cell cycling for integration. 53 In addition, it is very 

important to assess the kinetic profile of the CD34 + cell 

fractions which are believed to ensure permanent 

engraftment after PBSC allografting, such as cells phenotypically 

identified as CD34 + /CD38 – , CD34 + /CD33 – / 

HLA-DR – or very primitive progenitor cells capable of 

generating clonogenic precursors in secondary semisolid 

assay after 5 or more weeks of liquid culture, longterm 

culture-initiating cells (LTC-IC). In this regard, 

defective long-term repopulating activity of early BM 

cells induced to S-phase by cytokines has recently been 

shown. 54 

To further elucidate the phenotypic profile and functional 

and kinetic characteristics of G-CSF-mobilized 

hematopoietic progenitor cells, highly purified CD34 + 

cells from the apheresis products of normal individuals 

undergoing PBSC collections for allogeneic transplantation 

were recently analyzed. The results were then com- 


54 


Cell-cycle distribution of CD34+ cells (%) 

Figure 3. Analysis of cell-cycle distribution of PB and BM 

CD34 + cells. 

pared with those obtained on CD34 + cells enriched from 

the BM of the same donors under steady-state conditions 

and after G-CSF administration on the same day as 

PBSC harvest. 55 The results confirmed the expression of 

CD33 and CD13 antigens on a higher percentage of circulating 

CD34 + cells compared to BM cells (91±31% SD 

and 85.3±10% SD versus 51.1±21% SD and 64.6±25% 

SD, respectively; p


55 

adhesion molecules critical for mobilization and related 

to cytokine-induced cell-cycle transit. 62 Moreover, 

pharmacological doses of steel factor determine a redistribution 

of stem cells in mice 63 and reduce the avidity 

of 4 1 and 5 1 integrins on the MO7e cell line, with 

a consequent inhibition of the specific cell adhesion of 

MO7e cells to VCAM-164. Other adhesion molecules, 

like L-selectin and VLA4, might play a role in the mobilization 

of hematopoietic progenitors in primates. 65 

Figure 4. Recovery and proliferative status of CFC and LTC- 

IC in steady-state normal blood and bone marrow and in the 

leukapheresis products of cancer patients obtained after 

chemotherapy + G-CSF. The cells were cultured for 16 hrs 

in a medium containing serum substitutes, SF (100 

ng/mL), IL-3 (20 ng/mL) and G-CSF (20 ng/mL) in the presence 

or absence of 3 H-Tdr (panel B and A, respectively). 

SEM of BM), whereas the proportion of LTC-IC cycling 

was superimposable on that of BM. The frequency was 

not different in the two compartments (48.2±35 SEM 

and 62.5±54 SEM for 10 4 CD34 + cells in PB and BM, 

respectively; p = ns). Thus these data, coupled with previous 

observations from nonhuman primates on cellcycle 

status and response to CSF of cytokine-mobilized 

CD34 + cells, 61 suggest that many circulating progenitors 

are not deeply quiescent in G0-phase. Rather, they 

are actively cycling and their high clonogenic efficiency 

and prompt proliferative response to CSFs may indicate 

a faster progression through cell-cycle mediated, 

perhaps, by G-CSF priming. A kinetic and functional pattern 

of CD34 + cells similar to that observed in normal 

PBSC donors has been found in acute leukemia and multiple 

myeloma patients mobilized with chemotherapy 

and G-CSF, and in lymphoma individuals receiving G- 

CSF alone (Lemoli, unpublished observations). Thus, 

regardless of the mobilization protocol, the administration 

of G-CSF and/or the change of compartment (i.e. 

egress into peripheral blood) induces a profound effect 

on the characteristics of hematopoietic progenitor cells. 

Further studies are presently directed toward investigating 

the modulation of the expression of integrin 

Stem cells from umbilical cord blood: 

biological aspects 

More than 20 years ago it was described in this Journal 

that hematopoietic progenitors circulate between 

the fetus and the placenta during gestation. 66 However, 

placental/umbilical cord blood (CB) from human 

newborns was not considered as a source of stem cells 

for clinical use until Broxmeyer et al. 67 enumerated the 

number of CFU-GM that could be collected from the CB 

remaining in the placenta after birth and suggested that 

the total number was sufficient for transplantation in 

pediatric patients. The Fanconi anemia patient who in 

1988 first received a CB transplant from his HLAmatched 

sibling 68 is still alive at the present time and 

cured from the hematological point of view, thus 

demonstrating the long-term engraftment capability of 

CB-derived stem cells. 

In the past five years interest in the biological aspects 

and clinical applications of CB has grown since large 

CB banks for unrelated stem cell transplantation have 

been implemented in the USA and Europe, and more 

than 300 CB transplants have been performed. However, 

many aspects of the properties of CB stem cells are 

still elusive. It is remarkable that a unit of CB used for 

transplantation contains 1-810 6 CD34 + cells and 10- 

12010 3 CAFC/LTC-IC, 56,69-70 i.e. 1-2 logs fewer than the 

total number of CD34 + cells and CAFC usually infused 

into recipients of allogeneic BM or PBSC. On average, 

recipients of CB transplants are given 0.05-0.5×10 6 

CD34 + cells/kg b.w., while it has been suggested that 

recipients of allogeneic PBSC must receive at least 2.5- 

5×10 6 CD34 + cells/kg b.w. to obtain safe hematopoietic 

engraftment. 71 On the other hand, despite the delay 

in the reconstitution of the megakaryocytic lineage, the 

rate of engraftment failure in CB transplant recipients 

is similar to that observed after BM or PBSC transplants. 

72 These observations have prompted a number of 

investigators to focus on the proliferation potential of 

CB stem cells. In an elegant study, Lansdorp et al. 73 sorted 

CB-derived, CD34 + CD45RA lo CD71 lo cells, defined as 

stem cell candidates. In liquid cultures supplemented 

with SCF, IL-3,-6 and Epo, these purified cells generated 

a number of CD34 + and mature cells significantly 

greater than that obtained in cultures of purified CD34 + 

CD45RA lo CD71 lo cells obtained from adult donors. This 

advantage was clearly ontogeny-related, since the proliferative 

potential of purified CD34 + CD45RA lo CD71 lo 

cells collected from fetal liver was superior to that of 

CB-derived cells. In this context, Hows et al. 74 demon- 


56 


strated in long-term stroma culture that both progenitor 

cell cultures and the lifespan of cultures were greater 

in CB than in adult BM, and Payne et al. 75 showed that 

the proportion of CD34 + cells that are CD38 – (lin – ) is 

significantly higher in CB than in other stem cell sources. 

In contrast to what has been documented in adult BM, 

Traycoff et al. 76 demonstrated that LTC-IC and cells presumably 

capable of in vivo engraftment reside in the 

CD34 + HLA-DR + Rh123 dull fraction. The cycling status of 

CB progenitors is still a matter of debate. In fact, some 

investigators using the tymidine suicide technique 

reported a higher frequency of ceIls in S phase, 77 whereas 

others did not find any difference between CB and 

adult BM in the frequency of actively cycling progenitors. 

78 More insight into this area is especially necessary 

since CB is a very attractive target for the transfer of 

genes able to correct inherited or non-inherited diseases 

such as thalassemia, Fanconi anemia, ADA-deficiency, 

etc, 79 and since entering S phase is required for gene 

transfer through safety modified retroviruses. Interestingly, 

a higher efficiency of retrovirus-mediated gene 

trasfer has been reported in CB than in BM progenitors. 

78,80,81 One possible explanation is the particularly 

rapid exit from the G0/G1 phases of the cell cycle in 

response to cytokines described by Traycoff et al. 82 in 

CB-derived CD34 + progenitors, which might also justify 

the ontogeny-related advantage in proliferative 

potential. 

The functional meaning of these differences in the in 

vitro behavior of phenotypically defined CB and BM populations 

is not yet fully understood, but these findings 

represent an interesting parameter to consider when 

assessing the suitability of a CB unit for transplantation 

in pediatric or adult patients. So far, in fact, most CB 

transplant recipients have been pediatric patients 

weighing less than 50 kg. Sporadic reports of CB transplants 

in adult recipients have indicated that the time 

to myeloid lineage engraftment is comparable to that of 

BM recipients, whereas the delay in platelet reconstitution 

seems to be more pronounced than in pediatric CB 

transplant recipients. 83 As described in the CB processing 

section, ex vivo expansion of CB progenitors prior to 

trasplantation might be useful to hasten hematopoietic 

engraftment; however, since the long-term engraftment 

capability of ex vivo cultured cells might be lost or 

impaired, 84 more work seems necessary to reach this 

important goal. 

CB collection 

Established advantages of CB banks over BM donor 

registries include the immediate availability of the frozen 

CB unit, minimal donor attrition, the presence of CB 

donors from minority groups that are poorly represented 

in BM donors registeries, and a much lower incidence 

of CMV infected donors. In fact, the time from the 

request for a CB unit to finding a matched donor is on 

average less than 2 months, and less than 1% of CB units 

are contaminated by CMV. 85 

Worldwide, the creation of large CB banks has prompted 

investigators to improve the methods for CB collection 

and fractionation. The first method described by 

Broxmeyer et al. 67 included CB collection in heparinized 

tubes by gravity. Further studies 69,86 indicated that this 

open system is much more prone to bacterial contamination 

than closed systems based upon CB collection in 

bags, as first proposed by Gluckman et al. 87 Another 

approach, proposed by Turner et al., 88 includes catheterization 

of the umbilical vein. However, in a recent report 

this procedure was found to cause significant contamination 

of the CB collection by maternal cells, including 

potentially harmful T cells. 89 It has been demonstrated, 

moreover, that CPD/CPD-A have an advantage over ACD 

and heparin because the former can anticoagulate blood 

over a wider volume range. Figure 5 describes results 

obtained using the method of CB collection in closed 

bags while the placenta is still in utero. Briefly, after the 

birth the umbilical cord is doubly clamped 1-2 cm from 

the newborn and transected before the newborn is 

removed from the operative field. The free end of the 

cord must be accurately disinfected before CB collection 

by venepuncture of the umbilical vein. As shown in Figure 

5, there is a strict correlation between the time of 

umbilical cord clamping, the volume and the total number 

of nucleated cells collected. If the clamping procedure 

is delayed to the second minute after birth, it seems difficult 

to collect systematically a number of nucleated 

cells sufficient for clinical use of the CB unit. In fact, 

after the birth the newborn is frequently positioned 

below the level of the uterus, and this determines the socalled 

transfusion effect from the placenta to the newborn. 

90,91 Interestingly, when newborns are delivered following 

the Leboyer method there is no transfusion effect 

since the newborn is kept above the level of uterus. 92 

Under these circumstances, early clamping of the cord is 

not required for collection of CB for clinical use. At the 

present time there is no consensus among neonatologists 

and pediatricians about the more appropriate timing 

of umbilical cord clamping. However, cord clamping 

in the first 30-60” after birth seems adequate to most 

reviewers, 93-97 and recent data on the immediate followup 

of newborns who underwent early clamping of the 

the umbilical cord and CB collection support the safety 

of this procedure. 98 In this retrospective study, none of 

the newborns who had CB collected were reported to 

suffer from weight loss, fatigue while feeding, tachypnea 

and tachycardia, hypoxia, or cardiac or pulmonary 

disease with reduced arterial oxygen saturation. The sole 

significant difference between the group of 59 CB donors 

and the control group was a slight reduction of Hb values, 

which corresponded to a loss of about 15 to 20 mg 

of iron. 

CB processing 

As mentioned above, a CB unit to be used for transplantation 

contains remarkably fewer CD34 + progenitor 

cells than BM or PBSC collections. For this reason, during 

CB manipulation the loss of progenitor cells must be 

carefully avoided. In pilot projects for large scale bank- 



57 

ing, CB was in fact stored as unmanipulated whole 

blood. The high cost of this procedure, which requires 

large liquid nitrogen space, has fueled intense research 

to concentrate CB nucleated cells in a reduced volume. 

Among the methods recently proposed for CB processing, 

however, some included density separation by 

Ficoll 99 or red cell sedimentation by means of animal 

gelatin, 69,100 i.e. reagents that are currently not (and 

probably will never be) licensed for use in humans by 

regulatory agencies like the the FDA. Consequently, procedures 

involving the use of licensed products like 

HES 101,102 should be recommended and a maximum loss 

of 10-15% of progenitors accepted. In this context, it 

must be noted that a number of patients have already 

been successfully transplanted with red cell-depleted 

CB. 101,103 Conversely, data on the engraftment potential 

of purified, CB-derived CD34 + cells are still poorly reproducible 

in the SCID-hu mouse model 104 and totally lacking 

in humans, so for the time being the storage of purified 

CD34 + CB progenitors for clincal use is not recommended. 

However, it has been shown in vitro that the 

proliferation potential of purified CD34 + cells is markedly 

superior to that of the low density or Ficoll fraction. 

70,104 This finding has major implications for the possible 

ex vivo expansion of an aliquot of the CB unit prior 

to transplantation. Two different strategies have been 

evaluated: the goal of some authors was to obtain multiple 

lineage expansion of progenitors by means of 

cytokine combinations including SCF, FLT-ligand IL-1, 

IL-3, IL-6, IL-11, G- and GM-CSF, 104-106 while others were 

interested in selected-lineage expansion of the 

myeloid 107 or megakaryocytic lineage. 108 

Rubinstein et al. 101 have recently proposed a new procedure 

for washing the CB unit prior to transplantation. 

Advantages of this approach include removal of free Hb 

from lysed red cells and a significant reduction in the 

DMSO infused, a molecule which is particularly toxic 

for pediatric transplant recipients. 109 In vitro data indicate 

that the washing procedure may improve the 

engraftment potential of the transplanted cells, but this 

finding should be futher confirmed in an in vivo model. 

Figure 5. Effect of the time of umbilical cord clamping on the 

mean (±1 SD) volume and nucleated cell count of cord blood 

collections (n=67). As in most European OB/GYN units, 

soon after birth the newborn is kept below the level of the 

uterus, and delayed clamping of the umbilical cord is associated 

with a reduction of cord blood volume and cellular 

content. 

Immunological features of cord blood 

lymphocytes 

The immune system which develops during fetal life 

is not fully competent at birth and continues the differentiatation 

process after birth in response to various 

antigenic challenges. At least three important elements 

control the development of the immune system during 

fetal life, thus determining the peculiar characteristics 

of the cord blood lymphocyte (CBL)-mediated immune 

response: i) limited or even absent antigenic experience, 

ii) immaturity of the majority of lymphocyte populations, 

and iii) feto-maternal immunological interaction. 

110,111 These elements are believed to influence the 

immunological features most strictly related to cord 

blood transplantation (CBT) and, in particular, the 

capacity to develop alloantigen-directed reactivity, antimicrobial 

immunity and anti-tumor immune surveillance. 

As a consequence of poor antigenic experience during 

pregnancy, the majority of CBL are naive cells expressing 

the RA isoform of the CD45 molecule. 112 The most 

prominent immunoregulatory function of CD45RA + T 

lymphocytes is suppressor activity. 113 These peculiar features 

of neonatal lymphocytes explain their incapacity to 

develop, both in vivo 114 and in vitro, 115 an immune 

response directed towards recall antigens (i.e. tetanus 

toxoid, influenza virus). 

The immaturity of the CBL population is also a direct 

consequence of its poor antigenic experience. Compared 

to adult blood, the distribution of the most relevant CBL 

subpopulations is characterized by a reduced percentage 

of CD3 + mature T lymphocytes and by the presence 

of immature T and NK lymphocyte subsets which are 

not detectable in adult peripheral blood. 116,117 

B lymphocytes are present in a normal or even augmented 

percentage in cord blood as compared with adult 

blood, even though immunoglobulin production is limited 

to the IgM class. 118 Other CBL peculiar features related 

to their immaturity are low expression of adhesion/costimulation 

molecules such as CD11a (LFA-1), 

CD18, CD54 (ICAM-1), CD58 (LFA-3) antigens, 113,117,119 

poor expression of CD40 ligand on activated T lymphocytes, 

120 and reduced ability to secrete some cytokines 

(i.e. -interferon, tumor necrosis factor and interleukin- 

4). 117 

Spontaneous NK activity is reduced in cord blood as 

compared to adult blood, possibly because of the low 

expression of adhesion molecules, known to be useful in 

promoting the capacity of NK lymphocytes to adhere to 

target cells. 121 On the other hand, antibody-dependent 

cell cytotoxicity (ADCC) and lymphokine activated killer 

(LAK) activity of cord blood reach values comparable to 

or even higher than those observed in adult blood. 121 

Moreover, recent data demonstrate that the innate 


58 


immunity directed towards Epstein-Barr virus-infected 

cells is remarkably high in CBL collected from the majority 

of neonates. 122 The capacity of cord blood NK cells to 

be promptly activated in vitro suggests that innate 

immunity plays a key role in immune surveillance during 

fetal and perinatal life, as long as specific T-cell 

mediated adaptive immunity can be generated. 

From an immunological viewpoint, pregnancy can be 

considered as a successful HLA-mismatched transplantation. 

It is well known that the feto-maternal, anatomo-functional 

barrier allows the reciprocal transfer of 

lymphocyte subpopulations. It is thus conceivable to 

hypothesize that a very effective immunological network 

acts to prevent fetal rejection and graft-versushost 

reactions (GVHR). 111,123 Several lines of clinical and 

experimental evidence support this hypothesis, in particular: 

i) CBL preferentially display suppressor rather 

than helper immunological functions 111,113 and ii) both 

B and T lymphocytes maintain a state of hyporesponsivity 

towards noninherited maternal HLA molecules for 

a long time after birth. 124,125 A further confirmation of 

this peculiar state of tolerance derives from a recently 

reported observation 126 on the occurrence of acute 

GVHD in patients given CBT from donors who were disparate 

for the noninherited paternal allele, and on the 

absence of significant acute GVHD in recipients whose 

donors were disparate for the noninherited maternal 

allele. 

The fetal/neonatal period has been postulated to represent 

a crucial time in ontogeny, during which T and B 

lymphocytes learn to discriminate between self and nonself 

through the development of a state of tolerance 

toward antigens they encounter. 127 The concept of neonatal 

tolerance was recently re-examined in mice 128-130 

and it was demonstrated that induction of this phenomenon 

may depend on several elements, including 

the nature of the antigen-presenting cells, 128 the dose 

of antigen administered, 129 and the mode of immunization. 

130 

Poor antigenic experience, immaturity of lymphocyte 

subpopulations, feto-maternal immunological interactions, 

and neonatal tolerance may, altogether, contribute 

to the generation of a suppressive effect on CBL 

alloreactivity, thus permitting the use of HLA-partially 

matched donors for CBT. In agreement with this hypothesis, 

reduced proliferative and cytotoxic activity towards 

alloantigens was reported by several authors to be present 

in cord blood as compared with adult peripheral 

blood. 117,131-133 However, normal CBL alloreactivity has 

been documented in other studies. 134,135 The discrepancies 

observed between the above mentioned reports may 

depend on the high interindividual variability in the distribution 

of cord blood T and NK lymphocyte subpopulations. 

Interestingly, it has been recently reported that, 

even though proliferative response to alloantigen in a 

primary mixed lymphocyte culture (MLC) is comparable 

in adult and cord blood, restimulation in secondary MLC 

induces increased specificity and activity of adult alloreactive 

lymphocytes and a state of unresponsiveness in 

CBL. 136 These data strongly suggest that repeated in vitro 

stimulation with allogeneic cells amplifies the specific 

immune response of adult lymphocytes, while the same 

procedure induces a state of anergy in neonatal cells. 

Several clinical and experimental data obtained in the 

setting of allogeneic bone marrow transplantation 

(BMT) demonstrate that there is a strong correlation 

between GVHD and graft-versus-leukemia (GVL) effect. 

Thus, due to their low alloreactive capacity (responsible 

for the reduced GVHR), 126,135 CBL could be less efficient 

in mediating a GVL effect. As far as we know, no data 

concerning the identification of cord blood T lymphocytes 

capable of mediating specific anti-leukemic activity 

have been reported in the literature. This lack of 

information is not surprising since it is well known that 

the frequency of these cells is extremely low, even in the 

peripheral blood of healthy adult donors. However, some 

studies have recently demonstrated that innate antileukemic 

activity mediated by LAK cells and measured 

Table 3. Methods of T-Cell depletion in clinical trials. 

Method of T-Cell Depletion Cells Removed T-Cell Depletion 

(x log10) 

SBA lectin and E-rosette depletion 

T and B lymphocytes, monocytes, 

neutrophils 2.5 - 3.0 

Multiple E-rosette depletions T lymphocytes 2.0 

Mouse MoAb (anti-CD2, CD8) + rabbit C’ T lymphocytes 2.0 

Mouse MoAb (anti CD6) + human C’ T lymphocytes 1.5-2.0 

Rat MoAb(CAMPATH-1) + human C’ T and B lymphocytes, monocytes, 2.5 

Anti-CD5 immunotoxin-Ricin A Immunomagnetic T lymphocytes 2.0 

separation (anti-CD3, CD8) 

SBA lectin + immunomagnetic separation 

T and B lymphocytes, monocytes, 

3.1 

neutrophils 

AIS CD%/8T-CELLector T lymphocytes 2.5 

Autologous Immunorosettes 

(anti-CD2 and CD3 tetrametric complexes) 

T lymphocytes 2-3 



59 

against long-term tumor cell lines, is comparable in 

adult and cord blood. 119,137 Even though the above mentioned 

studies are interesting, more experimental data 

and clinical observations are required to better define 

the potential GVL effect of CBL. 

PBSC processing 

T-cell depletion 

The role of T lymphocytes in bone marrow transplantation 

is very complex. They are in fact responsible for 

GvHD, a major contributing factor correlated with morbidity 

and mortality in allogeneic bone marrow transplantation. 

138,139 Indeed when T-cells are removed from 

the graft before transplantation, the incidence of GvHD 

decreases sharply. 71,140-143 On the other hand, the possible 

beneficial roles of T-lymphocytes include sustaining 

engraftment 144 and preventing relapses through the 

graft-versus-leukemia effect. 145,146 They are also crucial 

in immune-hematological reconstitution after transplantation 

because slow or deficient reconstitution may 

lead to a high incidence of viral infections or other 

infectious complications. Ex vivo manipulation of the T- 

lymphocyte content is easier and T-cell depleted allogeneic 

transplants may in the future be followed by 

infusion of non-alloreactive T-lymphocytes or of specifically 

engineered lymphocyte clones exerting an antiviral 

or anti-neoplastic effect. 

Standard T-cell depletion techniques 

Over the past 15 years, several techniques have been 

developed for depleting T-cells from human marrow 

allografts. 147 Table 3 summarizes the principles on which 

they are based and the degree of T-cell depletion each 

provides. Unfortunately, these methods may be timeconsuming, 

cumbersome and difficult to standardize in 

different transplantation centers. Results are therefore 

often variable and no general consensus has emerged on 

the use and benefit of bone marrow T-cell depletion. 

Very few data are available on the use of standard T- 

cell depletion methods in heterogeneous nucleated cell 

populations collected by leukaphereses from the peripheral 

blood of donors previously stimulated by hemopoietic 

growth factors. 

Kessinger et al. 148 first reported allogeneic transplantation 

utilizing T-cell depleted peripheral blood 

mononuclear cells and a sheep erythrocyte rosetting 

technique. Engraftment was rapid and grade II acute 

GvHD was observed. 

In a preliminary study after monocyte lysis with L- 

phenylalanine methyl ester, Suzue et al. 149 depleted T- 

lymphocytes from apheresis products harvested after 

stimulation of healthy donors with G-CSF using both E- 

rosettes with sheep red blood cells and panning with 

flasks coated with anti CD5/CD8 monoclonal antibodies. 

They reported an unsatisfactory depletion of T-cells 

(99.5%) and a stem cell recovery of only 7.5%. 

Aversa et al. 71 employed soybean lectin agglutination 

followed by 2 to 4 rounds of E-rosetting with sheep red 

blood cells on the leukapheresis product from donors 

stimulated with G-CSF. This approach achieves approximately 

3×log 10 T-lymphocyte depletion, as measured 

by cytofluorimetric assays. The main drawbacks are its 

complexity and lengthy laboratory times. 

Stem cell positive selection 

In principle, reducing T lymphocytes in the leukapheresis 

product by positively selecting CD34 + hemopoietic 

progenitors appears to be a valid technical alternative. 

Table 4 shows the basis of the main techniques 

for positive selection of hemopoietic progenitor cells. 

All use one monoclonal antibody which identifies an 

epitope on the human CD34 antigen. Separation is 

effected by collecting the antibody-sensitized cells onto 

a solid phase such as magnetic beads, plastic plates or 

columns of non magnetic particles, while non-target 

cells remain in suspension. Systems that utilize high 

speed flow cytometry to sort CD34 + cell populations 

have also been developed. 150 

The CD34 + stem cell selection systems adopted in 

clinical use are based on immunoadsorption and indirect 

immunomagnetic beads. 

Most clinical trials to date have been carried out with 

a ® Ceprate Stem Cell Concentrator (CellPro Inc., Bothell, 

WA, USA), which employs biotinylated 12.8 monoclonal 

antibody. The sensitized cells are applied to a column 

of avidin-coated polyacrylamide beads. Cells expressing 

Company Method Antibody Detachment 

CellPro Immunoadsorption 12.8 Mechanical 

Baxter Magnetic beads indirect 9C5 Chymopapain 

Baxter Magnetic beads indirect 9C5 PR34+ oligopeptide 

Dynal Magnetic beads direct BI3C5 Anti-Antibody 

(anti Fab of mouse MoAb) 

AIS Panning ICH3 Mechanical 

Immunotech Magnetic latex beads direct QBEND10 Not required 

Milteny Magnetic colloid indirect QBEND10 Not required 

Terry Fox Laboratory Magnetic Colloid Indirect 8G12 Not required 

System x FACS (high speed) Various Not required 

Table 4. Methods 

available for stem 

cell positive selection. 


60 


the CD34 antigen are retained and unlabelled cells 

washed through the column with gentle mechanical 

agitation. The CD34 + cells are then removed from the 

beads and collected. 

Using this system on the leukapheresis product, Link 

et al. 151 recovered a mean of 30% CD34 + cells, with a 

purity of 70%. Peripheral blood CD3 + cells were reduced 

by 3 logs. Other investigators have reported similar 

results. 152-155 This degree of T-cell depletion is known to 

prevent severe GvHD in severe combined immune deficiency 

(SCID) patients after matched or mismatched 

bone marrow transplantation. 140 In leukemia patients 

undergoing matched transplants it may not be enough 

to eliminate GvHD completely without the concomitant 

administration of immunosuppressive drugs. The threshold 

number of clonable T-lymphocytes in the inoculum 

should be below 1×10 5 /kg b.w., 156 which is difficult to 

achieve with one-step positive selection of hemopoietic 

stem cells. For a successful mismatched bone marrow 

transplant the T-lymphocyte threshold must be < 3- 

5×10 4 /kg b.w. 157 in the inoculum because of the greater 

likelihood and increased severity of GvHD in these 

patients. On the other hand, infusing a number well 

below the threshold value could expose the patient to 

a high risk of graft failure. 

Because T-cell depletion with the ® Ceprate system 

applied directly on the leukapheresis product does not 

reduce T-lymphocyte content from the graft by more 

than 3 logs, an additional T-cell depletion step is 

required. 

In 10 patients with different types of leukemias, Aversa 

et al. 157 employed an E-rosetting procedure before 

positively selecting hemopoietic progenitors with the 

® 

Ceprate system. This combined method yielded a T-cell 

depletion of 4.3 logs in the graft and a mean CD34 + 

recovery of 50-60%. 158 

In small-scale experiments, Fernandez et al. 159 applied 

the E-rosetting procedure after positive selection of 

CD34 + cells with this same ® Ceprate system to obtain a 

mean log 10 T-cell depletion of 4. Slaper-Cortenbach et 

al. 160 achieved a median recovery of 42.7% CD34 + cells 

and a T-lymphocyte reduction of 2-3 logs in 13 haploidentical 

transplants for SCID and in leukemia patients 

by employing autologous immunorosettes after positive 

selection of CD34 + cells. 

CD34 + progenitor cell immunomagnetic selection 

(Baxter, Irvine, CA, USA) achieved a 3 log T-cell depletion 

in preclinical experiments. 40,161 However, the main 

problem with this methodology was bead release from 

the target CD34 + cells. In fact release mediated by chymopapain 

may cause intractable cell clumping, particularly 

when a large number of cells are processed. 

Recently the PR34+TM stem cell releasing agent, an 

oligopeptide competing with the anti-CD34 monoclonal 

antibody for the release of the CD34 + cells from the 

magnetic beads, has also been proposed. 161 Preliminary 

results showed a reduction of non-target T cells by a 

factor of 2-3 logs with yields of CD34 + cells ranging 

from 31.1 to 85%. 162 

In conclusion, positive selection of CD34 + cells with 

the ® Ceprate system reduces the graft T-lymphocyte 

content under the threshold of risk for GvHD only when 

combined with standard T-depletion techniques such as 

E-rosetting with sheep red blood cells or autologous 

immunorosetting. Indirect immunomagnetic systems 

have to be evaluated more precisely for the use as a T- 

cell depletion system. 

Immunogenic activity of CD34 + 

hematopoietic cells 

Autologous transplantation of selected CD34 + cells 

induces rapid and complete hematologic reconstitution 

in myeloablated patients. In addition, isolation of CD34 + 

cells can be considered as an ex vivo means of purging 

neoplastic cells from the marrow or peripheral blood of 

patients with solid tumors or hematologic malignancies. 

163-165 

In the allogeneic setting, selection of CD34 + cells may 

be aimed at depleting donor T-cells and professional 

antigen-presenting cells (APC) such as monocytes, activated 

B-cells and dendritic cells, which are very potent 

stimulators of T-cell responses. Dendritic cells constitutively 

express the B7-2 (CD86) costimulatory molecule 

and upregulate B7-1 (CD80), B7-2 (CD86) and other 

molecules upon activation. 166-171 Furthermore, they were 

recently shown to derive from CD34 + marrow or peripheral 

blood cells, and can be rapidly generated in vitro in 

the presence of a specific combination of growth factors. 

172-177 Since it has been demonstrated that T-cell 

receptor (TCR): antigen interaction, in the absence of 

Authors 

TNC 

(x 108/kg) 

CD34+ 

(x 106/kg) 

CD3+ 

(x 106/kg) 

NK 

(x 106/kg) 

Dreger et al. (26) 13.52 8.16 404 N.R. 

Weaver et al. (191)* 20.53 9.6 450 N.R. 

Körbling et al. (16) 16.5 10.7 300 64.3 

Schmitz et al. (17) 8.6 13.1 340 94.0 

Bensinger et al. (18) 10.6 13.1 385 N.R. 

Majolino et al. (27) 9 6.84 250 27 

Rambaldi et al. (203) 8 6.9 279 N.R. 

Table 5. Median value of nucleated 

cells, CD34 + cells, CD3 + cells and 

natural killer cells infused in 

patients undergoing allogeneic 

peripheral blood stem cell transplantation. 

Legend. *Syngeneic transplants. 

N.R.=Not reported. 



61 

appropriate costimulation, may induce T-cell unresponsiveness 

or even apoptotic deletion, 171,178-181 the alloantigen 

presenting function of CD34 + marrow cells was 

recently investigated to evaluate whether transplantation 

of purified CD34 + cells could minimize the immune 

sensitization of an allogeneic receipient. 182 CD34 + marrow 

cells have been purified to >98% by a two-step 

procedure consisting of a first enrichment on an 

immunoaffinity chromatography column, followed by 

fluorescence activated cell sorting. Cytofluorimetric 

analysis of purified CD34 + marrow cells revealed the 

expression of HLA-DR and CD86 on >95% and 6% of 

the cells, respectively. Primary mixed leukocyte cultures 

demonstrated that irradiated CD34 + marrow cells 

induce brisk proliferation of allogeneic T-cells isolated 

from HLA-DR incompatible donors. On the basis of previous 

reports, 183,184 expression of CD18, the common 

chain of a family of leukointegrins, was investigated on 

CD34 + marrow cells and CD34 + /CD18 – cells were sorted 

to investigate whether this cell population was enriched 

in early hemopoietic precursors incapable of immunostimulating 

activity. 

On average, 25% of CD34 + marrow cells were CD18 – 

by direct immunofluorescent analysis. Purified CD34 + , 

CD34 + /CD18 + and CD34 + /CD18 – marrow subsets were 

tested in bulk MLC with allogeneic T-cells, and it was 

observed that CD34 + , CD34 + /CD18 + and unseparated 

marrow mononuclear cells have a similar capacity to 

stimulate a T-cell response. Conversely, CD34 + /CD18 – 

cells do not elicit any T-lymphocyte proliferation. Moreover, 

limiting dilution assay (LDA) experiments showed, 

on a per cell basis, that CD34 + /CD18 – and CD34 + /CD86 – 

marrow cells have a very poor ability to induce a T-cell 

response, as opposed to CD34 + /CD18 + and CD34 + /CD86 + 

marrow cells. Since most marrow LTC-IC were included 

in the CD34 + /CD18 – cell fraction, it was concluded 

that CD34 + /CD18 – , or CD34 + /CD86 – marrow cells, may 

represent a useful source of progenitor cells for allogeneic 

transplantation because of their high stem cell 

activity combined with reduced immunogenicity. Data 

on normal human G-CSF mobilized CD34 + peripheral 

blood (PB) cells show that on average 30% of the cells 

express CD18 and only 3% express CD86, while functional 

in vitro results are consistent with what was previously 

observed in marrow. Thus, CD34 + PB cells can 

potently stimulate T cells, likely through the B7:CD28 

pathway, and CD34 + /CD18 – PB cells still have very weak 

immunostimulating activity. 

In a preliminary study sibling baboons were fully 

engrafted with allogeneic CD34 + marrow cells without 

GVHD, after receiving total body irradiation as conditioning 

regimen and standard GVHD prophylaxis. 184 

Development of mobilization regimens capable of 

increasing the number of peripheral blood hemopoietic 

stem cells in normal healthy donors allowed sufficient 

amounts of CD34 + PB cells to be harvested for allogeneic 

transplantation in humans. In fact, transplantation of 

enriched populations of G-CSF mobilized CD34 + cells 

resulted in rapid engraftment, similar to that observed 

in allogeneic PBSC transplants. 185-190 Purification of 

CD34 + cells on the Ceprate column obtains on average 

a 3 log depletion of CD3 + T cells in the graft; however, 

several studies reported contrasting rates of acute 

GVHD. In particular, > 80% of the patients transplanted 

with CD34 + PB cells in Seattle experienced aGVHD 

grade II-III after receiving a median number of 0.7×10 6 

T-cells/kg in the graft and GVHD prophylaxis with 

cyclosporin-A (CsA)± methotrexate (MTX). 187 Another 

study reported 2 cases out of 5 who died from aGVHD. 188 

By contrast, other groups reported a very low incidence 

of GVHD. 189,190 One of the reasons for these disparities 

may be that small numbers of patients, often with different 

malignancies and clinical characteristics, are 

included in these studies. Nevertheless, two hypotheses 

could be addressed: the first one suggests that infusion 

of as little as 0.5-1×10 6 CD3 + T cells/kg could be potentially 

capable of initiating GVHD, which would be prevented 

by further steps in T-cell depletion. 158 The second 

hypothesis, still to be tested, is whether APC in marrow 

or peripheral blood could play a role in the development 

of GVHD by presenting allogeneic peptides to donor T- 

cells. 

In this regard, a subset of CD34 + cells in the graft may 

induce the activation and proliferation of a limited number 

of T cells, such as those still present after CD34 

purification. 

Peripheral blood stem cells: 

immunological aspects 

Very few data are available on the effects of hemopoietic 

growth factors used to mobilize PBSC on peripheral 

blood lymphocytes. 

Weaver et al. 191 analyzed the influence of G-CSF on 

peripheral blood lymphocytes from 13 individuals (11 

autografts and 2 normal donors). In all cases they 

observed a slight increase in CD3, CD4, CD8, CD19 and 

CD20-positive lymphocytes, with a return to pretreatment 

values by days 4 and 5 of G-CSF administration. 

The change in the CD4/CD8 ratio was not statistically 

significant. 

The expression of CD2, CD3, CD4, CD7, CD8, CD20, 

CD25, CD57 and HLA-DR antigens was evaluated during 

administration of G-CSF (12 ug/kg/day for 5-7 days) 

to healthy donors. No significant variations were 

observed in the different lymphocyte subsets, in the 

CD4/CD8 ratio or in the expression of CD25 and HLA-DR 

antigens (unpublished data). G-CSF administration does 

not cause direct activation of T lymphocytes in vivo. This 

might be expected because lymphocytes do not possess 

the G-CSF receptor. 192 However, it is possible that activation 

could be caused by cytokine release from cells 

stimulated by G-CSF. 

Other important aspects of the PBSC allograft include 

the lymphocyte content, particularly T lymphocytes and 

natural killer cells, in the apheresis product. Table 5 

reports data on the total number of CD3 + lymphocytes 

derived from peripheral blood stem cells that were 

infused for allogeneic transplants. The number of 


62 


infused T lymphocytes was always 1.5-2 logs greater 

than that derived from bone marrow. 193 

The exact relationship between the T-lymphocyte 

content in the graft and the development and severity 

of GvHD remains unclear. A linear relationship between 

the number of T lymphocytes infused and the development 

of GvHD has long been hypothesized, 139,194,195 but 

this correlation has not always been confirmed. 196,197 

Findings in allogeneic peripheral blood stem cell transplantation 

seem to suggest that the number of T lymphocytes 

is less important than donor-cell specificity in 

triggering GvHD. 16,17 

Another aspect of peripheral blood stem cell transplantation 

concerns the number of natural killer cells 

(NK) infused (Table 5) since they are important effector 

cells in graft-versus-leukemia activity. 198 The number of 

infused NK cells is about 20 times greater in an allogeneic 

peripheral blood stem cell transplant than in a 

bone marrow graft. 16,17 The question of whether this will 

translate into more potent GvL activity in patients allografted 

with peripheral blood stem cells compared to 

unmanipulated bone marrow cannot be answered at 

this time, but needs further study. However, preliminary 

data from a murine model demonstrated strong GvL 

activity for allogeneic NK cells without the induction of 

GvHD. 199 

An important technical point is the effect of freezing 

and thawing of the graft or of keeping the apheresis 

product at 4°C overnight on T-lymphocytes inactivation. 

Van Bekkum 200 described selective elimination of 

immunologically competent cells from bone marrow 

after storage at 4°C. Eckardt et al. 201 also noted that cryopreservation 

of allogeneic marrow may reduce the risk 

of acute GvHD. Selective depletion or induction of anergy 

in GvHD-inducing cells was hypothesized. 201 Storage 

of the apheresis product at 4°C overnight does not modify 

the surface expression of the CD3, CD4, CD8, or the 

CD57 antigens in a significant manner (Tabilio, 1996, 

unpublished results); however, how cryopreservation 

affects the alloreactivity of peripheral blood stem cells 

needs to be investigated further. 

Conclusions 

Several studies have now shown that hematopoietic 

stem cells collected from PB after the administration of 

G-CSF, or from CB upon delivery, are capable of supporting 

rapid and complete reconstitution of BM function 

in allogeneic recipients. 16-20,204,205 The faster recovery 

of hematopoiesis as compared to BM-derived allografts, 

together with a lower incidence of aGVHD than 

expected with BM transplantation, raises the question 

of whether PBSC collections may differ from conventional 

BM harvests with respect to the number of stem 

cells and their functional characteristics, lymphoid cell 

composition and T-cell reactivity. Moreover, recent clinical 

studies on transplantation of CB-derived cells from 

unrelated HLA-mismatch donors support the notion that 

sources of hematopoietic stem cells other than BM represent 

a feasible alternative to conventional transplantation. 

In this paper, the phenotypic, functional and 

kinetic features of circulating and CB hematopoietic 

cells were reviewed. We also emphasized the technical 

aspects of CB collection and processing, as well as the 

protocols for PBSC mobilization and collection from normal 

donors. Notably, novel data on the immunogenic 

and kinetic profile of BM and PB CD34 + cells may shed 

new light on stem cell biology and may help clinical 

investigators to design future trials on transplantation 

of purified hematopoietic progenitors. 

It should be remembered that despite growing interest 

these procedures must still be considered as 

advanced clinical research and should be included in 

formal clinical trials aimed at demonstrating their definitive 

role in stem cell transplantation. In this regard, a 

large European randomized study is currently comparing 

PBSC and BM allografts. However, the possibility of 

collecting a large quantity of hematopoietic progenitor 

stem cells from PB, perhaps with reduced allo-reactivity, 

offers an exciting perspective for widening the number 

of potential stem cell donors and greater leeway for 

graft manipulation than is possible with BM. 

Acknowledgments 

Preparation of this manuscript was supported by 

grants from Dompé Biotec SpA and Amgen Italia SpA, 

Milan, Italy. 

This review article was prepared by a group of experts 

designated by Haematologica and by representatives of 

two pharmaceutical companies, Amgen Italia SpA and 

Dompé Biotec SpA, both from Milan, Italy. This co-operation 

between a medical journal and pharmaceutical 

companies is based on the common aim of achieving an 

optimal use of new therapeutic procedures in medical 

practice. In agreement with the Journal’s Conflict of 

Interest Policy, the reader is given the following information. 

The preparation of this manuscript was supported 

by educational grants from the two companies. 

Dompé Biotec SpA sells G-CSF and rHuEpo in Italy, and 

Amgen Italia SpA has a stake in Dompé Biotec SpA. This 

paper has undergone a regular peer-review process and 

has been evaluated by two outside referees. 

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donors increase the risk of acute graft-versushost-disease: 

effect of donor age and parity and analysis 

of cell subpopulations in the donor marrow inoculum. 

Br J Haematol 1986; 63:231-9. 

197. Jansen J, Goselink HM, Veenhof WFJ, et al. The impact 

of the composition of the bone marrow graft on 

engraftment and graft-versus-host-disease. Exp Hematol 

1983; 11:967-73. 

198. Murphy WJ, Reynolds CW, Tiberghien P, Longo DL. 

Natural killer cells and bone marrow transplantation. 

J Natl Cancer Inst 1993; 85:1475-82. 

199. Zeis M, Uharek L, Glass B, et al. Natural killer cells 

given after allogeneic BMT induce strong graft-vsleukemia 

(GVL) effects. Br J Haematol 1996; 93 (suppl 

2):76a. 

200. van Bekkum DW. The selective elimination of immunologically 

competent cells from bone marrow and 

lymphatic cell mixtures. Effect of storage at 4°C. 


201. Eckardt JR, Roodman GD, Boldt DH, et al. Comparison 

of engraftment and acute GvHD in patients 

undergoing cryopreserved or fresh allogeneic BMT. 


202. Dreger P, Suttorp M, Haferlach T, et al. Allogeneic 

granulocyte colony-stimulating factor mobilized peripheral 

blood progenitor cells for treatment of engraftment 

failure after bone marrow transplantation. 

Blood 1993; 81:1404-7. 

203. Rambaldi A, Viero P, Bassan R, et al. G-CSF-mobilized 


transplantation of resistant or relapsing acute 

leukemias. Leukemia 1996; 10:860-5. 

204. Kurtzberg J, Laughlin M, Graham ML, et al. Placental 

blood as a source of hematopoietic stem cells for 

transplantation into unrelated recipients. N Engl J 

Med 1996; 335:157-66. 

205. Laporte JP, Gorin NC, Rubinstein P, et al. Cord-blood 

transplantation from an unrelated donor in an adult 

with chronic myelogenous leukemia. N Engl J Med 

1996; 335:167-70. 


eview 

Clinical use of allogeneic 

hematopoietic stem cells from 

sources other than bone marrow 



Medica “L. & A. Seràgnoli”, Policlinico S. Orsola, via Massarenti 

9, 40138 Bologna, Italy. 

WILLIAM ARCESE,* FRANCO AVERSA,° GIUSEPPE BANDINI, # 

ARMANDO DE VINCENTIIS, @ MICHELE FALDA,^ LUIGI LANATA, @ 

ROBERTO M. LEMOLI, # FRANCO LOCATELLI, § IGNAZIO MAJOLINO, ** 

PAOLA ZANON, °° SANTE TURA # 

*Department of Cellular Biotechnology and Hematology, University 

“La Sapienza”, Rome; °Department of Clinical Medicine, 

Pathology and Pharmacology, Section of Clinical Hematology 

and Immunology, University of Perugia, Perugia; # Institute of 

Hematology and Medical Oncology "L. & A. Seragnoli", University 

of Bologna, Bologna; @ Dompé Biotec SpA, Milan; ^Division of 

Hematology, Ospedale S. Giovanni Battista, Turin; § Department 

of Pediatrics, University of Pavia and IRCC Policlinico S. Matteo, 

Pavia; ** Department of Hematology and BMT Unit, Ospedale “V. 

Cervello”, Palermo; °°Amgen Italia SpA, Milan, Italy 

Background and Objectives. Peripheral blood stem cells 

(PBSC) are being increasingly used as an alternative to 

conventional allogeneic bone marrow (BM) transplantation. 

This has prompted the Working Group on CD34- 

Positive Hematopoietic Cells to evaluate current utilization 

of allogeneic PBSC in clinical hematology. 

Evidence and Information Sources. The method 

employed for preparing this review was that of informal 

consensus development. Members of the Working 

Group met three times, and the participants at these 

meetings examined a list of problems previously prepared 

by the chairman. They discussed the single 

points in order to reach an agreement on different 

opinions and eventually approved the final manuscript. 

Some of the authors of the present review have been 

working in the field of stem cell transplantation and 

have contributed original papers in peer-reviewed journals. 

In addition, the material examined in the present 

review includes articles and abstracts published in 

journals covered by the Science Citation Index® and 

Medline®. 

State of the Art. Review of the current literature shows 

that unmanipulated allogeneic PBSC give prompt and 

stable engraftment in HLA-identical sibling recipients. 

Despite the much higher number of T-cells infused, the 

incidence and severity of acute GVHD after PBSC transplant 

seems comparable to that observed with bone 

marrow (BM) cells. In comparison to the latter, PBSC 

probably ensure faster immunologic reconstitution in 

the early post-transplant period. Controversial results 

on the incidence and severity of acute-GVHD have been 

reported when CD34 + selection methods are used. 

Prospective randomized trials are underway to compare 

the results of PBSC and BM allogeneic transplantation. 

In mismatched family donor transplants, T-cell depleted 

PBSC successfully engraft immune-myeloablated 

recipients through a mega-cell-dose effect able to overcome 

the HLA barrier. Experience with PBSC in the context 

of unrelated donor transplants is currently anecdotal 

and prospective trials should be completed before 

that practice becomes routine. Finally, there is also 

limited evidence that, following induction chemotherapy, 

the addition of PBSC to donor lymphocyte 

infusion (DLI) for treatment of leukemia relapse after 

BMT may improve the safety and effectiveness of DLI 

itself. Concerning cord blood (CB) transplants, the 

most interesting aspects are the ease of CB collection 

and storage, the low risk of viral contamination and the 

low immune reactivity of CB cells. This last property has 

its clinical counterpart in an apparently reduced incidence 

and severity of acute GVHD both in sibling and 

unrelated CB transplants, probably making the level of 

donor/recipient HLA disparity acceptable a greater 

degree with respect to what is required for transplants 

from other sources. 


Key words: hematopoietic stem cells, bone marrow, cord blood, peripheral 

blood, allogeneic transplantation, graft-versus-host disease 

In the field of allogeneic transplantation the use of 

alternative sources of stem cells, namely peripheral 

blood stem cells (PBSC) 1 and placental cord blood (CB) 

stem cells, 2 is rapidly expanding. The European Group for 

Blood and Marrow Transplantation (EBMT) registered 

only 12 allogeneic PBSC transplants in 1993, but this 

number increased to 180 in 1994 and to 571 in 1995. 3 

Concerning cord blood (CB) transplants, following initial 

attempts 4,5 considerable experience has now been 

achieved in the USA and Europe so that this modality is 

entering a phase of extensive clinical application, with 

hundreds of procedures registered both from sibling and 

unrelated donors, 6,7 The ease of collection and storage of 

CB stem cells and the apparent tolerance-inducing property 

of CB CD8 + suppressor cells 8 are the most interesting 

aspects of this latter source. 

Stem cells in peripheral blood and CB both possess a 

migratory status and differ in part from those found in 

bone marrow with respect to their biological and functional 

properties. However, while PBSC are envisaged as 

a means of improving results by increasing the number 

of cells available, placental CB stem cells open the realistic 

perspective of increasing the number of transplants 

thanks to the availability of thousands of cord samples 

for patients who lack a compatible donor among family 


70 

W. Arcese et al. 

members. 

This search for new stem cell sources also arises from 

the fact that allogeneic bone marrow transplantation still 

carries a high procedure-related mortality and disease 

recurrence rate. Cell dose has an influence on engraftment 

and chance of survival. In a retrospective study 

Bacigalupo et al. 9 showed that patients with hematologic 

malignancies who receive allogeneic bone marrow 

grafts with higher CFU-GM numbers have significantly 

higher platelet counts on day +80 and a lower mortality 

rate than those who receive fewer CFU-GM. The effect of 

CMV infection on platelet counts also appears to be less 

pronounced when the number of progenitor cells is higher. 

The use of more hematopoietic progenitors would then 

result in improved transplant outcome. 

The growing interest in allogeneic PBSC induced the 

Italian Bone Marrow Transplant Group (GITMO) to draw 

up a list of recommendations that were originally published 

in 1995 10 and recently revised in light of the 

increasing experience gained worldwide during the last 

two years. 11 Moreover, in a previous issue of this Journal 12 

a review article analyzed the biological and technical 

aspects of PB and CB stem cells. The key aspects dealt 

with were the mobilization and collection methods, the 

capacity for stable hemopoietic reconstitution, kinetic 

characteristics and immunological features. 

Historical background 

Interest in allogeneic transplantation of PBSC began 

thirty years ago. In the late sixties, based on an earlier 

demonstration that autologous PBSC were capable of 

restoring irradiation-myeloablated hematopoiesis, 13-15 

the Seattle group reported the first successful attempts 

at allogeneic PBSC transplantation in dogs 16,17 and nonhuman 

primates. 18 However, due to the high GVHD incidence, 

those experiments were unable to demonstrate 

long-term stability of the graft. Only a decade later did 

purification of PBSC and application of cytogenetic 

methods allow a group of German investigators 19,20 to 

document in dogs the stability of donor-derived hemopoietic 

function for more than ten years after PBSC allogeneic 

transplantation. 

A key issue at that time was the low number of progenitors 

in steady-phase peripheral blood, and clinical 

application of PBSC was limited to autologous transplantation 

in CML, 21 where hematopoietic progenitors, 

mostly of the leukemic counterpart, circulate in high 

numbers and can easily be collected by apheresis without 

any prior stimulation. A further step was the demonstration 

that the PBSC level increases dramatically during 

the post-chemotherapy recovery phase; 22 however, it 

was the advent of G-CSF and GM-CSF that provided the 

rapid expansion of PBSC technology and led to their use 

in allogeneic transplantation as well. The pioneer work 

of Socinski et al. 23 and Gianni et al. 24 established the ability 

of hematopoietic growth factors to expand the circulating 

progenitor cell pool either when used alone or 

in conjunction with chemotherapy. However, transferring 

growth-factor PBSC mobilization strategy from autograft 

patients to normal donors took some years. In fact, 

the safety of growth factors and the clinical applicability 

of allogeneic PBSC in terms of GVHD incidence and 

long-term engraftment represented serious reasons for 

caution. Clinical PBSC allogeneic transplantation began 

in 1989, when Kessinger et al. 25 reported the first attempt 

in an HLA-matched recipient with ALL. The patient was 

an 18-year-old man in third remission after CNS and 

testicular relapse. His sibling female donor preferred to 

donate PBSC rather than bone marrow, and she underwent 

10 apheretic procedures without any mobilization 

treatment. The apheresis product was T-depleted by 

sheep erythrocyte rosetting and infused after conditioning 

with high-dose Ara-C and TBI. The patient achieved 

full donor engraftment as demonstrated by cytogenetic 

studies but died on day +32, and sustained engraftment 

could not be demonstrated. 

Four years later, in 1993, Russell et al. 26 reported 

another transplant in a patient whose sibling donor presented 

an increased risk of complications from anesthesia. 

In this case, 10 mg/kg/day of G-CSF were given 

to mobilize PBSC. The cells were collected at two leukaphereses 

containing 36.8×10 4 /kg CFU-GM and infused 

without any prior manipulation. Engraftment occurred 

rapidly and GVHD did not develop despite the high T-cell 

content of the graft sample. The same year, a group of 

investigators from Kiel University 27 also successfully 

employed allogeneic PBSC. A 47-year old AML patient 

who failed to engraft after bone marrow transplantation 

from an HLA-identical sibling donor was infused with 

the unmanipulated product of 3 leukaphereses performed 

after treating the donor with 6 mg/kg/day G- 

CSF. Engraftment occurred on day +14, with moderate 

acute GVHD that responded to immunosuppression 

starting on day +18. Restriction fragment length polymorphism 

(RFLP) typing demonstrated full donor 

engraftment up to 60 days following transplantation. 

Another important step was the five PBSC transplants 

from syngeneic donors performed in Seattle and reported 

in 1993: 28 with a median of 9.6×10 6 /kg CD34 + cells 

infused, the patients engrafted 0.5×10 9 /L granulocytes 

on day 13 and 20×10 9 /L platelets on day 10. In 1995 

three separate reports appeared in the same issue of 

Blood, one from Seattle, 29 a second from Houston 30 and 

the other from Kiel; 31 a total of 25 patients were allografted 

with PBSC from their HLA-identical sibling 

donors. Acute GVHD was apparently not increased in 

those series. Molecular analysis of engraftment 30,31 furnished 

definitive proof of the experimental data 20 suggesting 

that allogeneic PBSC contain true long-term 

repopulating stem cells. The high engraftment potential 

of PBSC was exploited by the Perugia team 33 to successfully 

transplant leukemia patients from their haploidentical, 

three-loci-incompatible family donors 

through T-cell depletion. Finally, Ringdén et al. 34 recently 

reported the use of allogeneic PBSC in selected unrelated 

donor transplants. 

The kinetics of PBSC under cytokine mobilization was 

extensively analyzed in a previous review published in 


Clinical use of allogeneic hematopoietic stem cells 

71 

this Journal. 12 PBSC mobilization in healthy donors is 

best accomplished with G-CSF. The aspect of donor 

safety was analyzed in a cooperative GITMO study35 in 

which short-term side effects were shown to be minimal. 

Ten µg/kg/day of G-CSF for 5 days enabled the collection 

of >4×10 6 /kg CD34 + cells with two aphereses in 

85% of donors. Variations in blood counts included a 

sharp elevation of WBC and CD34 + cells and a moderate 

transitory thrombocytopenia. One problem, however, 

is the lack of data on the late effects of G-CSF. At the 

Geneva conference on allogeneic PBSC, Hasenclever and 

Sextro 36 presented a feasibility study of long-term risk 

analysis. In order to demonstrate a tenfold increase in 

leukemia risk, more than 2000 healthy PBSC donors 

would have to be followed for over 10 years. A control 

group of BMT donors of equal size would also be necessary. 

Such a study could only be carried out on a multi-national 

basis. 

Transplantation of allogeneic PBSC 

from HLA-identical siblings 

Conditioning regimens and GVHD prophylaxis 

Conditioning regimens employed in PBSC transplantation 

are the same as those used for bone marrow transplantation 

(BMT). As listed in Table 1, the majority of 

patients received CY-TBI or BU-CY with CY at 120 or 200 

mg/kg. Indeed, 33.8% and 24.5% of reported patients 

were conditioned with CY-TBI and BU-CY, respectively. 

Analogously to BM transplantation, patients with SAA 

received cyclophosphamide alone or in association with 

ATG. 

Recently, some innovative regimens have been developed 

in order to: i) increase antitumor activity; ii) reduce 

treatment-related toxicity. For instance, high dose Ara- 

C or VP16 has been employed for patients with more 

advanced disease. Others considered thiotepa, a potent 

myeloablative drug first introduced in conditioning by 

the Perugia group. 37 This latter compound was used along 

with classical BU-CY2 in a large series at the M.D. Anderson 

Cancer Center 38,39 or was associated with cyclophosphamide. 

40 In this study, thiotepa was introduced in the 

hope of reducing the liver toxicity of busulfan. 

It is interesting to note the recent introduction of fludarabine, 

a purine analogue initially proposed at conventional 

dosage by the M.D. Anderson group 41 and at 

a higher dose by the Perugia group in HLA-mismatched 

transplants. 42 Fludarabine has been associated with several 

other drugs or drug combinations including 

cyclophosphamide plus cis-platinum, high-dose (HD) 

Ara-C, idarubicine plus Ara-C or melphalan. 41,43 These 

fludarabine containing regimens were followed by full 

engraftment with complete donor chimerism in the 

absence of severe aplasia. Basically, these new regimens 

allow allograft even in older or medically infirm patients, 

since they reduce the toxicity but still maintain an effective 

graft versus leukemia reaction. For the same reason 

Adkins et al. 44 combined low-dose TBI (550 rad) at a 

high dose rate (30 cGy/min) with cyclophosphamide 120 

mg and methylprednisolone 2 g over two days. 

A non-myeloablative regimen with busulfan and 

methylprednisolone combined with immunosuppression 

with the CD3 monoclonal antibody was used by Tan et 

al., 45 while Slavin et al. 46 employed fludarabine and ATG 

as intensive immunosuppression associated with busulfan 

at 4 mg/kg/day over 2 days. Although these regimens 

are not specifically designed for PBSC transplantation, 

the high number of inoculated PB stem cells overcomes 

graft rejection, favoring rapid and stable chimerism. 

The large amount of stem cells in the inoculum might 

explain the full engraftment observed in a patient who 

could not complete the whole regimen and received 

busulfan alone as preparation. 47 

The large number of CD3 +ve cells present in the PBderived 

inoculum has raised some concerns about the 

severity of GVHD following PBSC allograft. However, 

GVHD prophylaxis has not been substantially modified 

from the standard regimens used for bone marrow transplants. 

Cyclosporin A (CsA) has been used alone (2.9%) 

or in association with either methotrexate (MTX) (49.8%) 

or methylprednisolone (MP) (21.2%) in 71% of patients 

(Table 1). 

New immunosuppressive regimens including tacrolimus 

(FK506) in association with methylprednisolone, 

methotrexate 39 or monoclonal antibodies 48 have been 

explored in 17% of cases. 

Finally, some centers have developed techniques for in 

vitro stem cell enrichment. However, using Ceprate-cell 

separation, a 2-3 log depletion of T-lymphocytes is not 

enough to avoid the risk of GVHD and further immunosuppression 

is generally required. 

In some institutions cryopreservation of collected PBSC 

is preferred to freshly collected material for several reasons. 

First, cryopreservation allows precise evaluation of 

the hemopoietic progenitor content in the harvested 

material. Second, the allograft may be scheduled at the 

proper time once adequate quantities of PBSC are collected. 

Finally, although still unproven, a reduced risk of 

acute GVHD in patients transplanted with cryopreserved 

BM cells has been suggested. 49 

Table 1. Conditioning regimens and GVHD prophylaxis: 301 

patients. 

Regimens No. % GVHD prophylaxis No. % 

CYTBI 102 33.8% CsA-MTX 150 49.8% 

BUCY 68 24.5% CsA-MP 82 21.2% 

TioBUCY 67 22.2% FK506-MTX 7 2.3% 

VP16TBI 10 3.3% FK506-MP 44 14.6% 

TioCY 8 2.6% CsA 15 2.9% 

CYATG 3 0.9% MTX 1 0.3% 

Others 43 16% Others 2 0.6% 


72 


Engraftment 

Engraftment kinetics following PBSC allograft has been 

extensively investigated. None of the studies include 

patients dying before day 21. The median time to reach 

an absolute neutrophil count(ANC) above 0.5×10 9 /L 

ranges between 10-16 days. The reported incidence of 

graft failure is definitely low: 1/59 in the EBMT survey, 50 

1/41 in the MD Anderson series, 51 1/26 in the Canadian 

experience. 52 The few rejections occurred in transplants 

with 1-2 antigen disparity. Platelet engraftment is also 

prompt, with median time to achieve an absolute platelet 

count (APC) of 20×10 9 /L ranging between 10 and 18 days 

in the reported series. 

Platelet more than neutrophil engraftment may be 

affected by acute GVHD or CsA toxicity as well as by early 

relapse or progressive disease. 50 In a recent report by 

the Genoa group a second infusion of PBSC without conditioning 

was required to achieve full engraftment of 

platelets in three out of thirty-one patients. 40 

The prompt engraftment offered by PBSC implies a 

reduction in transfusion need. The reported transfusion 

requirements range from 2 to 10 packed red cell units and 

from 3 to 12 platelet units. 

Furthermore, GVHD prophylaxis may adversely influence 

the time to engraftment. In particular, methotrexate 

given for GVHD prophylaxis delays neutrophil and 

platelet engraftment. 53,54 

Growth factors, mainly G-CSF, have been employed in 

several studies to speed-up engraftment. Urbano- 

Ispizua reported that G-CSF given to patients not receiving 

methotrexate accelerates neutrophil recovery 

(p=0.001); median time to >20×10 9 /L platelets was significantly 

delayed (p 0=0.01), although the time to reach 

5010 9 /L platelets was not affected. No difference in 

engraftment kinetics was seen between cryopreserved 

and fresh PBSC when G-CSF was administered following 

transplantation. 50,55 

The studies carried out so far are not sufficient to 

draw definitive conclusions about engraftment with 

PBSC as compared to engraftment with BM. Randomized 

studies are still in progress and results are not yet 

available. Most information comes from comparisons of 

PBSC results with historical data from BM transplants. 

A highly informative study was reported by the Seattle 

group, which compared 37 PBSC transplanted 

patients with a historical group of 37 bone marrow 

recipients. 56 Patients were well matched for diagnosis, 

disease stage, age and graft versus host prophylaxis. 

Faster neutrophil engraftment, 14 versus 16 days to 

reach more than 0.5×10 9 /L (p=0.0063), and earlier 

achievement of platelet transfusion independence, 11 

versus 15 days (p=0.0014), were observed in PBSC recipients 

compared to the BM control group. Consequently, 

the median number of platelet units transfused was 

24 versus 118 (p=0.0001) and the median number of red 

blood cell units transfused was 8 versus 17 (p=0.0005) 

in the PBSC group and in the BM group, respectively. 

Similar results have been reported by Russel et al.: 52 

duration of aplasia for both neutrophils (p=0.0002) and 

platelets (p=0.0003) was significantly reduced in 

patients receiving PBSC compared to BM recipients. 

Interestingly, the advantage of PBSC was also maintained 

if methotrexate was used as GVHD prophylaxis. 

A recent report by Rosenfeld et al. 57 evaluated 19 

patients transplanted with PBSC. No growth-factor was 

employed in the post-transplant phase. Significantly 

faster neutrophil recovery was observed in PBSC transplanted 

patients compared to historical control group 

transplanted with BM (p=0.01). However, the difference 

was not significant when the PBSC group was compared 

to BM recipients given G-CSF in the post-transplant 

phase. 

More recently, a prospective non-randomized study 

was carried out by the M.D. Anderson group. 39 The study 

included 74 adults transplanted with HLA-matched 

related donors. Thiotepa, busulfan and cyclophosphamide 

were employed as preparative regimen. The 

patients were divided into 3 cohorts: Group 1 received 

BMT using CsA and MTX as GVHD prophylaxis, Group 2 

received marrow using CsA and MP, and Group 3 

received PBSC with CsA and MP. All patients were given 

G-CSF post-transplant. Median time to neutrophils 

> 0.5×10 9 /L was 17, 9 and 10 days, and to platelets 

>20×10 9 /L was 32, 25 and 18 days in Groups 1, 2 and 

3, respectively. The use of CsA and MP for GVHD prophylaxis, 

rather than the source of engrafted cells was 

shown to be the most important factor for rapid neutrophil 

and platelet recovery. Provided that CsA/MP was 

used for GVHD prophylaxis, platelet transfusion requirement 

was found to be significantly lower in PBSC than 

in BMT recipients (p=0.04). Significant differences concerning 

regimen-related toxicity were seen for grade 2- 

4 stomatitis only between the BMT group using MTX in 

GVHD prophylaxis and the PBSC group using MP. 

Correlation between engraftment kinetics and quantity 

of PB cells infused is still an open question. The 

absolute number of nucleated PB cells or CD34 + cells did 

not correlate with time to neutrophils > 0.5×10 9 /L or 

with time to platelets > 20, > 50 or > 100×10 9 /L in a 

study by Rosenfeld et al. 57 Similarly, Urbano-Ispizua et 

al. did not find any correlation using several cut-off values 

of CD34 + cells at 2.5, 3, 4, 5.5 and 7×10 6 /kg. In contrast, 

Roy et al. 58 reported a correlation between CD34 + 

cells infused and engraftment using a mobilization regimen 

with G-CSF at a dose of 5 µg/kg. In a large series 

published by the M.D. Anderson group, 37 in univariate 

analysis of patients not given MTX prophylaxis the number 

of total nucleated cells infused positively affected 

ANC recovery. Moreover, platelet recovery was positively 

influenced by the number of CD34 + cells, as well as by 

young age and sex mis-matching. 

Immune reconstitution after transplantation 

of peripheral blood stem cells 

Patients undergoing allogeneic BMT experience a prolonged 

period of profound cellular and humoral immunodeficiency, 

mainly due to complete pre-transplant 

destruction of the host lymphohemopoietic system, the 



73 

use of immunosuppressive drugs for GVHD prophylaxis 

and the development of GVHD. 59-61 This immunodeficiency 

lasts until stem cells and mature lymphocytes 

contained in the transplanted marrow repopulate and 

reconstruct the hematopoietic and lymphopoietic systems 

which had been destroyed by the pre-transplant 

conditioning regimen. In particular, immunological 

reconstitution after BMT is considered to be dependent 

on two distinct phenomena. 59,60 In the early post-transplant 

period, there is an expansion of mature donorderived 

lymphocytes transferred with the graft, a 

process influenced by both the recipient’s environment 

and the cytokine storm 62 related to the transplant procedure. 

Thereafter, naive lymphocytes derived from the 

differentiation of donor hematopoietic stem cells colonize 

the lymphoid organs of the recipient and sustain 

the late immune response. 

The crucial role of the first step in immunological 

recovery is demonstrated by the observations that 

patients receiving a T-cell depleted transplant are at 

particular risk for infections and that patients transplanted 

using donors either recently vaccinated against 

or immune to a certain pathogen usually have a more 

rapid recovery of specific T-cell response than ones who 

received bone marrow from unprimed donors. 63-65 Formal 

proof of the contribution of transferred donorderived 

lymphocytes to recipient immune reconstitution 

has been recently reported. 62 In fact, using the combination 

of a cell culture method and a PCR amplified 

technique to study tetanus toxoid (TT)-specific T-cells 

clones, it was possible to demonstrate that patients after 

BMT display a small response that can be accounted for 

by a few donor-derived clones and that the T-cell clones 

transferred with the transplant were still detectable 

within the donor polyclonal T-cell lines for up to at least 

5 years after BMT. Moreover, the vaccination of donors 

with TT before BMT resulted in a more relevant transfer 

of antigen-experienced T-cells. 66 

The expansion of mature donor-derived lymphocytes 

transferred with the graft in recipients of peripheral 

blood stem cell (PBSC) transplantation could be expected 

to be more efficient than patients given BMT, in view 

of the higher number of donor lymphocytes transferred. 

However, at present, few reports specifically addressing 

the question of immune recovery after transplantation 

of PBSC are available. 

Ottinger et al. 67 demonstrated that, compared to BMT 

recipients, patients who were given a PBSC transplant 

had a more rapid recovery of both naive and memory 

CD4 + cells (expressing the RA and RO isoforms of the 

CD45 molecule, respectively) whose counts significantly 

exceeded those observed following marrow transplantation. 

This determined that in patients receiving 

PBSC transplantation the characteristic inversion of the 

CD4 + /CD8 + ratio observed after BMT was not encountered. 

Furthermore, the B-cell levels and, at least for the 

first 2 months after transplantation, the monocyte 

counts were augmented. Since monocytes of granulocyte 

colony-stimulating factor (G-CSF)-mobilized donors 

have been demonstrated to reduce the responsiveness of 

alloantigen specific T-cells, the increase in their count 

could contribute to the low incidence and reduced severity 

of acute GVHD reported after transplantation of 

PBSC. 68 Moreover, it must be noted that there is a prompt 

recovery of the lymphocyte counts after transplant of 

PBSC coupled with an enhanced in vitro response of lymphocytes 

to aspecific polyclonal activators (phytohemagglutinin 

and pokeweed mitogen) and to recall 

antigens (TT, Candida). The most likely hypothesis for 

explaining this accelerated recovery of helper T cells, B 

lymphocytes and monocytes is that the number of lymphocytes 

infused for each subset is more that one magnitude 

higher in recipients of PBSC transplant than in 

patients given BMT. However, alternative mechanisms 

cannot be excluded. 

Similar results in terms of more rapid recovery of CD4 + 

cells have also been reported by Bacigalupo et al. 40 in 

adults with advanced leukemia who received high-dose 

chemotherapy followed by G-CSF mobilized PBSC. More 

recently, two additional reports have further confirmed 

that recipients of PBSC transplants have a faster recovery 

of both naive and memory helper T cells. 69,70 Moreover, 

one of these studies documented that patients 

experiencing a more rapid recovery of the lymphocyte 

count had a significantly better probability of survival 

after transplantation. 69 

Whether the improved immune reconstitution 

observed after transplantation of PBSC is associated with 

a lower incidence of infectious complications still 

remains to be documented. In one of the previously mentioned 

studies, 69 the actuarial risk of reactivation of 

human cytomegalovirus (HCMV) infection in patients 

given a PBSC transplant was comparable to that 

observed in a historical control group of BMT recipients. 

This could be attributed to a greater viral load infused 

with the graft and correlated with the very large number 

of nucleated cells that can harbor HCMV transfused. 

Nonetheless, since the use of donor-derived adoptive 

immune therapy has been shown to be able to cure or 

prevent HCMV-related interstitial pneumonia and EBVinduced 

lymphoproliferative disorders, 71-73 it can be 

hypothesized that patients given PBSC transplants, with 

a more efficient transfer of antigen-experienced lymphocytes, 

may have a reduced incidence and/or reduced 

severity of infectious complications. Support for this theory 

is provided by the study reported by Bensinger et 

al. 74 in which a lower number of deaths from infectious 

complications was observed in patients given PBSC as 

compared to a historical group of BMT recipients. 

Acute and chronic GVHD 

PBSC collections contain a large number of T-cells – 

approximately 10 times more than unmanipulated marrow 

grafts. 75 Therefore concern for increased incidence 

and severity of GVHD after their infusion into an allogeneic 

host has been and still is a major issue after PBSC 

transplantation. Here we analyze the results reported 

so far in the most recent peer-reviewed studies pub- 


74 


lished. Because of the relatively short follow-up of these 

studies, the assessment of chronic GVHD (cGVHD) is less 

complete and less accurate than that of the acute form. 

Some of the studies in fact do not address the problem 

of cGVHD. Acute GVHD, on the other hand, can now be 

evaluated in a rather significant number of patients. We 

shall look first at the characteristics of the studies, then 

analyze acute and cGVHD separately and finally make 

comparisons between marrow and PB blood transplants. 

A set of tentative comments will be made at the end of 

the chapter. 

Type of studies. Selected studies of PBSC transplantation 

for hematological malignancies are reported in 

Tables 2 and 3. Several of them have been analyzed in 

the section on hematological recovery. Table 2 focuses 

on the main demographic characteristics, while Table 3 

gives details of the transplant procedure and results of 

acute and chronic GVHD where applicable. Six studies 

are from a single institution, 39,40,55,75-77 while three are 

from several centers; 50,52,54 one study from the EBMT 

Group, 50 multicentric in nature, also reports several 

patients included in four of the other studies – a typical 

example of double reporting – so that its results 

reinforce what has already been observed. The figures 

from this last study were not calculated in any further 

statistical analysis in order to avoid the error of counting 

some of the patients twice. However, they are useful 

for comparisons and have been left in the tables. The 

total number of patients is 212. None of the studies is 

prospective or randomized, but four 52,55,75,77 compare the 

results of PBSC with those of marrow, although using 

different methods. We shall have to wait some time 

before seeing the results of the two prospective randomized 

studies comparing marrow and PBSC transplantation 

which are now in progress in Europe and the 

US; for the moment, the reports analyzed here represent 

the best we have. The 8 studies took place recently, 

between late 1993 and 1995, and mostly dealt with 

adults (median age 38 yrs, with a range from 1-57), but 

some included pediatric patients. Transplants were from 

fully HLA-identical siblings in 96% of the cases, but a 

minority received cells from family donors mismatched 

for one HLA antigen; a minority of patients (5 to 10%) 

also received a second allo transplant, usually from the 

original sibling who had donated the marrow. Patients 

showed a typical spectrum of hematological malignancies 

for which transplant is indicated. The majority 

(median 83%) were in advanced phases of their diseases, 

although definitions are quite variable with the 

term high-risk being used as a synonym for advanced 

phase, but 17% had early phase or low-risk disease at 

the time of transplant. These proportions differed widely 

within studies, some including 100% advanced diseases 

and others only 60%, with many more early phase 

patients. Pre-transplant regimens were obviously different, 

but despite their apparent disparities they can be 

grouped into those based on busulphan (54%) or TBI 

(31%). Only two studies differ considerably from the 

rest of the series: the Genoa group 40 purposely employed 

a low intensity regimen based on thiotepa and 

cyclophosphamide to reduce toxicity in a rather old 

patient population. The MD Anderson Hospital, 39,55 on 

the other hand, used a very intensive regimen combining 

busulphan, thiotepa and cyclophosphamide in a pop- 

Table 2. Main characteristics of the studies reviewed. 

Ref Type Of study Period of study Median N° pts Median 2nd transplants Hla family Phase of the disease 

Follow-up days (range) age yrs (range) mismatches 

74 SC 12/93-11/95 nr 37 38 (20-52) NO NO Advanced 100% 

52 MC 5/93-6/95 nr 26 40 (1-54) 3 (11%) 6 (23%) High risk 23 (88%) 

Standard risk 3 (12%) 

40 SC nr 136 (6-228) 31 44 (19-55) NO 3 (10%) Advanced 28 (90%) 

Early 3 (10%) 

55 SC nr 270 (180-600) 25 43 (17-57) 1 (4%) NO Relapse 21 (84%) 

Remission 4 (16%) 

76 MC 3/94-7/96 nr 24* 37 (16-57) 1 (4%) 1 (4%) Early 10 (42%) 

Advanced 14 (58%) 

54 SC 1/94-4/95 nr 33 36 (12-53) 8 (24%) NO Early 12 (36%) 


39 SC 32 months nr 19 Not detailed 1 (5%) NO Early 18% 

Advanced 82% 

77 SC 3/94-4/95 111 (15-402) 17 33 (16-52) NO NO Early 6 (35%) 


50 MC 1994 nr 51° 39 (2-54) NO NO Early 15 (25%) 


LEGEND: SC = single center; MC = multicenter; n.r. = not reported; *includes 1 pt with SAA; °includes patients from studies #40, 76 and 54. 



75 

Table 3. Transplant modalities, aGVHD and cGVHD. 

Ref Fresh or Conditioning G-CSF GVHD Acute GVHD Chronic GVHD 

cryo- regimen* post TX prophylaxis n. grade grade GVHD related/ n. limited extensive 

preserved cells N° pts N° pts evaluable II-IV III-IV total deaths evaluable 

74 F TBI 32 no CsA/MTX 19 35 13 (37%) 5 (14%) 1/15 17 3 (18%) 4 (24%) 

Bus 5 CsA/PDN° 18 

52 C TBI 18 no CsA/MTX 26 nr 37% nr nr nr 53% overall 

40 F Thio-CTX 31 @ no CsA/MTX 31 31 17 (55%) 4 (13%) 4/12 28 15 (53%) 7 (25%) 

55 C Bus 25 yes CsA/PDN 25 25 11 (42%) 6 (22%) 3/7 nr nr nr 

76 F^ Bus 22 no Csa/MTX 23 22 10 (45%) 2 (9%) 0/7 16 1 (5%) 9 (50%) 

Other 3 CsA 1 

54 F 21 TBI 17 yes CsA 2 

C 1 Bus 16 (11 pts) CsA/MTX 22 32 11 (34%) 7 (22%) nr 11 4 (36% overall) 

CsA/PDN 9 

39 C Bus 19 yes FK-506/PDN 19 nr 22% 11% nr nr nr nr 

77 F Bus 17 no CsA/MTX 17 10 3 (33%) 4 (24%) 4/4 10 3 (33%) 0 

50 F 49 TBI 22 CsA/PDN 7 

Bus 22 yes CsA 6 57 30 (50%) 14 (23%) 7/29 49 17 (35%) 13 (26%) 

C 10 Thiotepa 11 (14 pts) Other 9 

Other 4 

*Conditioning regimen mainly based on; @ Regimen not including TBI or busulphan; CTX = cyclophosphamide; ^cells infused over 2 days: apheresis of day 1 stored at 4°C until infusion; 

°PDN=prednisone; n.r.=not reported 

ulation of similar age. Another difference is represented 

by the processing of the collected PBSC: in 130 cases 

(61%) they were infused fresh and in 82 cases (39%) 

they were cryopreserved instead until infusion. Finally, 

GVHD prophylaxis was not uniform: it was based on a 

combination of CsA and short-course methotrexate in 

130 (62%) of the cases, and on CsA plus prednisone in 

42 cases (20%); only one study 38 reports 19 patients 

(9%) who received a combination of tacrolimus and 

prednisone. CsA alone was used in three patients (1.4%). 

Acute GVHD. The incidence of aGVHD, grade II to IV, 

was about 40% on average. The Genoa study 40 reported 

a 55% incidence, but it also included the oldest patients 

in the series; the lowest incidence, 22%, was reported 

in the series from the MD Anderson Hospital where 

tacrolimus was used for GVHD prophylaxis. 39 The incidence 

of severe aGHVD, i.e. grade III and IV, was on average 

16% (range 11-24%). An interesting point is the 

fact that while most studies showed a direct correlation 

between the overall incidence of GVHD and severe 

GVHD – the latter being about half of the former – others 

did not. Two studies from Italy 40,76 which reported 

an overall incidence of over 50% also showed a low 

incidence of severe GVHD, which means that grade II 

accounted for the majority of the cases. No correlation 

could be made from the existing data between the variables 

known to influence aGVHD 78 and the results either 

within the single studies as discussed by their authors 

or by combining data as in this review. Of interest, on 

the specific issue of PBSC, no correlation was found with 

the number of T-lymphocytes infused or with the use of 

fresh or cryopreserved cells. However, it should be noted 

that the highest incidences of severe aGVHD (22- 

24%) were reported when prophylaxis was based on 

CsA/prednisone, 55 which is perhaps less effective than 

CsA/MTX or in the Spanish study which reported data 

from multiple institutions 54 with a good proportion of 

patients receiving CsA/prednisone for GVHD prophylaxis. 

Nevertheless, a high incidence was also reported in a 

study from Brazil where CsA/MTX was used in all cases. 

77 Mortality from aGVHD is not reported in all studies, 

as shown in Table 3; those giving the causes of death 

often do not mention, when infection was the main 

cause, whether GHVD was associated. However, considering 

the causes of death of 47 events analyzable in 

detail, GVHD was the main cause in 12 (25%). Incidentally, 

this figure is higher than the overall incidence of 

grade III-IV GVHD, but data on death are reported for 

fewer patients than data on GVHD. 

Chronic GVHD. The number of patients analyzable for 

cGHVD is smaller than for aGVHD; survival > 90, 100 or 

150 days is the requisite for evaluation. In addition, 

some studies give many details on cGVHD while others 

do not address the issue 55 or mention it very briefly. 39,52,54 


76 


We calculate that slightly more than 110 patients are 

evaluable. The overall incidence ranged from 36% to 

78%; considering the four studies which give detailed 

information, the extensive form of the disease occurred 

with nearly the same frequency or less than the limited 

form in three studies, 40,56,77 while one series reported a 

striking incidence of the extensive form, with the limited 

one being only minimally represented. 76 In that study 

cGVHD developed de novo in 5 out of 10 patients, at 

variance with the low incidence of aGVHD observed earlier. 

Another interesting observation is contained in a 

study from Seattle: 56 of 10 patients at risk, who had 

received CsA/prednisone for prophylaxis, 6 developed 

cGVHD, while only 1 of 7 given CsA/MTX did so. 

Comparisons between marrow and PBSC transplantation. 

Four studies 52,55,56,77 compared the incidence of GVHD 

after PBSC or marrow transplantation. These studies were 

carried out using matched-pair analysis with a historical 

control group of marrow recipients who were matched 

for diagnosis, disease and disease phase at transplant, 

age, GVHD prophylaxis 56 or age and disease status. 52 One 

study does not give the characteristics of the marrow 

recipients. 77 The Seattle study found a lower incidence of 

grade II-IV aGVHD for PBSC recipients – 37% vs 56% – 

severe GVHD (grade III-IV) was even more impressively 

lower in PBSC recipients, 14% vs 33% of marrow transplants. 

However, due to the small number of patients 

these data are not statistically significant. The overall 

incidence of cGVHD was similar in the two groups, with 

a tendency toward more severity in the PBSC than in the 

marrow group (42% vs 26% for any grade of clinical 

cGVHD), but again this was not statistically significant. 

The striking effect of MTX in the GVHD prophylaxis regimen 

observed in this study has already been mentioned. 

The multicenter Canadian study 52 reported a higher incidence 

of both aGVHD and cGVHD for the PBSC group 

than for the marrow recipients: 37% vs 21% grades II- 

IV aGVHD and 53% vs 48% for cGVHD, respectively. A 

different kind of comparison can be made in a study from 

the MD Anderson Hospital, 55 where patients with 

advanced hematological malignancies received, over a 3- 

year period, the same conditioning protocol but different 

forms of GVHD prophylaxis and different sources of allogeneic 

stem cells. The PBSC patients could be compared 

to the marrow group who received CsA/prednisone as 

GVHD prophylaxis: severe aGVHD was slightly less in the 

PBSC group, 22% vs 33%, but this difference was not statistically 

significant. No data on cGHVD were provided. 

The Brazilian study 77 reported more aGVHD in the PBSC 

than in the marrow group, grade III-IV 4/17 vs 3/21, but 

again this was not statistically significant. No comparative 

data on cGVHD were given. 

Comments. The fear of an unacceptably high rate of 

severe and perhaps uncontrollable acute GVHD after allo 

PBSC can now be allayed with a certain degree of confidence 

on the basis of the data analyzed here. In a population 

of adults with mainly advanced hematological 

malignancies who sometimes received second transplants 

or not fully HLA-identical grafts and, most important, 

were often not given the best GVHD prophylaxis 

available, acute GVHD was no more than what is expected 

with marrow transplants. Similar conclusions had 

been reached in an earlier review on the subject, 56 

although on a much smaller number of patients. It is 

more difficult to say whether GVHD is slightly more 

severe than after conventional marrow grafting, considering 

the wide variation in its occurrence, due to the 

multitude of factors which influence it. A broad comparison 

of the published data indicate that GVHD 

observed after PBSC is higher than in the best marrow 

series 79 but not worse than what is described in the large 

reports from registries. 80 Four studies have attempted a 

comparison with retrospective marrow transplants and 

none found significantly increased aGVHD incidence or 

severity. It is interesting to speculate why after infusion 

of 1 log more T-lymphocytes as compared to marrow 

aGVHD is not increased. One explanation is that any 

number of T-cells, once the 10 5 /kg threshold has been 

surpassed, 81 is already high enough to cause GVHD and 

even a 10-fold increase, with respect to the marrow, 

does not make any difference. Perhaps an extraordinarily 

large number of T-cells infused, that is usually not 

reached after G-CSF mobilization, for example 3 or 4 

logs, could be the next threshold above which more 

severe GVHD would regularly occur. Data from a study 

in Seattle, where 1500×10 6 /kg donor buffy-coat 

mononuclear cells were intentionally infused soon after 

BMT in patients with advanced disease to enhance a 

graft- versus-leukemia effect did show that acute, severe 

GVHD was indeed increased; in that study, unfortunately, 

GVHD prevention was based on MTX only so comparisons 

with today’s practices are not possible. 82 Other 

explanations for why aGVHD does not increase relate to 

the possible biological modifications of lymphokine production 

induced by G-CSF. For example, in mice G-CSF 

has been demonstrated to induce a polarization of T- 

lymphocytes towards the production of type-2 cytokines 

(namely IL-4 and IL-10), which display an anti-inflammatory 

effect. Such polarization was shown to be longlasting 

and was associated with a significant reduction 

in the severity of GVHD after transplantation of these 

cells into allogeneic mice recipients. 83 These results do 

not seem to be attributable to a direct effect of G-CSF 

on T-cells, since this subset of lymphocytes rarely 

expresses G-CSF receptors. Instead, these findings could 

be explained by the anti-inflammatory effects of G-CSF; 

in fact, administration of G-CSF decreases tumor necrosis 

factor (TNF) secretion. 84 Moreover, in normal subjects 

G-CSF is able to increase the production of two important 

cytokine antagonists such as soluble TNF-receptor 

and IL-1 receptor antagonists. 85 

Finally, leukapheresis products from G-CSF-mobilized 

donors contain a large number of monocytes. These cells 

have been demonstrated to significantly reduce the 

alloantigen specific proliferative response of T-lymphocytes. 

67 Also, monocytes from subjects treated with G-CSF 

or GM-CSF can induce the apoptosis of T-lymphocytes via 

the interaction of the FAS molecule with its ligand. 86 The 



77 

use of G-CSF may inhibit the function of monocytes as 

antigen presenting cells and this, in turn, may explain 

the ability of this cytokine to polarize T-cells towards an 

anti-inflammatory cytokine profile. These findings could 

also contribute to explaining the unexpectedly low incidence 

and reduced severity of GVHD after transplantation 

of PBSC. However, more studies on the characterization 

of G-CSF mobilized lymphocytes are needed. 

With regard to cGHVD, it appears from this analysis 

that there is a trend toward a slightly increased incidence 

after PBSC, although not all individual studies had the 

same results. The clinical presentation of cGVHD was 

reported as peculiar in two studies, with many de novo 

cases 31,75 but also with a high response to treatment. 49 It 

should be noted that early data from the M.D. Anderson 

on PBSC transplants also reported an increase in cGVHD, 

with more liver and gastrointestinal manifestations compared 

to the marrow. 87 At the time of this writing, several 

patients were still on immunosuppressive treatment so 

the full magnitude of cGVHD will be appreciated only in 

the future after withdrawal of CsA, which is a critical 

time for the development of the syndrome. Furthermore, 

in a study on aplastic anemia 88 the infusion of donor 

buffy coat cells was associated with a significant increase 

of cGHVD. However, this increase of cGVHD was not 

observed in malignancies in the study. 82 Clearly a longer 

follow-up of a much larger number of patients is needed 

to answer the question of cGVHD. 

Transplantation of enriched allogeneic 

CD34 + cells 

As reported above, allogeneic PBSC transplantation 

results in the infusion of approximately 1 log more T- 

cells than conventional BM transplantation. Thus, in order 

to reduce the potential risk of severe aGVHD, several 

investigators have attempted to remove T-lymphocytes 

from allogeneic grafts. The only technique that has been 

utilized so far for T-cell depletion has been the positive 

selection of hematopoietic CD34 + cells. 

The CD34 antigen is present on the earliest identifiable 

progenitor cells and committed myeloid precursors, 

whereas it is not expressed on mature myeloid and B- and 

T-lymphoid cells. 89 However, CD34 + cells co-expressing 

both T-lymphoid (CD2, CD3, CD7) and B-lymphoid markers 

(CD19) are likely to be the early precursors of the T- 

and B-lymphoid lineages. 90 Preclinical studies have also 

shown the capacity of positive selection of CD34 + cells to 

eliminate 3 to 4 logs of T-cells, coupled with a substantial 

recovery of hematopoietic progenitors. 91,92 More 

recently, transplantation of autologous CD34 + cells has 

been proven to reconstitute normal hematopoiesis in cancer 

patients treated with myeloablative regimens. 93-96 

Based on these premises, Link et al. 97 transplanted 5 

patients with unmodified marrow and CD34 + selected 

PBSC and 5 patients with enriched marrow and PB CD34 + 

cells. They concluded that hematopoietic recovery was 

accelerated with respect to marrow allografts without 

an apparent increase in aGVHD following conventional 

CsA and MTX prophylaxis. In a subsequent study, 98 the 

same authors transplanted 10 individuals with positively 

selected circulating CD34 + cells alone. The patients 

were grouped according two different regimens of 

aGVHD prophylaxis: CsA alone or CsA and MTX. The median 

grades of aGVHD were 3 in group I (CsA) and 1 in 

group II (CsA plus MTX). Two patients in group I died from 

aGVHD and 2 leukemic relapses occurred in group II. 

Complete and stable donor hematopoiesis was shown in 

all patients with a median follow-up of 370 days (range 

45-481). It was concluded that despite a 3-log reduction 

of T-cells by CD34 + cell enrichment, CsA alone was not 

sufficient to avoid severe aGVHD. 

More recently, Bensinger et al. 99 transplanted 16 

patients with advanced hematologic malignancies with 

HLA-identical highly enriched PB CD34 + cells. Prophylaxis 

against aGVHD was CsA alone for 5 patients and CsA 

plus MTX for 11. A median of 8.96×10 6 CD34 + cells/kg of 

patient body weight were infused with a median purity 

of 62%. Positive selection of stem cells resulted in a 

median 2.8-log reduction of T-cells. Despite the prompt 

and sustained engraftment, 8 out of 16 patients died 

between 3 and 97 days post-transplant of transplantrelated 

causes and 1 of progressive disease. Grade 2-4 

aGVHD occurred in 86% of patients and 6 out of 8 evaluable 

patients developed clinical chronic GVHD. 

More promising results have been reported by Urbano- 

Ispizua et al. (1997), who recently transplanted 20 acute 

and chronic leukemia patients with allogeneic CD34 + 

cells. The median number of CD34 + cells and CD3 + cells 

infused was 2.9×10 6 /kg and 0.42×10 6 /kg, respectively. 

The patients were conditioned with fractionated TBI (total 

dose 13 Gy in 4 fractions) and cyclophosphamide 120 

mg/kg. Additional GVHD prophylaxis included CsA and 

methylprednisolone. No patients developed grade II- IV 

aGVHD. The overall procedure was associated with low 

morbidity and no transplant-related deaths occurred 

within the first 100 days. Although the median followup 

(7.5 months) is rather short for a full evaluation of 

cGVHD incidence and disease relapse, the absence of 

extensive cGVHD and the low rate of disease recurrence 

(only 3 out of 20 patients relapsed) encourage further 

studies in this direction. In comparison with previous 

studies, 99 it should be noted that the median age of the 

patient population was 40 years and only 35% of the 

individuals were older than 45 years. Moreover, the 

majority of the leukemic patients were transplanted in 

the early phase of their disease. Both these parameters 

are generally associated with lower transplant-related 

mortality and a lower incidence of severe GVHD. 

Although further studies involving larger numbers of 

patients are currently in progress, these results, taken 

together, demonstrate that infusion of CD34 + selected 

PBSC results in rapid and stable engraftment. However, 

transplantation of purified stem cells may induce a higher 

rate of acute and chronic GVHD than expected, thus 

requiring full GVHD prophylaxis. Therefore this approach 

for T-cell depletion should be carefully evaluated in the 

setting of HLA-identical PBSC transplantation and 

weighed against the potential increased risk of disease 


78 


relapse, and perhaps delayed immunological reconstitution, 

and the increased cost of the procedure. 

Allogeneic PBSC from haploidentical 

familial donors: the mega-stem-cell 

dose concept 

Allogeneic BMT has been largely confined to patients 

who are HLA-identical to their donors. At present, only 

about 30-35% of patients who might benefit from allogeneic 

BMT have an HLA-identical sibling. The establishment 

of large registries of HLA-typed individuals 

during recent years has led to a substantial increase in 

transplants from unrelated donors. 100-102 Although 40 to 

50% of patients are successful in locating HLA-A, B, 

DR-matched unrelated donors, many patients still fail to 

find an appropriate donor. 103,104 In contrast, nearly all 

patients have an HLA-haploidentical relative (parent, 

child, sibling,) who could serve as a donor. 

The feasibility and safety of transplants from partially 

matched family members have been investigated and 

the results of these studies have demonstrated that HLA 

matching is a critical and limiting factor in marrow 

transplantation. 105-107 In published works on mismatched 

transplants, there has been no large study involving 

patients mismatched with their donors by one full haplotype. 

These experiences have been limited because the 

problems of transplant increase with the number of 

antigenic disparities between donor and host. 106 In 2- or 

3-antigen mismatched transplants, studies by the Seattle 

program 105 and the International Bone Marrow Transplant 

Registry 108 reported graft failure in 20 to 30% of 

cases. The reported incidence of acute GvHD (grade II or 

greater) varied from 34% to 100% overall, but in 2- and 

3-antigen mismatched patients the incidence was at 

least 80%. 105,106 Severe GvHD was a greater problem 

than graft rejection, preventing more widespread use of 

mismatched related transplants during the latter 1970s 

and 1980s. 

By contrast, extensive experience in severe combined 

immunodeficiency (SCID) patients has shown that GVHD 

is largely preventable, even in 3-antigen mismatched 

transplants, when a 3-log T-cell depletion of the donor 

bone marrow is achieved. 109,110 In 1981 Reisner et al. 

reported the first case of leukemia treated with a T-cell 

depleted marrow transplant from a haploidentical, 3- 

loci incompatible, parental donor. 111 There was full 

engraftment and no GVHD. Subsequently, clinical trials 

in mismatched-sibling BMT for patients with leukemia 

were begun using the lectin or other T-cell depleting 

methods which included monoclonal antibodies with 

complement or conjugated to toxins, and counterflow 

centrifugal elutriation (reviewed in ref. #112). It was 

determined that the threshold dose below which GVHD 

was not seen in matched patients was 2.0×10 5 T 

cells/kg. 113 However, early enthusiasm for all methods 

was soon tempered by an increased incidence (> 50%) 

of graft rejection. 114 

In exploring the problem of failure in mismatched 

grafts, the inadequacy of immunosuppression was documented 

by the observation of residual host lymphocytes 

in patients who failed to engraft after being conditioned 

with conventional preparative regimens and 

given T-cell depleted mismatched transplants. 114 Work 

in esperimental models has shown that incompatible T- 

cell depleted transplants can be successfully performed 

by manipulating the conditioning regimen and/or the 

graft composition. 115 The immunologic response of the 

remaining host immune system against the graft can 

be overcome by increasing the total dose of TBI 116 or by 

adding selective anti-T measures with minimal toxicity, 

such as splenic irradiation 117 or in vivo treatment with 

anti-T monoclonal antibodies. 118 Engraftment is also 

improved by increasing the myeloablative effect of the 

conditioning regimen through the use of dimethylmyleran, 

busulfan or thiotepa, given with TBI. 119,120 

Different cytoreductive agents or radiation regimens 

were therefore added to the basic conditioning protocols 

used for conventional BMT. Although a marked beneficial 

effect was found in recipients of T-cell depleted 

HLA-identical bone marrow upon adding ATG and 

thiotepa to TBI and cyclophosphamide, 121 none of these 

agents were found to be useful in recipients of T-cell 

depleted haploidentical 3-antigen incompatible transplants. 

Others, using the monoclonal antibody Campath- 

1G instead of ATG, have observed similarly disappointing 

rejection rates. 122 

Concerning the composition of the graft, Lapidot et al. 

showed that megadoses of T-cell depleted incompatible 

bone marrow inoculum could obtain full donor-type 

engraftment in mice treated with sublethal irradiation, 

or presensitized with donor lymphocytes or partially 

reconstituted before the transplant by adding back a 

controlled number of host-type mature thymocytes. 123 

The means of overcoming graft failure elucidated in 

the experimental model can be applied in the clinical setting 

by combining approaches that increase both the 

conditioning of the host and the size of the stem cell 

inoculum. The major advance that finally made full haplotype-mismatched 

transplantation possible in leukemia 

patients was the availability of rhG-CSF 124 and the experience 

in autologous transplants in which G-CSF was used 

to mobilize high numbers of stem cells into the blood of 

patients without significant side effects. 125 Their employment 

made it feasible to increase the number of donor 

stem cells to a level which, in animal models, made transplantation 

across the histocompatibility barrier possible. 

117 On the basis of these concepts, the BMT team at 

the University of Perugia first introduced the megadose 

cell transplant in full haplotype-mismatched leukemia 

patients. 126 After a conditioning regimen which included 

8 Gy TBI in a single fraction at a fast dose rate (16 cGy/m), 

thiotepa (10 mg/kg), rabbit ATG (20 mg/kg in 4 days) and 

cyclophosphamide (100 mg/kg in 2 days), advanced 

leukemia patients, mostly adults, were given the combination 

of marrow and G-CSF-mobilized blood stem cells. 

Donors compatible with the patients for only one haplotype 

and 3-antigen disparate on the other haplotype 



79 

underwent bone marrow harvest followed within a few 

days by treatment with G-CSF (12 µg/kg/d × 7 days). Four 

leukaphereses of progenitor cells were performed starting 

on the fourth day. The marrow as well as the leukapheresis 

product were each depleted of T-cells using soybean 

lectin agglutination and E-rosetting. 127 Both the 

CD34 + cells and CFU-GM were increased 7- to 10-fold 

over bone marrow alone, and the average number of CD3 + 

cells infused was 2.2×10 5 /kg recipient body weight. Following 

conditioning and stem cell infusion, patients 

received no additional GvHD prophylaxis. The results of 

the first 17 patients were reported in 1994 126,128 and subsequently 

27 additional leukemia patients, most of them 

in chemoresistant relapse at the time of transplant, were 

treated. For the first time a very rapid hematopoietic 

engraftment was observed in more than 80% of patients 

and, without any post-transplant prophylaxis, acute 

GVHD occurred in only 27%, and there was no significant 

chronic GvHD. As for survival, 7 patients are currently 

alive and disease free at a median follow-up of more than 

3 years. The major complications observed in this pilot 

study were interstitial pneumonitis, which occurred in 

43%, and infections in the setting of GvHD. Both were 

responsible for the 60% transplant-related mortality. 

This pilot experience showed that the megadose cell 

strategy, together with a highly immunosuppressive and 

myeloablative conditioning, resulted in a high incidence 

of durable engraftment with significantly reduced GvHD 

comparable to historical experience with unmanipulated 

transplants. It also confirmed that in humans, as in 

mice, the stem cell dose plays a crucial role in overcoming 

HLA-histocompatibility barriers. 129 This concept 

is also supported by the recent work by Rachamin et 

al. 130 demonstrating that purified CD34 + cells have a 

very powerful veto activity. They are able to specifically 

reduce, in a mixed lymphocyte culture, the frequency 

of CTL precursors against the stimulatory cells of the 

same subject and thereby help to overcome allogeneic 

rejection and enhance their own engraftment. 

The approach to haplotype-mismatched transplants 

has evolved since Aversa et al. 126,128 originally proposed 

the megadose cell concept. With their initial protocol 

GvHD was decreased but not eliminated, and it contributed 

to transplant-related mortality, which was significantly 

greater than in matched patients receiving 

similar conditioning (Aversa et al., unpublished data). 

These remaining problems were addressed in a subsequent 

trial, where it was possible to completely abrogate 

GVHD by improving the T-cell depletion method. 131 By 

processing the peripheral blood progenitor cells with an 

initial debulking of both mononuclear and T-cells with 

one-round E-rosetting followed by positive selection of 

CD34 + cells with the Ceprate stem cell concentrator 

(CellPro Inc. Bothell, WA, USA), it was possible to infuse 

a median of 3×10 4 CD3 + cells/kg and 13×10 6 CD34 + 

cells/kg in 24 high-risk leukemia patients. Conditioning-related 

toxicity was also reduced by modifying pretransplant 

chemotherapy. As a substitute for cyclophosphamide, 

which was considered a possible factor in 

the early mortality in the first pilot study, fludarabine 

was tested. In fact, it had been shown to have powerful 

immunosuppressive effect in patients treated for 

lymphoproliferative disorders, even at doses which were 

not associated with significant extra-hematologic toxicity. 

132 Furthermore, in a mouse model TBI+fludarabine 

(40 mg/m 2 /d × 5) was shown to provide an immunosuppressive 

effect comparable to TBI+cyclophosphamide. 

133 

At present, a regimen including TBI in a single fraction, 

thiotepa, fludarabine and ATG followed by the infusion 

of T-depleted bone marrow plus T-depleted CD34- 

selected blood cells is being evaluated for toxicity and 

efficacy. The preliminary results of this study were 

recently presented at the American Society of Hematology 

meeting in Orlando. 134,135 As hoped, with the 

decrease in the number of T-cells infused and the modifications 

in conditioning, the problem of GvHD was 

largely prevented (only 2 patients developed grade II 

acute GvHD and one progressed to chronic GvHD); the 

engraftment rate was 95% and there was a decrease in 

transplant-related mortality to 29% compared to the 

previous 60%. 

A more recent update on 48 patients was presented 

in Mannheim. 136 The abstract reports that 22/28 patients 

were in chemoresistant relapse at the time of transplant; 

age ranged from 4 to 53 years (median 27). Forty 

of 48 patients engrafted, grade II-IV acute GvHD 

occurred in only two patients and no one developed 

chronic GvHD. Twenty patients were alive and disease 

free at a median follow-up of 5 months (range 1-16). 

There were 11 relapses and 17 nonhematologic deaths. 

Transplant-related mortality was 35%. 

An unsolved problem remains the slow immunologic 

recovery of engrafted patients that is responsible for 

infections. Counting of peripheral blood lymphocytes 

which exhibited a phenotype of NK cells (CD56 + ), helper 

T cells (CD3 + /CD4 + ), cytotoxic T-cells (CD3 + /CD4 + ), cytotoxic 

T-cells (CD3 + /CD8 + ) and B-cells (IgM + ) revealed early 

recovery (within 2-4 weeks) of NK cells and extremely 

delayed recovery of T cells. In particular, CD4 + cells 

reached near normal values after 10 to 12 months. 137 In 

addition, the frequencies of T-cells responding to polyclonal 

activators in a sensitive limiting dilution assay 

were approximately 1 in 100 within the first post-grafting 

month and 1 in 10 at 10 months post-transplant 

(control responder cell frequencies are in the range of 1 

in 2). 137 The low number of T-cells, combined with their 

functional peculiarities (i.e. failure to respond to TcR 

stimulation) are certainly implicated in the high frequency 

of infectious complications and are strongly 

indicative of a markedly distorted T-cell maturational 

process. 

Interestingly, looking at post-transplant immune 

reconstitution, Albi et al. observed a large donor-type 

TcR- + CD8 + cell population that co-express NK-like 

receptors for specific MHC class I alleles. 137 NK cells 

expressed multiple, clonotypically distributed membrane 

receptors with different specificities for families of MHC 


80 


class I alleles (termed killer cell inhibitory receptors, KIR). 

The interaction between these receptors and the appropriate 

alleles produces a signal which inhibits killing of 

the target cells. Analysis of more than 900 clones 

revealed that 40% to 80% of these KIR + T-cells exhibit 

NK-like functions, i.e. they were able to lyse class I-negative 

targets and were functionally blocked by the 

expression of specific class I alleles on target cells. Furthermore, 

these cells do not lyse autologous hemopoietic 

cells, but are able to lyse fresh leukemic cells. 137 

This might suggest that they could provide a graft-versus-leukemia 

effect without causing GVHD. 

In a period of twenty years transplants across the histocompatibility 

barrier have advanced from being experimentally 

to clinically possible. The principles outlined 

at the beginning – adequate cell dose, adequate 

immunosuppression and myeloablation, avoidance of 

GvHD – have been successfully combined. Two other 

groups have recently reported on successful engraftment 

in haplotype mismatched transplants by combining bone 

marrow and G-CSF-mobilized blood stem cells after 

CD34-positive selection for patients with advanced 

leukemia. 138,139 Refinements of this protocol should make 

haplotype mismatched transplants an attractive therapeutic 

option for patients with high-risk leukemia without 

a matched related or unrelated donor. 

Furthermore, there are enormous potential applications 

of the concept of the stem cell dose for the future 

treatment of non-neoplatic diseases like aplastic anemia, 

Fanconi’s anemia, SCID, thalassemia, and for induction 

of tolerance in organ transplantation. This approach 

should be applicable not only in mismatched transplants 

but also for overcoming problems which remain in the 

matched transplant setting, such as rejection in aplastic 

anemia, regimen-related toxicity in Fanconi’s anemia 

and thalassemia. 

Transplantation of allogeneic PBSC 

from unrelated donors 

Two retrospective studies have recently suggested 

that the number of hematopoietic cells present in BM 

harvest correlates with the clinical outcome in the setting 

of stem cell transplantation from both HLA-identical 

siblings and from HLA-matched unrelated donors. 9,140 

In the latter case, the number of cells infused has proven 

to be the most potent prognostic factor for survival. 

Therefore, given the much higher number of progenitor 

cells collected in primed PB as compared to conventional 

BM harvest, the use of PBSC appears to be a 

promising alternative for improving the results of transplantation 

from unrelated donors. In this regard, Ringden 

et al. 34 and Stockschlader et al. 141 recently reported 

their preliminary experience with transplantation of 

allogeneic PBSC from full-matched or 1-antigen mismatched 

unrelated donors. In particular, Ringden et al. 

transplanted 6 patients with high-risk hematologic disease. 

Four of them received allogeneic PBSC as primary 

treatment while 2 others were treated after a BM graft 

failure. Five PBSC collections were infused without any 

manipulation; in 1 case Campath-1 monoclonal antibody 

was used for T-cell depletion. In the German study, 

1 AML patient in 2 nd CR received purified CD34 + cells 

from an HLA-matched unrelated donor. The total number 

of patients transplanted is too small and the followup 

too short to draw any conclusion; however, these 

preliminary data showing a rapid rate of engraftment 

are encouraging, whereas the role of T-cell depletion 

remains to be clarified. 

Transplantation of umbilical cord blood 

progenitor cells 

The existence of hematopoietic progenitors circulating 

between the fetus and the placenta during gestation 

was first described in this Journal more than 20 years 

ago, 142 but their clinical application began only when it 

became evident that the progenitor cell content of CB 

was sufficient for bone marrow repopulation in pediatric 

patients given myeloablative chemo-radiotherapy. 143 In 

1988, a patient affected with Fanconi’s anemia was first 

transplanted with CB progenitor cells from his HLAmatched 

healthy sibling. 4 Subsequently, successful CB 

transplants (CBT) were sporadically reported in patients 

affected by both malignant and nonmalignant disorders. 

5,144-147 The establishment of large CB banks in 

Europe and USA, and improvement of the methods of 

cell collection, manipulation and freezing have permitted 

a rapidly increasing use of CB progenitor cells, which 

are now extensively employed for allogeneic transplantation. 

148-150 

The biological and functional characteristics of CB 

hematopoietic stem cells have been already reviewed 

by the Working Group. 12 

Clinical results after cord blood 

transplantation 

As mentioned above, the use of human umbilical CB 

hematopoietic progenitors represents an alternative 

modality of transplantation. Advantages of CBT include 

ease of hematopoietic stem cell collection, absence of 

donor risks, low risk of viral contamination (cytomegalovirus, 

Epstein-Barr virus, etc.) and, for transplantation 

among unrelated individuals, prompt availability of 

hematopoietic stem cells. Over the past decade, placental 

blood has been used to transplant hundreds of 

patients (mainly children) and information on the rate 

and kinetics of engraftment and on the risk of severe 

acute or chronic GVHD is now available for CBT recipients 

from both related and unrelated donors. 

In the two largest cohorts of patients transplanted 

from an HLA-identical sibling reported to date, 7,148 the 

probability of engraftment of donor hematopoiesis was 

79% and 85%, respectively, even though it must be 

underlined that in the cohort analyzed by Wagner and 

colleagues rejections were mainly observed in patients 

affected by bone marrow failure syndromes or hemoglobinopathies, 

which are diseases with a high risk of 

graft failure. In the cohort reported by Wagner et al., the 



81 

median time to achieve granulocyte (PMN >0.5×10 9 /L) 

and platelet (PLT >50×10 9 /L) recovery was 22 and 49 

days, respectively; these values were greater than those 

observed with BMT. Comparable time for PMN and PLT 

recovery were observed in the European experience. 7 In 

particular, in this latter report, patients receiving a higher 

number of nucleated cells (i.e. more than 37×10 6 /kg) 

experienced faster engraftment than those given a lower 

number of cord blood progenitors, suggesting that 

the number of cells infused is the main factor influencing 

the rate of hematologic recovery. More prolonged 

periods of profound leukopenia and thrombocytopenia 

have also been described in children receiving CBT from 

unrelated donors. In fact, in the first two series of 

patients transplanted from an unrelated donor, 149,150 

PMN recovery occurred in a median of 22 and 24 days, 

respectively, whereas the median time for PLT recovery 

was 82 and 67 days, respectively. The importance of the 

number of cells infused on the kinetics of PMN and PLT 

engraftment in the Eurocord Transplant Group experience 

was also observed in the group of patients given 

an unrelated CBT. Moreover, unlike BMT, where the use 

of hematopoietic growth factors has been demonstrated 

to hasten myeloid recovery significantly, 151,152 administration 

of these cytokines has produced conflicting 

results in CBT recipients. In fact, in the cohort of patients 

receiving CBT from HLA-identical or disparate family 

donors studied by Wagner et al., the use of G-CSF or 

GM-CSF did not influence the kinetics of PMN reconstitution. 

142 In contrast, in a group of children transplanted 

using unrelated CB units reported by the same 

authors, patients receiving hematopoietic growth factors 

experienced faster myeloid recovery than those who 

were not given the cytokines. 150 

The delayed rate of neutrophil engraftment and the 

conflicting data mentioned above could be explained by 

the infusion of fewer progenitor cells with CBT with 

respect to BMT, as suggested by the European experience, 

or, alternatively, by the particular characteristics 

of the proliferative, self-renewing and differentiating 

capacity of CB cells. A practical consequence of the 

above observation is that specific attention should be 

paid to the risk of infectious complications in children 

receiving CBT. 

During the first few months after transplant CBT 

recipients show a steady, impressive increase in HbF 

whose values are significantly higher than those 

observed in patients receiving BMT. Moreover, the subsequent 

decline is usually less pronounced than that 

observed in normal children in the first year of life (Figure 

1). 153,154 This preferential production of chains in 

erythroid progenitors seems to reproduce the normal 

ontogeny of erythropoiesis, even though the persistence 

of HbF levels higher than those observed in the first year 

of age suggests a more delayed switch from fetal to 

adult hemoglobin synthesis. 

The dose of CB progenitor cells necessary to ensure 

early and sustained hematopoietic engraftment and 

favorable clinical outcome has still not been precisely 

defined. Wagner et al. 148 claimed that the lowest dose 

of CB nucleated cells reported to be capable of yielding 

complete and sustained engraftment is 1×10 7 /kg of 

recipient body weight. However, as previously mentioned, 

the Eurocord Transplant Group documented that 

a dose of nucleated cells available before thawing of 

fewer than 3.7×10 7 /kg recipient body weight was highly 

predictive of both graft failure and poor survival after 

CBT. 7 The importance of this value also emerges from 

Kurtzberg et al’s experience. 149 Ten out of 13 patients 

undergoing CBT from an unrelated donor and having 

Figure 1. HbF levels observed in the 5 CBT recipients in 

the first year after transplant. Normal percentiles of HbF 

values (dotted line, mean±2SD) observed in the first year 

of age (Galanello et al., 1981) are also reported (F. 

Locatelli, personal data). 


82 


received fewer than 3.710 7 /kg nucleated cells failed to 

benefit from the procedure. Although the importance 

of this cellular dose appears evident from these two 

reports, it should be noted that rarely is such a number 

of cells available in the case of adult patients. In fact, 

since the average leukocyte count in placental blood is 

about 10×10 6 /mL and the average volume of donated 

blood is about 80 mL, the average number of nucleated 

cells before thawing in one cord blood unit may reach 

800×10 6 . About 30% of nucleated cells are lost during 

the thawing and washing procedure, and even though 

the loss mostly involves mature cells which have no role 

in transplantation, it is reasonable to expect fewer than 

3.7×10 7 /kg viable cells for patients with body weight 

greater than 30-40 kg. 

The reduced immune reactivity of cord blood cells 

found a clinical counterpart in 38 children reported by 

Wagner et al. 148 who received CBT from an HLA-identical 

or 1-antigen mismatched sibling. In these patients, 

the incidence of grade II-IV acute GVHD and limited 

chronic GVHD was 3% and 6%, respectively, with no 

patient dying of GVHD. Confirmatory results were 

obtained by the Eurocord Transplant Group, which 

reported a 9% incidence of grade II-IV acute GVHD in 

CBT recipients from an HLA-identical relative. However, 

it is noteworthy that the same group documented a 

50% incidence of grade II-IV acute GVHD in patients 

transplanted from an HLA nonidentical family donor. 

In CBT performed between unrelated subjects with, in 

some cases, a disparity of 2-3 HLA antigens, the incidence 

of acute grade III-IV GVHD is reduced (approximately 

10-20%) 7,149,150 with respect to that observed 

after unmanipulated BMT between unrelated subjects 

for whom, notwithstanding complete HLA identity 

between recipient and donor, the observed risk of acute 

grade III-IV GVHD reaches at least 30-40%. In particular, 

in the cohort of patients given CBT from an unrelated 

donor reported by Kurtzberg et al., 149 no patient developed 

grade IV acute GVHD or experienced hepatic 

involvement or died of acute GVHD, and only 4 out of 65 

patients given an unrelated CBT reported by the Eurocord 

Transplant Group showed grade IV acute GVHD. 

From the data collected up to now, therefore, it clearly 

appears that CBT, from both familial and unrelated 

donors, is associated with a reduced risk of acute and 

chronic GVHD. 148-150 In view of this observation, different 

centers tend to adopt less intensive schemes of 

GVHD prophylaxis. Typically, children transplanted with 

a CB unit collected from an HLA-identical sibling receive 

GVHD prophylaxis consisting of CsA alone, whereas for 

patients undergoing unrelated CBT the most widely used 

regimens are those based on a combination of CsA with 

either low- or high-dose steroids. 149,150 The association 

of CsA with short-term methotrexate as proposed by 

the Seattle group in BMT recipients 155 is not generally 

employed due to concerns about the prolongation of 

time required for engraftment and possible damage to 

hematopoietic progenitors with a reduction in the 

potential for marrow repopulation. Procedures involving 

T-cell depletion of CB cells are also discouraged. 

The reported low incidence of GVHD 7,148-150 might, on 

the other hand, be a major drawback to the use of CB as 

a source of stem cells for allogeneic transplantation in 

leukemic patients. In fact, since the role of allogeneic 

lymphocytes in the control and/or eradication of malignancy 

is well established, the potential absence of GVL 

activity could represent a theoretical concern in leukemic 

subjects given CBT. Currently available data do not conclusively 

establish whether CBT really predisposes 

patients to an increased risk of leukemia relapse. However, 

considering the concern mentioned above, the 

choice of less intensive GVHD prophylaxis schemes could 

represent a possible means for partially sparing the 

immune-mediated GVL effect, which may significantly 

contribute to preventing regrowth of leukemia cells. 

Immunological reconstitution following 

cord blood transplantation 

Although immunological reconstitution after BMT has 

been extensively studied, 58,59 few data are available on 

the kinetics of immune recovery in CBT recipients. 

144,153,156 After CBT, recovery of T-cell immunity, as 

well as that of natural killer subpopulations, mimicks 

what is described in BMT recipients. 153 In particular, in 

the early post-transplant period recovery of CD8 + lymphocytes 

seems to be faster than that of CD4 + cells, 

determining a characteristic inversion of the ratio 

between the two subpopulations during the first 6 

months after CBT, similarly to what is described in BMT 

recipients. Considering the much lower number of lymphoid 

cells transferred with CBT as compared to BMT, 

the recovery of T-lymphocyte number and function 

towards normal must be considered rapid. The prompt 

recovery of T-cell immunity following CBT could be positively 

influenced by the reduced incidence and severity 

of both acute and chronic GVHD, which per se 

adversely affects the acquisition of lymphocyte function. 

However, it must be noted that the prompt recovery 

of lymphocyte function in vitro does not necessarily 

correlate with effective in vivo immunity. In fact, at 

present there are insufficient data to prove that this 

rapid T-cell recovery translates into a low incidence of 

viral and fungal infections after CBT. 

In contrast to what is observed in BMT recipients, 58,59 

an impressive increase in the percentage and absolute 

number of B-lymphocytes, apparently not related to 

viral infections, has been documented in children receiving 

CBT. 153,157 Possible hypotheses to account for this 

observation could involve the physiological characteristics 

of B-cell ontogeny in the first year of life and/or 

different distribution of mature memory lymphocytes in 

bone marrow and CB. 158,159 

Ethical problems of cord blood 


Like any other innovative treatment, CBT also poses 

some ethical questions that have still not been completely 

resolved. In particular, these ethical considera- 



83 

tions can be subdivided into those concerning transplants 

between HLA-compatible siblings and those 

regarding CBT from unrelated donors. 

The two main ethical problems regarding transplantation 

from a family donor are those of conceiving a sibling 

with the hope of producing a compatible donor for 

a previous child who requires transplantation of hematopoietic 

stem cells, and of his/her HLA typing in utero. 

Of course, any decision to conceive a child for the sole 

purpose of making it become a cord blood donor entails 

belittling the value of the individual to be born. However, 

it cannot be ignored that it is extremely difficult to 

separate the reasons that lead to conceiving a child solely 

for the joy of procreating from those linked to the 

possibility of saving a living, sick child. On the other 

hand, even this last reason does not lessen the importance 

of the future child who will bring happiness to the 

family in addition to being the person who, in the case 

of successful transplantation, allowed the family to save 

the life of a child who would have otherwise been lost. 160 

In the meantime, it is important to stress the inappropriateness 

of performing HLA typing in utero; because 

of the increased abortion rate due to the procedure 

(about 1-2%), it entails the risk of causing the death of 

a healthy human being and would in any case be deeply 

despicable if it were used to dispose of a conceived child 

found to be HLA-incompatible with the sick patient. 

From the point of view of the unborn child, HLA typing 

in utero quite obviously poses critical problems and offers 

no advantages, but only tangible risks for that unborn 

child’s survival. HLA typing in utero should be carried out 

only when other, far more important reasons (for example, 

advanced age of the mother with consequent higher 

risk of chromosome-21 trisomy for the fetus) suggest 

performing prenatal diagnostic procedures. 

Since the donor is a newborn infant, the use of cord 

blood for an unrelated sick patient has raised many questions 

of ethical interest. These ethical aspects go beyond 

the scope of this review, but we would like to comment 

briefly on some of them. Particular attention has been 

devoted to the problems raised by tests required to determine 

whether cord blood is suitable and usable without 

the risk of transmitting to the recipient any disease carried 

by the donor cells (namely infectious diseases and 

genetically transmissible disorders). In fact, for this specific 

aspect the ethical question is: what kind of behavior 

should be adopted by the medical operator who works 

with a woman (or with the parents of a child) if a disease 

for which there is no therapy is detected in the 

infant? 161 Such possibly dramatic news must cause as 

little damage as possible. We must by all means prevent 

our increasingly profound biological awareness of our 

selves from leading to a culture of anguish. One might 

recall in this regard that, for example, it has been stated 

that minors should not be tested for abnormal genes 

unless there is an effective curative or preventive treatment 

that must be instituted early in life. 162 

Another heavily debated problem regarding unrelated 

transplants is the case of a cord blood unit assigned 

to allotransplantation, making these cells unavailable 

in the case the donor needs them for an autologous 

transplant. However, to ensure that every CB donor has 

the right to use the donated blood for himself if necessary, 

there would be no way to provide cord blood units 

for allotransplants. Therefore the very nature of the 

technique originally conceived for allotransplants would 

be profoundly transformed, and this would punish all 

donation ethics at their very core. 

Strictly linked to these considerations is the problem 

of private banking of cord blood cells. 163 In any case, we 

firmly believe that involving money-making aspects in 

CB transplantation technology is unacceptable. In particular, 

as stated by other authors as well, 164 no part of 

the human body should be commercialized and CB 

should not be used for the benefit of financial speculators. 

Rational use of PBSC in the treatment 

of leukemic relapse after allogeneic 

transplant 

The cure rate of patients receiving an allogeneic transplant 

for hematological malignancies is negatively 

affected by relapse. The incidence and time of disease 

recurrence depend on several factors such as diagnosis, 

disease phase at the time of transplant, conditioning 

regimen, GVHD prophylaxis and T-cell content of the 

infused graft. 165 

The response rate and clinical outcome of relapsing 

patients with acute leukemia treated with chemotherapy 

alone are extremely poor. 166,167 Interferon therapy 

significantly prolongs the survival of CML patients 

relapsed after transplant, but its benefit is not durable 

over long-term follow-up. 168-170 Finally, a second transplant 

offers some possibilities of cure for relapsed 

patients but it carries high morbidity and regimen-related 

mortality. 171-173 

During the past few years the therapeutic approach to 

post-transplant relapse has been substantially modified. 

Following its first report by Kolb et al., 174 donor lymphocyte 

infusion (DLI) is currently being used as a form 

of adoptive immunotherapy for patients with hematological 

malignancies who relapse after transplant 175-188 

or develop EBV lymphoproliferative disorders. 72,189 

A number of observations in allogeneic transplants 

support the evidence that a GVL effect, whether associated 

or not with GvHD and mediated by donor 

immunocompetent cells, contributes to the eradication 

of the neoplastic clone. 82,190-195 

The rationale for the use of DLI in post-transplant 

relapse is based on two main factors: 1) the persistence 

of an immunotolerant status versus donor cells in the 

relapsing host; 2) the cytotoxic activity exerted by HLAunrestricted 

NK and LAK cells or by HLA-restricted T- 

cells of donor origin against host malignant cells. 188,196 

However, the effectiveness of DLI therapy is variable 

since it greatly depends on the type of disease and its 

stage at the time of relapse. Following DLI, a high pro- 


84 


portion of CML patients with molecular, cytogenetic or 

chronic phase hematologic relapse will likely experience 

a long-term disease free survival, but the success rate 

is substantially lower in recurrent AML and virtually 

absent in patients relapsing with ALL or blast crisis 

CML. 186 Furthermore, the therapeutic success of DLI is 

counteracted by related severe complications such as 

GVHD and myelosuppression, which occur in up to 90% 

and 50% of cases, respectively. The mortality due to DLI 

may approach 20%. 

Therefore in order to optimize adoptive immunotherapy 

with DLI and to improve the general management of posttransplant 

relapse, several biological and clinical conditions 

should be considered. 

One of the most important factors is the time required 

for GVL to destroy the host neoplastic cells. In early stage 

CML this time seems to be enough to allow a GVL reaction 

to build up and eliminate residual CML cells. By contrast, 

neoplastic growth is so fast in acute leukemia that 

it may not be challenged by the GVL effect. 

A further variable influencing the response to DLI is the 

potential of the neoplastic clone to mature and differentiate 

into dendritic cells, which contribute to the GVL reaction 

by enhancing the antigen presentation capacity of 

tumor cells. This property is spontaneously attained by 

CML cells in chronic phase and, to some extent, by AML 

cells, particularly when cell differentiation follows the 

tumor reduction induced by chemotherapy. 197 Dendritic 

cells derived from bone marrow or produced in vitro by 

CD34 + cell cultures in the presence of cytokines 198,199 can 

exert their action through an HLA restricted mechanism. 200 

Therefore, in treating leukemia relapse weakly expressing 

HLA or tumor-specific antigens, donor hematopoietic 

progenitor cells may improve the immunologic effect 

mediated by DLI. 

Finally, bone marrow chimerism detected by PCR prior 

to DLI may predict either response to treatment or the 

occurrence of myelosuppression. Although long-term persistence 

of donor T-cells in the peripheral blood during 

relapse has been reported, 184 this observation does not 

provide any prognostic information on the post-DLI clinical 

outcome. Southern blot RFLP analysis, erythrocyte 

phenotype and cytogenetics have been employed to 

detect residual donor cells, but no correlation was found 

among pre-DLI BM chimerism, response to treatment and 

the risk of myelosuppression. However, pre-DLI BM 

chimerism assessed by quantitative PCR of VNTR 

sequences in relapsed CML patients is associated with 

cytogenetic and molecular remission and strongly predicts 

the development of aplasia, thereby providing an 

early indication for the reinfusion of PBSC from the 

donor. 201,202 

Donor PBSC reinfusion has frequently been adopted as 

from rescue of DLI-associated myelosuppression. As to the 

combined use of donor PBSC and DLI, the reported experiences 

are limited to a small number of patients who 

relapsed with acute leukemia. 203-205 

In these studies, donors were stimulated with G-CSF at 

doses ranging from 2.5 mg/kg for 10 days to 16 mg/kg for 

5 days. The apheresis products obtained over 1 to 3 consecutive 

days contained a median of 4×10 8 /kg CD34 + cells 

and a median of 3.5×10 8 /kg CD3 + cells. All patients 

received chemotherapy prior to PBSC infusion and most 

of them achieved CR with prompt hematopoietic reconstitution 

which in the cases analyzed originated from 

donor cells. The majority of patients developed acute or 

chronic GVHD and related complications. In one of the 

reported series, the median duration of CR after this combined 

treatment was longer than the median time from 

transplant to relapse. 206 These results compare favorably 

with those recently reported in patients receiving DLI 

alone for relapse of acute leukemia or myelodysplasia after 

BMT. 187 Of the eight patients receiving this treatment, only 

one achieved CR and 7 died of progressive disease. 

In conclusion, these preliminary experiences suggest 

that patients relapsing with acute leukemia or advanced 

phase CML after BMT should be treated with intensive 

chemotherapy regimens, not necessarily including 

immunosuppressive drugs, followed by donor mobilized 

PBSC. 

This approach might result in certain therapeutic 

advantages such as: 1) reduction of the tumor burden; 2) 

slowing down of the neoplastic growth; 3) acceleration of 

donor hematopoiesis recovery and promotion of dendritic 

cell differentiation; 4) the possibility for the immunocompetent 

donor cells to express their GVL activity to a 

greater extent. Whether the additional administration of 

cytokines (IFN, IL-2, G-CSF, GM-CSF) would improve the 

efficacy of chemotherapy and PBSC is unknown at present 

and awaits further investigation. 

Finally, the GVL reaction exerted by donor lymphocytes 

against CML cells which retain biological features of early 

stage disease is potent enough that patients might be 

spared a repetition of previous chemotherapy. However, 

donor PBSC infusion should be considered for CML 

patients with either cytogenetic or chronic phase relapse 

who show minimal (< 10%) or no BM chimerism. 206 In 

such cases the use of donor PBSC is mainly indicated to 

counteract the risk of severe BM aplasia following the 

infusion of DLI alone. 

Contributions and Acknowledgments 

All authors equally contributed to the conception and 

writing of this review article. 

Disclosures 

Conflict of interest: this review article was prepared 

by a group of experts designated by Haematologica and 

by representatives of two pharmaceutical companies, 

Amgen Italia SpA and Dompé Biotec SpA, both from 

Milan, Italy. This co-operation between a medical journal 

and pharmaceutical companies is based on the common 

aim of achieving an optimal use of new therapeutic 

procedures in medical practice. In agreement with the 

Journal’s Conflict of Interest Policy, the reader is given 

the following information. The preparation of this manuscript 

was supported by educational grants from the 

two companies. Dompé Biotec SpA sells G-CSF and rHuEpo 

in Italy, and Amgen Italia SpA has a stake in Dompé 



85 

Biotec SpA. 

Redundant publications: no substantial overlapping 

with previous papers. 

Manuscript processing 

Received on July 29, 1997; accepted on November 28, 

1997. 

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91 

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Self-renewal and differentiation 

Marrow and blood hematopoietic cells are heterogeneous 

and belong to different lineages at different 

stages of maturity. The structural and functional 

integrity of the hematopoietic system is maintained by 

stem cells that, by definition, comprise a relatively small 

cell population, located mainly in the bone marrow, 

which can (i) undergo self-renewal to produce stem 

cells or (ii) differentiate to produce progeny which is 

progressively unable to self-renew, irreversibly committed 

to one or other of the various hematopoietic linreview 

Ex vivo expansion of hematopoietic 

cells and their clinical use 


Correspondence: Sante Tura, M.D., Istituto di Ematologia ed 

Oncologia Medica "L. & A. Seragnoli", Policlinico S. Orsola, 

via Massarenti 9, 40138 Bologna, Italy 

MASSIMO AGLIETTA,* FRANCESCO BERTOLINI,° CARMELO 

CARLO-STELLA, # ARMANDO DE VINCENTIIS, @ LUIGI LANATA,^ 

ROBERTO M. LEMOLI, § ATTILIO OLIVIERI,** SALVATORE 

SIENA,°° PAOLA ZANON,^ SANTE TURA § 

*Hematology Oncology, Ospedale Mauriziano, University of 

Turin, Turin; °Department of Oncology, IRCCS Fondazione 

Maugeri, Pavia; # Hematology and Bone Marrow Transplantation, 

University of Parma, Parma; @ Dompé Biotec SpA, Milan; 

^Amgen Italia SpA, Milan; § Institute of Hematology and Medical 

Oncology "L. & A. Seragnoli", University of Bologna, 

Bologna; **Division of Hematology, University of Ancona, 

Ancona; and °°Istituto Clinico Humanitas, Milan, Italy 

Background and Objectives. Hematopoietic stem cells 

are being increasingly used for treatment of malignant 

and nonmalignant disorders. Various attempts have 

been made in recent years to expand and manipulate 

these cells in order to increase their therapeutic potential. 

A Working Group on Hematopoietic Cells has analyzed 

the most recent advances in this field. 

Evidence and Information Sources. The method used 

for preparing this review was an informal consensus 

development. Members of the Working Group met three 

times, and the participants at these meetings examined 

a list of problems previously prepared by the 

chairman. They discussed the single points in order to 

achieve an agreement on different judgments, and 

eventually approved the final manuscript. Some 

authors of the present review have been working in the 

field of stem cell biology, processing and transplantation, 

and have contributed original papers in peerreviewed 

journals. In addition, the material examined in 

the present review includes articles and abstracts published 

in journals covered by the Science Citation 

Index® and Medline®. 

State of Art. Over the last decade, recombinant DNA 

technology has allowed the large scale production of 

cytokines controlling the proliferation and differentiation 

of hemo-lymphopoietic cells. Thus, in principle, ex 

vivo manipulation of hemopoiesis has become feasible. 

The present review covers three major area of interest 

in experimental and clinical hematology: manipulation 

of hematopoietic stem/progenitor cells, cytotoxic effector 

cells and antigen presenting dendritic cells. Preliminary 

data demonstrate the possibility of using, in a 

clinical setting, ex vivo expanded hematopoietic cells 

with the aim of reducing, and perhaps abrogating, the 

myelosuppression after high-dose chemotherapy. Concurrently, 

other important potential applications for ex 

vivo manipulation of hematopoietic cells have recently 

been investigated such as the generation and expansion 

of cytotoxic cells for cancer immunotherapy, and 

the large scale production of professional antigen presenting 

cells capable of initiating the process of 

immune response. 

Conclusions and Perspectives. Present and future challenges 

in this field are represented by the expansion of 

true human stem cells without maturation, to extend 

this strategy to allogeneic stem cell transplantation as 

well as the manipulation of cycling of primitive progenitors 

for gene therapy programs. The selective outgrowth 

of normal progenitor cells over neoplastic cells 

to achieve tumor-free autografts may ameliorate the 

results of autologous transplantation. The selective production 

of cellular subsets to manipulate the graft versus-host 

and graft versus-tumor effects and anti-tumor 

vaccination strategies may be important to improve 

cellular adoptive immunotherapy. 


Key words: hematopoietic stem cells, bone marrow, cord blood, 

dendritic cells, peripheral blood, allogeneic transplantation 

Hematopoietic stem cells 

Following the discovery that bone marrow transplantation 

could be used to rescue irradiated mice, the 

identification and characterization of the hematopoietic 

stem cell has become essential in order to achieve 

new developments in stem cell expansion and transplantation 

(SCT). 1 The potential for using stem cells as 

vehicles for gene therapy has further increased the 

efforts of a number of research groups working on stem 

cell identification, characterization, cloning and manipulation. 

2 


Clinical use of hematopoietic stem cells 

93 

eages, and able to generate clones of up to 10 5 lineagerestricted 

cells that mature into specialized cells. 3 

Although in recent years, (i) the development of in vitro 

and in vivo assays for hematopoiesis, (ii) the identification 

and characterization of hematopoietic growth 

factors, and (iii) the development of strategies for 

enriching hematopoietic cells have expanded our knowledge 

and understanding of hematopoiesis, the definition 

of the stem cell, originally proposed by Lajtha 4 and 

McCulloch, 5 has not substantially changed. 

In addition to self-renewal and differentiation, a number 

of properties are ascribed to hematopoietic stem 

cells, including a high migratory potential, the ability to 

undergo asymmetric cell divisions, the capacity to exist 

in a mitotically quiescent form and extensively regenerate 

the different cell types that constitute the tissue 

in which they exist. 6 

The issues of asymmetrical and symmetrical cell divisions 

and the regulation of self-renewal/differentiation 

process are crucial when analyzing stem cell behavior 

and the potential for stem cell manipulation. Asymmetric 

cell divisions produce one differentiated daughter 

(progenitor cell) and another daughter that is still a stem 

cell (Figure 1A). When all cell divisions are necessarily 

asymmetric and controlled by cell-intrinsic mechanisms, 

no amplification of the stem cell size is possible. 7 Asymmetric 

divisions are referred to unequally distributed 

transcription factors in daughter cells, 8,9 and have been 

shown to be possible in hematopoietic progenitors by 

clone-splitting experiments. 10 Symmetric cell divisions 

produce either two progenitor cells or two stem cells 

according to a 0.5 probability of self-renewing versus 

differentiative divisions (Figure 1B). In this case, it can be 

assumed that the size of the stem cell pool can be modified 

by factors affecting the 0.5 probability value, i.e., 

factors that control the probability of self-renewing versus 

differentiative divisions. 7 A third model postulates 

that individual cell divisions can be, but not necessarily 

are, asymmetric with respect to daughter cell fate (Figure 

1C). This model also implies that daughters behave 

differently due to different local environments. Although 

it is not known whether a single cell can switch from an 

asymmetric to a symmetric mode of cell division, available 

evidence in the hematopoietic stem cell system 

favors a predominance of symmetric cell divisions. 

The decision of a stem cell to either self-renew or differentiate 

as well as the selection of a specific differentiation 

lineage by a multipotent progenitor during commitment 

have been proposed to be regulated according 

to either stochastic or deterministic (inductive) models. 

Based on computer simulation and the distributions of 

colony-forming units in spleen (CFU-S) in individual 

spleen colonies, stochastic models postulate that the 

decision of a stem cell to self-renew (birth) or to differentiate 

(death) is randomly regulated by a probability 

parameter "p" which is equal to 0.5 in steady-state conditions. 

11 Deterministic models postulate the existence 

of lineage-specific anatomic niches that direct the differentiation 

of uncommitted progenitors. 12 There is experimental 

evidence suggesting that the hematopoietic system 

may employ both stochastic and deterministic 

strategies, probably depending upon the stage of lineage 

differentiation. 

Based on a number of studies performed in the last 

three decades, the regulation of self-renewal, commitment, 

proliferation, maturation, and survival can be 

assumed to reflect highly integrated processes under control 

of extracellular mechanisms, including regulatory 

molecules and microenvironment, as well as intracellular 

mechanisms, including protooncogenes, cell cycle regulators, 

tumor suppressor genes, transcription factors. 

Regulatory molecules include positive (growth factors) 

and negative (interferons, TGF-, MIP-1) factors which 

interact in complex ways (synergism, recruitment, antagonism). 

13,14 Molecules that maintain the stem cell state 

are beginning to be identified. These include ligands of 

the Notch family receptors that act from outside the cell 

as regulators of proliferation or maintenance of the 

undifferentiated state, 15 and factors like PIE-1 that act 

from within the cell. 16 However, despite the efforts which 

have been devoted to elucidating the issue of self-renewal 

control, no factors have yet been identified that are 

Figure 1. Possible patterns 

of stem cell division. “S” 

indicates a stem cell; “C” 

indicates a committed progenitor 

cell. 


94 


capable of maintaining self-renewing divisions and the 

molecular basis of self-renewal capacity remains to be 

elucidated. Growth factors so far identified more probably 

act as regulators of proliferation and survival. Theoretically, 

growth factors and cell-cell interactions can 

influence the outcome of fate decisions by stem and 

progenitor cells in a selective or instructive manner. 

According to selective mechanisms, the stem cell commits 

to a particular lineage independently of the growth 

factors and the factors act to control survival and proliferation 

of committed progenitors. In instructive mechanisms, 

growth factors cause the stem cell to choose one 

lineage at the expense of others. The relative contribution 

of these two mechanisms to hematopoietic regulation 

remains controversial, but experimental evidence 

suggests that at least some subsets of stem/progenitor 

cells can be instructed by growth factors to choose one 

differentiation pathway at the expense of others. 17 In the 

absence of still unidentified instructive signals, it can be 

hypothesized that environmental signals may act by 

increasing or decreasing the probability of choosing a 

particular fate, rather than promoting or repressing it in 

an all-or-none manner. 2 

Although growth factors play a key role in stem/progenitor 

cell proliferation and differentiation it seems 

improbable that hematopoiesis is regulated by a random 

mix of growth factors and responsive cells. Indeed, it is 

likely that regulatory molecules and localization phenomena 

within marrow stroma are required to sustain 

and regulate hematopoietic function. 18 Stromal cells of 

the hematopoietic microenvironment provide the physical 

framework within which hematopoiesis occurs. They 

play a role in directing the processes by synthesizing, 

sequestering or presenting growth-stimulatory and 

growth-inhibitory factors, and also express a broad repertoire 

of adhesion molecules which mediate specific interactions 

with hematopoietic stem/progenitor cells. 19 Differential 

expression of adhesion molecules could cause 

different stem cell subsets to home to different marrow 

microenvironments capable of differently affecting selfrenewal. 

While much progress has been made in identifying 

cytokines and stromal factors, little is known about intracellular 

mechanisms regulating hematopoietic stem/progenitor 

cells self-renewal and differentiation. 20 Structure-function 

analysis of growth factor receptors as well 

as identification of novel signal transduction molecules 

have provided new insights into the processes involved in 

signal transmission pathways. Post-translational modifications 

of pre-existing proteins, in particular tyrosine 

phosphorylation, play a key role in transmitting signals 

and thereby linking extracellular signals to the activation 

of nuclear effector molecules which govern gene expression. 

20 Accumulating evidence points to transcription factors 

such as AML-1, Ikaros, SCL/Tal-1, Rbtn-2, Tan-1, 

GATA-2, and HOX homeobox genes as important regulators 

of these processes. Overexpression of HOXB4 in 

murine bone marrow cells markedly increases the regenerative 

potential of long-term repopulating cells and 

causes an expansion in clonogenic progenitor cell numbers, 

without altering their ability to differentiate normally 

into mature myeloid, erythroid and lymphoid cells. 21 

In contrast, overexpression of HOXB3 causes defective 

lymphoid differentiation and progressive myeloproliferation. 

22 The glucocorticoid receptor, in combination with 

an activated receptor tyrosine kinase, seems to be a key 

regulator of erythroid self-renewal. 23 Shc overexpression 

increases GM-CSF sensitivity and prevents apoptosis of 

the GM-CSF-dependent acute myeloid leukemia cell line 

GF-D8, thus suggesting that Shc is an important regulator 

of cell survival and proliferation. 24 Different levels of 

protein kinase C modulate progenitor cell phenotype by 

favoring myelomonocytic or eosinophil differentiation. 25 

Recently, it has been shown that telomerase expression 

correlates with hematopoietic self-renewal potential. 

Hematopoietic stem cells show decreasing telomere 

length with increasing age. 26 Thus, telomerase may regulate 

self-renewal capacity by reducing the rate of DNA 

shortening. Overall, intracellular mechanisms of hematopoietic 

control result in the repression or de-repression 

of lineage-specific genes regulating growth factor 

responsiveness and/or proliferation potential. 27 The exact 

knowledge of these mechanisms will greatly modify our 

approach to stem/progenitor cell manipulation. 

In summary, stem and progenitor cell behavior is the 

result of highly integrated phenomena based on extracellular 

signals triggering intracellular transduction phenomena. 

The properties of self-renewal and differentiation 

give stem cells their remarkable ability to repopulate 

the hematopoietic tissue of lethally irradiated or 

genetically defective recipients. Understanding the 

interplay between extracellular and intracellular regulatory 

factors in controlling lineage determination 

remains an important challenge for the future clinical 

use of hematopoietic cells. 

Stem cell antigen(s) 

CD34 is a surface glycophosphoprotein expressed on 

early lympho-hematopoietic stem and progenitor cells, 

small-vessel endothelial cells, as well as embryonic 

fibroblasts. 28 CD34 + hematopoietic cells are morphologically 

and immunologically heterogeneous and functionally 

characterized by the in vitro capability to generate 

clonal aggregates derived from early and late 

progenitors and the in vivo capacity to reconstitute the 

myelo-lymphopoietic system in a myeloablated host. 29 

The CD34 + cell population contains virtually all the 

myeloid and lymphoid progenitors as well as a small 

subset of cells that can initiate and maintain stromal 

cell-supported long-term cultures. Expression of the 

CD34 marker has dominated attempts to isolate, purify 

and characterize human hematopoietic stem cells by a 

variety of immunologic means. 

Several monoclonal antibodies (MoAbs) assigned to 

the CD34 cluster identify a transmembrane glycoprotein 

antigen of 105-120 kd expressed on 0.5-2% normal BM 

cells, 0.01-0.1% peripheral blood cells and 0.1-0.4% 

cord blood cells. 30 The function of the CD34 antigen is 



95 

not yet known, although it seems that CD34 is involved 

in stem/progenitor cell localization/adhesion in the marrow. 

31 CD34 antigen expression is associated with the 

concomitant expression of several markers, including 

the lineage non-specific markers Thy1, CD38, HLA-DR, 

CD45RA, CD71 as well as T-lymphoid, B-lymphoid, 

myeloid and megakaryocytic differentiation markers. 30 

Analysis of the expression of CD38, Thy-1, CD71, the 

isoforms of CD45, and uptake of rhodamine-123 have 

resulted in a consensus stem cell phenotype which is 

CD34 bright , Thy-1 + , CD38 – , CD45RA – , rh-123 dull , Hoechst 

33342 dull , Lin – . CD34 + cells also express receptors for a 

number of growth factors classified as tyrosine kinase 

receptors, such as the stem cell factor receptor (SCF-R) 

or the stem cell tyrosine kinase receptors (STK), and 

hematopoietic receptors, not containing a tyrosine 

kinase domain. 32 Tyrosine kinase receptors are of particular 

relevance since their ligands might represent new 

factors able to selectively control stem cell self-renewal, 

proliferation and differentiation. 33 Recently, CD34 – 

cells have been shown to have functional characteristics 

associated with stem cells and differentiate, in vivo, 

to CD34 + cells. 34 

Stem/progenitor assays to evaluate 

engraftment potential 

Different types of progenitors can be measured directly 

by multiparameter phenotyping of CD34 + cells or subpopulations. 

Although this approach has the significant 

advantage that the results may be quickly available and 

can be used to guide clinical decisions, correlations 

between progenitor cell phenotype and functional activity 

are not yet refined enough to be clinically applicable. 

In addition, although CD34 antigen is expressed by virtually 

all progenitor cells, the percentage of CD34 + cells 

with clonogenic activity in vitro ranges from 10 to 50%. 

Non-clonogenic CD34 + cells include lymphoid progenitors 

as well as subsets of cells which are unresponsive to conventional 

growth factors and might require the presence 

of still unknown factors able to activate stem cell specific 

genes. 14 Figure 2 shows a schema of the cellular organization 

of hematopoiesis based on in vitro and in vivo 

functional properties of progenitor cells. 

Recently, a new human hematopoietic cell, termed the 

SCID repopulating cell (SRC), that is capable of extensive 

proliferation and multilineage repopulation of the bone 

marrow of non-obese diabetic (NOD)/SCID mice has been 

identified. 35,36 The SRCs which are detected exclusively in 

the CD34 + CD38 – cell fraction have been shown to be 

biologically distinct from CFC and most LTC-IC. 35,36 With 

the exception of transplantation of human cells into 

immunodeficient mice, the identification of putative 

human stem cells has essentially relied on in vitro assays. 

Short-term in vitro assay systems require appropriate 

nutrients and growth factors and are particularly suitable 

for measuring quantitative changes of the different 

progenitor cell types as well as for evaluating growth 

factor responsiveness or investigating differential effects 

of regulatory molecules on progenitors at different 

stages of differentiation or on different hematopoietic 

pathways. 37 Short-term assays are not suitable for analyzing 

self-renewal or interactions of hematopoietic 

progenitors with stromal cells. 

By using the long-term culture (LTC) technique, a sustained 

production of myeloid cells can be readily achieved 

in vitro, provided that a stromal layer is present, when 

marrow (or blood) is placed in liquid culture at relatively 

high cell concentration, with appropriate supplements, 

temperature and feeding conditions. 37 

The LTC system, based on the re-establishment in vitro 

of the essential cell types and mechanism responsible 

for the localized and sustained production of hematopoietic 

cells in the marrow in vivo, offers an approach able 

to investigate not only the proliferative and differentiative 

events but also self-renewal of any clonogenic cell 

types. A 5- to 8-week time period between initiating 

cultures and assessing clonogenic progenitor numbers 

allows a very primitive, self-renewing human cell, the 

Figure 2. Cellular organization 

of hematopoiesis. 


96 


so-called long-term culture-initiating cell (LTC-IC) to be 

quantified. 38,39 Limiting dilution assays allow the frequency 

of LTC-IC and their proliferative potential (number 

of CFU-GM generated by each LTC-IC) to be calculated. 

Another assay system, the cobblestone area-forming 

cell (CAFC), uses a pre-formed stroma as a support 

for hematopoiesis. 40 In this system, the primitive cells 

are measured directly by their ability to form characteristic 

colonies of cells resembling cobblestones. 

With the possibility of studying not only the differentiation 

but also the self-renewal of primitive progenitors, 

the LTC system will play an increasingly important role in 

the design and assessment of new strategies involving 

the genetic engineering of hematopoietic cells and marrow 

stromal cells. Hematopoietic cells that can generate 

active hematopoiesis for weeks in vitro or months in vivo 

after transplantation are considered stem cells. This seems 

a clinically useful criteria, because it characterizes those 

cells which are important for sustained hematopoietic 

recovery following SCT. However, it reflects an oversimplification 

of the rather complex process of hematopoietic 

function. In fact, the ability of a cell to provide longterm 

hematopoietic activity can either be due to a long 

period of quiescence after the initiation of the culture or 

be a function of the probability of stem cell self-renewal 

which influences the long-term survival of stem cell 

clones. 41 Thus, the number of primitive cells measured in 

an LTC assay will be the product of the number of stem 

cells present at the onset of the culture and the probability 

of stem cell self-renewal. Although LTC assays will 

likely predict the in vivo repopulating activity of the graft, 

the clinical definition of a stem cell does not consider 

those stem cells that differentiate and die soon after 

transplantation or initiation of a culture. 

Preparation of hematopoietic cells for 

ex vivo expansion 

In the vast majority of cell culture systems, the presence 

of inhibitory mature and accessory cells limits the 

degree of ex vivo expansion of the progenitor cell compartment. 

Thus, a higher production of total cells, clonogenic 

cells and more immature hematopoietic progenitors 

has been observed when purified progenitor cells 

(namely CD34 + cells) rather than the whole BM, cord 

blood. 42 or peripheral blood stem cell (PBSC) collections 43 

are cultured ex vivo. Haylock et al. 43 selected and cultured 

1,000 CD34 + cells in presence of a 10-fold excess 

of contaminating CD34 – , CD3 – , CD14 – cells and found no 

differences in total cell production after 14 days of culture, 

as compared to the production of 1,000 CD34 + cells 

grown alone. However, when CD34 + cells were mixed 

with increasing concentrations of CD3 + CD14 + cells, a 

marked decrease in the total cell output was observed 

after two weeks of culture suggesting an inhibitory activity 

of monocytes and T-cells. Despite the lack of information 

on CFU-C production, these results point out that 

the purity of the starting population is an important variable 

and it was recommended that at least 50% of cells 

should be CD34 + in the initial cellular input. 43 There are 

Table 1. Hematopoietic cell separation systems. 

Recovery Purity Clinical grade 

(% of initial cells) (% CD34+ cells) device 

Immunomagnetic beads 

Negative selection 20-50 20-60 Yes 

Positive selection 30-60 50-90 Yes 

MACS >80 >90 Under 

evaluation 

Panning 30-50 40-70 No 

Avidin-biotin 40-60 50-90 Yes 

immunoabsorption 

FACS (high-speed cell sorting) 30-50 >95 Yes 

also practical advantages in using a purified stem cell 

population as starting material for ex vivo manipulation, 

such as the ease of cell handling and the amount of 

cytokines consumed in the culture. Other variables that 

play an important role in stem cell expansion, which will 

be discussed in detail in the following paragraphs are: 

initial cell density, different cytokines used and their concentration, 

the presence of stromal cells, the composition 

of the culture medium and the refeeding schedule. 

Conversely, it has been recently demonstrated 44 that 

CD34 + cells can be safely and efficiently processed after 

cryopreservation suggesting that the availability of fresh 

hematopoietic cells may not be an essential prerequisite 

for ex vivo expansion. Moreover, whereas the majority of 

preclinical and clinical studies 45 have attempted to optimize 

the expansion of highly enriched CD34 + cells, under 

certain circumstances it may be advisable to select earlier 

subfractions of progenitor cells such as CD34 + DR – 

cells in chronic myelogenous leukemia (CML) or CD34 + 

Thy-1 + lin – cells in multiple myeloma (MM). In these diseases 

the CD34 + cell population is still contaminated, to 

various degrees, by malignant cells; 46-49 therefore, isolation 

of primitive progenitors prior ex vivo expansion may 

provide a starting cell population with a high proliferative 

potential free of tumor cells. 

Methods for hematopoietic progenitor cell 

(HPC) enrichment 

Several methods have been proposed for purification of 

HPC (Table 1). Their final target is a cell population with 

optimal purity, viability and high proliferative potential 

obtained by means of a low cost, rapid and simple separation 

technique. Early attempts toward the purification 

of HPC were based on the cell physical properties. Density-gradient 

centrifugation, velocity sedimentation and 

elutriation are methods that separate cells based on cell 

size and buoyant density. More recently, immunologic 

selection techniques which take advantage of the expression 

of specific antigens on HPC membrane, have allowed 

a much better degree of enrichment. Specifically, the 



97 

demonstration of the presence of the CD34 antigen on 

HPC 28 has led to a number of positive selection systems 

which use MoAbs to purify hematopoietic precursors. 

Alternatively, CD34 + cells can be enriched by depletion 

of CD34 – accessory and mature cells. 

Fluorescence activated cell sorter (FACS) 

Flow cytometry can be used to separate HPC from a 

heterogeneous population after incubation of cells with 

fluorochrome-conjugated MoAbs directed to cell-surface 

markers. Moreover, multiparameter enrichment can 

be obtained by combining physical properties such as 

cell size and cytoplasmic granularity and intracellular 

characteristics indicating cellular function (e.g. propidium 

iodide to determine cell viability; rhodamine-123 to 

assess metabolic quiescence and nucleic acid dyes to 

evaluate cycling status). This cell sorting technique can 

yield a highly purified (> 99%) cell population combining 

positive and negative markers for HPC using clinically-graded 

MoAbs. The main criticisms to the use of 

flow cytometry for selecting large numbers of cells are 

the low recovery of target cells and the length of time 

required to process the whole BM harvest or the leukapheresis 

products. The development of multiparameter 

high-speed cell sorting has been described and recently 

upgraded for clinical use. 50,51 Viable cells have been 

sorted at rates as high as 40,000 cells/sec as compared 

to 2,000-5,000 cells/sec of commercially available cell 

sorters. Thus, the sample processing time can now be 

reduced to 8-12 hours. The sorted cell fraction also 

maintains its hematopoietic potential based on the presence 

of CFU-C, more immature CAFC and long-term 

repopulating cells in mice. 51 Presently, selection of HPC 

(i.e. CD34 + Thy-1 + lin – cells) from clinical samples is 

directed toward the purification of cell populations 

highly enriched for HPC free of contaminating malignant 

cells in myeloma patients. 47 

Panning 

Anti-CD34 MoAbs bound to the bottom of cell culture 

flasks have been used to select CD34 + cells. 52 The 

target cell population present in a heterogeneous cell 

suspension is blocked on the plastic surface while CD34 – 

cells remain in the supernatant and can be easily eliminated. 

Despite the good results reported, the availability 

of more efficient methods of cell separation have 

made this technique largely redundant. 

Immunomagnetic systems 

A variety of magnetic cell-separation methods have 

been described. 53 Some of these systems are commercially 

available and have been used in clinical trials. The 

main differences between the currently used magnetic 

cell-separation methods are the composition and size of 

the magnetic particles used for labeling the cells and the 

separation process. Superparamagnetic beads can be 

equally used for negative and positive cell separation 

depending on the specificity of MoAbs. The rosetted target 

cells can be easily isolated from unlabeled cells by 

a magnet applied on the outer wall of the test tube or 

Table 2. High efficiency of Mini-MACS separation system for 

the enrichment of BM or circulating CD34 + cells. 

Enrichment 

Source Pre Post Recovery CE* LTC-IC 

(%) (%) (%) (x10 4 CD34 + ) 

BM 2.3±1 97±3 88±9 3.6±0.3 62.5±54 

PB 0.7±0.4 98.9±1 90±8 3.9±0.4 48.2±35 

*Abbreviations: CE, clonogenic efficiency; BM, bone marrow; PB, peripheral blood. 

The results are expressed as mean±SD. 

blood bag. Large magnetic beads (diameter >0.5 µm) 

have been used clinically for the purging of neuroblastoma 

and lymphoma cells from stem cell harvests prior 

to autologous transplantation. 53,54 Immunomagnetic 

beads coupled with anti-CD34 MoAb can be used for 

positive selection of HPC. 55 However, before the clinical 

use of the enriched cell fraction, the cell-bound particles 

must be removed to avoid damage to the cells 

and/or toxic events to the patient. Beads can be released 

using chymopapain or a peptide competing with the 

CD34 Ab. In alternative, HPC can be enriched by negative 

depletion of mature and accessory cells targeting 

lineage-specific antigens. 

More recently, the magnetic cell-sorting (MACS) system 

has been proposed as an efficient and more manageable 

alternative to flow cytometry for cell separation. 

56 It uses colloidal-sized superparamagnetic particles 

made of dextran and iron oxide with 60 nm of 

diameter. The use of very small beads minimizes unspecific 

binding and allows the efficient isolation of rare 

cells. In addition, the magnetic particles are readily 

internalized by the labeled cells without affecting their 

physical, phenotypic and functional capacity. 57 Table 2 

reports the results of a large number of experiments 

(=14) comparing the efficiency of the Mini-MACS system 

for selecting CD34 + cells from two different cellular 

sources (R.M.L., unpublished observations). 

High-affinity chromatography based on 

avidin-biotin immunoabsorption 

This technique relies on the high affinity between the 

protein avidin and the vitamin biotin whose interaction 

has an extremely high dissociation constant (= 10 -15 M). 

In this system, a heterogeneous cell population is incubated 

with a biotinylated antibody to the CD34 antigen. 

The cell mixture is then passed through a disposable column 

containing avidin-coated polyacrylamide beads. 

CD34 + cells are retained on the column due to the high 

affinity binding of biotin to avidin while negative cells 

are washed away. Target cells are then recovered by 

mechanical agitation of the column which disrupts the 

antibody-antigen link. Thus, bound cells are eluted from 

the column mainly free of antibody. An automated version 

of the device controlled by a computer which guar- 


98 


antees reproducibility and reduces risks of operator errors 

has been developed and used in the setting of autologous 

and allogeneic stem cell transplantation. 

46, 58 

Dynamic systems 

In addition to positive or negative selection of CD34 + 

cells, effective ex vivo expansion of hematopoietic progenitor 

cells and, perhaps, putative stem cells can also be 

achieved in the presence of unselected cell populations. 

In this case the use of dynamic perfusion cultures is 

strictly required. As stated above, the production of 

inhibitory factor(s) by mature and accessory cells rather 

than the availability of growth promoting factors, is 

probably the main limitation to successful stem cell 

expansion. For instance, it is well known that suboptimal 

cell expansion occurs when CD34 + cells are cultured 

at concentrations exceeding 10 4 cells/mL. Moreover, the 

presence of stromal feeder-layer cells seems to be 

important for effective BM stem cell expansion induced 

by exogenous cytokines. Therefore, an artificial capillary-perfusion 

system (Bioreactor) in which nutrients 

consumed by proliferating cells are continuously replenished 

by exchange of the nutrient-depleted medium 

with fresh culture medium, has been tested for ex vivo 

expansion of hematopoietic cells co-cultivated with 

stroma cells or stromal cell lines. 59 Cytokines such as IL- 

3, IL-6 and GM-CSF have been added to the culture to 

optimize cell expansion and to provide growth factors 

which are not produced by stromal cells (i.e. IL-3). In this 

system, cultured cells are confined in a small compartment 

separated from a large medium reservoire. Medium 

exchange is optimized when there is a maximal 

membrane surface area per unit volume across which 

medium and nutrients can pass by diffusion. This is best 

achieved by a capillary-perfusion module. Three important 

requirements for optimal expansion of hematopoietic 

cells are: the capillary porous size, the capacity of 

supporting the attachment of stromal cells by the module 

and the minimal cell activation by the materials of 

the module. In fact, mature myeloid cells such as macrophages 

release ,upon surface activation, tumor necrosis 

factor(s) (TNFs), interferon(s) (IFNs) and other substances 

which negatively affect stem cell expansion. 

Bioreactors have induced a remarkable expansion of 

committed hematopoietic progenitor cells coupled with 

a modest increase in the number of LTC-IC, 59 a population 

of primitive cells which correlates most positively 

with the long-term reconstituting capacity of autologous 

and allogeneic grafts. 

Ex vivo expansion of myeloid 

progenitor cells 

Ex vivo expansion of hemopoietic progenitors might 

result in: 

• amplification of the population of committed progenitors 

due to an extensive, although controlled, differentiation 

process; 

• amplification of the stem cell pool through extensive 

self-renewal of the early progenitor cell population. 

Obviously, both processes can take place simultaneously 

mimicking, in vitro, the complex interplay of regulatory 

mechanisms that allows hemopoiesis in vivo. The 

latter situation, so far, has never been obtained in vitro, 

whereas different approaches have permitted the first 

goal to be reached (at least, to a certain extent) and 

some recent data suggest that relevant self-maintaining 

processes can be triggered. 

The clinical relevance of extended differentiation versus 

self-renewal is obviously different. The induction of 

an increased in vitro production of committed progenitors 

might hasten the early phases of hemopoietic reconstitution 

which occur after myeloablative treatment and 

stem cell transplantation. Moreover an increased number 

of infused cells might modulate graft versus-host 

disease (GVHD) intensity in the allogeneic setting. 60 

Although relevant, the potential clinical benefit of 

techniques allowing only committed progenitor cell 

expansion is outweighed by the possibility of triggering 

the self maintenance, and perhaps amplification, of early 

hemopoietic progenitors. In this situation, starting 

from a limited number of progenitors, long-term reconstitution 

of hematopoiesis might become feasible. 

Moreover, ex vivo manipulation of primitive hemopoietic 

cells could be performed under experimental conditions 

suboptimal for the growth of neoplastic cell contaminating 

autologous grafts. Thus, a purging effect 

could be obtained. 

Several attempts of in vitro expansion of hemopoietic 

progenitors have been published in the last years. 

Recent reviews 61, 62 summarize early experiences. 

The first studies on ex vivo generation of hematopoietic 

progenitors involved liquid culture in the presence 

of cytokines such as SCF, IL-1, IL-3, IL-6, G-CSF, GM-CSF 

and Epo. These experiences showed that a relevant 

increase (from 10 to 1,000 fold) of CD34 + cells and of 

committed progenitors can be obtained. The expansion 

of committed progenitors does not mean, however, that 

the long-term reconstitution of hemopoiesis is achievable. 

Indeed, several data support the concept that an 

uncontrolled commitment decreases the stem cell pool. 

Yonemura et al. 63 have reported that IL-3 or IL-1 abrogates 

the reconstituting ability of hematopoietic stem 

cells. Furthermore, Peters et al. 64 have demonstrated 

that ex vivo expansion of murine marrow cells with IL- 

3, IL-6, IL-11 and SCF leads to impaired engraftment in 

irradiated hosts. 

As indicated by Traycoff et al. 65 ex vivo expansion of 

hematopoietic cells using SCF, IL-1, IL-3 and IL-6 generates 

classes of cells possessing different levels of BM 

repopulating potential based on their cycling status. 

Along this line, Young et al. 66 correlated a higher proliferative 

capacity with a quiescent status after ex vivo 

expansion in the presence of SCF, IL-3, IL-6 and LIF. 

Taken together, these data suggest that cultures in the 

presence of cytokine combinations based upon SCF, IL- 

1 and IL-3 involve differentiation of BM or mobilized 

CD34 + cells entering the S phase. Different results were 

obtained by Di Giusto et al., 67 who found that ex vivo 



99 

expanded cord blood CD34 + cells repopulated the marrow 

of immunodeficient mice as well as non-expanded 

cells. However, it must be remembered that cord blood 

is rich in hemopoietic progenitors 68 that have an 

increased proliferation potential. 69 

New strategies to induce the expansion of CD34 + cells 

with little (or no) differentiation might involve different 

approaches. The use of stirred suspension 70 or hollow 

fiber 71 bioreactors has been proposed in order to grow 

cells in a more physiologic environment, and inhibitors 

such as TGF- and MIP-1 have been the object of 

intense studies. Recently, MIP-1 has been found to 

exert a weak inhibitory effect on CD34 + CD38 – cells and 

to enhance the proliferation of CD34 + CD38 + cells, 

whereas TGF- strongly inhibits both cell populations. 

72,73 The most promising results, however, have 

been obtained with the recent introduction of FL and 

Tpo. FL, a recently discovered member of the class III 

tyrosine kinase receptor family, 74 is able to induce proliferation 

of very early hematopoietic progenitors that 

are non-responsive to other early acting cytokines, and 

to improve the maintainance of progenitors in vitro. 75-77 

This is also supported by the finding that FL significantly 

reduced the number of cultured cells undergoing 

apoptosis. 78 Analysis of the effects of 16 cytokines on 

CD34 + CD38 – cells showed that FL, SCF and IL-3 produced 

a 30-fold amplification of the input of LTC-IC. 79 

Yonemura et al. 80 compared FL- and SCF-driven ex vivo 

expansion. They reported that both cytokines, in combination 

with IL-11, enhanced the production of progenitors, 

but with different kinetics. In fact, the maximal 

expansion by FL required a longer incubation than with 

SCF. Interestingly, in these studies the combination of 

SCF/IL-11, together with IL-3, reduced the ability of cultured 

cells to reconstitute hematopoiesis in irradiated 

hosts. 80 Other recent data have compared the effect of 

FL and SCF. FL acts as a self-renewal or proliferation/expansion 

signal for CD34 + -low cells while the 

effect of SCF is more likely to transduce a differentiation 

signal, resulting in more rapid repopulation at the 

expense of cell expansion. 81 Gene transfer studies in 

mice have also demonstrated that FL maintains the ability 

of human CD34 + cells to sustain long-term hematopoiesis. 

In fact, incubation of CD34 + cells with FL before 

transduction was associated with long-term provirus 

expression, whereas provirus expression declined in 

recipients of CD34 + cells transduced in the absence of 

FL. 82 The expansion ex vivo of early progenitors seems to 

be affected at the single cell level by changes in cytokine 

concentrations. In a recent paper by the Vancouver 

group, 83 maximal LTC-IC expansion was obtained in the 

presence of 30 times more FL, SCF, IL-3, IL-6 and G-CSF 

than could concomitantly stimulate the near-maximal 

amplification of CFC. 

Tpo, the ligand of the mpl receptor expressed on both 

early and committed hematopoietic progenitors, 84 is 

known to support megakaryocytopoiesis. 85 Moreover, it 

has been shown to be capable of enhancing ex vivo 

expansion of early/committed progenitor cells. As single 

factors, FL and Tpo stimulated a net increase of LTC-IC 

generated from CD34 + CD38 – cells within 10 days. 79 Furthermore, 

as demonstrated in mice recipients of BM 

cells transduced with the mpl receptor, 86 Tpo does not 

induce lineage-restricted commitment of mpl-receptor 

positive pluripotent progenitors but permits their complete 

erythroid and megakaryocytic differentiation. Tpo 

has also been found to increase the multilineage growth 

of CD34 + CD38 – cells from 3%, in absence of the 

cytokine, up to 40% when Tpo is added to SCF and FL. 87 

The presence of additional cytokines such as IL-3, IL-6 

and Epo does not significantly enhance clonal growth 

above that observed in response to Tpo, SCF and FL. 87 

lnterestingly, the soluble form of Tpo receptor and G-CSF 

receptor directly stimulate the proliferation of primitive 

hematopoietic progenitors of mice in synergy with SCF 

and FL. 88 

A step toward extensive ex vivo amplification of early 

human progenitor cells has been reported by Piacibello 

et al. 89 They first demonstrated that IL-3 induces 

an early production of committed progenitors but is not 

able to sustain true self maintenance of hemopoietic 

stem cells even in the presence of other early acting 

cytokines (FL, Tpo, SCF). Afterwards, several combinations 

of early acting cytokines were tested for their ability 

to sustain long-term hematopoiesis in stroma free 

cultures. Among the various combinations tested on 

purified cord blood CD34 + cells, the mixture of Tpo + FL 

was found to be able to maintain early progenitors up 

to six months. 89 These data indicate the enormous 

potential of cord blood progenitors and the key role of 

Tpo and FL in the regulation of early hematopoiesis. 

However, several issues remain to be clarified: 

• is it true self-renewal or a slow differentiation of cord 

blood cells, which are rich in immature progenitors? 

• what is the in vivo repopulating capacity of ex vivo 

expanded cells? 

• is such expansion possible using CD34 + cells obtained 

from the marrow or peripheral blood of adult subjects? 

In this view, while a number of papers have already 

reported that committed progenitors can be generated 

and safely administered to transplant recipients, there 

are no reports on expansion of cells with long-term 

repopulating capacity in humans. 

Brugger et al. 45 reported the successful reconstitution 

of hematopoiesis in ten cancer patients transplanted 

with autologous cells generated from CD34 + cells cultured 

in the presence of SCF, IL-1, IL-3, IL-6 and Epo. 

However, the conditioning regimen given to these 

patients was not fully myeloablative, and this study 

offered no insight into the long-term engraftment 

potential of cells generated in this fashion. A similar 

approach was followed by Alcorn et al. 44 In ten patients 

with malignancy, an aliquot of the PBSC harvest was 

recovered from liquid nitrogen and CD34 were selected. 

Cells were cultured for 8 days in the presence of the 

same cytokine combination. A mean of 379×10 6 expanded 

cells were reinfused in addition to unmanipulated 


100 


cells. The authors reported that the total LTC-IC number 

was not increased and most of the CD34 + cells were 

differentiated in front of an average 15-fold CFU-GM 

expansion (range 4-39). Similarly, averages of 71 (range 

27-151)-fold megakaryocytic cell expansion and 1,040 

(19-16.000)-fold erythroid cell expansion were reported. 

Although adverse reactions were not reported, no 

difference in the kinetics of engraftment was observed 

in comparison with historical controls. 

Different results come from preclinical studies where 

the infusion of large numbers of ex vivo expanded committed 

hematopoietic progenitors, together with unmanipulated 

cells might speed engraftment after 

chemotherapy and/or total body irradiation (TBI). Data 

from Uchida et al. 90 have suggested in the past that most 

of the short- as well as the long-term engraftment 

potential resides in uncommitted progenitors. More 

recently, Szilvassy et al. 91 have demonstrated that partially 

differentiated ex vivo expanded cells accelerate 

hematologic recovery in myeloablated mice transplanted 

with highly enriched long-term repopulating stem 

cells. In humans, Williams et al. 92 reinfused 9 breast cancer 

patients with unmanipulated apheresis products 

together with a mean of 44×10 6 /kg mature CD15 + cells 

generated ex vivo by CD34 + cells cultured in the presence 

of PIXY-321. No toxicity was observed after reinfusion, 

and time of white cell recovery was similar to 

that observed in the retrospective control group. In a 

more recent study, 78 megakaryocytic progenitors (MP) 

were obtained from CD34 + cells cultured in serum-free 

medium in the presence of Tpo, FL, SCF, IL-3, -6, -11 and 

MIP-1. Proliferation peaked on day 7 in culture, and a 

8±5-fold expansion of CD34 + /CD61 + cells, a 17±5-fold 

expansion of CFU-MK and a 58±14-fold expansion of 

the total number of CD61 + cells was obtained. Ten cancer 

patients undergoing autologous PBPC transplant 

received MP generated ex vivo (range 1-21 CD61 + cells 

×10 5 /kg) together with unmanipulated PBSC. Platelet 

transfusion support was not needed in 2 out of the 4 

patients receiving the highest dose of cultured MP and 

this result compared favorably with a retrospective control 

group of 14 patients, all requiring platelet transfusion 

support. A major concern is the potential expansion 

of contaminating tumor cells along with hematopoietic 

progenitors. In fact, it has been demonstrated that 

CD34 + cell selection decreases (but does not abrogate) 

neoplastic cell contamination from aphereses of myeloma 

patients. 46 For instance, in the majority of B cell lymphoma 

patients CD34 + cell selection does not eliminate 

contaminating t(14;18) + cells. However, during ex vivo 

expansion residual lymphoma cells do not proliferate 

and become undetectable by molecular analysis in the 

majority of cases. 93 Similarly, Vogel et al. recently indicated 

that exogenously mixed epithelial tumor cell lines 

might have a relative disadvantage over CD34 + cells during 

ex vivo expansion. 94 

Future directions 

Future challenges in this field are represented by the 

expansion of true human stem cells without maturation, 

to extend this strategy to allogeneic stem cell 

transplantation, and especially cord blood allograft, as 

well as the manipulation of cycling of primitive progenitors 

for gene therapy programs. 

Selective amplification of specific myeloid lineages (e.g. 

platelets or granulocytes) may improve the results of 

autologous transplantation. Moreover, although early 

results need confirmation, the amplification of early/committed 

hematopoietic cells coupled with the removal of 

neoplastic cells contaminating autologous grafts appears 

to be feasible. 

Expansion of cytotoxic effectors 

Human cytotoxic effector (CE) cells can be divided in 

two major groups: 

1. cells requiring prior antigen sensitization, which recognize 

their target in the context of the major histocompatibility 

complex (MHC) molecules; 

2. cells not requiring prior antigen sensitization being 

spontaneously cytotoxic against tumor target cells 

(e.g. K-562 cell line) in a non-MHC restricted setting. 

While the first group includes only some subsets of T- 

lymphocytes (CD8 + or CD4 + cells), the second one is 

more heterogeneous and includes both T-cells and natural 

killer (NK) cells, expressing the CD56 antigen. 95 

The so-called antibody-dependent cellular cytotoxicity 

(ADCC) can be mediated by cells expressing the Fc 

receptor II and the Fc receptor III (e.g. NK cells and 

CD3 + /CD16 + cells). Although this activity is not exhibited 

by non MHC-restricted cells, it cannot be considered 

aspecific and it is also exerted by monocytes. 

The lymphokine-activated killer cells (LAK) are capable 

of killing NK-resistant cellular targets (e.g. Daudi cell 

line). Although some tissue-resident lymphocytes may 

have spontaneous LAK activity, normal blood mononuclear 

cells do not show any LAK activity, which can be 

acquired after incubation with Interleukin-2 (IL-2). 96 

Therefore the LAK assay is a measure of the capacity 

of T and NK cells to become activated and to express 

cytolytic function. 

Killing mechanisms of cytolytic effectors 

Cytotoxic T-Lymphocytes (CTL) and NK cells possess at 

least two distinct, fast-acting, lytic mechanisms: 

97, 98 

1. the granule exocytosis pathway involves the secretion 

of perforin and granzymes which penetrate 

throughout the target cell pores, inducing cell death; 

2. a non-secretory mechanism which is mediated by 

the interaction between the Fas-ligand, expressed 

by the killer-cell and Fas (CD95) which triggers a 

cascade of proteolytic enzymes leading to apoptosis 

of both the killer and the target cell. 

A third cytolytic pathway, involving TNF, has recently 

been described. 99 

A series of apoptosis-resistant clones of human lym- 



101 

phoma cells has been described. These cells express 

fas/APO-1 receptors lacking the intracytoplasmic signaling 

domain. 100 

NK cells, as well as CTL, can recognize MHC class I 

molecules. However, recognition of MHC on target cells 

downregulates the NK cell function, suggesting the 

presence of inhibitory receptors. 101 

LAK cells are not MHC-restricted and are also capable 

of killing freshly isolated tumor cells. Similarly to 

CTL and NK cells, their activity is mediated by both lytic 

pathways (perforin/granzyme) and Fas-mediated 

apoptosis. 

Cytokines involved in CE function 

Several cytokines affect CTL and NK cell response. In 

particular, IL-2 expands the pool of alloreactive CTL precursors 

and IL-15 (produced by monocytes) mimics IL- 

2 action by inducing -IFN production, the activation of 

memory T cells and CTL proliferation. 102 

In addition, GM-CSF can affect certain T-lymphocyte 

functions by enhancing their cytotoxicity and -IFN 

production. This multifunctional cytokine can also augment 

NK cell function and the expression of adhesion 

molecules on the surface of leukemic cells. 103,104 Moreover, 

it has been hypothesized that the association of 

GM-CSF/IL-2 can also be useful for the activation of 

cytotoxic effectors by circulating progenitors, preserving 

the clonogenic potential of normal hemopoietic precursors. 

105 

Figure 3. NK cells differentiation pathway. 

Modified from Miller et al, Blood 1994; 83: 2594-601. 

IL-12 elicits the production of -IFN by CTL thus 

enhancing their antineoplastic efficacy and promotes the 

differentiation of T-helper-1. 106,107 Finally, IL-7 seems to 

be critical for the development of CTL and for a fast 

immune reconstitution after bone marrow transplantation 

(BMT). 108 

In conclusion, these cytokines play a pivotal role in the 

immune response against tumor cells by expanding, activating 

and recruiting CE and secondary effector cells 

(macrophages) or by directly inhibiting tumor cell growth. 

Role of cytotoxic effectors in immunosurveillance 

There are several in vitro and in vivo data supporting 

the role of immunosurveillance in tumor growth control. 

109 The graft-versus-leukemia (GVL) effect has been 

demonstrated to play a critical role in the eradication of 

minimal residual disease (MRD) after allogeneic 

trasplantation and there is evidence supporting the role 

of both T cells and NK cells in preventing disease 

relapse. 110 

Today, there is no doubt that CTL have a major role in 

killing allogeneic tumor cells in a MHC-restricted manner. 

For example, in CML, the higher relapse incidence 

after T-cell depleted allogeneic BMT 111 and the dramatic 

effect of donor T-lymphocyte infusion after relapse 

following BMT, 112 strongly support the importance of 

MHC-restricted GVL. 

Unfortunately, a selective GVL effect (separated from 

GVHD) can be obtained very rarely in patients receiving 

allogeneic BMT. Moreover, although animal models indicate 

that autologous GVHD exists and could generate a 

significant antitumor effect, the high incidence of relapse 

in patients receiving autologous BMT demonstrates that 

autologous GVL is often clinically ineffective. 113 

Rationale for cytotoxic effectors’ 

expansion 

There are many important reasons to increase the 

number, the efficacy and the specificity of cytolytic 

effectors both in the allogeneic and in the autologous 

setting. The main clinical goals are the following: 

1. to reduce the relapse-incidence after autologous 

BMT, which is still relevant in acute leukemia, lymphoma 

and breast cancer; 

2. to cure diseases in which autologous BMT can only 

prolong the survival (multiple myeloma, CML, metastatic 

chemosensitive cancers); 

3. to reduce the incidence of relapse after allogeneic 

BMT especially in patients transplanted with great 

tumor burden; 

4. to accelerate the immune reconstitution after BMT, 

in order to reduce the morbidity and transplantrelated-mortality 

caused by serious infections (e.g. 

CMV and systemic mycosis). 

The CE which are investigated for in vivo or ex vivo 

expansion are mainly NK cells in the autologous setting 

and CTL in the allogeneic setting. 


102 


Development and expansion of NK cells 

NK cells belong to the naive part of the immune system 

and begin to appear into PB early after allo and 

auto-BMT. These cells express the N-CAM homologous 

CD56 antigen, but lack T-cell receptor / complexes. 

They are also characterized by low affinity receptors for 

IgG (CD16) 114-116 and their binding to malignant cells is 

mediated by CD18 molecule. 117 The most important 

organ for NK differentiation is the BM. Several data suggest 

that a common precursor of T cells and NK cells 

does exist. Fetal NK cells express the TCR , , , subunits 

while fetal B precursors do not express TCR subunits 

(Figure 3). 

These bipotential T/NK precursors do not have TCR 

rearrangement and share CD34/CD33/CD7 antigens. 

They differentiate to NK in the presence of SCF, IL-7, IL- 

2 and stromal feeder cells. The first step of NK differentiation 

is stroma-dependent while the second step of NK 

maturation is stroma-independent and is strongly 

potentiated by the association of IL-2 with IL-7. 118 In all 

studies IL-2 is required for NK cell differentiation from 

CD34 + cells. This finding suggests that a fraction of 

CD34 + cells expresses IL-2 receptors and that activated 

T-cells (as IL-2 source) should be detectable in the cellular 

milieu. However, T-cell deficient mice have normal 

NK cell development and humans lacking the -chain 

subunit of IL-2R lack NK activity. These unexpected 

observations have recently been supported by the 

demonstration that IL-15, produced by BM stromal cells, 

can directly induce CD34 + cells to differentiate into 

CD3 – /56 + NK cells in the absence of IL-2. 119 

The last step of NK development, after the expression 

of CD16, is characterized by the appearance of the CD56 

molecule. The intensity of CD56 expression directly correlates 

with the proliferative potential and the killing 

ability of NK cells. 120 

There is clear evidence that mature NK elements have 

a clonally-distributed ability to recognize their target 

cell by class I MHC alleles and a precise correlation has 

been established between the expression of p58 receptors 

on NK cell surface and class I MHC alleles. These 

receptors transduce an inhibitory signal upon interaction 

with MHC class I antigens, to prevent NK cells from 

killing target cells expressing certain (self) HLA alleles. 

These findings are consistent with a self-tolerance 

mechanism exerted by the NK population which can be 

disrupted as a consequence of tumor transformation or 

viral infection or any other events inducing (or masking) 

class I molecules. 121,122 

After incubation with IL-2, NK cells become LAK cells 

capable of killing otherwise NK-resistant target cells. 

These (NK) activated cells express new markers such as 

CD25, MHC class II and fibronectin which can be useful 

for the evaluation of their functional state. The NK cell 

compartment is heterogeneous and distinct subsets 

have been characterized. The most informative functional 

differences are based on relative CD56 fluorescence: 

only CD56 +bright , but not CD56 +dim NK cells, express 

the high-affinity IL-2 receptor. As a consequence, they 

respond to low concentrations of IL-2 and expand 10 

times more than CD56 +dim . This subset seems to be significantly 

reduced in leukemic patients. A remarkable 

reduction of CD56 +bright NK cells has been observed in 

CML patients coupled with a significant decrease of 

their spontaneous cytotoxicity against the K-562 line. 

However, this defect was corrected by 18 hours incubation 

with 1000 U/mL of recombinant IL-2. 120 These 

data strongly suggest that during tumor progression, 

the NK compartment (and particularly the small fraction 

of NK-CD56 +bright with high proliferative ability) is progressively 

suppressed even though the exogenous 

administration of IL-2 can partially reverse this phenomenon. 

The strong correlation between functional 

capacity of the NK cell compartment and tumor progression 

has often been reported as well as the efficacy 

of the administration of LAK cells plus IL-2 in restoring 

an anti-tumor response. 123 

Along this line, more than 90% of patients with acute 

leukemia in complete remission do not show spontaneous 

cytotoxicity against autologous blast cells. However, 

ex vivo treatment with IL-2 restores cytolytic activity 

in 37.5% of these patients. 124 

The first attempts to generate and expand LAK activity 

either in vivo or in vitro (after ex vivo incubation with 

IL-2) have been clinically disappointing especially in 

patients autotransplanted for acute lymphoblastic 

leukemia (ALL). 125 Nevertheless, there is now a renewed 

interest in the use of activated NK cells in hematologic 

malignancies, based on the optimization of different 

approaches: 

• administration of IL-2 in vivo to expand functionally 

active CE in patients with low tumor-burden, in 

order to reach an optimal effector/target ratio; 

• harvesting and culturing large amount of NK cells for 

additional ex vivo expansion/activation with IL-2. 

Expanded cells should be reinfused in the early phase 

after BMT; 

• sequential combination of both techniques (Figure 

4). 126 

The systemic administration of IL-2 for in vivo expansion 

and activation of the NK compartment may theoretically 

have some advantages: 

1. high number of CE precursors in the body; 

2. possibility of activating CE residing in the tumor 

bulk; 

3. more feasible and cheaper strategy than the generation 

and administration of LAK cells. 

On the other hand, the main drawbacks of this 

approach are the high toxicity of systemic administration 

of IL-2 and the high variability of anti-tumor 

response. 

Sources of cytotoxic effectors and 

modalities of NK cell expansion 

Human NK progenitor cells can normally be found in 

the BM, 127,128 and originate from CD34 + hematopoietic 

progenitors. 129 So far, the generation of NK cells from 

CD34 + precursors has been described on a small scale 



103 

A 

B 

Figure 4. Strategies for generation and activation of NK cells in vitro and in vivo. Modified from Klingemann et al., Exp Hematol 

1993; 21:1263-70. 

basis but not in large scale experiments. 130 

High numbers of functionally active NK cells can be 

easily demonstrated in mobilized cells from patients 

receiving chemotherapy plus G-CSF. Silva et al. 131 have 

shown a 5.4-fold expansion of NK cells from leukapheresis 

products incubated in the presence of IL-2 for 

6-8 days without affecting the CD34 + cell content. 

However, decreased function of NK cells has recently 

been described in the PB of normal donors after G-CSF 

administration. 132 Circulating NK progenitors showed a 

decreased killing capacity and diminished proliferative 

ability in response to IL-2, as compared to their 

unprimed BM counterpart. 

Based on previously published LAK trials, 123, 133 it can be 

estimated that about 10 10 -10 11 activated NK cells are 

needed to stimulate an anti-tumor response. A 100-fold 

ex vivo expansion of these cells from a standard leukapheresis 

collection would, therefore be required to obtain 

such a high number of effectors. 

Beaujean et al. 134 reinfused autologous BM cells incubated 

for 10 days with IL-2 in 5 ALL patients following 

a myeloablative treatment. This procedure resulted in 

an important loss of hemopoietic progenitors with 

delayed engraftment. Moreover, in spite of this attempt 

to induce an autologous GVL, all patients eventually 

relapsed. 134 

Wong et al. 135 compared the ability of IL-2 alone or 

combined with IL-7 or IL-12 to stimulate NK activity in 

BM or PB samples. They found that IL-2/IL-12-activated 

blood cells suppressed the growth of the leukemia cell 

line K-562 about eight-fold more efficiently than BM 

cells. They also found that cryopreservation and subsequent 

stimulation of BM and PB cells did not significantly 

decrease the activity of NK cells. Finally, the combination 

of IL-2 and IL-12 showed a synergistic effect on both 

BM and PB elements. 135 Large-scale ex vivo expansion 

of NK cells for adoptive immunotherapy not only 

requires an optimal source of precursors, but also clinically 

approved materials and procedures. In this context, 

Miller et al. 136 obtained a 21-fold expansion of NK 

cells using a 21-day large scale NK culture performed in 

gas-permeable bags. Pierson et al. 137 observed a 352- 

fold expansion of NK cells after 33 days of incubation 

in a bioreactor. Their starting population was NK precursors 

enriched by negative panning with anti-CD5 and 

anti-CD8 antibodies. The activated NK population was 

highly purified (>90%) in CD56 + /CD3 – cells and maintained 

a powerful cytotoxicity against K-562 cells. 

The use of a homogeneous NK cell fraction for celltherapy 

programs seems to be advantageous because 

activated NK cells have more specific lytic activity than 

heterogeneous LAK populations. 138 However, creating 

such a fraction requires a first step of enrichment (e.g. 

by eliminating CD8 + /CD5 + cells) and long-term cultures 

carry the risk of fungal or bacterial contamination. 


104 


Cytokine induced killer cells (CIK) 

expansion 

In 1986 Lanier described a subset of CD3 + T cells coexpressing 

the CD56 antigen which is a typical NK marker. 

139 A remarkable expansion of this cellular subset has 

recently been obtained by Schmidt et al. 140 following a 

16-day incubation in the presence of IFN, IL-1, IL-2 

and a monoclonal antibody against CD3 as the mitogenic 

stimulus. The ability of CIK cells to deplete 

leukemic cells from CML marrow was then investigated 

by the same group. 141 While standard LAK cells were, in 

most cases, unable to lyse CML cells, CIK cells were toxic 

to both autologous and allogeneic CML blasts without 

affecting normal hemopoietic progenitors. 

CTL expansion for adoptive therapy 

It is well known that the GVL effect can be transferred 

with donor-buffy-coat (BC) lymphocytes. 142 The 

antitumor effect of the CTL contained in the BC has 

been shown to be more potent than that induced by NK 

cells, even though NK cells exert a GVL activity different 

from GVHD. 143 

In a murine model, a single dose of 210 7 CTLs in 

tumor bearing mice (DBA/2) resulted in the eradication 

of primary cancer and metastases without causing 

severe GVHD. 144 Unfortunately, this GVL effect cannot be 

easily separated from GVHD in humans. As a matter of 

fact, in clinical studies the beneficial effect of CTL 

administration is often offset by the severity of GVHD or 

marrow aplasia. 

Although leukemic cells share common antigens with 

other tissues of the host, there is also the chance that 

distinct leukemic antigens may be recognized by specific 

allogeneic CTL. 145 

Leukemia-specific T-cell clones have been isolated 

from HLA-identical siblings 146 and this finding may 

explain the high incidence of CR, without GVHD, in 

patients with CML relapsed after allo-BMT and treated 

with donor BC. 147 

The subset of donor-lymphocytes involved in the GVL 

effect is not entirely defined. Both CD4 + and CD8 + GVL 

effectors have been described in animal models. Recent 

studies in man suggest a prominent role of CD8 + cells 

in acute leukemia and CD4 + cells in CML. 148 The therapeutic 

index of this approach may be increased by 

treating the donor lymphocytes, previously activated 

with recipient PHA-stimulated blast, with anti-CD25 

ricin-conjugated antibodies. 149 This procedure gives origin 

to a CTL population which retains over 75% of its 

antileukemic activity with only 10% of the initial 

responsiveness against the non-leukemic cells of the 

recipient. 

Strategies for generation and expansion of 

specific CTL 

A very attractive system for generating and expanding 

CTL is based on the selection and isolation of tumorspecific 

peptides (e.g. those encoded by bcr-abl or PML- 

RAR fusion genes) and to presenting them to T-cells to 

stimulate a specific T-cell response. The responding T- 

clones can then be amplified and selected by limiting 

dilution techniques. Unfortunately, in many cases the 

tumor-specific peptide is not presented by leukemic cells 

making the generation of peptide-specific CTL useless. 150 

In this regard, the transfection in tumor cells of DNA 

sequences encoding for co-stimulatory molecules (B7-1) 

or cytokines such as GM-CSF has greatly enhanced the 

anti-tumor response of T-cells. Alternatively, the use of 

professional antigen presenting cells (APC), primed with 

tumor specific antigens (e.g. tumor specific idiotype in 

low-grade B-cell lymphoma) has proved to be effective 

for the generation of tumor-specific CTL clones in vivo 

capable of inducinga measurable anti-tumor response. 151 

Allo-CTL have also been used against EBV-related lymphoma 

developed in allograft recipients and HIV patients. 

EBV infection is controlled in normal individuals by specific 

CTL which lyse EBV-infected B-cells upon recognition 

of viral peptides presented on the cell membrane in 

association with MHC class I molecules. 

EBV-specific CTL have been isolated from normal 

donor leucocytes and expanded ex vivo by Rooney et 

al. 152 Following the reinfusion of 1.2×10 8 CTL/m 2 into an 

allograft recipient, the complete resolution of an EBVrelated 

immunoblastic lymphoma was observed. 

Ex vivo expanded CTL can also be used to restore CMVspecific 

responses in immunodeficient individuals receiving 

allogeneic BMT. Walter et al. 153 treated 14 patients 

with infusions of CD8 + CTL directed to CMV proteins 

obtained from bone marrow donors. In this study, CMVspecific 

CTL were expanded by stimulation with anti-CD3 

antibodies coupled with autologous CMV-infected 

fibroblasts in IL-2-containing culture. This approach to 

adoptive immunotherapy was well tolerated by the recipients 

and not associated with severe GVHD. 


Preliminary clinical data suggest that the efficacy of 

donor BC infusion for the treatment of leukemic relapse 

can be significantly improved by the administration of IL- 

2 in vivo after reinfusion and by a brief incubation of BC 

with IL-2 before the reinfusion. 154 The antitumor efficacy 

of T-lymphocytes can also be enhanced by transfection 

of cytokine genes or new receptors. An interesting 

approach is represented by the binding of TCR to a specific 

anti-tumor antibody Fab fragment or the use of 

bispecific (anti-tumor and anti-CD3) antibodies capable 

of recruiting and expanding tumor-specific CTL at the 

tumor site. 155 Recently, a significant autologous GVHD 

effect has been obtained by the addition of -IFN to 

cyclosporin-A in order to upregulate MHC class II molecules 

. 113 Alternatively, GM-CSF seems to enhance the 

anti-tumor response by stimulating professional APC 

(see below). 156 

In conclusion, there is clear evidence that CTL exert 

their cytotoxic effect through the recognition of minor 

HLA antigens. 157 However, in some patients a very low 

frequency of specific antileukemic CTL, responsible for 

a GVL effect distinct from GVHD, have been isolated. 

Moreover, genetic approaches, such as the transduction 



105 

of donor lymphocytes with a suicide gene, have been 

proposed to control GVHD occurring after CTL administration. 

158 

Ex vivo generation of human dendritic APC 

Clinical investigators are keenly interested in the role 

of APC in the initiation of immune responses because of 

the potential to exploit these cells for immunotherapy 

of cancer and viral diseases. Pioneer studies in mammals 

by Steinman et al. 159 have demonstrated that the specialized 

system of APC is constituted by BM-derived 

dendritic cells (DC). DC are distinguished by their unique 

potency and ability to capture, process, and present 

antigens into peptide-HLA complexes to naive T lymphocytes 

and to deliver the co-stimulatory signals necessary 

for T lymphocyte activation and proliferation. 

Here, we summarize the main experimental evidence 

supporting the working hypothesis that individuals vaccinated 

with DC expanded ex vivo and engineered to 

present tumor associated antigen(s) can mount tumorspecific 

humoral and cellular responses. This can lead to 

tumor regression as well as protective immunity against 

tumor growth in vivo. 159-162 

Identification of dendritic cells 

DC are leukocytes derived from hematopoietic stem 

cells along the myeloid differentiation pathway (Figure 

5). The differentiation of DC is a stepwise process: 163 originating 

from myeloid progenitors in the BM, immature DC 

distribute via blood to tissues where they have the capacity 

to take up and to process antigens. As migratory DC, 

they transit through the lymph or blood to lymphoid 

organs, where they become mature DC, which lose antigen-processing 

ability and acquire superior antigen-presenting 

capacity for T lymphocytes. 163 In humans, DC at 

different developmental stages circulate in PB and they 

are found in virtually all tissues of the body where, 

depending on the location, they are referred to as interstitial 

DC (heart, kidney, gut, and lung), Langerhans cells 

(skin, mucous membranes), interdigitating DC (thymic 

medulla, secondary lymphoid tissue); or veiled cells (lymph, 

blood). 163 DC are regarded as distinct from monocytes/macrophages, 

although they share a common progenitor 

after the CFU-GM stage. However, this has been 

questioned as mixed colonies of dendritic cells and 

macrophages are generated in vitro from single CD34 + 

hematopoietic progenitors more commonly 163-175 than 

pure DC colonies. 176 

DC can be distinguished from other APC by a) morphology; 

b) cell-surface membrane phenotype; and c) 

the strong capacity to present antigens to T cells, usually 

assessed in the allogeneic mixed leukocyte culture 

(reviewed in ref. #163). 

Cutaneous DC, as well as most of the DC generated ex 

vivo from human CD34 + progenitors cells express high 

levels of the surface membrane CD1a antigen (Figure 6). 

Although CD1a antigen can be found on cortical thymocytes 

and some B lymphocytes, its presence (noted by 

immunofluorescence and flow cytometry) is the most 

useful way to quantify the ex vivo generation of DC from 

early precursors. In addition to the CD1a antigen, DC 

express peculiarly high levels of class I and class II histocompatibility 

complex structures, co-stimulatory molecules 

for T-lymphocytes such as B7-1 (CD80) and B7- 

2 (CD86), and adhesion molecules such as ICAM-1 

(CD54) and ICAM-3 (CD50) which are involved in DC- 

Figure 5. Scheme of DC 

development in BM, PB 

and thymus. 

Abbreviations: ly, lymphocyte; 

NK, natural killer 

cells; NSE, non-specific 

esterase; PPSC, pluripotent 

stem cells; CFU-DL, 

dendritic/Langherans cell 

colony forming unit. Modified 

from Reid CA, Br J 

Haematol 1997; 96:217- 

33. 


106 


Figure 6. Flow cytometry evaluation of cell surface phenotype of DC derived ex vivo from CD34 + progenitors on day 12 of culture 

in the presence of GM-CSF, TNF-, SCF and FL. 

dependent T-lymphocyte proliferation. DC lack monocyte/macrophage- 

and lymphocyte-lineage-restricted 

antigens with the exception of the CD4 antigen. 170 As 

shown in Table 3, relevant co-stimulatory (B7-1 and B7- 

2) and adhesion molecules (ICAM-1) are expressed on all 

CD1a + cells derived from CD34 + progenitors, but on fewer 

CD1a + cells derived from monocytes. 177,178 More 

recently, the CD83 cell surface antigen has been recognized 

as a valuable tool for detecting blood DC. 179 

Ex vivo expansion of dendritic cells 

Although DC circulate in the PB and are found in virtually 

all tissues of the body, it is difficult to obtain 

enough cells for ex vivo manipulation because of their 

scattered locations and low number in the blood where 

they account for approximately 0.1% of all leukocytes. 163 

For this reason, it has been of crucial interest to know 

that: a) TNF- cooperates with GM-CSF to generate DC 

from CD34 + hematopoietic progenitors from BM, cord 

blood or PB; 166, 168-176 and b) IL-4 cooperates with GM- 

CSF in the development of DC from circulating monocytes. 

169,180 A detailed description of the methods utilized 

to obtain human DC from myeloid precursors has been 

recently reported. 163 However, in evaluating these methods 

in view of a clinical trial, at least three issues should 

be taken into consideration: 

a) the type of DC generated either from monocytes 

(monocyte-derived DCs) or from CD34 + hematopoietic 

progenitors (CD34 + derived DC); 

b) the source of serum for DC growth in culture; 

c) the combination of cytokines required for optimal ex 

vivo expansion of functional immunostimulatory DC. 

Monocyte-derived DC are being employed in patients 

with advanced stage malignancies in phase I-II clinical trials. 

The trials are particularly aimed at evaluating toxicity 

and immune responses after subcutaneous administration 

following DC pulsing ex vivo with either melanoma 

tumor-associated peptides 181-183 or with B-cell lymphoma 

and myeloma idiotype proteins from autologous 

serum. 151,161 Early reports from clinical studies, in patients 

with melanoma who are HLA-A1 positive and whose 

malignant cells express the MAGE-1 gene, show that in 

vivo immunization with autologous monocyte-derived 

DC pulsed with MAGE-1 gene coded nonapeptide, is not 

toxic and can induce peptide-specific autologous 

melanoma reactive CD8 + cytotoxic T-lymphocyte 

responses in situ at the vaccination site and at distant 

tumor sites 181 as well as in PB. 182 From a technical point 

of view, in these studies the generation of DC from PB 

mononuclear cells is dependent on a culture medium 

necessarily containing GM-CSF without serum or with 

human pooled donor serum. Under these conditions the 

production of DC is quite scarce in comparison with that 

achieved with fetal calf serum, as reported in early studies. 

169 However, the presence of fetal calf serum in the 

culture medium induces undesired DC-mediated immune 

responses to xenogenic proteins as observed in murine 184 

and human preclinical studies. 170 In this regard, experimental 

data suggest that CD34 + cell-derived DC can also 

be generated in the absence of serum if the culture 

medium containing GM-CSF and TNF- is supplemented 

with TGF-1. 185 

Modalities for the large-scale procurement of functional 

DC from CD34 + hematopoietic progenitors in 

patients with cancer have been evaluated. 170 It was 

found that mobilized PB progenitors currently utilized in 

phase III trials 186 include a fraction of CD34 + DC precursors 

which give origin ex vivo to a progeny with the char- 



107 

acteristics of professional APC i.e., typical DC morphology 

and immunophenotype undistinguishable from cutaneous 

Langerhans cells and DC from cord blood and BM 

CD34 + cells. Most importantly, these ex vivo generated 

DC retained the capacity to process and present antigens 

to T lymphocytes as demonstrated by elicitation of HLA 

class II and class I-restricted activation of CD4 + and CD8 + 

autologous T lymphocytes in response to xenogenic antigens 

of fetal calf serum 170 or melanoma tumor-associated 

antigen peptides, 177, 178 respectively. Quantification 

of progenitors of DC by limiting dilution analysis of 

CD34 + cells sorted from blood cell autografts showed 

that they are approximately 140-fold more numerous 

than in steady-state control autograft. To obtain this 

favorable result, blood cell autografts were collected at 

the time of maximal mobilization of CD34 + cells into PB 

as occurs after treatment with high-dose cyclophosphamide 

and cytokines. 

In a systematic search for culture conditions capable 

of ameliorating the ex vivo generation of DC dendritic 

cells, a variety of exogenous stimuli have been evaluated 

as well as monocyte-derived versus CD34 + cell-derived 

DC. 170,177,178 In this respect, it has been shown that GM- 

CSF plus TNF--dependent generation of DC from mobilized 

CD34 + cells is 2.5 fold enhanced by either FL or SCF, 

and 5-fold enhanced by a combination of these growth 

factors. In addition, autologous high-dose chemotherapy 

recovery phase serum rather than fetal calf serum or 

human donor pooled AB serum has been shown to be the 

optimal serum for the generation of DC. Regardless of 

the precise mechanism of action of FL and SCF in association 

with GM-CSF and TNF- on the enhancement of 

DC differentiation and proliferation, these findings have 

provided new advantageous tools for the large-scale generation 

of DC from mobilized CD34 + cells in patients 

undergoing cancer treatment. In fact, the stimulation of 

CD34 + cells from an average blood cell autograft should 

permit the generation of a median of 0.610 9 /kg DC from 

Table 3. Phenotype of CD1a + dendritic cells generated from 

mobilized CD34 + progenitors or from monocytes. 

CD1a + cells derived from 

Antigens CD34 + progenitors Monocytes 

CD14 2% 3% 

CD80 100% 84% 

CD86 100% 67% 

CD54 100% 67% 

MHC Class I 

HLA-A0201 100% 100% 

MHC Class II 

HLA-DR 100% 100% 

HLADQ 60% 89% 

Modified from Mortarini et al., Cancer Res, in press. 

an average 65 kg individual, i.e., almost 4010 9 DC. In 

contrast, differentiation of DC from monocytes in the 

presence of autologous high-dose chemotherapy recovery 

phase serum plus GM-CSF and IL-4 is not associated 

with a comparably high outgrowth of DC. 177,178 These 

observations, together with the weaker expression of costimulatory 

molecules in monocyte-derived DC in comparison 

with CD34 + cell-derived DC 187 may favor the utilization 

of the latter source of APC for the development 

of active immunization programs involving DC in humans. 

The comparative efficiency as APC of DC derived from 

monocytes versus CD34 + hematopoietic progenitors has 

recently been studied with DC isolated from blood of 

patients with melanoma. In particular, it has been shown 

that DC derived from G-CSF-mobilized CD34 + cells are 

more efficient than those derived from monocytes in 

inducing melanoma tumor-associated antigen peptide 

specific activation of autologous CD8 + cytotoxic T-lymphocytes. 

Interestingly, in the same experiments the latter 

cells were also capable of lysing a panel of melanoma 

cell lines sharing the same HLA class I alleles with the 

patients from whom CD8 + cytotoxic T-lymphocytes were 

generated with tumor-associated antigen peptide pulsed 

autologous DC. 177,178 Moreover, CD34 + cells mobilized into 

PB by G-CSF were shown to be capable of generating a 

higher number of mature and fully functional DC than 

their BM counterparts. 188 However, it should be pointed 

out that, at present, clinical studies utilizing DC as vehicles 

for anti-tumor vaccination have been carried out 

with monocyte-derived DC either freshly isolated from 

PB 152 or cultured ex vivo. 161, 181-183 In addition, culture conditions 

which allow the large scale production of terminally 

differentiated and fully functional monocytederived 

DC have recently been described. 189 

Dendritic cells for antitumor cell therapy 

An extensive review on the clinical use of DC is beyond 

the scope of this paper, however, a few remarks in this 

regard are needed. 

The goal of vaccination is the induction of protective 

immunity. Originally, vaccinations were used in the setting 

of infectious diseases, but are now expanding to 

include the treatment of allergy, autoimmune diseases, 

and tumors. A rational approach to vaccination involves 

3 steps: a) the identification of the protective effector 

mechanisms, b) the choice of an antigen that can induce 

a response in all individuals, and c) the use of an appropriate 

way to deliver the vaccine to induce the proper 

type of response. 159-162 

It has now been demonstrated that certain tumor cells 

are antigenic by expressing tumor-associated antigens 

that can be recognized by T lymphocytes in a syngeneic 

host. However, they are often poorly immunogenic, at 

least in part because they lack the cellular armamentarium 

for specific T-lymphocyte recognition, activation, 

and co-stimulation typical of APC especially DC. 190,191 

Different mechanisms may account for the ability of 

tumor cells to evade immune responses. Tumor cells may 


108 


display low immunogenicity through low MHC and/or 

tumor-speciifc antigen expression or may downregulate 

Fas and constitutively express Fas-Ligand, which binds to 

Fas on cytotoxic T-lymphocytes, resulting in apoptosis of 

the latter. 192 Furthermore, it has recently been shown 

that vascular endothelial growth factor produced by 

tumors inhibits the functional maturation of CD34 + cellderived 

DC. 193 When considering the use of the unique 

antigen-presenting capacity of DC to prime specific antitumor 

T-lymphocytes, this phenomenon should be taken 

into account, as it may result in poor recovery and 

function of DC directly recovered from the blood of cancer 

patients. In contrast, dendritic APC expanded ex vivo 

in the presence of cytokines and in the absence of 

inhibitory factors released by tumors would probably be 

functional. 193-195 However, it should also be considered 

that published data demonstrate that whereas in vivo 

administration of DC loaded with low doses of tumor 

antigen enhances antitumor immunity, DC pulsed with 

high doses of antigen or high numbers of tumor antigenpulsed 

DC may inhibit development of immunity. 196 This 

finding supports the notion that stimulation of DC-mediated 

antigen presentation in vivo may act in a tolerogenic 

or immunogenic fashion depending on a variety of 

partially understood factors. 197 

Basically, there are at least two approaches to tumor 

vaccination (Figure 7). The first is to identify a tumorassociated 

antigen to be used as a vaccine, the second 

is to increase the immunogenicity of tumor cells and let 

the immune system decide which antigen to target. 

Indeed, in experimental models with the appropriate 

manipulation exploiting the physiologic function of 

antigen-presenting DC, the immune system can be 

induced to mount responses that can kill tumor cells 

and also protect animals from subsequent challenge 

even with a poorly immunogenic tumor. 185,198-203 

Given the richness of recently identified tumor associated 

antigens and their corresponding peptide epitopes 

recognized by MHC-restricted CD8 + or CD4 + T 

lymphocytes (Table 4), investigators are currently evaluating 

the clinical efficacy of specific tumor-associated 

antigen-based vaccines for the treatment of various 

malignancies. Recently, in a cooperative clinical trial it 

was observed that partial tumor regressions can occur 

in HLA-A1 + patients with melanoma treated with a 

naked MAGE 3 peptide epitope vaccine even in the 

absence of any engineering of antigen-presenting cells 

or adjuvant cytokine(s). 204 This clinical evidence induces 

the belief that the effectiveness of peptide-based vaccines 

is likely to benefit further from administration of 

appropriate cytokines 156,205,206 or cellular adjuvants (e.g. 

DC) capable of promoting cellular immunity. 

Among hematologic malignancies, CML is being 

intensively evaluated as a possible target of dendritic 

APC-based immunotherapy. It is well known that CML 

is characterized by a specific translocation of the c-abl 

oncogene (9q34) to the bcr region on chromosome 22 

(22q11). Alternative recombination sites involving either 

the second or third exon of the bcr gene splicing to exon 

Table 4. Tumor antigens capable of eliciting T-lymphocyte 

responses. 

Activated oncogene products 

Mutated: 

Rearranged: 

Overexpressed: 

Tumor suppressor gene products 

p53 mutations 

Position 12 point mutation of 21 ras 

bcr-abl (b3a2 peptide) 

HER-2/neu 

Reactivated embryonic gene products 

MAGE family (at least 12 genes) 

BAGE 

GAGE 

Melanocyte differentiation antigens 

Tyrosinase protein 

Melan-A/MART1 

gp100 

gp75 

Viral gene products 

Idiotype epitopes 

Human papilloma virus antigens (E6, E7) 

EBV EBNA-1 gene products 

Ig and TCR hypervariable regions 

2 of the abl gene yield two potential fusion gene transcripts, 

b2a2 and b3a2, respectively. The translated 210- 

kd bcr-abl fusion protein, which has abnormal tyrosine 

kinase activity, includes a new potentially antigenic 

sequence at the fusion site: a new amino acid is generated 

at the junctional site by the fusion event; in the 

b2a2 fusion a glutamic acid (E) is encoded, whereas in 

the b3a2 recombination event a lysine (K) is generated. 

Interestingly, a bcr-abl peptide from the b3a2 fusion 

region has been found to be immunogenic in mice. 207 In 

humans, binding of b3a2 peptides to various HLA class 

I alleles 208 and priming of CD8 + cytotoxic T-lymphocytes 

in vitro has been described although the capacity of 

these peptide-specific CD8 + T-lymphocytes to lyse CML 

cells has not been determined. 209 In contrast, in a recent 

study it has been demonstrated that CD4 + T-lymphocytes 

can be identified that proliferate in an HLA class- 

II restricted manner in response to a 11mer 

(GFKQSSKALQR) b3a2 peptide especially when the latter 

is presented by purified CMRF-44 + blood DC. 210 In the 

same study the peptide-specific CD4 + T-lymphocytes 

were able to respond to the whole protein in crude 

extract from CML cells. Intriguingly, dendritic antigenpresenting 

cells in CML patients can be derived from 

the malignant clone and these malignant dendritic cells 

can induce antileukemic reactivity in autologous T lymphocytes 

without the necessity of additional exogenous 

antigens. 211 

Although, the above observations cannot be extrapolated 

a priori to other malignancies carrying specific 

translocations and corresponding fusion genes and 

products, 212 further investigations on the possible clin- 



109 

ical application of bcr-abl peptide(s) presented by autologous 

dendritic antigen-presenting cells are warranted. 

The translation into the clinical setting of the above 

experimental hints favoring the use of DC pulsed ex vivo 

with synthetic tumor-associated antigen peptides for 

effective cell therapy in humans is likely to be hampered 

by: a) the limited availability of patients with HLA typing 

compatible for the utilized tumor-associated antigen 

peptide(s); b) the occurrence of independent mechanisms 

of tumor escape in vivo such as loss of expression 

of tumor-associated antigens or of HLA class I during 

tumor progression; and c) the short duration of the 

immune responses thus requiring annoying boost vaccinations. 

It has been suggested 213 that these limitations 

may be overcome by transduction of genes encoding 

relevant proteins into DC or their progenitors so that 

DC could tailor peptides to their own HLA molecules 

thus obviating the need to synthesize tumor-specific 

peptides most of which have stringent HLA restrictions. 

A further advantage of the transduction approach may 

be the stable long-term expression of the antigen by 

the DC, which would allow its presentation to the 

immune system for longer periods without the concerns 

about the turnover of preformed peptide/HLA complexes 

in vivo after immunization. 214 

Based on the above experimental body of evidence, a 

pioneer clinical trial has evaluated the ability of autologous 

monocyte-derived DC pulsed ex vivo with non 

Hodgkin’s lymphoma-specific idiotype protein to stimulate 

host immunity when infused as a vaccine. 151 In this 

study active immunotherapy of patients with B-cell lymphoma 

against idiotypic determinants led to anti-tumor 

immunity that correlated with improved clinical outcome 

in some patients. 

Regardless of the type of cell manipulation (Figure 7) 

(ex vivo pulse with tumor-associated antigen peptides 

versus transduction with tumor associated antigen 

genes versus immunization with fusions of dendritic and 

carcinoma cells 215 ) that will be successful in clinical 

applications the necessity of methods of generating 

large numbers of functional DC is implicit for the evolution 

of such studies. 


Since DC have been shown to be intimately involved 

in the generation of CD4 + and CD8 + T-lymphocyte mediated 

tumor-specific immunity, it is attractive to speculate 

that vaccination with DC pulsed or engineered ex 

vivo to present tumor antigen(s) may be effective in generating 

tumor immunity in vivo. Among the recently 

prospected sources of DC, namely BM, neonatal cord 

blood, and adult PB, the last is certainly the richest and 

most accessible in all patients with cancer, although it 

remains to be confirmed whether functional differences 

will favor the utilization of monocyte- versus CD34 + cellderived 

DC. Thus, in the clinical setting of adoptive 

immunotherapy for patients with malignancies, a therapeutic 

protocol could be envisioned involving the mobilization 

of CD34 + cells into PB with hematopoietic 

growth factors, with or without prior intensive chemotherapy. 

Thus, enrichment of hematopoietic progenitor 

cells could be followed by ex vivo generation of DC 

pulsed or engineered to present tumor antigen(s), to be 

reinfused as a potential secondary tumor-specific immunotherapy 

or vaccination. It remains to be established 

whether the latter effect could be further enhanced by 

in vivo adjuvant cytokines such as GM-CSF and/or FL, as 

occurs in murine models. 

A potential advantage of ex vivo immune cell therapy 

over direct in vivo immune intervention, is the lack 

of functional inhibition that may occur in vivo. This 

hypothesis is based on a recently proposed mechanism 

of tumor escape/resistance from the host immune system, 

in which cancer cells produce a vascular endothelial 

growth factor that impairs antigen presentation 

required to induce specific antitumor immune responses 

in vivo. 193 

Figure 7. Sources of tumor antigens 

for DC-based cancer vaccines. 


110 



All the authors equally contributed to the manuscript 

and they are listed in alphabetical order. 

Disclosures 

Conflict of interest. This review article was prepared by 

a group of experts designated by Haematologica and by 

representatives of two pharmaceutical companies, Amgen 

Italia SpA and Dompé Biotec SpA, both from Milan, Italy. 

This co-operation between a medical journal and pharmaceutical 

companies is based on the common aim of 

achieving optimal use of new therapeutic procedures in 

medical practice. In agreement with the Journal’s Conflict 

of Interest policy, the reader is given the following 

information. The preparation of this manuscript was supported 

by educational grants from the two companies. 

Dompé Biotec SpA sells G-CSF and rHuEpo in Italy, and 

Amgen Italia SpA has a stake in Dompé Biotec SpA. 


Manuscript received March 11, 1998; accepted June 

10, 1998. 

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eview 

Cell therapy: achievements and 

perspectives 


Correspondence: Prof. Sante Tura, Istituto di Ematologia e Oncologia 

Seragnoli, Policlinico S. Orsola, Via Massarenti 9 40138 Bologna, Italy 

Phone: +39-051-390413 – Fax: +39-051-398973 – E-mail: 

tura@orsola-malpighi.med.unibo.it 

CLAUDIO BORDIGNON,* CARMELO CARLO-STELLA,° MARIO PAOLO 

COLOMBO, @ ARMANDO DE VINCENTIIS,^ LUIGI LANATA,^ ROBERTO 

MASSIMO LEMOLI, § FRANCO LOCATELLI, ** ATTILIO OLIVIERI,°° 

DAMIANO RONDELLI, § PAOLA ZANON,^ SANTE TURA § 

*Institute of Hematology, S. Raffaele Hospital, Milan; 

°Division of Oncology, Istituto Nazionale dei Tumori, Milan; 

@ 

Experimental Oncology D, Istituto Nazionale dei Tumori, 

Milan; ^Dompé Biotec, Milan; § Institute of Hematology and 

Medical Oncology, University of Bologna and Policlinico S. 

Orsola Bologna; °°Division of Hematology, University of 

Ancona, Ancona; ** Department of Pediatrics, University of 

Pavia Medical School and IRCCS Policlinico S. Matteo, Pavia; 

and Amgen Italia, Milan, Italy 

Background and Objectives. Cell therapy can be considered 

as a strategy aimed at replacing, repairing, or 

enhancing the biological function of a damaged tissue or 

system by means of autologous or allogeneic cells. There 

have been major advances in this field in the last few 

years. This has prompted the Working Group on 

Hematopoietic Cells to examine the current utilization of 

this therapy in clinical hematology. 

Evidence and Information Sources. The method employed 

for preparing this review was that of informal consensus 

development. Members of the Working Group met three 

times, and the participants at these meetings examined a 

list of problems previously prepared by the chairman. They 

discussed the single points in order to reach an agreement 

on different opinions and eventually approved the 

final manuscript. Some of the authors of the present 

review have been working in the field of cell therapy and 

have contributed original papers in peer-reviewed journals. 

In addition, the material examined in the present 

review includes articles and abstracts published in journals 

covered by the Science Citation Index and Medline. 

State of the Art. Lymphokine-activated killer (LAK) and 

tumor-infiltrating lymphocytes (TIL) have been used since 

the ’70s mainly in end-stage patients with solid tumors, 

but the clinical benefits of these treatments has not been 

clearly documented. TIL are more specific and potent 

cytotoxic effectors than LAK, but only in few patients 

(mainly in those with solid tumors such as melanoma and 

glioblastoma) can their clinical use be considered potentially 

useful. Adoptive immunotherapy with donor lymphocyte 

infusions has proved to be effective, particularly in 

patients with chronic myeloid leukemia, in restoring a 

state of hematologic remission after leukemia relapse 

occurring following an allograft. The infusion of donor T- 

cells can also have a role in the treatment of patients with 

Epstein-Barr virus (EBV)-induced post-transplant lymphoproliferative 

disorders. However, in this regard, generation 

and infusion of donor-derived, virus specific T-cell lines or 

clones represents a more sophisticated and safer 

approach for treatment of viral complications occurring in 

immunocompromized patients. Whereas too few clinical 

trials have been performed so far to draw any firm conclusion, 

based on animal studies dendritic cell-based 

immunotherapy holds promises of exerting an effective 

anti-tumor activity. Despite leukemic cells not being 

immunogenic, induction on their surface of co-stimulatory 

molecules or generation of leukemic dendritic cells may 

induce antileukemic cytotoxic T-cell responses. Tumor 

cells express a variety of antigens and can be genetically 

manipulated to become immunogenic. The main in vitro 

and in vivo functional characteristics of marrow mesenchymal 

stem cells (MSCs) with particular emphasis on 

their hematopoietic regulatory role are reviewed. In addition, 

prerequisites for clinical applications using cultureexpanded 

mesenchymal cells are discussed 

Perspectives. The opportuneness of using LAK cells or 

activated natural killer (NK) cells in hematologic patients 

with low tumor burden (e.g. after stem cell transplantation) 

should be further explored. Moreover the role of new 

cytokines in enhancing the antineoplastic activity of NK 

cells and the infusion of selected NK in alternative to CTL 

for graft versus leukemia (GVL) disease (avoiding graft 

versus host disease (GvHD) seems very promising. Separation 

of GVL from GvHD through generation and infusion 

of leukemia-specific T-cell clones or lines is one of the 

most intriguing and promising fields of investigations for 

the future. Likewise, strategies devised to improve 

immune-reconstitution and restore specific anti-infectious 

functions through either induction of unresponsiveness to 

recipient alloantigens or removal of alloreactive donor T- 

cells might increase the applicability and success of 

hematopoietic stem cell transplantation. Cellular 

immunotherapy with DC must be standardized and several 

critical points, discussed in the chapter, have to be 

properly addressed with specific clinical studies. Stimulation 

of leukemic cells via CD40 receptor and transduction 

of tumor cells with co-stimulatory molecules and/or 

cytokines may be useful to prevent a tumor escaping 

immune surveillance. Tumor cells can be genetically modified 

to interact directly with dendritic cells in vivo or 

recombinant antigen can be delivered to dendritic cells 

using attenuated bacterial vectors for oral vaccination. 

MSCs represent an attractive therapeutic tool capable of 

playing a role in a wide range of clinical applications in 

the context of both cell and gene therapy strategies. 


Key words: cell therapy 


118 

C. Bordignon et al. 

The role of lymphoid cells in rejecting solid tumors 

transplanted into animal models was strongly suggested 

in the first decades of this century by J.B. 

Murphy (1926) 1 who, nonetheless, did not demonstrate 

it formally. Following his revolutionary findings on the 

immunologic mechanisms of allogeneic skin tolerance 

and rejection, in 1958 P.B. Medawar 2 coined the term 

“immunologically competent cell” to define a cell that is 

“fully qualified to undertake an immunological 

response”. Forty years after Medawar’s definition, the 

development of molecular and biological research has 

enormously improved our understanding of the complex 

regulatory mechanisms of proliferation, differentiation 

and function of the cells involved in the immune 

response. The concomitant evolution of biotechnology 

has also progressively given new opportunities to isolate 

and/or expand cell subsets, or to develop new molecules, 

in order to amplify or modify specific cell functions. 

Thus, the possibility of exploiting a specific cell 

function, in vivo or ex vivo, to obtain a therapeutic 

effect, such as an anti-tumor cytotoxic activity, or complete 

immune reconstitution, is part of the definition of 

cell therapy that is herein reviewed. 

In a general context, cell therapy can be considered as 

a strategy aimed at replacing, repairing, or enhancing the 

biological function of a damaged tissue or system by 

means of autologous or allogeneic cells. For instance, in 

the hematopoietic system cell therapy may include: a) 

removal or enrichment of various cell populations; b) 

expansion of hematopoietic cell subsets; c) expansion or 

activation of lymphocytes for immunotherapy; and d) 

genetic modification of lymphoid or hematopoietic cells, 

when these cells are intended to engraft permanently or 

transiently in the recipient and/or be used in the treatment 

of a disease. 

This review contains extensive considerations on the 

clinical use of lymphocytes and/or natural killer (NK) cells 

as a strategic weapon in preventing or curing the neoplastic 

relapse after chemotherapy and/or hematopoietic 

stem cell transplantation, the infusion of T-cell clones or 

lines able to restore a specific anti-viral activity, the in 

vivo and ex vivo potential use of dendritic cells to generate 

a tumor-specific cytotoxic activity, and the innovative 

use of donor stromal cells in conjunction with stem 

cell transplantation. Even tumor cells engineered to 

express cytokine or co-stimulatory molecules and representing 

the entire antigenic repertoire of a certain neoplasia 

can be used as a cancer vaccine. On the other hand, 

a broad definition of cell therapy at this time should 

include autologous and allogeneic transplants of purified 

hematopoietic stem cells, which, however, have been 

extensively reviewed in previously published reports. 3-5 

Tumor escape from immune surveillance 

Although several mechanisms allowing tumor cells to 

escape the host immune protection have been recently 

described, it is conceivable that others remain still 

undiscovered. However, tumor cells often fail to induce 

specific immune responses because of their inability to 

function as competent antigen presenting cells (APC). 

Professional APC, in fact, are fully capable of delivering 

two signals to T cells: 6 the first is antigen (Ag) specific 

and is mediated by the interaction of MHC molecules 

carrying antigenic peptides with the T-cell receptor 

(TCR), and the second signal, or co-stimulatory signal, is 

not Ag-specific and is principally mediated by members 

of the B7 family, namely B7-1 (CD80) and B7-2 (CD86), 

via their T-cell receptors CD28 and CTLA-4, and/or by 

CD40 via CD40L binding. 7-8 

The lack of a suitable tumor-associated antigen (TAA), 910 

or defective antigen processing, 11 or production of 

immunologic inhibitors, 12 or lack of co-stimulatory signaling 

by tumor cells, 13 as other mechanisms, can all contribute 

to prevent or abrogate an anti-tumor immune 

response. Moreover, neoplastic cells within the same 

tumor may show different reactivity with monoclonal 

antibodies (mAbs), cytotoxic T-lymphocyte (CTL) clones 

and lymphokine-activated killer (LAK) or tumor infiltrating 

lymphocyte (TIL) populations. Furthermore, despite 

many tumors having TAA and potentially being capable 

of stimulating T cells, in some cases they fail to induce 

an adequate CTL frequency in vitro. In other cases the 

antigen loss can be one of the mechanisms for escaping 

immune protection. 14 Private TAA often result from 

mutated gene products 15 and are potentially useful for 

developing tumor vaccines. These Ags, however, can be 

down-regulated or modified by point mutations, inducing 

a consistent reduction or the abrogation of peptidebinding 

by specific CTLs. Another critical issue for preventing 

immune responses is the absence, or the downregulation 

of MHC molecules on neoplastic cells, as 

shown in animal models, 16 or in human lung cancer. 17 

The pivotal role of B7 molecules in the immune 

response has been demonstrated in a variety of experimental 

models showing that after TCR signaling, binding 

of CD28 induces T-cell interleukin-2 (IL-2) secretion, 

proliferation and effector function, whereas presentation 

of the antigen in the absence of co-stimuli 

induces T-cell unresponsiveness either by anergy or 

clonal deletion. Therefore, since most neoplastic cells 

lack co-stimulatory molecules, it is likely that they can 

deliver the first signal through the MHC:TCR binding, 

but not the second one, thus driving host T-cells to tolerate 

the tumor. 18 Potential strategies to prevent or to 

reverse T-cell tolerance by CD28 or CD40L stimulation, 

or IL-2 receptor triggering, are under investigation. 

Further mechanisms impairing immunologic responses 

include the suppression of cytotoxic activity by the 

release of soluble factors or by direct cell-contact. In 

fact, tumor cells may secrete cytokines, such as MIP-1, 

or TGF-, or IL-10, that may be capable of inhibiting T 

cell activity. 12 Alternatively, tumor cells may induce T- 

cell apoptotic clonal deletion by increasing Fas:Fas-L 

ligation. 19 A schematic example of the main defects 

described in the tumor cell: T cell interaction is shown 

in Figure 1. 

Finally, since normal lymphocytes can bind to venular 

endothelial cells through adhesion receptors, such as 


Cell therapy 

119 

L-selectin or / integrins, and then by rolling out they 

can reach tissues, lack of adhesion receptors on tumor 

vessels might prevent lymphocytic infiltration and contact 

with neoplastic cells. 20 In this case even the best 

strategies aimed at modifying the immunogenicity of 

tumor cells may not be successful at overcoming the 

lack of an antitumor immune surveillance. 

Figure 1. Main mechanisms for tumor escape of immune 

surveillance. 

Lymphokine-activated killer and tumorinfiltrating 

lymphocytes: 

past and present 

Natural killer cells and lymphokine-activated 

killer phenomenon 

Since 1970 NK cells have been recognized as a functionally 

distinct subset of cytotoxic effectors (Table 1). 

NK cells from rodent or from human peripheral blood kill 

a wide range of tumor cells and virus-transformed cells 

without the need for prior sensitization. 

In 1975 Heberman et al. 21 described a phenomenon of 

normal unstimulated lymphoid cells lysing cultured 

tumor-cell lines in a short in vitro assay. This cytolytic 

activity was subsequently shown to be neither MHC 

restricted nor mediated by the T-cell receptor complex. 

Such ability to eliminate tumor cells, but not normal 

tissues suggests that NK cells are not only involved in 

the control of cancer, but also that their presence and 

state of activation are important in the outcome of the 

disease and finally in the treatment of tumors. 22 

Mature NK have a clonally-distributed ability to recognize 

their target cell by class I MHC alleles. Karre et 

al. 23 demonstrated in a murine model that leukemia cell 

Table 1. Characteristics of cytotoxic effectors useful for adoptive immunotherapy of cancer. 

Effector type CTLs TILs NK cells LAK cells CIKs 

Source Peripheral Metastatic Peripheral blood NK cells Subset of 

blood lymphocytes lymph nodes and bone marrow and CTL activated T-lymphocytes 

by IL-2 

activated by cytokines 

Culture conditions: 

Tumor stimulation Yes None None None None 

need of IL-2 for response ++++ ++++ ++++ (CD56 dim ) - ++++,IFN-, 

+ (CD56 bright ) IL-12, anti-CD3 antibody 

Duration of culture 6 weeks 4 weeks 2-3 weeks 2-5 days 2-5 weeks 

target cells in vitro allogeneic cells autologous K-562 Raji, Daudi autologous and 

tumoral cells 

allogeneic tumoral cells 

In vitro cytolytic activity : 

MHC restricted to none: spontaneous none: lyse cytotoxic activity 

allogeneic cells lysis of virus-infected cells, a wide spectrum of superior to LAK; 

Specificity Restricted to autologous tumoral cells, tumor cells lyse whether 

MHC not restricted, autologous tumor allogeneic tumoral cells including cells CML autologous 

toward opsonized (MHC and/or that are or allogeneic blasts 

cells (ADCC) tumor associated) antibody-dependent resistant to NK; but do not lyse 

antigens) cell-mediated cytotoxicity 

(ADCC) specificity 

normal hematopoietic 

progenitors 

Effector phenotype -CD3+/4+ ,CD3+/8+ CD3+/8+/56+ CD3-/CD16+/CD56+ CD56+ CD3+/56+ 

-CD3+/8+/16+. 

CD25+ 

CTL: cytotoxic T-lymphocytes; TIL: tumor inflitrating lymphocytes; NK: natural killer; LAK: lymphokine-activated killer; CIK: citokine-activated killer. 


120 


lines lacking certain MHC class I molecules were killed 

by NK cells, while parental H-2 bearing line were not. In 

humans both NK and a subset of cytotoxic T-lymphocytes 

express receptors for MHC HLA class I molecules 

which exert an inhibitory effect on cell-mediated cytotoxicity. 

These surface molecules, belonging to the 

immunoglobulin superfamily, have been termed killercell 

inhibitory receptors (KIRs). Two distinct KIR families 

have been described: a) KIRs with IG-like domains, recognizing 

HLA-A, B and C alleles; and b) the CD94/NKG2A 

subtype, with a lectin domain, recognizing peptides 

related to the HLA-E class I system. 24 The interaction 

between KIRs and the corresponding MHC class I antigens 

prevent NK from killing target cells expressing self 

HLA alleles. 25 In addition some NK also express receptors 

that induce lysis of target cells expressing foreign HLA 

class I alleles. 26 

These findings explain the mechanism of self-tolerance 

in the NK population, which can be disrupted as a 

consequence of tumor transformation or viral infection 

or any other events inducing a loss or a substantial modification 

of class I molecules. These transformed cells 

can easily escape detection by T-lymphocytes by down 

regulating MHC antigens, but are normally destroyed 

by autologous NK cells. 27 

The NK cell compartment is heterogeneous and distinct 

NK subsets have been characterized. The most 

informative functional differences are based on relative 

CD56 fluorescence: only CD56 +bright , but not CD56 +dim NK, 

express the high-affinity IL-2 receptor, and respond to 

the low IL-2 concentration. They also expand 10 times 

more than CD56 +dim . 28 

NK progenitors differentiate into immature NK in 

presence of SCF, IL-7, IL-2 and bone marrow stromal 

cells producing IL-15. This last cytokine can directly 

induce CD34 + cells to differentiate into NK cells in the 

absence of IL-2. 29 The second step of NK maturation is 

stroma-independent and is characterized by the appearance 

of CD56 molecules: the intensity of CD56 expression 

reflects the proliferative potential and the killing 

ability of the NK. 30 

The effects of IL-2 on NK precursors appears to be 

stage-specific, confirming that, while mature NK precursors 

readily respond to IL-2, more immature progenitors 

need complete mixtures of cytokines and stromal 

cells. NK cells, after incubation with IL-2, become lymphokine-activated 

killer cells: LAK cells kill NK-resistant 

cell targets (e.g. Daudi cell line) and a wide spectrum of 

different fresh tumor cells in both autologous and allogeneic 

settings, while fresh normal tissues are resistant 

to LAK-mediated lysis. 31 

Although some tissue-resident lymphocytes may have 

spontaneous LAK activity, normal blood mononuclear 

cells (MNC) do not show any LAK activity, which can be 

acquired only after incubation with interleukin-2. 32 

These NK activated cells express new markers such as 

CD25, MHC class II antigens and fibronectin. 33 LAK activity 

can be generated not only in peripheral blood MNC, 

but also in the thymus, spleen, bone marrow and in MNC 

from lymph nodes. Many experimental data suggest that 

most LAK precursors are present in the null lymphocyte 

population. 

In humans LAK activity was much more evident in the 

MNC population after depletion of macrophages, T and 

B-cells. Residual MNC were CD16 + and did not show T- 

cell markers. 34 

LAK cells: experimental observations and 

clinical trials 

In animal models the combined administration of IL- 

2 and LAK has proved to be more efficacious than either 

component alone. In murine models the administration 

of high-dose IL-2 alone or in conjunction with LAK cells 

induced the regression of lung, liver and subdermal 

metastases. The antitumor effect correlated both with 

the IL-2 dose and the number of LAK cells administered; 

finally at different doses of IL-2, the concomitant 

administration of LAK cells resulted in increased reduction 

in established metastases. 35,36 

LAK cells are capable of inhibiting acute myeloid 

leukemia (AML) progenitor growth, and leukemia incidence 

is higher in people with deficiency of NK cells. 37 

In the large majority of patients at diagnosis or in relapse 

blasts appear resistant to lysis by autologous LAK cells. 

Moreover, about 90% of patients with acute leukemia in 

complete remission do not show spontaneous cytotoxicity 

against autologous blast cells, but ex vivo treatment 

with IL-2 restores cytolytic activity in 37.5% of these 

patients. 38 In a population of 42 patients with AML in 

complete remission, LAK cytotoxicity against autologous 

leukemic blasts was not significantly different from LAK 

of normal subjects. 39 However, multivariate analysis for 

prognostic factors showed that patients whose LAK had 

more lytic activity on leukemic blasts had significantly 

less risk of relapse than patients with poor LAK activity. 

In the first National Cancer Institute trial endstage 

cancer patients received high-dose bolus IL-2 therapy 

for 3 to 5 days. 35 Lymphocytes harvested during the systemic 

treatment with IL-2 were cultured in the presence 

of IL-2 for 2 to 4 days, in order to expand the LAK cell 

number; autologous LAK cells were then reinfused into 

patients in combination with the high-dose intravenous 

bolus IL-2 administration. Of 72 patients with renal cancer 

who were treated, 33% obtained an overall response, 

8 with complete response (CR) and 17 with partial 

response (PR); of 48 patients with metastatic melanoma 

21% responded with 4 CR and 6 PR; responses were 

also observed in patients with colorectal carcinoma and 

non-Hodgkin’s lymphoma. 40 The ILWG used the same 

strategy, obtaining an overall response rate of 19% in 

patients with melanoma and 16% in those with renal 

carcinoma. 41 After these initial trials the original schema 

of the National Cancer Institute was modified with the 

use of IL-2 in continuous infusion rather than bolus 

injection in order to reduce the systemic toxicity. 42 

The first randomized study, comparing IL-2 alone to 

IL-2 plus LAK cells, was published by McCabe. 43 This trial 

included patients with either renal carcinoma or 


Cell therapy 

121 

melanoma; no significant difference in response rate 

between the two groups was reported. A second randomized 

study at the National Cancer Institute followed 

these pioneering experiences, comparing IL-2 alone to 

IL-2/LAK cells: 44 181 patients were enrolled in this study 

(90 in the IL-2 plus LAK arm and 91 in the IL-2 alone). 

A total of 10 CR were observed in the IL-2/LAK arm as 

compared to only 3 in the IL-2 alone arm. The overall 

response rates were similar, but there was a survival 

trend (p=0.07) in favor of the IL-2/LAK arm: the actuarial 

survival for patients receiving IL-2/LAK was 31% 

compared to 17% for those receiving IL-2 alone. Toxicity 

was virtually equivalent in both arms and the majority 

of toxic effects were due to IL-2 administration, 

while the only complication associated with LAK therapy 

was transient hepatitis A, due to contamination of 

the culture medium. 

A third randomized trial, comparing IL-2 alone versus 

IL-2/LAK therapy was published in 1995. 45 In this study 

only patients with advanced renal carcinoma were 

treated and IL-2 was administered as a continuous infusion 

rather than bolus injection. Seventy-one patients 

entered (36 vs. 35) this trial and only 6% overall 

obtained a major response, with a median survival of 13 

months; the difference between the two groups was not 

significant. Therefore it may be concluded that LAK cells 

did not improve the activity of IL-2 in patients with 

advanced renal carcinoma. 

The last randomized trial published was conducted in 

174 primary lung carcinoma patients after surgery, comparing 

the adjuvant treatment with IL-2 plus LAK (for 

two years) with conventional treatment. 46 The 5- and 9- 

year survival rates were significantly superior in patients 

receiving IL-2/LAK therapy, but no comparison was 

planned between Il-2 alone and IL-2/LAK therapy. The 

impressive results obtained in terms of overall survival 

also in non-curative cases after surgery (OS: 52% at 5 

years) should probably be interpreted as due to fact that 

in this study patients received the immunotherapy after 

consistent tumor debulking. 

Other clinical trials (non-randomized) were conducted 

with IL-2 with or without LAK cells, and the overall 

response rate was similar for both the immunotherapy 

modalities. 47,48 The detailed review of other (non-ran- 

Table 2. Clinical trials with LAK cells. 

Treatment schedule 

Author Year Patients Kind of tumor IL-2 (dose and schedule) LAK cells Response 

Rosenberg 1987 157 Melanoma Randomize: IL-2 vs. IL-2+LAK CR: 2.2% vs 7.5% 

PR: 10.9% vs 14.2% 

mR: 2.2% vs 9.4% 

West 1987 40 Miscellaneous 1-7x10 6 U/m 2 /day CI CR+PR: 22-28% 

Yoshida 1988 23 Brain tumor Direct injection of LAK into recurrent tumor Regression: 26% 

cavity + IL-2 (50-400 U); multiple treat 

Fisher 1988 29v Renal carcinoma 12.9 MIU/kg (median 10 doses) 7x10 10 cells OR: 16% 

West 1989 30 Renal carcinoma 3x10 6 U/m 2 /day CI NR 22-28% 

Dutcher 1989 32 100,000 U/kg q8h 8.9x10 10 CR+PR: 19% 

Paciucci 1989 24 Miscellaneous 1-5x10 6 U/m 2 /day CI 5.6x10 9 CR+PR: 20.8% 

Neqrier 1989 51 Renal carcinoma 3x10 6 U/m 2 /day CI 1.2x10 10 CR+PR: 27% 

Stahel 1989 23 Miscellaneous 3x10 4 U/kg q8h 5.1x10 10 CR+PR: 17% 

Rosenberg 1993 181 Metastatic cancer Randomized: IL-2 vs IL-2+LAK CR: 5% vs 11.76% 

PR: 15.2% vs 16.5% 

OS (3 yrs): 17% vs 31% 

(p2=0.089) 

Bajorin 49 Renal carcinoma Randomized: IL-2 vs IL-2+LAK (73x10 9 ) No difference 

(3 MU/m 2 ) 

Keilholz 1994 9 Liver metastic carcinoma IL-2 CI into the splenic artery LAK transfer into the CR+PR: 33% 

or intravenous infusion 

portal vein or the 

hepatic artery 

Murray Law 1995 66 Renal carcinoma Randomized: IL-2 vs IL-2+LAK (NR) CR+PR: 9% vs 3% 

(3x10 6 U/m 2 /day) (p=0.61) 

Kimura 1997 82,788 Resected lung carcinoma Randomized: IL-2+LAK vs. Standard therapy OS (5 yrs): 54.4% vs 52% 

(7x10 5 U/day x 3 days) (1-5x10 9 cell) OS (9 yrs): 33.4% vs 24.2% 


122 


domized) experiences using these two different 

immunotherapies suggests that LAK cell reinfusion 

slightly increased the number of CR and the duration of 

response, especially in patients with metastatic 

melanoma (Table 2). 49,50 

In hematologic malignancies the first attempts to generate 

and expand LAK activity by using IL-2 in vivo were 

clinically disappointing especially in patients autotransplanted 

for ALL; after transplantation patients were randomly 

assigned to treatment with systemic IL-2 (without 

LAK cell administration) or no treatment, but the 

disease-free survival was similar in the two arms. 51 The 

use of LAK cells has also been proposed after autologous 

transplantation for hematological malignancies, but the 

very small series of patients reported does not allow any 

definitive conclusion to be drawn about its clinical benefit. 

52 Beaujean et al. 53 reinfused, after myeloablative 

therapy, BM incubated with IL-2 into 5 ALL patients, 

observing a very marked delay of the engraftment and 

the recurrence of disease in all patients. Recently there 

has been a report of 61 women with breast cancer autotransplanted 

with IL-2 activated PBPC and treated with 

low dose IL-2 starting from PBPC reinfusion, without 

graft failures or major toxicity; there are no data concerning 

the outcome of patients and this experience only 

confirms the feasibility of the approach. 54 

In a very preliminary experience a sustained major 

cytogenetic response to immunotherapy with GM- 

CSF+IL-2 and LAK infusion was observed in chronic 

myeloid leukemia (CML) patients after autologous transplantation. 

55 However, a renewed interest in this 

approach has led to new research pursuing different 

directions: 

a. selection of patients with low tumor-burden and 

with significant in vitro LAK activity against autologous 

tumor cells, in order to reach an optimal effector/target 

ratio; 

b. harvest of large amounts of NK cells (for additional 

ex vivo expansion/activation with IL-2) to be reinfused 

in the early phase after BMT; 56 

c. direct activation of leukapheresis, after priming with 

chemotherapy followed by cytokines, in order to 

reinfuse, after HDT, a product richer in cytotoxic 

effectors and probably less contaminated; 57 

d. identification and selection of more efficient NK 

progenitors (e.g. adherent NK) by eliminating undesired 

accessory cells which could inhibit their killing 

and proliferative ability; 

58, 59 

e. generation and expansion of other CE subsets with 

more powerful activity against autologous tumor 

cells, e.g. cytokine-induced killer cells (CIK); 60 

f. use of other cytokines in association with IL-2, in 

order to potentiate the activity and/or improve the 

selectivity of activated peripheral blood MNC. 

Tumor infiltrating lymphocytes 

The disappointing results of adoptive immunotherapy 

with blood-derived LAK cells led to a search for more 

specific CE cells. Tumor infiltrating lymphocytes (TIL) are 

T-lymphocytes with unique tumor activity that infiltrate 

some tumors and can be expanded by long-term culture 

with IL-2 at low-intermediate concentrations. 61 In 

murine models TIL have exhibited a stronger anti-tumor 

effect than LAK cells on a per-cell basis; in humans TIL 

have been isolated with variable frequency from different 

solid tumors and very often (about 30% of cases) 

from patients with melanoma. Phenotypic analysis 

showed that TIL consisted mainly of CD4 + cells in colon, 

breast and urothelial tumors, while in melanoma CD8 + 

cells are prevalent. 62,63 CD3 – CD16 + NK cells have also 

been isolated from several tumors, confirming the large 

heterogeneity of tumor infiltrates. 64 The mechanism of 

the antitumor action of TIL is unknown; there is some 

evidence that these cells secrete cytotoxins and 

cytokines which are capable of killing tumor cells and 

recruiting other CE. 

Experimental models and clinical trials 

Mice carrying spontaneous metastases, treated with 

IL-2 plus tumor-derived T-cells, obtained from splenocytes 

after mixed lymphocyte-tumor cultures, had a better 

survival than those treated with LAK cells; previous 

tumor debulking (with chemotherapy and/or radiotherapy) 

was needed to maximize the efficacy of TIL-therapy. 

65 

Unfortunately large amounts of TIL can be collected 

very rarely, and the large scale expansion of this population 

is crucial in order to obtain relevant clinical 

responses; this step of ex vivo manipulation is not 

always successful, because the need for prolonged culture 

of TIL (from 6 to 8 weeks with IL-2) may abrogate 

the selectivity against the tumor; moreover only a small 

fraction of the readministered human TIL is able to concentrate 

in the tumor sites. 66 

Wong et al. 67 showed in a mouse model that TIL preferentially 

localize in the liver and lungs. In contrast trafficking 

studies employing TIL radiolabeled with In 111 , 

have shown that TIL do traffic to tumor sites; 68 this homing 

property should produce high concentrations of TIL, 

and probably their permanence, in the area of a tumor. 

Human TIL transfected in vitro with the neomycinresistance 

gene and reinfused intravenously, have been 

detected by polymerase chain reaction (PCR) techniques 

from 6 to 60 days in patients affected by metastatic 

melanoma. 69 Aebersold et al. 70 observed a strong correlation 

between the tested tumor cytotoxicity in vitro and 

the in vivo response, in a small cohort of patients with 

metastatic melanoma. A similar relationship was 

observed in a murine model in which the in vivo therapeutic 

effect of TIL correlated with secretion of IFN and 

tumor necrosis factor (TFN). 71 

In order to increase their specificity and potency, TIL 

have been engineered with genes encoding cytokines or 

cytotoxins such as TNF or IFN- or IL-2. 69,72 However, 

some experimental observations suggest that these high 

concentrations of cytokines can cause systemic toxicity 

and in some cases could even make the tumor more 

aggressive. 73,74 


Cell therapy 

123 

Table 3. Clinical trials with LAK cells and IL-2. 

Treatment schedule 

Author Year Patients Kind of tumor IL-2(dose and schedule) TIL cells Response 

Rosenberg 1988 20 metastatic melanoma 110 5 U/kg every 8h; CPM 25 mg/kg 20.510 10 cell Regress: 60% 

Kradin 1989 38 miscellaneous 1-310 6 U/m 2 CIx 24h OR: 26% 

Rosenberg 1990 5 metastatic melanoma TIL gene modified 

Aoki 1991 10 advanced or recurrent OR: 70% 

ovarian cancer TIL after single CI CPM Long term: 57% 

Dillman 1991 21v metastatic melanoma 1810 6 IU/m 2 /day CI 10 11 cell OR: 24% 

expensive, difficult 

Arienti 1993 12v metastatic melanoma 13010 6 IU/m 2 /day CI 6.810 9 cell RR:33% 

Belldegrun 1993 10v metastatic renal 210 6 IU/m 2 /day in 96h (IL-2) TIL CR: 30% 

cell carcinoma 

610 6 IU/m 2 /day (IFN-) 

Schwartz– 1994 41 melanoma IL-2 TIL CR+PR: 21.9% 

entruber 

Pockaj 1994 38 metastatic melanoma 7.210 5 IU/kg every 8h 1.3-2.210 11 cell OR: 38.5% 

and CPM 25 mg/kg 

Chang 1997 20v advanced melanoma and IL-2 anti-CD3 vaccine primed OR:33.3% 

renal cell cancer lymph node cells PR:9.1% 

activated 

Curti 1998 solid tumor and NHL 9x10 6 UI/m 2 /day x 7 days CI T CD4+ cell+ anti CD3 some tumor regression 

Ridolfi 1998 32 miscellaneous 12-6 MIU/ day 5.8x10 10 TIL no response in patients 

(West's schedule) 

with advanced cancer 

ev: evaluable; PR: partial response; OR: overall response. 

In addition to their potential therapeutic use as 

cytolytic effectors, the ability of some TIL to recognize 

unique antigens on tumor cells has made the study of the 

biologic characteristics of these antigens more feasible. 

Melanomas from different patients who share MHC antigens 

are often cross-recognized by allogeneic TIL, as 

could be expected for an MHC-restricted T-cell response; 

the presence of shared antigens in different patients with 

melanoma suggests the possibility of using these antigens 

in an active immunization program for this disease. 75 

When adoptively transferred into patients, TIL showed 

significant therapeutic efficacy in patients with advanced 

melanoma, but not in renal carcinoma patients. In a 

phase II trial patients with malignant melanoma were 

treated with IL-2 and TIL following chemotherapy: 76 39% 

of them achieved some sort of response, including those 

who had previously experienced a failure of IL-2 therapy. 

Kradin et al. 77 treated some patients with a combination 

of chemotherapy, IL-2 and TIL, obtaining 23% of 

responses in those affected by melanoma and 29% in 

those with renal carcinoma, but none in patients with 

non-small cell lung carcinoma. 

A summary of most clinically relevant clinical trials 

with TIL is given in Table 3. 

The lack of important clinical trials with TIL is probably 

due to the difficulties in finding TIL at diagnosis and 

especially because the techniques for TIL priming and 

expansion are time-consuming and not completely 

standardized. TIL therapy is still young, but its very interesting 

potential has not yet been thoroughly investigated. 

New approaches with LAK or TIL cells 

Allogeneic setting. Whereas it is widely accepted that 

graft-versus-host disease (GvHD) is initiated by donor T 

cells recognizing foreign host antigens, other factors 

including toxicity of conditioning regimens and cytokine 

dysregulation are involved in the pathogenesis of 

GvHD. 78,79 Data from murine experiments show that NK 

cells play an active role both in GvHD and in garft-versus-leukemia 

(GVL) events: in a recently published model 

100% of SCID mice bearing human leukemic cells, 

and transplanted with NK + T-cells, died of acute GvHD; 

but while animals which received only T-cells developed 

clinical GVL associated with relevant chronic GvHD, NKtransplanted 

animals showed the same degree of protection 

from leukemia, experiencing only mild-moderate 

acute GvHD without chronic GvHD. 80 These data 

suggest that in order to optimize the GVL effect while 

minimizing the severity of acute GvHD, donor grafts 


124 


should be manipulated by adding a moderate dose of T- 

cells in the early phase and using purified NK cells in the 

late phase after transplantation. 

Preliminary data suggest that in normal donors, after 

G-CSF mobilization, NK progenitors have decreased 

killing capacity and diminished proliferative ability in 

response to IL-2, compared to the unprimed bone marrow 

counterpart. 81 In contrast, after an HLA incompatible 

transplant, a progressive expansion of NK and CTL 

with NK like function (CD3 + /CD56 + ) has been observed; 

recipients received the T-cell depleted graft without 

developing GvHD, but in most cases a significant GVL 

effect could be demonstrated both in vitro and in vivo; 

these data support the critical role of CTL KIR + in this 

particular subset of transplanted patients. 82 

Concerning the expanding role of cord blood transplantation, 

even though the content of NK in this source 

seems normal, the decreased IL-12 production by cord 

blood MNC, reducing IFN- stimulation, may contribute 

to reduce NK and LAK cytotoxicity; these data suggest 

one possible explanation for cord blood immaturity and 

their clinical implications such as decreased GvHD and 

GVL, which could be enhanced by IL-12 administration. 83 

Autologous setting. Considering the impressive results 

observed in the allogeneic setting using donor-buffy coat 

lymphocytes for treatment of relapse, CML seems an 

attractive field for testing the efficacy of adoptive 

immunotherapy in the autologous setting too; some 

experimental data support this hypothesis. The MNC of 

patients with CML contain a population of benign NK 

cells which can be expanded and activated by IL-2, generating 

a CE population capable of killing both NK-sensitive 

and NK-resistant tumor targets. 84 Both number 

and functional activity of activated NK (ANK) in CML 

patients decrease with the progression of the disease. 85 

In vitro data show that autologous ANK inhibit both 

committed and very early Philadelphia positive progenitors 

in a MHC-unrestricted manner. 86 In these experiments 

CML progenitor cell killing by autologous and allogeneic 

ANK (after T-cell depletion) was comparable. 

Finally the CML blast killing was not dependent of soluble 

factors because it was abrogated by a transwell 

membrane, but was mediated by cell-to-cell contact 

being significantly blocked by anti-integrin antibodies. 87 

In 1986 Lanier and Phillips described a subset of CD3 + 

T cells co-expressing the CD56 antigen which is a typical 

NK marker (CIK). 88 More recently Schmidt-Wolf et al. 89 

obtained large expansion of this subset in a 16-day liquid 

culture containing IFN-, IL-1, IL-2 and a monoclonal 

antibody against CD3 as the mitogenic stimulus. The 

same group tested the ability of this population to purge 

bone marrow in patients with CML; they found that while 

standard LAK cells were in most cases unable to lyse CML 

cells, CIK cells were able to lyse both autologous and allogeneic 

CML blasts, without affecting normal hemopoietic 

progenitors. 90 Recently it has been reported that CIK 

administration in SCID mice bearing human CML induced 

the disappearance of Ph’+ cells in the spleen of 12/14 

animals. 91 

Another interesting potential application of autologous 

LAK is the treatment of EBV-related lymphomas 

arising in organ-transplanted patients; a preliminary 

description of four complete responses after treatment 

with autologous peripheral MNC incubated with IL-2 

seems very promising. 92 Recently in thyroid cancer 

patients Katsumoto et al. 93 generated cytotoxic CD4 + 

lymphocytes from TIL after non-specific in vivo stimulation 

with OK-432 (which induces severe local inflammation 

in the draining lymph nodes) and low-dose IL-2, 

obtaining large amounts of cytotoxic CD4 + (Th1) cells, 

producing high levels of IFN- and TNF- in the supernatants. 

These CE lysed a wide spectrum of tumor cell 

lines; anti-TCR antibodies did not inhibit their killing 

activity, which was in favor of a non-MHC restricted 

lysis, while antibodies anti-ICAM-1 completely inhibited 

the activity. 

Tsurushima et al. 94 induced autologous CTLs directly 

from peripheral blood MNC by preparing a co-culture of 

minced tissue fragments of glioblastoma multiforme 

with a mixture of cytokines (IL-1, 2, 4, 6 and IFN-) for 

2 weeks. At the end of culture the population contained 

mainly CD4 + and CD8 + lymphocytes able to kill 82 to 

100% of the glioblastoma cells while normal LAK cells 

killed only 33%. 

Finally, in follicular lymphomas freshly isolated TIL, 

normally lacking tumor-specific cytotoxicity, were stimulated 

with lymphoma cells, in the presence of IL-2 and 

CD40 ligand; these T-TIL were capable of proliferating 

in response to follicular lymphoma cells; moreover TIL 

could be further expanded in the presence of IL-4, IL-7 

and IFN-. 95 

The potential role of new cytokines 

Several cytokines affect CTL and NK response: first of 

all IL-2 which expands the precursor pool of alloreactive 

CTL; IL-15 (producted by monocytes) mimics IL-2 

action by inducing IFN- production, T-cell memory 

activation and CTL proliferation. 96 IL-12 shares certain 

functional properties with IL-2, but using a different, 

IL-2 independent pathway. 97 In addition IL-12 enhances 

the lytic activity of human peripheral blood MNC against 

a wide spectrum of tumors. 98,99 Recently it has been 

observed that the combination of IL-2 and IL-12 is capable 

of inducing lysis of blasts resistant to IL-2-activated 

effectors, even in the autologous setting. 100 

Therefore the association of IL-2 plus IL-12 could potentially 

become an important tool to increase the antitumor 

efficacy both ex vivo, by generating large amounts of 

CIK, 101 and in vivo, by systemic administration. 

GM-CSF is a cytokine capable of inducing a pleiotropic 

immunostimulatory effect and also increases the immunogenicity 

of tumors; in a model for ex vivo expansion of LAK 

cells from leukaphereses in order to obtain contemporaneously 

a decontaminated harvest and a large amount of 

CE to reinfuse after myeloablative therapy, the association 

GM-CSF+IL-2 obtained a 5-fold expansion of the NK compartment 

while sparing the clonogenic potential of hemopoietic 

progenitors. 102 


Cell therapy 

125 

Biodistribution and targeting of LAK and TIL 

At present adoptive immunotherapy with LAK or IL-2 

activated TIL has had limited success in patients with 

advanced cancer. Although a well-defined mechanism 

remains to be established, numerous in vitro findings and 

in vivo data suggest that the cancer-specific cytotoxicity 

of CE is obtained in multiple steps; a prerequisite, 

however, is optimal delivery of CE to the target tissues 

while minimizing systemic cytotoxicity. Two major areas 

currently requiring investigation are the survival and 

localization of adoptively transferred CE in the tumorbearing 

host, and the detailed mechanism of tumor 

regression. The major goals in this area concern the optimal 

administration of systemic cytokines together with 

CE, and (finally) the ways to enhance localization and 

transcapillary migration of the infused cells. 

Experimental evidence together with theoretical considerations 

based on CE functions indicate that the ability 

of adoptive immunotherapy to eradicate an established 

tumor is quantitatively determined by the initial 

tumor burden, growth pattern, and the magnitude of 

immunologic response generated by CE and other accessory 

cells at the site of the tumor. 103,104 Thus, to achieve 

tumor eradication and minimize systemic toxicity, the 

explanation of the mechanisms underlying lymphocyte 

biodistribution and the factors governing effector cell 

uptake in tumor sites is critical, but unfortunately data 

about CE biodistribution in humans are scarce. 

Although a physiologically based kinetic modeling 

approach has been applied to the pharmacokinetics of 

drugs and antibodies, there has been no effort to extend 

this approach to cell biodistribution, probably because 

of its complexity. 

One interesting attempt to apply this method to adoptive 

immunotherapy has, however, recently been published. 

105 The importance of lymphocyte infiltration from 

surrounding normal tissues into tumor tissue was found 

to depend on lymphocyte migration rate, tumor size, 

and host organ. 

It is likely that therapy with CE has not been as effective 

as originally promised, in part because of the very 

low CE concentration in the systemic circulation; this 

was mainly due to lung entrapment. Reducing this phenomenon 

by decreasing the attachment rate or adhesion 

site density in the lung by 50%, the tumor uptake could 

be increased by 40% for TIL to 60% for adherent NK 

cells. 

Theoretical models indicate that intra-arterial administration 

has a dramatic advantage over intravenous 

delivery, with more than a 1,000-fold higher CE accumulation 

in the tumor site. Indeed experiments in murine 

models show that it is possible to eliminate liver metastasis 

by loco-regional administration of human IL-2 ANK 

or by systemic adoptive transfer. 106 

Finally the differences in biodistribution between different 

lymphocyte populations, mainly due to the different 

attachment rates in the tumor and the lung, 

should be carefully considered. ANK cells are more easily 

trapped than CTL in lung vessels due to their larger 

diameter and greater rigidity. 107 A greater accumulation 

of TIL was expected in the spleen as a result of their 

stronger adhesion at this site through the lymphocyte 

homing receptor. 108 Although this model has limitations 

related to the sensitivity of analysis of parameters such 

as adhesion-site density, lymphocyte attachment and 

arrest rate, it could be considered a useful basis for 

designing new experimental models to increase the concentration 

and recirculation of CE in tumor sites, reducing 

effector cell rigidity or blocking adhesion molecules. 

The so-called antibody-dependent cellular cytotoxicity 

(ADCC) could be mediated by cells expressing Fc 

receptor II and Fc receptor III (e.g. NK cells and 

CD3 + /CD16 + cells). This kind of cytotoxicity, even though 

exhibited by non MHC-restricted cells, cannot be considered 

aspecific and is also exhibited by monocytes. 

LAK cells are extremely potent mediators of ADCC 109 

and thus the use of LAK plus IL-2 in combination with 

monoclonal antibodies will probably become a powerful 

tool for treating some immunogenic tumors. This 

approach has been tested in patients with colorectal 

cancer, 110 but could be also proposed for treating some 

immunogeneic hematologic malignancies such as follicular 

lymphoma or multiple myeloma. 

Donor lymphocyte infusion for treatment 

of leukemia relapse and as a means for 

accelerating immunologic reconstitution 

in patients given transplantation of 

hematopoietic progenitors 

Manipulation of the immune system after hematopoietic 

stem cell transplantation (HSCT) to reverse 

leukemia relapse or to reduce its incidence remains one 

of the most fascinating, even though difficult, challenges 

for successful cure of patients with hematologic 

malignancies. In fact, over the last 10-15 years, evidence 

has emerged from clinical transplantations to 

suggest that the anti-leukemia effect of allogeneic HSCT 

cannot merely be ascribed to the myeloablative therapy 

employed during the preparative regimen, donor lymphocytes 

playing a pivotal role in the eradication of 

malignant cells. Adoptive immunotherapy with donor 

lymphocyte infusion (DLI) in patients relapsing after 

HSCT has provided one of the most effective demonstrations 

of the importance of the graft-versus-leukemia 

effect in the cure of patients with hematologic malignancies. 

111,112 

Even though DLI may sometimes be burdened by complications 

that endanger the patient's life, mainly myelosuppression 

and GvHD, in individuals with CML experiencing 

relapse in chronic phase after an allograft approximately 

70% complete remissions can be obtained with 

this treatment. 112-116 Most of these remissions are sustained 

over time, this proving the capacity of DLI to eradicate 

clonogenic leukemia cells or control their regrowth. 

DLI has also been extensively employed to 

reverse relapse in patients with acute leukemia, non- 

Hodgkin’s lymphoma and multiple myeloma. However, 


126 


the response rate of patients with other hematologic 

malignancies, especially acute leukemia, is significantly 

lower. 115, 116 In fact, only 20-30% of patients with AML 

achieve a hematologic remission after DLI and the value 

for patients with ALL is even lower. Patients with 

acute leukemia experiencing recurrence following an 

allograft have a higher probability of response with DLI 

if treated after having achieved a state of complete 

remission with chemotherapy, that is in a condition characterized 

by a limited tumor burden. 117 

The most important factor predicting response to DLI 

in patients with CML is the type of relapse. In fact, as 

already mentioned, patients suffering from cytogenetic 

relapse or hematologic relapse in chronic phase have a 

high probability of response to DLI, while patients with 

more advanced disease (accelerated phase or blast crisis) 

respond less frequently (20-25% of cases). 112-116 

Relapse occurring in the first 1-2 years after allograft, 115 

little or no acute and chronic GvHD after transplantation 

or removal of T-lymphocytes before HSCT 116 are also 

associated with a higher probability of benefitting from 

DLI. In patients with CML responding to DLI, the median 

time to obtain hematologic remission has been reported 

to be about 6-8 weeks, 115 whereas a longer time (in 

the order of 11 months) is needed for molecular remission, 

this documenting that clearance of leukemia cells 

is a dynamic, progressive phenomenon. 118 The number of 

T-cells to be infused and the best schedule of DLI for 

optimal response without concurrent development of 

severe GvHD are still to be conclusively established since 

they depend on several variables, such as degree of HLAcompatibility 

between donor and recipient, original disorder, 

and type of relapse. 112 Some authors have claimed 

that infusion of no more than 110 7 donor-derived T- 

cells per kg of recipient body weight or CD8-depleted 

lymphocytes can induce a state of remission and substantially 

prevent GvHD occurrence. 119 However, recently, 

the Hammersmith Hospital group reported that the 

response in CML patients relapsing after HSCT and given 

graded increments of donor lymphocytes seems to be 

less sustained over time than that observed after infusion 

of a larger number (i.e. >110 8 /kg of recipient body 

weight) of T-cells (Dazzi F, personal communication, 

1999). Support to the importance of the number of cells 

infused is also given by the results of Lokhorst et al., 120 

who observed that, in multiple myeloma, patients given 

more than 110 8 T-cells/kg had the highest probability 

of benefitting from DLI. In some of these patients, the 

response was complete with disappearance of myeloma 

proteins. 

The two major complications occurring after DLI are 

myelosuppression and GvHD. Myelosuppression is experienced 

by approximately 50% of the patients treated 

with DLI for CML in hematologic relapse, while it occurs 

much less frequently in patients with cytogenetic recurrence, 

112 this indicating that such a complication is 

observed in situations characterized by a predominance 

of host-type hematopoiesis. Therefore, myelosuppression 

can be explained by a direct effect of the transfused 

donor lymphocytes on hematopoietic cells of the recipient, 

similarly to that observed in transfusion-associated 

GvHD. The majority of patients experiencing myelosuppression 

after DLI recover a normal blood cell count 

spontaneously: nevertheless, myelosuppression may be 

fatal in approximately 10% of patients, with death being 

caused by infection or bleeding. 115,116 Infusion of a huge 

number of donor-derived peripheral blood hematopoietic 

progenitors, mobilized through hematopoietic 

growth factors such as granulocyte colony-stimulating 

factor (G-CSF), can alleviate the problem of pancytopenia 

in some selected cases, hastening the recovery of 

neutrophil and platelet counts. 

Grade II-IV acute GvHD develops in almost half of 

patients given DLI, 115,116 the highest incidence being 

observed when the donor is an unrelated volunteer. 121 

Incidence and severity of GvHD after DLI does not appear 

to correlate with GvHD after the original transplant and 

it may occur with a high incidence since donor lymphocyte 

therapy involves the infusion of large numbers of T- 

cells, whose immunocompetence is not usually modulated 

by cyclosporin A and/or methotrexate. Even though 

GvHD occurring after DLI is well-correlated with disease 

response as proved by the observation that most patients 

obtaining a hematologic remission after this treatment 

developed acute and/or chronic GvHD, GvHD may not be 

sufficient to induce GVL. Moreover, some patients not 

experiencing GvHD after DLI achieve hematologic remission, 

this indicating the existence of a GVL effect separate 

from development of GvHD. 113,116,117,122 

GVL effect occurring after HSCT and DLI is considered 

to be mediated by HLA-unrestricted NK or LAK cells or by 

T-lymphocytes that recognize leukemia cells in an HLArestricted 

fashion. 123,124 In particular, when patient and 

donor are HLA-identical, it is believed that recipient non- 

MHC-encoded minor histocompatibility antigens (mHAg) 

are recognized by donor CTL. While widely distributed 

mHAg account for the GVL effect associated to GvHD, tissue 

restricted or leukemia-specific antigens can elicit a 

specific GVL reaction 108-113 and it has been demonstrated 

that both CD4 + and CD8 + CTL recognizing mHAg in a classical 

MHC-restricted fashion can be generated in vitro. 

124,125 In particular, mHAg-specific CD8 + CTL can display 

strong lysis of mature leukemia cells, as well as suppress, 

together with CD4 + mHAg-specific CTL, the growth 

of clonogenic leukemia precursor cells. 126,127 Production 

of cytokines (such as -interferon and tumour necrosis 

factor ) able to induce the apoptotic death of leukemia 

cells can also contribute to the GVL effect. 128,129 This said, 

it is not surprising that several efforts have been directed 

towards the identification of strategies capable of 

selecting and/or amplifying specific GVL response, not 

associated with development of GvHD. Since it has been 

documented in humans that CTL directed against allogeneic 

leukemic blasts can be detected in the peripheral 

blood of healthy donors 130 and that CTL specifically reactive 

towards recipient leukemic blasts can emerge and 

persist over time in children given allogeneic HSCT 131 a 

possible intriguing approach is that of generating and 


Cell therapy 

127 

expanding clones or cell lines that are leukemia-reactive. 

The first elegant demonstration of the feasibility and efficacy 

of this sophisticated strategy has been recently 

reported by Falkenburg et al., 132 who, through the infusion 

of donor-derived in vitro cultured CTL specifically recognizing 

leukemia progenitor cells, induced a complete 

hematologic and cytogenetic response in a patient with 

CML who had relapsed after an allograft and was resistant 

to DLI treatment. 

A diverse, but equally elegant, approach proposed to 

abrogate the DLI-associated GvHD and its relevant morbidity 

and mortality is the infusion of thymidine kinase 

gene-transduced DLI followed by treatment of the recipient 

with ganciclovir if GvHD occurs. 133 In a study reported 

by Bonini et al. 133 this strategy proved to be able to 

control GvHD in 3 patients experiencing this complication 

after DLI; two of them, who had achieved a complete 

hematologic remission before ganciclovir administration, 

remained in full remission after disappearance of 

the transduced lymphocytes. If confirmed in a larger 

number of patients with a longer follow-up, genetic 

manipulation of donor lymphocytes, through the transfer 

of a suicide gene for specific and selective elimination 

of effector cells responsible for GvHD, could demonstrate 

the possibility of separating GvHD from GVL effect, 

thus sparing the anti-leukemia activity of DLI. 

One of the most important, still unsolved problem of 

DLI is that concerning the much lower efficacy of GVL in 

patients with acute leukemia than in those with CML. An 

immediate explanation for this observation may be that 

the more rapid growth kinetics of blast cells, which 

occurs in patients with acute leukemia during the lag 

period between leukocyte infusion and GVL development, 

may hamper the immune-mediated effect played by 

donor lymphocytes in controlling disease progression. In 

fact, response to DLI occurs after weeks and hence the 

exponential expansion of leukemia cells in vivo may 

exceed the immune response. 112,113,124 The more encouraging 

results obtained when DLI is used as consolidation 

therapy for patients who have obtained a complete 

remission after chemotherapy provide support for this 

interpretation. However, other hypotheses, involving different 

intrinsic susceptibility of acute leukemia to adoptive 

immunotherapy must be considered. In particular, 

since patients with ALL have the lowest chance both of 

responding to DLI and of benefitting from the GVL effect 

after bone marrow transplantation, 134 a peculiar resistance 

of lymphoid leukemia to immunotherapy cannot 

be excluded. 

As peptides differentially expressed within the 

hematopoietic system can trigger and act as a target of 

the GVL reaction, 112,124 it could be hypothesized that, for 

example, the presence of these antigens on myeloid 

blasts, but not on lymphoid leukemia cells accounts for 

the low response of ALL to donor lymphocytes. The 

reported demonstration of CTL response directed 

towards peptides derived from proteinase 3, which is 

expressed by myeloid cells (including blast cells), 135 is a 

typical example of the possible differential susceptibility 

to the immune-mediated anti-leukemia effect of different 

types of hematologic malignancies. 

Several other possibilities exist to explain why acute 

leukemia (and in particular ALL) can escape the GVL 

effect. For example, leukemia cells may have defective 

expression of HLA-class I or II molecules on their surface 

such that they do not present antigens or, alternatively, 

the mechanisms of antigen processing and transport may 

be impaired. 112,129 Moreover, leukemia blasts may product 

cytokines (such as transforming growth factor , IL- 

10) capable of suppressing T-cell activation, expansion 

and effector function or may express on their cell surface 

molecules, such as FAS ligand, able to mediate T- 

cell apoptosis. 112,129 One of the most interesting fields of 

investigation for explaining why in some patients a sustained 

anti-leukemia response in vivo fails to be induced 

is that of co-stimulatory molecules. As previously 

described, full activation of T-cells requires two distinct 

but synergistic signals. 136 In fact, in the absence of costimulatory 

signals, a T cell encountering an antigen 

becomes unresponsive to the appropriate stimulation 

(anergic) 137 or undergoes programmed cell death (apoptosis). 

138 Leukemia cells lacking these co-stimulatory 

molecules have a poor capacity of inducing a T-cell specific 

immune response and induction of CD80 and CD86, 

by signalling through the CD40 molecule, is able to 

restore T-cell co-stimulation via CD28 and to generate 

both allogeneic and autologous CTL, which could contribute 

to inducing or maintaining a state of hematologic 

remission. 139,140 

Some clinical strategies have been devised to improve 

the efficacy of adoptive immunotherapy in patients with 

acute leukemia. An approach for ameliorating the efficacy 

of DLI which has produced interesting results is that 

recently reported by Slavin et al., 106 who documented that 

the success rate of this adoptive immune therapy may be 

increased in patients with both acute and chronic 

leukemia by activation of donor peripheral blood lymphocytes 

with IL-2 both in vivo and/or in vitro. In particular, 

a relevant proportion of patients who had not 

responded to DLI were induced into remission only after 

in vivo administration of IL-2 or in vitro activation of 

donor lymphocytes. If further confirmed, the results 

obtained make it possible to hypothesize that this strategy 

could be employed as first-line treatment of patients 

with acute leukemia relapsing after an allograft, since 

ALL and to a lesser extent AML patients do not greatly 

benefit from DLI alone. Another reasonable attempt for 

improving the response to DLI in patients with acute 

leukemia is to use this adoptive immunotherapy in individuals 

with minimal residual disease, as determined by 

cytogenetic investigations or sensitive molecular tools, 

that is in conditions characterized by a limited tumor 

burden, in which the GVL effect has demonstrated its 

greatest efficacy. 

Unmanipulated DLI may also provide a means of compensatory 

T-cell repletion for the prevention of leukemia 

recurrence in patients given a T-cell depleted marrow 

transplantation from a relative. This approach has been 


128 


recently proposed 141 and studies enrolling larger cohorts 

of patients are necessary to define whether this strategy 

can be useful to prevent the increased risk of relapse 

associated with the removal of donor T-cells. However, 

the main indication of adoptive infusion of donor 

immune cells to accelerate immune reconstruction after 

HSCT is transplants from HLA-disparate family donors. 

Infusion of a high number of T-cell depleted, peripheral 

blood hematopoietic progenitors from these donors 

has been demonstrated to be associated with a high 

chance (>95%) of donor hematopoietic engraftment. 142 

The significant delay in immune reconstitution, due 

mainly to removal of mature T cells from donor marrow 

and HLA disparity between donor and recipient, remains 

the major problem of HSCT from HLA-disparate donors. 

In fact, it is responsible for the dramatic incidence of 

leukemia relapse and life-threatening viral and fungal 

infections observed after this type of HSCT. A possible 

strategy to improve the process of immune recovery is 

to infuse donor T-lymphocytes selectively rendered nonreactive 

towards alloantigens of the recipient, but maintaining 

the capacity to generate an immune response 

against viruses, fungi and leukemia cells. In this regard, 

as previously mentioned, the manipulation of co-stimulatory 

molecules is an extremely promising field of 

investigation, since the absence of a second signal 

induces anergy rather than activation of T lymphocytes. 

Drugs and monoclonal antibodies blocking co-stimulatory 

pathways have been demonstrated to be able to 

prevent T-cell activation in response to alloantigens and 

to induce a state of anergy. 143 In particular, it was 

recently documented that the combination of monoclonal 

antibodies blocking CD80/CD86 molecules and 

cyclosporin A was able to generate a state of selective 

in vitro unresponsiveness of T cells towards allo-antigens, 

not reversed by adding IL-2. 144 Since the induction 

of this state of unresponsiveness was associated with 

the maintenance of in vitro capacity to respond toward 

virus antigens and leukemia cells, 145 the relevance of 

this approach is evident for strategies of donor T-cell 

add-back after T-cell depleted transplant of hematopoietic 

progenitors from HLA-partially matched donors 

aimed at accelerating the process of immune reconstitution. 

A different, but equally promising, method of deletion 

of unwanted alloresponses is based on the elimination 

of alloreactive T-cells after specific activation through 

their killing 146 or fluorescence-activated cell sorting, 147 

while sparing T cells with other functions. In a human 

pre-clinical study, it was demonstrated that allospecific 

T-cell depletion by using an immunotoxin directed 

against the p55 chain of IL-2 receptor, was feasible and 

specific. 146 The spared T-cells were still able to proliferate 

against third-party cells, Candida and cytomegalovirus 

antigens, 148 as well as to kill both leukemia blasts 

and autologous EBV-B lymphoblastoid cell lines. 149 

Moreover, in vivo studies in a murine animal model 

showed that this particular T-cell depletion was efficient, 

at least partially, in preventing both graft rejection 

and GvHD in a complete haplotype mismatched 

combination. 150 

Finally a brief mention should be made of the generation 

and infusion of T cells with suppressive and regulatory 

activity. A particular subset of these cells called 

Tr1 has recently been described by Groux et al., 151 who 

in an animal model demonstrated the ability of this population 

to prevent, through their activity on naive cells, 

the occurrence of ovo-albumin induced inflammatory 

bowel disease. Whether these cells will have a role in 

promoting a true state of tolerance in transplant of 

hematopoietic progenitors or solid organs (in which the 

immune response to alloantigens is mainly sustained by 

memory cells) remains to be proved in specific pre-clinical 

and clinical studies currently underway. 

Adoptive immunotherapy for the treatment 

of viral infections in immunocompromised 

patients 

Prevention or treatment of viral infections in immunecompromised 

patients through the infusion of specific T- 

cell lines or clones is one of the most sophisticated 

examples of adoptive immunotherapy approaches. 152 In 

fact, it implies the elaboration of true cellular-engineering 

strategies able to generate, select and expand lymphocyte 

subsets, which display a specific function. The 

first study in humans to evaluate the efficacy of adoptively 

transferred T-cell clones for reconstitution of specific 

immunity was performed in recipients of allogeneic 

HSCT at risk of developing human cytomegalovirus 

(HCMV) infection and/or disease. 153 Even though preemptive 

therapy of HCMV infection based on monitoring 

of antigenemia 154 and prophylaxis of seropositive 

HSCT recipients using antiviral drugs (i.e. ganciclovir and 

foscarnet) 155 have significantly reduced the number of 

patients experiencing HCMV disease, this viral infection 

still represents a major life-threatening complication of 

stem cell allograft. The capacity to recover from a severe 

HCMV infection in transplanted patients is directly correlated 

with the ability of the host to generate virusspecific 

class I HLA-restricted CD8 + cytotoxic cells and 

during the first 100 days after HSCT approximately 50% 

of patients are persistently deficient in CD8 + cytotoxic T- 

lymphocytes specific for HCMV. 156,157 It is not surprising 

that, to evaluate the efficacy of adoptive immunotherapy 

in this viral infection, HCMV-specific CD8 + T-cell 

clones of donor origin were generated and infused in 

HSCT recipients. 153,158 These cells, generated through a 

highly complex expansion strategy using irradiated 

donor-origin skin fibroblasts infected with a strain of 

HCMV, proved to be efficient in the prophylaxis against 

HCMV infections that can complicate allogeneic HSCT. 

Moreover, the cloning strategy allowed selection of T 

cells which lacked significant alloreactive capacity and, 

thus, did not cause clinically relevant GvHD or toxicity. 

These clones, directed towards either pp65 or pp150 (two 

abundant viral tegument proteins presented for recognition 

by cytotoxic T-lymphocytes), restored HCMV-spe- 


Cell therapy 

129 

cific cytotoxicity, which persisted for several weeks. 158 In 

fact, through a PCR technique able to detect the V and 

V T-cell receptor rearrangements specific for the donor 

clones, it was possible to prove the donor origin of these 

cells formally and to document the persistence of the 

adoptively transferred HCMV-specific T-cells for at least 

12 weeks. Unfortunately, these clones persisted in the 

circulation at high levels only in patients experiencing an 

endogenous recovery of CD4 + virus-specific cells. 158 By 

contrast, in patient lacking this spontaneous recovery of 

HCMV-specific CD4 + lymphocyte, the donor-origin, 

adoptively transferred cytotoxic T-cell activity progressively 

declined and eventually disappeared. This observation 

emphasizes the importance of CD4 + lymphocytes 

in promoting sustained restoration of antigen-specific 

immunity and suggests that the use of polyclonal T-cell 

lines containing both CD4 + and CD8 + cells could be 

preferable to the infusion of cytotoxic T-cell clones. 

In this regard, the use of T-cell lines for prevention 

and/or treatment of Epstein-Barr virus-induced lymphoproliferative 

disorders (LPD) has represented a further, 

equally sophisticated, evolution of the approaches 

of adoptive immunotherapy for the restoration of 

virus-specific immunity. EBV-LPD have emerged as a 

significant complication for both HSCT and solid organ 

transplant recipients. 159-161 In the former cohort, the use 

of HLA-partially matched family and unrelated donors, 

as well as selective procedures of T-cell depletion sparing 

B-lymphocytes, are risk factors for the development 

of EBV-LPD. 160-162 In HSCT recipients these disorders are 

of donor origin and usually present in the first 4-6 

months after transplantation, whereas in patients given 

a solid organ allograft they usually develop from the 

recipient B-lymphocytes months to years after transplantation. 

160,161 High levels of EBV-DNA in blood and in 

vitro spontaneous growth of EBV-lymphoblastoid cell 

lines predict development of these lymphoproliferative 

disorders. 163 They often present as high-grade diffuse 

large cell B-cell lymphomas, which are oligoclonal or 

monoclonal and express the full array of EBV antigens 

including EBNA-1 through EBNA-6 and the latency 

membrane proteins LMP-1 and LMP-2. 161 The lymphomas 

which develop in immunocompromised hosts 

not only invade the hematopoietic system, but also the 

lung, nasopharynx and central nervous system. The therapeutic 

approaches proposed to date (i.e. discontinuation 

of immunosuppression, -IFN, antiviral agents and 

cytotoxic chemotherapy) have been applied with varying, 

but overall unsatisfactory, results; moreover, graft 

rejection, GvHD and toxicity are frequent complications 

of these strategies, and mortality rate due to EBV-LPD 

remains high. 160,161 

Normal EBV seropositive individuals have a high frequency 

of circulating virus-specific cytotoxic T-lymphocytes 

precursors, which control outgrowth of EBV-infected 

B-cells. Since EBV-LPD in immunocompromised hosts 

appears to stem from a deficiency of virus-specific cytotoxic 

activity, it is reasonable to hypothesize that an 

adoptive immunotherapy approach with donor-derived 

T-lymphocytes could be able to prevent unchecked lymphoproliferation 

and eradicate established disease. In 

1994, the Sloan Kettering group first demonstrated that, 

through the infusion of unselected peripheral blood 

mononuclear cells from a donor, 5 patients given HSCT 

with post-transplant EBV-LPD obtained remission of the 

disease. 164 However, this treatment was associated with 

development of clinically relevant GvHD and 2 patients 

of inflammatory-mediated lung damage, leading to respiratory 

failure. 

A further refinement of this approach was achieved by 

Rooney and colleagues, who generated EBV-specific T- 

cell lines from donor lymphocytes and infused them as 

prophylaxis against EBV-LPD in patients given T-cell 

depleted HSCT from HLA-disparate family or unrelated 

donors, and, thus, considered at high risk for this disease. 

165 The infusion of these polyclonal T-cell lines 

proved to be safe and effective in the prevention of EBV- 

LPD. Moreover, these cytotoxic cells may also have a 

role in the treatment of established disease. 165 The most 

recent update of this experience confirms that the infusion 

of EBV-specific T-cell lines is highly effective for the 

prevention of EBV-LPD, since none of 39 patients given 

a T-cell depleted allograft and treated with this adoptive 

immunotherapy developed the disease, as compared 

to 7 out 61 transplanted patients not receiving the prophylactic 

treatment. 166 Gene marking studies have 

shown the persistence of these donor-derived EBV-specific 

cell cytotoxic lines in patient’s peripheral blood for 

months after infusion and their re-appearance after 

periods of apparent non-identifiability during episodes 

of viral reactivation, this further stressing the importance 

of helper T-cell function in the persistence of 

transferred CD8 + cells. 167 

The profound immunosuppression necessary for graft 

survival carries a well-recognized predisposition to the 

development of viral complications, in particular EBV- 

LPD, also in recipients of solid organ transplantation. 159 

An immunotherapy approach to EBV-LPD using autologous 

in vitro generated EBV-specific cytotoxic lines could 

be an appealing strategy in this cohort of patients. Support 

for this hypothesis is given by the recently described, 

although not unexpected, possibility of generating, from 

pre-transplantation blood samples of EBV-seropositive 

solid organ transplant recipients, virus-specific T-cell 

lines which are effective in controlling EBV replication 

post-transplantation. 168 However, generation and storage 

of cytotoxic lines for each patient undergoing solid 

organ transplantation requires enormous, unavailable 

levels of funding, laboratory facilities and workforce. A 

more rational strategy is to generate, expand and infuse 

autologous EBV-specific cytotoxic lines from the peripheral 

blood of organ transplant patients presenting 

increased EBV-DNA levels after transplantation, which, as 

previously mentioned, are a risk factor for EBV-LPD development. 

The feasibility of generating autologous EBVspecific 

cytotoxic lines from the peripheral blood of organ 

transplant patients receiving in vivo immunosuppression 

for prevention of graft rejection has been recently 


130 


proved. 169 Moreover, these cytotoxic T-lymphocytes were 

demonstrated to be able to display EBV-specific killing in 

vivo, as proved by prompt viral DNA clearance, without 

augmenting the probability of graft rejection. A peculiar 

problem, fortunately not particularly common, is that of 

EBV-seronegative patients, who develop primary EBV 

infection after solid organ transplantation. In fact, in 

these patients, in vitro generation of virus-specific T-cell 

lines able to control EBV-driven B-cell proliferation can 

be particularly complicated, time-consuming and sometimes 

unsuccessful. 

Autologous EBV-specific cytotoxic lines with demonstrated 

anti-viral activity in vitro and in vivo may also 

have a role in the treatment of other EBV-associated primary 

malignancies: for example, 40-50% of patients 

with Hodgkin’s disease tumor cells are EBV-antigen positive 

and may therefore be suitable targets for virus specific 

cytotoxic lymphocytes. 160,170 A recently reported 

study provides further support for this possibility, documenting 

that, although more complicated than in normal 

donors, generation of EBV-specific cytotoxic lines is 

feasible in a relevant proportion of patients with EBVpositive 

Hodgkin’s disease. 171 These lines retained their 

potent antiviral effects in vivo and persisted for more 

than 13 weeks in patients with relapsed Hodgkin’s disease. 

171 Whether this approach of adoptive immunotherapy 

will become an adjunctive treatment option for 

patients failing to gain benefit from conventional 

chemotherapy remains to be proved in prospective clinical 

trials. 

Finally, it should be mentioned that adoptive transfer 

of cytotoxic T-cell response could be of value also in 

the prevention or treatment of other viral infections that 

cause morbidity and mortality in immunocompromised 

patients. In this regard, pre-clinical studies are underway 

to establish systems for generating cytotoxic T-cell 

responses to adenovirus. 160,172 

Genetically engineered donor lymphocyte 

infusion for treatment of leukemia 

relapse and as a means of accelerating 

immunologic reconstitution in patients 

given transplantation of hematopoietic 

progenitors 

Tumor recurrence is the major cause of treatment failure 

of autologous bone marrow transplantation. 173,174 

Indeed, the rate of tumor relapse is lower when transplantation 

is performed between matched unrelated or 

mismatched family member donor and recipients. It is 

now established that the curative potential of allo-BMT 

is represented by the additional effect of high dose 

chemo-radiotherapy in addition to the presence of allogeneic 

T-lymphocytes that are responsible for the 

GVL. 175,176 However, the therapeutic impact of allogeneic 

BMT is limited by the inevitable occurrence of 

GvHD. 177 Severe GvHD can be circumvented by the in vitro 

removal of T-lymphocytes from the BMT. 175 However, 

recipients of depleted marrow have delayed immune 

recovery, and increased incidences of viral infections 

and tumor relapse. 178,179 

Recent studies have shown the clinical efficacy of the 

adoptive transfer of immune effectors specific for viral 

antigens 153,195,167 in patients who underwent BMT. In this 

context gene transfer of a marker gene provides a means 

of evaluating the survival, homing and efficacy of the 

infused cells. 

In marrow-transplanted recipients, lymphoproliferative 

disorders associated with EBV, a human herpes virus 

that normally replicates in epithelial cells of the oropharyngeal 

tract, occurs in 5-30% of the treated patients. 

EBV-LPD are usually malignant B-cell lymphomas of 

donor origin, which may be either polyclonal or monoclonal. 

The latter have a rapidly progressive, fulminating 

and fatal course. 180,181 The transformed B cells express 

virus-encoded latent cycle nuclear antigens, latent membrane 

proteins, and a number of cell adhesion molecules. 

Most of these viral proteins are recognized as antigens 

by the immune system of a normal individual. 182 In the 

normal host, in fact, EBV-induced lymphoid proliferation 

is controlled by EBV-specific and MHC-restricted T- 

lymphocytes, MHC-unrestricted effectors and by antibodies 

directed toward specific viral antigens. Since a 

limited number of specific cytotoxic T-lymphocytes is 

required for controlling EBV-transformed B-lymphocytes 

in normal individuals, the administration of donor lymphocytes 

for the occurrence of EBV-LPD in recipients of 

T-cell depleted bone marrow transplantation could control 

this severe complication by providing the patient 

with donor immunity against EBV. 164,183 Successful 

regression of the disease, documented histologically and 

by full clinical remission, has been achieved by the infusion 

of unmanipulated donor leukocytes. 164 However, 

acute or mild chronic GvHD developed in all the patients 

who responded to the treatment. 164 

To prevent GvHD, Brenner’s group has evaluated the 

use of EBV-specific CTL rather than unmanipulated T 

cells. Donor derived EBV-specific CTL have been generated 

in vitro by stimulation with irradiated donorderived 

EBV-infected lymphoblastoid cell lines (LCL). 184 

The polyclonal effector populations were predominantly 

CD8 + with a varying number of CD4 and showed specific 

cytotoxic activity toward the EBV-infected target 

cells. In order to investigate the long-lasting survival of 

the injected cells, the anti-EBV effectors were marked 

with the neo-gene before administration. 

Neo-marked cells were detected in circulation for at 

least 10 weeks after the injections. 165 Moreover, the 

infusions allowed the establishment of a population of 

CTL precursors that could be activated to proliferate by 

in vivo or in vitro challenge with the virus. 166 The authors 

showed that EBV-specific CTL lines expressing the neomarker, 

could be derived from patient's peripheral blood 

lymphocytes (PBL) for up to 18 months, by in vitro restimulation 

with the autologous EBV-lines. 166 

These findings support a more widespread use of antigen-specific 

CTL in the treatment of infections and cancer. 

Their use may extend in the near future to other dis- 


Cell therapy 

131 

eases which express well-known antigens that could 

serve as target of CTL therapy (e.g. Hodgkin’s disease and 

nasopharyngeal carcinoma). 

The adoptive transfer of in vitro stimulated effectors 

achieves clinical results without causing the appearance 

of GvHD. However, the application of this strategy 

to a large number of allo-BMT treated patients, especially 

in prophylaxis protocols has some limitations 

related to the in vitro manipulation necessary for the 

generation of specific effectors (e.g. availability of 

donor-EBV lines; in vitro stimulation and expansion of 

antigen-specific effectors). An alternative approach was 

proposed in 1994 by the S. Raffaele Hospital group. 185,186 

Their protocol was aimed at maintaining the potential 

of the infusion of polyclonal cell lines while providing a 

specific means to control acute GvHD. To this aim they 

transduced donor lymphocytes by a retroviral vector 

containing a suicide gene for in vivo selective elimination 

of the infused lymphocytes. 

It was previously shown that introduction of a gene 

encoding for a susceptibility factor, a so-called suicide 

gene, makes transduced cells sensitive to a drug not 

ordinarily toxic. 187,188 A series of retroviral vectors carrying 

a suicide gene for ganciclovir-mediated in vivo selective 

elimination of the infused lymphocytes was 

designed. The vectors carried either an HSV-thymidinekinase-neo 

(Tk-neo) fusion gene, coding for a chimeric 

protein for both negative and positive selection, or the 

HSV-Tk gene alone. 189 

A crucial prerequisite for the application of this strategy 

in the clinical context is the transduction of all 

infused donor lymphocytes. For this purpose, the 

designed retroviral vectors also carried a gene encoding 

a modified (non-functional) cell surface marker not 

expressed on human lymphocytes. Positive immunoselection 

of the transduced cells 190 by the use of the 

cell surface marker resulted in virtually 100% genemodified 

lymphocytes. 

Based upon the preclinical data described above, a 

clinical protocol was developed 186 for the use of donor 

lymphocytes transduced by the SFCMM-2 retroviral vector 

for transfer and expression of two genes: the HSV- 

Tk gene that confers to the transduced PBL in vivo sensitivity 

to the drug ganciclovir, for in vivo specific elimination 

of cells potentially responsible for GvHD; and a 

modified (non-functional) form of the low affinity 

receptor for the nerve growth factor gene (∆LNGFr), for 

in vitro selection of transduced cells and for in vivo follow-up 

of the infused donor lymphocytes. 

Increasing doses (beginning at 110 6 /kg) of donor 

PBL were infused into several patients affected by 

hematologic malignancies who developed severe complications 

following a T-cell depleted BMT from their 

HLA-identical related donors. After the infusion, the 

transduced lymphocytes could be detected in the blood 

of patients by cytofluorimetric and PCR analyses. In particular 

one patient affected by an EBV-LPD, showed a 

progressive increase in the number (up to 13.4% of the 

total PBL) of infused marked lymphocytes that was 

accompanied by a complete clinical response. However, 

signs of acute GvHD, confirmed by skin biopsy, were 

observed approximately four weeks after the infusion 

of the transduced-donor lymphocytes. The intravenous 

(i.v.) administration of two doses of ganciclovir (10 

mg/kg/day) quickly resulted in elimination of marked 

donor PBL, and near resolution of all clinical and biochemical 

signs of acute GvHD. 187 

As mentioned before, when comparable preparative 

regimens are employed, the rate of tumor recurrences 

after autologous BMT is significantly higher than the 

rate observed after allogeneic BMT. GvHD develops in 

50-70% of patients undergoing allogeneic BMT. The 

effectors of such response are thought to be mature 

donor lymphocytes from the marrow graft that respond 

to the foreign major and/or minor histocompatibility 

antigens of the recipient and also recognize and destroy 

the tumor cells. In fact, patients who underwent mature 

T-cell-depleted allogeneic BMT have a lower rate of 

GvHD but also a higher rate of leukemia relapses. 178,179 

The infusion of donor lymphocytes, early after T-celldepleted 

allogeneic BMT, increases the incidence of 

GvHD without improving the control of leukemia. 191 

However, a delayed transfusion of donor lymphocytes, 

when graft tolerance is established, seems to be more 

effective in preventing and treating tumor relapses. 

Indeed the delayed administration of donor lymphocytes 

has recently become a new tool for treating 

leukemic relapse after BMT. Patients affected by post- 

BMT recurrence of chronic myelogenous leukemia, acute 

leukemia, lymphoma, and multiple myeloma could 

achieve complete remission after the infusion of donor 

leukocytes without requiring cytoreductive chemotherapy 

or radiotherapy, 106,192-194 even though the response 

rate of patients with acute leukemia, non-Hodgkin’s 

lymphoma and multiple myeloma is significantly lower 

than that of patients affected by chronic myelogenous 

leukemia. Although the delay in the administration of T 

lymphocytes is expected to reduce the risk of GvHD, this 

risk is still present at higher doses of donor T-cells. 116 

Therefore, as described above, a clinical protocol was 

developed, for the use of donor lymphocytes transduced 

by the SFCMM-2 retroviral vectors 186 for transfer and 

expression of the HSV-Tk gene, and the cell surface 

marker ∆LNGFr, for in vitro selection of 100% transduced 

cells and for in vivo follow-up of the infused 

donor lymphocytes. 190 

In a phase I-II study, eight patients affected by hematologic 

malignancies who developed severe complications 

following an allogeneic T-cell depleted BMT, 

received escalating doses of donor PBL transduced by 

the described retroviral vector. 133 After gene transfer, 

transduced cells were selected for the expression of the 

cell surface marker ∆LNGFr by the use of specific 

immunobeads and the proportion of transduced cells 

was assessed by cytofluorimetric analysis. 190 In this study, 

we made the following observations: 1) transduced cells 

survived long-term in vivo and were detectable by cytofluorimetric 

analysis and PCR in high proportions (up to 


132 


13.4% of circulating PBL) and long-term (up to 6 

months); 2) three patients showed complete response, 

three patients had partial response, one progressed with 

no response, and one patient could not be evaluated; 3) 

three patients developed GvHD that required ganciclovir 

treatment; 4) ganciclovir-mediated elimination of transduced 

cells resulted in near resolution of all clinical and 

biochemical signs of acute GvHD. Data from this study 133 

indicate that genetically modified cells maintain their in 

vivo potential to develop both anti-tumor and GvHD 

effect, and may represent a new potent tool for exploiting 

anti-tumor and anti-host immunity, while providing 

a specific means for eliminating acute GvHD, in the 

absence of any immunosuppressive drug. 

A potential limitation of the clinical approaches 

described could be the development of a specific 

immune response against vector-encoded proteins, 

which might allow the selective elimination of the 

transduced cells by the host immune system. For some 

gene products, such as the hygromycin-thymidine 

kinase (Hy-Tk) fusion protein, a specific immune 

response, able to eliminate large numbers of transduced 

cells in less than 48 hours, has been described in HIVpatients. 

195 

We observed that immune recognition and killing of 

cells transduced by retroviral vectors is a more general 

phenomenon related to the foreign nature of the proteins 

expressed by the injected cells. Indeed, cells 

expressing the widely used marker gene neo and the 

HSV-Tk gene are targets of a strong immune response, 

while the endogenous proteins (e.g. the cell surface 

marker ∆LNGFr) are not recognized, even if ectopically 

expressed in a context which is otherwise extremely 

immunogenic. 196 The relative immunogenicity detected 

for the three vector-encoded components (none by 

∆LNGFr, low by HSV-Tk, high by neo) clearly outlined 

the modifications of this type of gene therapy. Since neo 

is the only component not-strictly necessary for the 

strategy and can be efficaciously replaced by the surface 

marker for all in vitro handling and selection, 189,197 the 

immunogenicity of the new neo-less vectors should be 

reduced. 

The clinical results obtained with gene modified donor 

lymphocytes, for the treatment of hematologic relapses 

and EBV-lymphoproliferative disorders, suggest the 

potential use of this approach. 133 The transfer of a suicide 

gene, that allows selective and specific elimination 

of effector cells of GvHD may allow full advantage to be 

taken of the beneficial effect of allogeneic lymphocytes 

with the possibility of eliminating all unwanted effects 

of GvHD in the absence of toxic side effects. A large 

scale application of this strategy will increase the number 

of patients who could potentially benefit from allogeneic 

BMT by allowing the use of less compatible marrow 

donors. 

With regard to the immune recovery associated with 

the genetically-engineered donor lymphocytes, our group 

has recently obtained in vitro data demonstrating that 

genetically-engineered donor T-cells maintain a normal 

TCR V immune repertoire and retain antigen-specific 

lytic activity against an allogeneic target or an autologous 

EBV cell line at cytotoxic T-cell precursor frequencies 

comparable to unmodified lymphocytes. In the light 

of this in vitro evidence, and our previous clinical application, 

133 a clinical trial, based on the prophylactic infusion 

of 110 7 /kg HSV-Tk transduced T-cells six weeks 

after T-cell-depleted bone marrow transplantation, was 

developed. In the first five treated patients we documented 

the presence of various proportions of transduced 

cells in the peripheral blood. In particular, geneticallyengineered 

donor lymphocytes were responsible for antiviral 

immune reconstitution in one patient. CD3 + lymphocytes 

began to appear in the circulation of this 

patient two weeks after the infusion of HSV-Tk T-cells. All 

the CD3 + lymphocytes were genetically engineered as 

documented by the expression of the cell surface marker 

∆LNGFr. These cells retained a polyclonal TCR repertoire 

and were probably responsible for the clearance of 

a persistent CMV antigenemia. Indeed, the CMV antigenemia 

dropped below levels which could be detected 

by PCR shortly after the appearance of circulating genetically-engineered 

CD3 + T cells in the absence of any 

antiviral drug therapy. 198 These data, if confirmed in a 

larger number of patients with longer follow-up, suggest 

that in addition to the anti-tumor activity, the infusion 

of genetically-engineered donor lymphocytes may play a 

role in restoring immunity against opportunistic infections 

early after allogeneic BMT. 

Dendritic cells as natural adjuvants in 

cancer immunotherapy 

Among professional antigen presenting cells (APC), 

dendritic cells (DC) are specialized in capturing and processing 

antigens into peptide fragments that bind to 

major histocompatibility complex molecules. DC are the 

most potent stimulators of T-cell responses and they 

are unique in that they stimulate not only memory but 

also naive T-lymphocytes. Thus, DC appear critical 

(nature adjuvants) for the induction of B-and T-cellmediated 

immune responses. Recent evidence in experimental 

models supports the role of DC for immunization 

strategies aimed at stimulating specific anti-tumor 

immunity. 

In this section we will briefly review: 

1. the biological characterization of DC; 

2. different strategies for ex vivo generation of DC; 

3. methods for the efficient delivery of tumor associated 

antigens (TAA) to DC; 

4. the use of DC for cellular immunotherapy. 

Biological characterization of dendritic 

cells 

DC are widely distributed in the body and are particulary 

abundant in tissues that interface the environment 

(i.e. Langerhans cells in the skin and mucous membranes) 

and in lymphoid organs (interdigitating DC) 

where they act as sentinels for incoming pathogens. 


Cell therapy 

133 

Inflammatory signals such as TNF- and IL-1 as well 

as bacteria, bacterial products (LPS) and viruses induce 

migration of antigen-loaded DC from the peripheral tissues 

to secondary lymphoid organs. During migration, 

DC mature and upregulate MHC, adhesion and co-stimulatory 

molecules, thus strongly augmenting their ability 

to prime T-cells. 199-204 

The functional activity of DC derives from a number 

of properties of these cells (Figure 2). Their dendritic 

shape, along with the high level of expression of certain 

adhesion molecules and integrins (LFA-3, ICAM-1, 

ICAM-3), increases the area of contact with the effector 

cells of the immune system. 205 DC strongly express 

the HLA class II molecules -RD, -DQ and -DP and costimulatory 

molecules (CD80, CD86 and CD40) which 

activate their ligands on T-cells (CD28, CTLA-4 and 

CD40L), thus providing the second signal strictly necessary 

to induce a proliferative response, rather than tolerance, 

upon antigen recognition. 199 In addition, DC produce 

a number of cytokines including IL-12 which promotes 

a cytotoxic immune response by inducing the differentiation 

of TH0 cells to IFN- and IL-2-producing 

TH1 cells. 206,207 It has recently been demonstrated that 

upon Ag recognition, T-helper cells activate DC via 

CD40-CD40L interaction and activated DC are then able 

to trigger a cytotoxic response from T-killer cells. 208-210 

However, DC are present in peripheral tissues in an 

immature state unable to prime T-cells. At this stage of 

differentiation, they can very efficiently take up soluble 

antigens, particles and micro-organisms by phagocytosis, 

macropinocytosis or by the macrophage mannose receptor, 

Fc and Fc receptors, 211 but they lack all the accessory 

signals for T-cell activation. Antigen uptake induces 

DC to maturation by up-regulating MHC and co-stimulatory 

molecules as well as DC-associated Ag (e.g. CD83 

and p55) whereas the capacity to capture and process Ag 

is lost. However, full activation of DC is dependent upon 

the contact with T-cells by the CD40-CD40L interaction 

which induces the production of IL-12. Thus, the key 

functions of DC (antigen uptake, T-cell stimulation) are 

strictly segregated to subsequent stages of differentiation 

(Figure 3). It is noteworthy that IL-10 212 and vascular 

endothelial growth factor (VEGF), secreted by cancer 

cells, 213 prevent the maturation of DC thus inhibiting the 

efficient priming of T-cells. 

Different strategies for the generation of 

DC ex vivo 

Circulating CD14 + monocytes represent the most readily 

available source of DC if incubated with appropriate 

cytokines such as GM-CSF, IL-4 and TNF-. 214, 215 Moreover, 

DC precursors have been isolated within the CD34 + 

cell fraction in bone marrow, cord blood and steady state 

or mobilized peripheral blood. 216-221 Also in this case the 

differentiation of CD34 + cells into fully functional DC is 

strictly dependent upon stimulation with certain 

cytokines such as GM-CSF, TNF-, SCF, FLT3-L and IL-4. 

Figure 2. Phenotypic and functional characteristics of dendritic cells. Modified from ref. #199 (Bancherau and Steinman, 

Nature, 1998). 


134 


An extensive review of the different types of human DC 

and their ex vivo generation is beyond the scope of this 

chapter. However, in view of the clinical use of DC a few 

critical points should be stressed. GM-CSF and IL-4 

induce the differentiation of non-proliferating CD14 + 

monocytes to immature DC with a low level of expression 

of CD83 and p55 Ag and are largely incapable of priming 

naive T-cells. These immature DC are not fully differentiated 

and revert to an adherent state if the cytokines 

are removed from the culture medium. 222,223 The addition 

of inflammatory cytokines such as TNF-, IL-1 or PGE2 

for 1-2 days to the medium containing GM-CSF and IL- 

4 promotes the maturation of DC and increases the ability 

of stimulating T-cells. A potential bias toward the 

clinical use of this culture system is the requirement of 

fetal calf serum (FCS), a xenogenic protein that is contraindicated 

for human use. An innovative culture system 

for the generation of mature and functional DC from circulating 

monocytes that uses FCS-free conditions has 

recently been described. 222,223 In this system, adherent 

peripheral blood (PB) cells are cultured for 6-7 days with 

GM-CSF and IL-4 in the presence of FCS, which is then 

washed out, and subsequently exposed to macrophageconditioned 

medium (Mo-CM) and 1-5% autologous 

plasma for 1-3 days. Mo-CM is very efficient in inducing 

the terminal maturation of DC and is prepared by growing 

T-cell-depleted PB cells on immunoglobulin (Ig)-coated 

Petri dishes for 24 hours. 

Taken together, these findings lead to the conclusion 

that immature DC generated from CD14 + cells in the 

presence of GM-CSF and IL-4 are well equipped for capturing 

and processing soluble TAA. However, they do 

require a further maturation stimulus (Mo-CM, TNF-) 

to exert their stimulatory effect on T-cells. Immature DC 

are the ideal targets for genetic manipulation using viral 

or bacterial vectors which infect non-replicating cells 

(see below). In this case, the modified pathogens can 

induce by themselves the full maturation of DC. In alternative, 

mature DC could be used in vaccination protocols 

involving TA peptides as DC also prime T-cells to foreign 

Ag that bind directly to MHC molecules without prior 

processing. 224 

As reported above, CD34 + cells can be induced to differentiate 

into fully functional DC which resemble cutaneous 

Langherans cells. 218 The issue of the large scale 

production of DC from CD34 + precursors has been discussed 

in detail elsewhere. 5 However, very recently the 

phenotypic and functional characteristics of DC derived 

from CD34 + cells mobilized into PB or from BM progenitors 

have been formally compared. 225 The published 

results indicate that G-CSF mobilizes DC precursors (CFU- 

DC) with an increased frequency and a higher proliferative 

capacity than their BM counterparts. This finding 

translates into a higher number of mature DC generated 

in liquid culture. Despite pre-treatment with G-CSF, these 

cells maintain the same functional capacity of stimulating 

allogeneic T-cells as BM-derived DC. CD34 + cellderived 

DC are also capable of processing and presenting 

soluble Ag to autologous T-cells for both primary and 

secondary immune responses. The potential clinical usefulness 

of autologous serum in place of FCS 220 was also 

confirmed in the same study. Of note, IL-4 was shown to 

be capable of modulating DC differentiation from bipotent 

CD34 + cells during the later stages of the culture as 

previously demonstrated for monocyte-derived DC. 226 

Thus, mobilized CD34 + cells may represent the optimal 

source for the generation of DC for cancer immunotherapy 

rather than BM precursors. Very recent data indicate 

the mobilization of large numbers of DC precursors 

by GM-CSF 227 and FLT-3L. 228 However, it remains to be 

Figure 3. Functional proporties 

of dendritic cells at different 

stages of differentiation. 

Pathogens or inflammatory 

cytokines induce the 

maturation of dendritic cells 

which become activated 

upon interaction with T-cells 

via CD40-CD40L. 


Cell therapy 

135 

established whether circulating CD34 + elements are an 

equivalent source of DC to CD14 + monocytes. In this view, 

it has recently been demonstrated 229 that CD34 + cellderived 

DC are more efficient than monocyte-derived DC, 

from the same patients, in stimulating a specific CTL 

response to Melan-A/Mart-1 peptides. 

Delivery of TAA to DC 

Several methods for the efficient delivery of TAA to DC 

have been described so far (Figure 4). Their rationale is 

based on the finding that tumor cells are often poorly 

immunogenic due to the lack of T-cell recognition, activation 

and co-stimulation typical of professional APC. To 

this end, Gong et al. 230 fused murine DC with the carcinoma 

cell line MC38 to provide tumor cells with the 

functional characteristics of DC. The fusion cells showed 

all the phenotypic features of DC and were shown to be 

capable of preventing tumor growth when the mice 

were challenged with the cell line. Moreover, treatment 

with fusion cells induced the rejection of pulmonary 

metastates. 

Several TA peptides which are presented to T-cells in 

association with HLA class I molecules have been 

recently identified and proved to be useful in stimulating 

an autologous CTL response in vitro and in vivo. 

However, pulsing DC with peptides may not be optimal 

for clinical application because of the strict MHC restriction 

of the immune response and their limited stability. 

In addition, pulsing with peptides may not induce a T- 

cell response directed toward tumor cells expressing the 

relevant Ag. Although DC can be loaded with a cocktail 

of peptides from different Ag derived from the same 

type of cancer (see below), this vaccination approach is 

likely to limit patient selection on the basis of HLA phenotype. 

An attractive alternative is the use of unfractionated 

tumor-derived proteins, when available (see 

below), apoptotic cells 231 or tumor lysates. In the last 

case the obvious disadvantage is the possibility of inducing 

immune responses against self-Ag expressed in tissues 

other than tumor cells. 

A further possibility is the transduction of DC with 

expression vectors encoding for TAA genes (Figure 4). DC 

can be engineered by different means which differ in 

their capacity of targeting quiescent cells, stable integration 

in the genome, infection efficiency and stimulation 

of anti-tumor immunity (Figure 5). Retrovirallytransduced 

DC constitutively express the relevant 

sequence and are potent stimulators of a specific T-cell 

response. 232 However, retroviral vectors have a relatively 

low efficiency of transduction, they can only infect 

actively replicating cells and carry the theoretical risk of 

oncogenic transformation of target cells. Conversely, adenoviruses 

infect both quiescent and proliferating cells 

and do not integrate into DNA. 233 Moreover, supernatants 

with a high titer of the virus can be easily obtained. 

Recently, DC have been transduced with an adenovirus 

combined with cationic liposomes showing an infection 

efficiency close to 100%. 234 The major limitation to the 

clinical use of adenoviruses is their high immunogenicity 

which induces the production of neutralizing antibodies 

and the rapid development of CTL directed at infected 

cells. 

Vaccinia virus vectors are not oncogenic, do not integrate 

into genome and can be manipulated to carry 

large fragments of heterologous DNA. 235 However, these 

viruses are toxic for target cells and the viability of DC 

is approximately 50%. Nonetheless, antigen-specific 

inhibition of tumor growth has been observed in murine 

models using vaccinia vectors encoding for CEA and 

Mucin-1. 236,237 Two phase I clinical trials have been conducted 

to assess the safety of vaccinia virus vectors 

engineered to express HPV and CEA genes and to asses 

their capacity of stimulating an immune response. 238,239 

More recently, maturation of DC with neo-biosynthesis, 

translocation and stabilization of MHC molecules on 

the cell surface and efficient induction of both CD4 and 

CD8 T-cell activation has been induced by infection with 

bacterial vectors. 240 As a result, a model Ag (ovalbumin) 

expressed on the surface of recombinant Streptococco 

Figure 4. 


136 


Gordonii, is processed and presented on MHC class I 

molecules 10 6 times more efficiently than soluble OVA 

protein. Therefore, bacterial vectors are potentially useful 

means of delivering exogenous Ag to DC for stimulating 

a tumor-specific CTL response. A different 

approach has been taken by Boczkowsky et al. 241 who 

transfected DC with the total RNA extracted from tumor 

cells and combined it with cationic lipid to enhance the 

infection efficiency. Similarly to the use of tumor lysates, 

this strategy can be applied in those situations in which 

a tumor-specific antigenic marker is lacking; the major 

concern is the increased risk of autoimmune reactivity. 

DC for cellular immunotherapy 

The central role of DC in stimulating a tumor-specific 

immune response is well established in vitro and in 

vivo in animal models. 232,241-246 Whereas murine DC 

pulsed with TA-proteins or peptides or transduced with 

TAA genes have induced both the rejection of challenge 

tumor cells and the regression of established cancers, it 

remains to be determined which of the several strategies 

proposed for cellular immunotherapy is the most 

efficient. It may well be that different tumors require 

different approaches. 

In humans, initial studies were performed in patients 

with melanoma using DC pulsed with MAGE peptide. 

247,248 The infusion of loaded DC induced the migration 

of MAGE-specific CTL to the site of injection and 

increased the frequency of circulating tumor-specific 

CTL. More recently, Nestle et al. 249 have treated advance 

stage melanoma patients with intranodal injection of 

peptides or tumor lysates-pulsed DC according to the 

HLA profile of the patient. The authors reported the 

stimulation of a peptide-specific T-cell response in all 

cases. Moreover, in 5/16 patients an objective clinical 

response was observed. In this study, DC were generated 

ex vivo from monocyte precursors in the presence of 

IL-4 and GM-CSF and directly injected into an inguinal 

lymph node to reach T-cell rich areas. 

Tumor-specific peptides (fragments of prostate specific 

antigen, PSA) have also been used to pulse autologous 

DC in prostate cancer patients refractory to hormone-therapy. 

250 Seven out of 51 patients showed a partial 

response while none of the patients in the control 

group, injected with peptides alone, showed any clinical 

benefit. In B-cell malignancies, the patient-specific idiotype 

(Id) gene sequence and its protein product represent 

the optimal targets for vaccination strategies as previously 

shown in murine models 251,252 and humans. 253 Hsu 

et al. 254 have reported on the treatment of 4 patients 

with low-grade non-Hodgkin’s lymphoma (NHL), resistant 

to conventional chemotherapy or who had relapsed, 

with DC pulsed with the Id as soluble antigen. A tumorspecific 

T-cell-response was observed in all cases coupled, 

in one case, with the regression of tumor burden. 

At the time of writing, 16 patients have been treated 

and a tumor-specific cellular response has been found in 

8 individuals (R. Levy, personal communication). The 

same strategy of targeting the Id has been proposed by 

the same group for inducing a T-cell immune response 

in multiple myeloma patients. 255 

In contrast to the strategy used by Nestle et al. 249 in this 

preliminary trial DC were freshly isolated from the PB by 

subsequent enrichment steps and were reinfused intravenously. 

Although a much larger number of DC were 

injected in NHL patients compared to melanoma patients 

(3-2010 6 DC vs 110 6 ), this approach raises concerns 

about both the efficacy of uncultured PB DC of efficiently 

Figure 5. 


Cell therapy 

137 

stimulating T-cells and the capacity of Id-loaded APC to 

reach secondary lymphoid organs to prime T-cells, escaping 

the entrapment of the pulmonary apparatus. 


The few clinical data available so far have barely provided 

the proof of principle that autologous DC generated 

ex vivo and reinfused into cancer patients are 

effective in stimulating an anti-tumor immune response. 

This is the result of the complexity of the interplay 

between different cellular populations involved in tumor 

immunity. In addition, cellular immunotherapy with DC 

has yet to be standardized. As mentioned above, crucial 

issues such as 1) the choice of the most suitable TAA to 

stimulate an immune response; 2) the use of soluble 

proteins/peptides or DC engineered with expression vectors; 

3) the optimal source for the generation of DC and 

the number of APC needed to promote a clinical effect; 

and 4) the most effective route of administration of DC, 

are points which still need to be solved. At this stage, 

relying for the most part on animal studies, we can only 

conclude that DC-based immunotherapy holds promises 

of exerting a potent anti-tumor effect in humans. 

Oral vaccination by in vivo targeting of DC 

A simple approach to targeting APC in vivo is to use 

attenuated bacterial vectors, such as those commonly 

developed to control infectious diseases. They usually 

enter the host through the oral route and selectively 

replicate within macrophages and DC. Listeria monocytogenes 

is a promising vaccine carrier that naturally 

infects APC, and may deliver immunogens to both MHC- 

I and II pathways of antigen processing and presentation. 

256 Furthermore, this bacterium may constitute per 

se an excellent danger signal for the immune system, 

since it stimulates the innate immune response to produce 

cytokines (e.g. IL-12) and mediators (e.g. nitric 

oxide) that enhance antigen presentation. In addition, it 

promotes a TH1-type cellular response, which is mainly 

associated with the eradication of tumors and intracellular 

parasites. Most of these features are also shared 

by Salmonella typhimurium-based carriers. 

The ideal vaccine carrier should maintain its immunogenicity 

intact, being attenuated enough to allow its 

use in humans. However, the safety profile of a vaccine 

destined for human use also requires the absolute stability 

of the mutant phenotype, which can only be guaranteed 

by the generation of chromosomal deletion 

mutants. Furthermore, the release of recombinant microorganisms 

under uncontrolled conditions makes the lack 

of antibiotic resistance markers essential. Mutation of 

genes involved in bacterial spread and survival are the 

best targets for attenuation. 

The recent progress in Listeria and Salmonella genetic 

manipulation and the availability of suitable in vitro 

and in vivo models, make these micro-organisms very 

attractive vaccine delivery systems. 

For example, attenuated Listeria monocytogenes carrier 

strains expressing the -galactosidase (-gal) model 

antigen can prevent outgrowth of an experimental 

tumor in BALB/c mice by inducing a specific immune 

response against the -gal TAA. 257 Similarly, a live attenuated 

AroA- auxotrophic mutant of Salmonella typhimurium 

(SL7207) has been used as a carrier for the 

pCMV vector that contains the -gal gene under the 

control of the immediate early promoter of cytomegalovirus 

(CMV). After a primary immunization and three 

orally administered boosts at 15-day intervals, a Salmonella-based 

vaccine induced both cell-mediated and 

systemic humoral responses to -gal. These experiments 

suggested that insertion of a plasmid containing an 

expression cassette into a Salmonella-carrier allowed 

DNA immunization and specific targeting of antigen 

expression to APC, in vivo, through oral immunization. 

To prove that the transgene was actually expressed by 

APC cells as a function of a eukaryotic promoter the 

green fluorescent protein (GFP) was placed under the 

control of either the eukaryotic CMV or a prokaryotic 

promoter and spleen cells from treated mice were analyzed 

by cytofluorometric analysis. 

GFP was detectable in both macrophages and DC, but 

not in other splenocytes, of mice treated with Salmonella 

containing the CMV-plasmid, 28 days after the 

first vaccine administration, whereas it was undetectable 

in spleen cells of mice receiving the Salmonella 

containing the constitutive prokaryotic promoter 

which directs GFP synthesis only within the carrier. 258 

GFP expression in DC highlights the possibility of loading 

DC without the need for ex vivo manipulations and 

opens up the possibility of administering a cancer vaccine 

orally. Oral vaccination is viewed as an easier and 

more acceptable strategy for patients especially in a 

phase in which they are disease-free. 

Leukemic cells as antigen presenting 

cells 

Tumors may escape immune detection and killing 

through a variety of mechanisms affecting the capacity 

of either presenting tumor antigens or fully activating T- 

cells. 258,260 In particular, tumor cells are likely to prevent 

a clinically evident cytotoxic T-cell response because of 

the absence of a specific antigenic tumor peptide, or 

because they lack HLA molecules, or co-stimulatory molecules 

on their surface. In this last case the patient’s T- 

cells might become anergic and tolerate tumor cells. 

Alternatively, neoplastic antigens may induce a clonal 

deletion of thymocytes, 261 or tumor cells expressing Fas 

molecule may be responsible for an apoptotic T-cell deletion 

through Fas:FasL interaction. 262 So far, different 

immunologic strategies aimed at overcoming these 

defects by inducing or improving the antigen presenting 

function of tumor cells have been demonstrated in experimental 

models, 263,264 and the hypothesis that leukemic 

cells may become efficient APC by changing their phenotype 

or by differentiating into DC-like cells has been 

tested. A first example was shown in B-cell neoplasms 

since it is well known that normal B-cells may present 

antigen to T-cells 265 and that cognate interactions 

between B- and T-cells may induce either a T-cell prolif- 


138 


eration and an enhanced T-helper activity to cytotoxic T- 

cells, 266 or T-cell clonal unresponsiveness. 267 The triggering 

of the CD40 receptor on the surface of APC increases 

the expression of adhesion and co-stimulatory molecules 

both in vitro and in vivo. 269,269 Thus, the possibility 

of modifying the phenotype and the APC function of 

CD40 + -chronic lymphocytic leukemia B (CLL-B) cells 

through the CD40:CD40L interaction was demonstrated 

showing that this pathway induces the upregulation of 

CD80 and CD86 on CLL-B cells and the triggering of a T- 

cell proliferative response. 270,271 These results support the 

idea that induction of B7 molecules on CLL-B cells, either 

by T-cell-contact and growth factors, 270, 272 or by gene 

transfer methods 273 may be a potential clinical vaccinetherapy 

capable of eliciting efficient anti-leukemic 

immune responses. Similar approaches may also apply to 

B non-Hodgkin’s lymphomas (B-NHL). Studies in experimental 

models indicated that CD40 stimulation may 

result in the inhibition of lymphoma cell growth in vivo, 274 

and in the up-regulation of adhesion receptors and costimulatory 

molecules on lymphoma cells in vitro. 275,276 

Interestingly, follicular B-NHL cells which express CD40 

and low levels of B7-2 fail to present alloantigen, but 

after activation via CD40 they express higher levels of B7- 

1 and LFA-3 and alloreactive T-cells respond to tumor 

cells efficiently. 276 Finally, encouraging results have also 

been obtained in pre-B acute lymphoblastic leukemia 139 

in which approximately 50% of the cases blast cells have 

been reported to express CD86 but not to induce tumor 

rejection, and B7-blasts determine an immunologic tolerance 

of the tumor. Nonetheless, this study showed that 

pre-activation of blast cells via CD40, or cross-linking 

CD28, or signaling through the common chain of the 

IL-2 receptor on T-cells can prevent T-cell tolerance. The 

authors hypothesize at least two possible mechanisms to 

explain the induction of lymphocyte unresponsiveness: 

first, they propose that at the time of initial transformation, 

clonogenic pre-B acute leukemia cells may not 

express CD86 thus inducing a T-cell anergy that could not 

be reversed by following expression of CD86 on a blast 

cell fraction; second, they suggest that marrow microenviroment 

may play a role in modulating T-cell immunity 

by secreting negative regulators, as previously shown in 

experimental models. 277,278 However, after co-stimulation 

by either B7 transfectants or professional APC, autologous 

antileukemic cytotoxic marrow T cells can be generated 

upon contact with CD40-stimulated pre-B acute 

leukemia cells. 140 

All these data on B-cell neoplasms strongly suggest 

that poor tumor immunogenicity may depend on both 

the quality and the quantity of accessory molecules 

required for T-cell stimulation. However, future therapeutic 

strategies aimed at stimulating the CD40 receptor, 

or at directly transducing B7 molecules on chronic or 

acute leukemia B-cells will facilitate the ex vivo expansion 

of specific anti-tumor cytototoxic T-cells. Normal 

myeloid CD34 + progenitors include a small subset of 

APC279, 280 

that are committed precursors of the 

macrophage/dendritic lineage. 281 In fact, both marrow 

and peripheral blood CD34 + cells, and circulating monocytes 

can be utilized to obtain large numbers of dendritic 

cells in vitro. Due to the relevance of co-stimulatory 

molecules on tumor cells for the generation of anti-tumor 

immune responses, the hypothesis of whether even acute 

or chronic myelogenous leukemic cells might differentiate 

into dendritic cells in vitro and become immunogenic 

has been addressed by several groups. Alternatively, 

transduction of co-stimulatory molecules on leukemic 

myeloblasts has been attempted in experimental models 

to generate specific cytotoxic responses. Both these 

approaches require that TAA are expressed and exposed 

on HLA molecules, and it is likely that genetic alterations, 

such as chromosomic translocations, might result in the 

appearance of pathologic peptides, specific for each 

acute or chronic leukemia and potentially immunogenic. 

Chronic myelogenous leukemia may represent an optimal 

candidate for antitumor vaccine strategies since several 

reports have shown that the bcr-abl fusion protein can 

bind to defined HLA class I and class II molecules 282-286 

and also that dendritic cells generated in vitro from CML 

patients still carry the t(9;22). 287,288 In this latter study, in 

fact, CML cells that were incubated with GM-CSF, IL-4 

and TNF- developed DC phenotypic and functional characteristics 

inducing autologous cytotoxic T-cells capable 

of directly lysing leukemic cells and of inhibiting CML 

colony growth in vitro. Further studies suggested that 

CML DC-stimulated anti-leukemic T-cell reactivity is due 

to an oligoclonal T-cell response and develops in an HLArestricted 

manner. 289 Dendritic cells can be generated 

even from CD34 + CML marrow progenitors in the presence 

of GM-CSF, TNF- and IL-4, and after 7-10 days of 

culture they are Ph + , express high levels of HLA molecules 

and co-stimulatory receptors and induce a T-cell 

proliferation 10-30 fold higher than unprocessed marrow 

cells. 290 Nonetheless, it is likely that different culture systems 

may be required for efficient in vitro generation of 

DC when using CML-CD34 + cells rather than normal 

progenitors, since the former show a lower DC clonogenic 

activity but both their expansion and their differentiation 

can be significantly improved by prolonging the 

duration of culture in the presence of specific growth 

factors. 291 

When a neoplastic event affects undifferentiated or 

more mature progenitors of the granulocytic and/or 

macrophage lineage an AML develops, and we can distinguish 

different subtypes of AML on the basis of morphologic 

and phenotypic characteristics. The identification 

of AML cells with some phenotypic affinities to DC, 

such as the expression of the CD1a marker, 292 or deriving 

from a monocytic/dendritic cell progenitor, 293 has been 

attempted in the past. Indeed in this latter study, cells 

from an AML, FAB M2 patient were shown to differentiate 

into terminal DC with potent alloantigen presenting 

capacity after in vitro culture with GM-CSF, TNF-, SCF 

and IL-6. Similar results were achieved by culturing freshly 

isolated AML cells with GM-CSF, IL-4 and IL-13 for 7 

days. 294 Alternatively, restoration of anti-tumor immune 

control can be attempted by identifying peptides, such as 


Cell therapy 

139 

PR-1 derived from proteinase 3, 135 that could be capable 

of inducing HLA-restricted cytotoxic T-lymphocytes to 

lyse fresh leukemic cells, or by engineeing leukemic cells 

to induce either the expression of co-stimulatory molecules 

or the production of cytokines. The role of B7-1 in 

developing protective immunity was initially tested in a 

mouse model in which the injection of a myeloid cell line 

transfected with the bcr/abl gene was rapidly lethal, 

while prolonged survival was observed only in mice that 

received the cell line co-transfected with the B7-1 

gene. 295 Moreover, the same model was used to test the 

role of both B7-1 and B7-2, suggesting that B7-1 may be 

more effective than B7-2 in obtaining an efficient in vivo 

anti-leukemic response. 296 The potential advantage of B7- 

transduced blasts was confirmed by using primary AML 

cells instead of a cell line; a CD8 + T-cell dependent and 

B7:CD28-mediated anti-leukemia activity was documented. 

297 A recent study compared the in vitro immunogenic 

activity of human AML cells cultured with GM-CSF, 

IL-4 and TNF-, or transfected with CD80. 298 Both these 

approaches resulted in an enhanced T-cell response in a 

mismatched primary MLR, however, only B7-1 transduced 

AML cells stimulated a strong immune response of 

T-cells from an HLA identical bone marrow donor, and 

generated leukemia reactive CD4 + T-cell lines and clones. 

Interestingly, this model allowed the authors to observe 

CD80 + AML-mediated T-cell responses that can be directed 

against the patient’s minor histocompatibility antigens 

or tumor-specific antigens. 

Although B7-1 and B7-2-engineered tumor cells 

could play a pivotal role in anti-leukemia immunotherapy 

strategies, there is evidence that transduction 

of other receptors 299 or cytokines 300-303 might, at least, 

co-operate with B7 molecules in the antigen presenting 

capacity of neoplastic cells. 

Genetically modified cells as vaccine 

for the active immunotherapy of cancer 

Non-specific approaches to cancer immunotherapy 

probably date back to the beginning of the 18 th century 

and originated from the observation of sporadic, 

spontaneous remission of tumors in patients who suffered 

severe bacterial infection. This observation 

prompted Dr. William B. Coley to begin, in 1891, to treat 

patients with soft tissue sarcoma with a mixture of 

Gram positive and negative bacteria: Coley’s toxins. 

This empirical approach was enforced by Shear’s discovery 

that endotoxins were active components responsible 

for tumor hemorrhagic necrosis. Furthermore, the 

finding that bacillus Calmette-Guérin (BCG) increased 

resistance to tumor transplants in mice led to clinical 

application of BCG which, together with Streptococcusderived 

OK-432, is a strategy used to this day. 

The anti-tumor effects obtained by treatment with BCG 

and derivatives are largely dependent on indiscriminate 

necrosis of tissues containing mycobacterium (the Koch 

phenomenon). The discovery of cytokines explained most of 

the phenomena induced by microbial products and 

cytokines were then used with the initial hope of copying 

the positive effects of such bacterial products while avoid- 

Known, limited 

repertoire of TAA 

Large repertoire of 

unknown antigens 

Gene 

Gene, protein/peptide 

Cell fragments RNA 

Whole tumor cells 

Loading DC 

Ex vivo 

Directly in vivo 

DNA vaccination 

Needs adjuvant: 

plasmid sequences 

muscle injury 

Bacterial vectors 

Active immunization for cancer: 

strategies are based on whether tumor antigens 

have been molecularly defined. 

Figure 6. 


140 


ing the negative ones. 

More recently, the discovery of Th1 and Th2 distinct 

pathways of T-cell maturation helped to explain protective 

and non-protective BCG-induced cell-mediated 

immune reactions in tuberculosis, phenomena that have 

correlates with protection against cancer. In the presence 

of a Th1 deflected immune response, the effect of 

TNF- is not that of large necrosis which is, rather, the 

characteristic of inflamed tissues of a Th2 type of 

response, in this case extremely sensitive to TNF-. 304 

Cytokines deflecting the immune response to a Th1 or 

Th2 type of response may drive the type of immune 

response to cancer cells, escaping the simple definition of 

Th1 promoting and Th2 inhibiting anti-tumor immunity. 

Rather, a strong Th1 as well as a strong Th2 response 

may induce tumor destruction and immune memory with 

the same efficacy although through different mechanisms 

(see below). Moreover, genetic background may 

influence the ability to mount a Th1 or Th2 response, as 

shown in murine models. 

Microbial products have mainly local effects which 

may be reproduced and improved by local injection of 

recombinant cytokines. Experiments in non-tumor systems 

have shown that IL-2 offsets antigen recognition 

and overcomes tolerance. Thus cytokines could be used 

not only to stimulate tumor destruction but also to 

impair tolerance and activate effective and specific 

immune recognition of TAA. 

Identification and cloning of the long elusive TAA, 

especially from human melanomas, 305 pointed tumor 

immunotherapy to a general systemic response and, thus, 

the use of cytokines shifted from that of being responsible 

for local tumor debulking to that of being an aid to 

triggering and boosting the immune response to TAA. 

In addition to antigens triggering the T-cell-receptor 

(TCR) of T-lymphocytes, optimal T-cell response also 

requires co-stimulatory molecules, as detailed above. 

Cytokines, co-stimulatory molecules and several 

cloned TAA are now available: how can we use them to 

provide an effective immunotherapeutic approach to 

cancer patients? 

Two major strategies are envisaged (see Figure 6): one, 

already described, takes advantage of antigen availability 

in the forms of genes, proteins or peptides and of 

the standardized methods of obtaining DC from peripheral 

blood in large quantities to be loaded with the antigen 

and reinfused in vivo; the other strategy still considers 

the tumor cells representative of the entire antigenic 

repertoire of a certain neoplasia; such cells, genetically 

modified to produce cytokines and/or co-stimulatory 

genes, could be injected into patients as a cellular 

vaccine. In the latter case a pool of cell lines derived 

from different patients with the same type of tumor 

could increase the antigenic repertoire and avoid 

immunoselection that certain antigens may have 

encountered in some patients. Unmatched MHA are not 

a problem in terms of antigen presentation since injected 

cells are destroyed and represented by host APC. 

Moreover if different sets of alloantigens are selected 

from different pools, the risk of repeated alloimmunization 

during booster vaccination would probably be 

avoided. The background and prospectives of genetically 

modified tumor cell vaccines are presented below. 

Cytokines at the tumor site 

In initial studies recombinant cytokines were injected 

at the tumor site or cytokine genes were inserted 

into somatic cells to be injected at the tumor site. All 

these studies collectively established that most of the 

cytokines accumulated at the tumor site were able to 

induce tumor destruction and the reaction they induced 

was sometimes strong enough to eradicate a tumor 

antigenically unrelated to the cytokine-releasing cells. 

The obtained tumor debulking was often followed by a 

systemic tumor-specific immune memory. It should be 

underlined, however, that tumor debulking may occur 

through non-specific immune reactions or so fast as to 

prevent efficient T-cell priming, this being reminiscent 

of the dichotomy described for BCG: indiscriminate 

necrosis versus protective immunity. 

Engineered tumor cell vaccines 

Engineering of tumor cells with the gene of a particular 

cytokine is an efficient way of ensuring that this 

cytokine will be durably present at the tumor site. 

Repeated local injections would, of course, have the 

same effect. Bolus administration, however, does not 

provide a constant supply of cytokine. Its effects are 

much less evident than those achieved by the injection 

of engineered tumor cells 306 that can ensure the provision 

of antigen and continued local accumulation of the 

cytokine until a physiologic or a pharmacologic threshold 

is reached, and the biological activity of the cytokine 

can begin. 

The immunogenicity that tumor cells can acquire 

upon cytokine-gene transduction may stem from 

recruitment by released cytokines, of particular repertoires 

of inflammatory cells, whose differing abilities to 

influence TAA presentation and secrete secondary 

cytokines may shape both immunogenicity and deflection 

of the ensuing immune memory towards a Th1 or 

Th2 type of response. A cytokine may be simultaneously 

involved in tumor rejection, leukocyte recruitment 

and activation of memory mechanisms. 

Many experimental studies have been performed in 

mice over the last seven years and cytokine genes from 

IL-1 to IL-18 have been tested. Most of those studies 

described whether a certain cytokine gene, upon transduction, 

can inhibit tumor growth in vivo; some also 

described whether the cytokine induced protective 

immunization against challenge by parental cells whereas 

only a few studies described efficacy in a therapeutic 

setting. It is clear that the way cytokines modify 

tumor oncogenicity, immunogenicity and curative effect 

is not only dependent on the cytokine employed but also 

on the tumor model utilized. The immune mechanisms 

responsible for inhibition of tumor growth may not be 

the same as those required for immune memory or those 

necessary for eradication of an established tumor. 


Cell therapy 

141 

Translation of animal studies into a clinical setting 

faces a substantial difference, that is the fast growth of 

transplanted tumors and therefore the short time window 

in which immunization can be performed before 

the animal’s death. In murine models, the so-called 

established tumor is a tumor that has been injected one 

to three days before the beginning of vaccination. This 

contrasts with phase I/II clinical studies in which enrolled 

patients have advanced disease. Clear evidence of therapeutic 

effects is not expected in these patients, therefore 

tumors with antigens whose genes have been cloned 

and are recognized by CTL should be used to allow, at 

least, an immunologic follow-up that could prove the 

effect of vaccination. This confines the choice to those 

carrying the MAGE, GAGE and BAGE family genes and to 

melanomas, which also express antigens of the melanocyte 

lineage, such as tyrosinase, gp100 and MART- 

1/Melan-A. 305 The choice is further restricted by the difficulty 

of obtaining cells and cell lines from tumors that 

are not melanomas to be transduced and then employed 

for immunologic evaluation. Melanoma is thus the tumor 

most frequently chosen for vaccination studies. 

Nevertheless, vaccination with cytokine-transduced, 

freshly isolated cells, which should retain the tumorantigen 

repertoire, could be a way of generating tumorspecific 

T-lymphocyte lines and clones with which to 

identify antigens expressed by tumors other than 

melanomas. 

In a few cases only, the antigens associated with the 

murine tumors employed in pre-clinical studies were 

characterized; the majority of studies designed to discover 

the immunologic mechanisms associated with 

tumor rejection utilized proteins not classifiable as 

tumor-associated antigens, such as -galactosidase 244 

and influenza nucleoprotein. 307 Most of these animal 

studies were carried out in the syngeneic system, that 

in humans corresponds to the autologous situation, in 

which a tumor cell line was both the cell vaccine and the 

tumor to be cured. Autologous application is actually 

difficult, since it requires tumor cell cultures from every 

patient for both gene transduction and immunologic 

follow-up. Each patient’s cell vaccine should then be 

checked for safety, and a great variability in terms of 

cytokine production other than adhesion molecules and 

antigenic phenotypes may exist between cell vaccines. 

The use of allogeneic cell lines, on the other hand, has 

the advantage of employing vaccines well-characterized 

in terms of tumor antigen, MHC and adhesion molecules, 

as well as the constant amount of cytokine 

released; these parameters in combination may provide 

a standard reagent for clinical studies. 

Both syngeneic and allogeneic tumor cells expressing 

a common TAA are processed by host APC such that TAA 

derived peptides are presented in association with host 

MHC in either case. 307 Nevertheless, in most clinical protocols 

the expression of the MHC class I allele, which 

presents TAA derived peptide(s), on the immunizing 

tumor cells is preferred. If cross-priming occurs efficiently, 

this should not be necessary, but it is still unclear 

whether vaccination with transduced tumor cells actually 

primes the host or boosts already present activated 

T-lymphocytes. This observation indicates that co-stimulatory 

molecules, such as B7, in addition to cytokines 

may be transduced in cell vaccines in order to amplify 

the boosting effect, since is not clear whether B7 transduced 

cells prime the host directly. 

Clinical vaccination protocols using IL-2 or IL-4 genetransduced 

allogeneic melanoma cells have been performed 

at the Istituto Nazionale Tumori in Milan, Italy. 

An HLA-A2 melanoma cell line expressing Melan- 

A/MART-1, tyrosinase, gp100 and MAGE-3 has been 

transduced and irradiated before the treatment of 

advanced HLA-A2+ melanoma patients. 308 In the first 

protocol, patients were injected subcutaneously on days 

1, 13, and 26 with IL-2 gene-transduced and irradiated 

melanoma cells at doses of 5 (3 patients) and 15 (4 

patients) 10 7 cells. Mixed lymphocyte-tumor cultures 

(MLTC) and limiting dilution analyses were performed to 

compare pre- and post-vaccination PBL. While MLTC 

revealed an increased but MHC-unrestricted cytotoxicity, 

in two cases the frequencies of melanoma-specific 

CTL precursors were clearly augmented by vaccination. 

In one patient, HLA class II-restricted effectors were 

found to be involved in the recognition of autologous 

tumor. Which antigen(s) was involved in the recognition 

by PBL of vaccinated patients remains unclear. In 3 out 

of 5 cases studied, pre- and post-vaccination PBL could 

not recognize any melanoma peptide tested or known to 

be restricted by HLA-A2 allele. 308 Among other possible 

explanations, this might be due to a tumor associated 

antigenic repertoire that exceeds the limited number of 

antigens whose genes have been cloned so far. 

This indicates that vaccination with cell lines is advantageous 

because the cell lines stimulate the host with 

the entire repertoire of known and unknown antigens. In 

the allogeneic system it is then easy to rotate the transduced 

cell line within the protocols and so maximize the 

chances that a relevant tumor antigen is present in the 

vaccine. Some antigens, in fact, may be negatively 

selected and lost in one patient-derived line, but not in 

others. In addition, selection of allogeneic cell lines displaying 

various MHC reduces the interference of repeated 

boosting with strong alloantigens. Indeed vaccination 

with a pool of three melanoma cell lines commenced 

before the cloning of known melanoma associated antigens, 

resulted in increased survival correlated with the 

level of antibody against the GM2 ganglioside, indicating 

possible involvement of a humoral response; correlation 

with the CTL response was not investigated. 309 

Going back to animal studies in which vaccination 

therapy with cytokine-transduced tumor cells was successful, 

it should be underlined that it was not clear 

which of the measured immune responses was responsible 

for the therapeutic effect since, generally, induction 

of cytotoxic T-lymphocytes was, per se, insufficient 

to produce a cure. In keeping with this statement, vaccination 

of 13 evaluable patients with MAGE-3.A1 peptide 

resulted in 3 clinical regressions, although no CTL 


142 


precursors were found in the PBL of these responders. 310 

Refined animal studies performed to identify which 

immune responses correlate with the therapeutic activity 

indicated that both T- and B-cells should be properly 

activated. 

311, 312 

These observations may suggest that while a patient 

could be immunized against a tumor, the immunity thus 

induced might be insufficient to fight the established 

tumor growing within its own stroma. The combination 

of poor immune function and large tumor burden makes 

patients with advanced disease dubious predictors of 

clinical response. 

The general idea is that cytokine engineered tumor 

cells should be used as vaccines in minimal disease settings. 

313 A new form of treatment would thus be available 

for combination with conventional management 

of patients after surgical removal of their tumor, 

patients with minimal residual disease, or patients 

expected to manifest tumor recurrence after a significant 

apparently disease-free interval. When compared 

with conventional forms of management, vaccination is 

a soft, non-invasive treatment, unlikely to cause particular 

distress or side-effects, and could be administered 

after resection of a primary tumor when recurrence is 

expected. 

Use of mesenchymal cells for treatment of 

neoplastic and non-neoplastic disorders 

In addition to hematopoietic stem cells which can differentiate 

to produce progenitors committed to terminal 

maturation, 314 human bone marrow also contains 

stem cells of non-hematopoietic tissues which are currently 

referred to as mesenchymal stem cells (MSC), 

because of their ability to differentiate into cells that 

can roughly be defined as mesenchymal, or as marrow 

stromal cells because they appear to arise from the complex 

array of supporting structures found in marrow. 315 

Stromal cells of the marrow microenvironment include 

fibroblasts, endothelial cells, reticular cells, adipocytes, 

osteoblasts and macrophages, the last, although of 

hematopoietic origin, being considered functional components 

of the regulatory stroma. 316 The heterogeneous 

populations of mesenchymal cells and their associated 

biosynthetic products have the unique capacity to regulate 

hematopoiesis. 317 

Environmental components can modify the proliferative 

and differentiative behavior of hematopoietic cells by 

means of (i) cell-to-cell interactions, (ii) interactions of 

cells with extracellular matrix molecules, and (iii) interactions 

of cells with soluble growth regulatory molecules. 

316 All these regulatory modalities participate in 

stromal cell-mediated regulation of hematopoiesis. In 

fact, marrow stromal cells provide the physical framework 

within which hematopoiesis occurs, play a role in directing 

the processes by synthesizing, sequestering or presenting 

growth-stimulatory and growth-inhibitory factors, 

and also produce numerous extracellular matrix proteins 

and express a broad repertoire of adhesion molecules 

that serve to mediate specific interactions with 

hematopoietic stem/progenitor cells of both myeloid and 

lymphoid origin. 318, 319 Although growth factors play key 

roles in stem/progenitor cell proliferation and differentiation 

it seems improbable that hematopoiesis is regulated 

only by a random mix of growth factors and responsive 

cells. Rather, it is likely that regulatory molecules 

and localization phenomena within marrow stroma are 

required to sustain and regulate the function of the 

hematopoietic system. 320 

Although it is commonly accepted that stem cells are 

capable of homing to the marrow and docking at specific 

sites, the exact role of microenvironmental cells, adhesion 

molecules and extracellular matrix molecules in regulating 

the localization and spatial organization of 

hematopoietic stem cells in the marrow and driving 

myeloid and lymphoid regeneration following stem cell 

transplantation remains a matter of hypothesis. 321 Studies 

in animals demonstrated that stem and progenitor 

cells have different distributions across the femoral marrow 

cavity of mice, thus suggesting that marrow stroma 

is organized into functionally discrete environments, such 

as primary microenvironmental and secondary microenvironmental 

areas, allowing distinct differentiation patterns 

of hematopoietic stem cells. 322 The stem cell niche 

hypothesis, proposed by Schofield 323 suggested that certain 

microenvironmental cells of the marrow stroma 

could maintain the stem cells in a primitive, quiescent 

state. Another mechanism supporting the concept of specialized 

microenvironmental areas is stroma-mediated, 

compartimentalized growth factor production. Growth 

factor produced locally by stromal cells may bind to the 

extracellular matrix and be presented to immobilized target 

cells which recognize each growth factor through 

specific receptors. 320 This mechanism may provide the 

opportunity for localizing distinct growth factors at relatively 

high concentrations to discrete sites. As yet, relatively 

little is known of the nature of the factor(s) produced 

by different stromal cell types which modulate lineage 

development. However, a growing body of evidence 

suggests that marrow stroma is involved not only in regulating 

myeloid cell growth, but also in T- and B-cell lymphopoietic 

development. 324-327 Distinct adhesion molecules 

and cytokines are known to regulate stroma-dependent 

T- and B-lymphopoiesis, 328, 329 suggesting that marrow 

stroma may function as a site of T- as well as B-cell 

lymphopoiesis. 

The existence of self-renewing MSC is supported by 

several in vitro and in vivo data. 330 At the functional level, 

MSC residing within marrow microenvironment, establish 

marrow stroma both in vitro and in vivo and have 

multilineage differentiation capacity, being capable of 

generating progenitors with restricted development 

potential which include fibroblast, osteoblast, adipocyte, 

chondrocyte and myoblast progenitors (Figure 7). 331-333 

Putative stromal cell progenitors have been identified in 

human marrow by their ability to generate colonies of 

fibroblast-like cells originating from single clonogenic 

progenitors termed fibroblast colony-forming units (CFU- 

F). 334 These progenitors, which belong to the osteogenic 


Cell therapy 

143 

Figure 7. 

stromal lineage, play a central role in establishing the 

marrow microenvironment both in vitro and in vivo. 335-337 

Under appropriate culture conditions and supplementation 

with specific stimuli, a proportion of marrow CFU-F 

can be induced to either adipogenesis 333 or osteoblastogenesis. 

338 Studies involving ectopic transplantation of 

individual fibroblastic clones grown in vitro from mouse 

marrow beneath the renal capsule of syngeneic hosts 

demonstrated that approximately 15% produced a marrow 

organ containing the full spectrum of stromal cell 

types of hematopoietic microenvironment, thus suggesting 

that CFU-F have multilineage differentiation capacity 

and supporting the stromal stem cell hypothesis. 339 

Based on these findings, CFU-F can be identified as multipotent 

stromal progenitors rather than lineage-restricted 

fibroblast progenitors. 

CFU-F can be enriched from adult bone marrow by 

means of the STRO-1 monoclonal antibody that identifies 

essentially all assayable marrow CFU-F. 340 STRO-1 + 

cells do not express the CD34 antigen and fail to generate 

hematopoietic progenitors, thus facilitating a clean 

separation between hematopoietic and stromal progenitors. 

341 Flow-sorted STRO-1+ cells grown under longterm 

culture conditions generate adherent stromal layers 

consisting of fibroblasts, osteoblasts, smooth muscle 

cells and adipocytes. 340 These stromal layers are capable 

of supporting hematopoiesis in long-term cultures initiated 

with CD34 + cells. In addition to STRO-1, other monoclonal 

antibodies, such as SH-2, have been described 

which specifically detect mesenchymal progenitors. 342 

In vivo data generated in animal models support the 

functional regulatory role of the marrow microenvironment. 

In the fetal sheep model of in utero stem cell 

transplantation, co-transplantation of stem cells with 

marrow stromal cells has been shown to improve levels 

of donor cell engraftment. 343 In the NOD/SCID mouse 

model of in utero stem cell transplantation, fetal stem 

cells have a nine times greater engraftment potential 

but this advantage is abrogated if the recipients are irradiated 

prior to transplant, indicating that the marrow 

microenvironment is important in driving myeloid and 

lymphoid engraftment. 344 

The importance of stromal cells in hematopoiesis has 

also been demonstrated by several studies in humans. 

Despite normal peripheral blood counts, levels of primitive 

and committed progenitors in the bone marrow of 

patients who have received allogeneic stem cell transplantation 

remain subnormal for many years. 345 Furthermore, 

cultured stromal cells from patients who have 

received allogeneic stem cell transplant (SCT) show significant 

impairment in their ability to support the growth 

of hematopoietic progenitors from normal marrow. 346 

Decreased CFU-GM production and defective stroma production 

have been demonstrated following autologous 

SCT 347 as well as after induction chemotherapy. 348 

The role of marrow stroma in hematopoietic regulation 

and the peculiar functional characteristics of stromal 

cells raise the possibility that the delivery of ex vivo 

expanded marrow MSC into a hematopoietically-compromised 

marrow might promote hematopoiesis. Bone 

marrow stromal cells are a quiescent, non-cycling population 

with low cell turn-over, as demonstrated by the 

resistance to irradiation. Based on these characteristics, 

methods have been developed which allow for gene 

delivery into stromal cells. 349 Since stromal cells are metabolically 

active they also provide a suitable means of 

secreting therapeutic proteins, including coagulation factors 

or adenosine deaminase. 350 Recent data showing that 

MSC suppress allogeneic T-cell responses in vitro suggest 

a role for stromal cells in modulating allogeneic 


144 


transplant rejection and graft-versus-host disease. 351 

It must be emphasized that because of the limited 

knowledge of MSC biology, clinical applications of stromal 

cells, although exciting, essentially remain a matter 

of hypothesis to be carefully tested in the appropriate 

clinical setting. Essential prerequisites for clinical 

applications using culture-expanded mesenchymal cells 

as a supplement for hematopoietic SCT are (i) the possibility 

of isolating mesenchymal progenitors and 

manipulating their growth under defined in vitro culture 

conditions 352 and (ii) the demonstration of the possibility 

of efficiently introducing cultured stromal cells back 

into patients. 

Studies in rodents and dogs have clearly demonstrated 

that if sufficient stromal cells are reinfused, they not 

only seed the bone marrow but also enhance 

hematopoietic recovery. 353-357 Although demonstrated 

in several mouse models, the transplantability of marrow 

stromal elements remains a controversial issue in 

humans. 358, 359 The majority of data so far generated in 

recipients of HLA-identical marrow transplants has 

failed to demonstrate any contribution of donor cells to 

marrow stroma regeneration. 358 Although many factors 

may affect the transplantability of stromal elements, 

the low frequency of stromal progenitors in conventional 

marrow harvests may explain the failure of mesenchymal 

cell transplanttion in humans. 

Indeed, during the last decade, SCT methodology has 

changed substantially, particularly as a result of the 

increasing use of peripheral blood transplants. The existence 

of a circulating stromal progenitor has been 

demonstrated by using a NOD/SCID model and this is 

extremely relevant to stromal cell therapy. 360 By using 

the X-linked human androgen receptor (HUMARA) gene 

and fluorescent in situ hybridization analysis for the Y 

chromosome, the transplantability of stromal progenitors 

in a proportion of recipients of haploidentical HLAmismatched 

T-cell-depleted allografts reinfused with a 

combination of bone marrow and mobilized peripheral 

blood cells has recently been demonstrated (Carlo-Stella 

and Tabilio, unpublished observations, 1999). Taken 

together, these findings allow the hypothesis that MSC 

are transplantable in man provided that an adequate, 

but as yet unidentified, number of CFU-F is reinfused. In 

addition, these data allow the planning of clinical studies 

using culture-expanded, gene-marked mesenchymal 

cells in order to investigate a number of issues, including 

(i) dose of marrow stromal progenitors necessary to 

achieve a transplant; (ii) duration of post-transplant 

marrow stromal cell function; (iii) role of stromal cells 

in myeloid, B- and T-lymphoid reconstitution following 

SCT. 

A limited number of clinical trials using ex vivo generated 

MSC are currently underway. So far, the only 

published phase I clinical trial using MSC reported that 

the systemic infusion of autologous MSC appears to be 

well tolerated. 361 MSC can be explored as vehicles for 

both cell therapy and gene therapy (Table 4). MSC could 

be used to replace marrow microenvironment damaged 

Table 4. Potential clinical applications of mesenchymal 

stem cells. 

• Replacement of chemotherapy-damaged stroma 

• Enhancement of myeloid recovery following hematopoietic stem cell 


• Enhancement of T- and B-cell reconstitution following allogeneic 

stem cell transplantation 

• Compartimentalized growth factor/cytokine production 

• Modulation of GvHD 

• Delivery of exogenous gene products 

by high-dose chemotherapy in order to either improve 

hematopoietic recovery from myeloablative chemotherapy 

or to treat late graft failures or delayed platelet 

engraftment. Based on their functional characteristics, 

MSC are attractive vehicles for gene therapy in that they 

are expected not to be lost through differentiation as 

rapidly as hematopoietic progenitors. Examples of diseases 

in which stromal cell-mediated gene therapy 

might be appropriate include factor VIII and factor IX 

deficiencies and the various lysosomal storage diseases. 

Interestingly, compared to skin fibroblasts or leukocytes, 

marrow-derived mesenchymal cells produce significantly 

higher levels of -iduronidase, an enzyme 

involved in type II mucopolysaccharidoses (Danesino and 

Carlo-Stella, unpublished data). In addition, stromal cells 

might also be transduced with cDNA of various 

hematopoietic growth factors or cytokines. This 

approach might allow high levels of compartimentalized 

growth factor production and might be used (i) to stimulate 

hematopoiesis in patients with congenital or 

acquired hematopoietic defects, (ii) to improve B- and 

T-cell recovery following allogeneic SCT, (iii) to accelerate 

myeloid reconstitution in recipients of cord blood 

transplants. 

In conclusion, MSC appear to be an attractive therapeutic 

tool capable of playing a role in a wide range of 

clinical applications in the context of both cell and gene 

therapy strategies. However, a number of fundamental 

questions about MSC still need to be resolved before 

they can be used for safe and effective cell and gene 

therapy. 

Conclusions 

Although most of the new therapeutic approaches of 

cell therapy are experimental and have not yet been validated 

by phase III clinical trials, they appear to hold a 

high therapeutic potential. Separation of GVL from GvHD 

through generation and infusion of leukemia-specific T- 

cell clones or lines is one of the most intriguing and 

promising fields of investigations for the future. Likewise, 

strategies devised to improve immune reconstitution 

and restore specific anti-infectious functions 

through either induction of unresponsiveness to recipient 

alloantigens or removal of alloreactive donor T-cells 


Cell therapy 

145 

might increase the applicability and success of 

hematopoietic stem cell transplantation. Cellular 

immunotherapy with DC must be standardized and several 

critical points, discussed in this review article must 

be properly addressed with specific clinical studies. Stimulation 

of leukemic cells via CD40 receptors and transduction 

of tumor cells with co-stimulatory molecules 

and/or cytokines may be useful in preventing tumor 

escape from immune surveillance. Tumor cells can be 

genetically modified to interact directly with dendritic 

cells in vivo or recombinant antigens can be delivered to 

dendritic cells using attenuated bacterial vectors by oral 

vaccination. MSC represent an attractive therapeutic 

tool capable of playing a role in a wide range of clinical 

applications in the context of both cell and gene therapy 

strategies. 


All authors gave substantial contributions to analysis 

and interpretation of literature data and drafting the 

article or revising it critically. CB was primarily responsible 

for the section on genetically engineered donor lymphocyte 

infusion for treatment of leukemia relapse, CCS 

for the section on mesenchymal cells, MPC for the sections 

on oral vaccination and engineered tumor vaccines; 

RML for the section on dendritic cells DR. FL was primarily 

responsible for sections on adoptive immunotherapy, 

AO for the sections on LAK and TIL and DR for the section 

on tumor escape from immune surveillance and on 

leukemic cells as APC. The authors are listed in alphabetical 

order. 

Disclosures 

Conflict of interest. This review article was prepared by 

request from Haematologica. The authors were a group of 

experts and representatives of two pharmaceutical companies, 

Amgen Italia SpA and Dompé Biotec SpA, both 

from Milan, Italy. This co-operation between a medical 

journal and pharmaceutical companies is based on the 

common aim of achieving optimal use of new therapeutic 

procedures in medical practice. In agreement with the 

Journal’s Conflict of Interest policy, the reader is given 

the following information. The preparation of this manuscript 

was supported by educational grants from the two 

companies. Dompé Biotec SpA sells G-CSF and rHuEpo in 

Italy, and Amgen Italia SpA has a stake in Dompé Biotec 

SpA. 

Redundant publications: no overlapping with previous 

papers. 


Manuscript received May 10, 1999; accepted August 

30, 1999. 

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eview 

Antitumor vaccination: 

where we stand 


Correspondence: Prof. Sante Tura, Istituto di Ematologia e Oncologia 

Medica “Seràgnoli”, Università di Bologna, Policlinico S.Orsola- 

Malpighi, via Massarenti 9, 40138 Bologna, Italy. Phone: international 

+39-051-6363680 – Fax: international-39-051-6364037 – 

E-mail: tura@orsola-malpighi.med.unibo.it 

MONICA BOCCHIA,* VINCENZO BRONTE,° MARIO P. COLOMBO, # 

ARMANDO DE VINCENTIIS, @ MASSIMO DI NICOLA,^ 

GUIDO FORNI, § LUIGI LANATA,** ROBERTO M. LEMOLI,°° 

MASSIMO MASSAIA, ## DAMIANO RONDELLI,°° PAOLA ZANON,** 

SANTE TURA°° 

*Department of Hematology, University of Siena; °Department 

of Oncology, University of Padova; # Division of Oncology, Istituto 

Nazionale dei Tumori, Milan; @ Dompè-Biotec, Milan; ^Bone 

Marrow Transplant Unit, Istituto Nazionale dei Tumori, Milan; 

§ 

Department of Clinical and Biological Sciences, University of 

Turin; **Amgen Italia, Milan; °°Institute of Hematology and 

Medical Oncology “Seràgnoli”, University of Bologna; ## Division 

of Hematology, University of Turin, Italy 

Background and Objectives. Vaccination is an effective 

medical procedure of preventive medicine based on 

the induction of a long-lasting immunologic memory 

characterized by mechanisms endowed with high 

destructive potential and specificity. In the last few 

years, identification of tumor-associated antigens (TAA) 

has prompted the development of different strategies 

for antitumor vaccination, aimed at inducing specific 

recognition of TAA in order to elicit a persistent 

immune memory that may eliminate residual tumor 

cells and protect recipients from relapses. In this 

review characterization of TAA, different potential 

means of vaccination in experimental models and preliminary 

data from clinical trials in humans have been 

examined by the Working Group on Hematopoietic 

Cells. 

Evidence and Information Sources. The method 

employed for preparing this review was that of informal 

consensus development. Members of the Working 

Group met four times and discussed the single points, 

previously assigned by the chairman, in order to 

achieve an agreement on different opinions and 

approve the final manuscript. Some of the authors of 

the present review have been working in the field of 

antitumor immunotherapy and have contributed original 

papers to peer-reviewed journals. In addition, the 

material examined in the present review includes articles 

and abstracts published in journals covered by the 

Science Citation Index and Medline. 

State of the art. The cellular basis of antitumor 

immune memory consists in the generation and extended 

persistence of expanded populations of T- and B- 

lymphocytes that specifically recognize and react 

against TAA. The efficacy of the memory can be modulated 

by compounds, called "adjuvants", such as certain 

bacterial products and mineral oils, cytokines, 

chemokines, by monoclonal antibodies triggering costimulatory 

receptors. Strategies that have been shown 

in preclinical models to be efficient in protecting from 

tumor engraftment, or in preventing a tumor rechallenge, 

include vaccination by means of soluble proteins 

or peptides, recombinant viruses or bacteria as TAA 

genes vectors, DNA injection, tumor cells genetically 

modified to express co-stimulatory molecules and/or 

cytokines. The use of professional antigen-presenting 

cells, namely dendritic cells, either pulsed with TAA or 

transduced with tumor-specific genes, provides a useful 

alternative for inducing antitumor cytotoxic activity. 

Some of these approaches have been tested in phase 

I/II clinical trials in hematologic malignancies, such as 

lymphoproliferative diseases or chronic myeloid 

leukemia, and in solid tumors, such as melanoma, 

colon cancer, prostate cancer and renal cell carcinoma. 

Different types of vaccines, use of adjuvants, timing of 

vaccination as well as selection of patients eligible for 

this procedure are discussed in this review. 

Perspectives. Experimental models demonstrate the 

possibility of curing cancer through the active induction 

of a specific immune response to TAA. However, while 

pre-clinical research has identified several possible targets 

and strategies for tumor vaccination the clinical 

scenario is far more complex for a number of possible 

reasons. Since experimental data suggest that vaccination 

is more likely to be effective on small tumor burden, 

such as a minimal residual disease after conventional 

treatments, or tumors at an early stage of disease, 

better selection of patients will allow more reliable 

clinical results to be obtained. Moreover, a poor 

correlation is frequently observed between the ability of 

TAA to induce a T-cell response in vitro and clinical 

responses. Controversial findings may also be due to 

the techniques used for monitoring the immune status. 

Therefore, the development of reliable assays for efficient 

monitoring of the state of immunization of cancer 

patients against TAA is an important goal that will 

markedly improve the progress of antitumor vaccines. 

Finally, given the promising results, identification of 

new or mutated genes involved in neoplastic events 

might provide the opportunity to vaccinate susceptible 

subjects against their foreseeable cancer in the next 

future. 


Key words: antitumor vaccination. 


Antitumor vaccination 

157 

Antitumor vaccines: the meaning 

As illustrated in a previous paper of this series, many 

strategies are being used to try to cure cancer, each one 

based on different theoretical and experimental 

grounds. 1 Active immunotherapy strategies elicit specific 

or non-specific antitumor reactions by stimulating the 

patient’s immune system. Alternatively, lymphocytes 

collected from patients are stimulated in vitro and reinjected 

into the patient (adoptive immunotherapy). 

Lastly, passive immunotherapy consists in the administration 

of antitumor antibodies to the patient. However, 

dealing with such a dramatic issue as is cancer, emotional 

empiricism spurs the adoption of distinct strategies 

or their mixing in an apprehensive pursuit of efficacy. 

Indeed, emotional empiricism has been and still is 

a deadly sin of tumor immunology. In the long run, only 

rational considerations lead to clinical progress. 

The issue 

Vaccination is an effective medical procedure characterized 

by being a) predominantly a maneuver of preventive 

medicine; b) based on the induction of a longlasting 

immunologic memory that is c) characterized by 

mechanisms endowed with high destructive potential 

and specificity. 2 It rests on an artificial encounter of a 

sham pathogen with the immune system. The sham 

pathogen elicits a strong host reaction and leaves a persistent 

memory of this first artificial fight. If the real 

pathogen enters the immunized organism it becomes the 

target of a much stronger and precise reaction than that 

put up by a non-immunized organism. If the pathogen 

had a small possibility of escaping the reaction of a naive 

immune system, very seldom would it evade a memory 

reaction. 3 The cellular basis of immune memory consists 

in the extended persistence of expanded populations of 

T- and B- lymphocytes that specifically recognize and 

react against the pathogen. Memory lymphocytes are also 

experienced veterans, able to detect a pathogen promptly 

and fight effectively against it. 

There is a large universe of sham pathogens that are 

used for vaccination, named antigens or immunogens. 

A killed or inactivated pathogen, or a non-pathogenic 

organism sharing critical molecules with the real thing 

can be a good immunogen. Memory can also be induced 

by a protein from the pathogen. In addition, a virus engineered 

with the DNA coding for a protein of the 

pathogen or even the mere naked DNA induces an effective 

memory. 4 

The efficacy of the memory is modulated by numerous 

compounds, called adjuvants and danger signals. 5,6 

These provide additional activation signals, recruit reactive 

leukocytes at the immunization site and delay antigen 

catabolism. Bacterial products and mineral oils are 

typical conventional adjuvants. Cytokines and chemokines 

also act as adjuvants. As will be discussed in detail, 

their use allows the induction of selective mechanisms 

of immune memory. 7 

Antitumor vaccination has a defined goal: to provoke 

specific recognition of tumor-associated antigens (TAA) 

in order to elicit a persistent immune memory. Many 

experimental data have shown that following immunization, 

the growth of tumor cells expressing the same 

TAA as the vaccine can be impaired. In patients, the 

immune memory elicited by vaccines is sometimes fast 

and strong enough to hamper the growth of their 

tumor. 8 

A brief history 

Interest in antitumor vaccination arose around 1900 

when a series of microbial vaccines proved to be effective. 

The idea was straightforward: to apply the same 

intervention to tumor. «If .........it is possible to protect 

small laboratory animals in an easy and safe way against 

infectious and highly aggressive neoplastic specimens, 

then it will be possible to do the same for human 

patients». These words of 1897 by Paul Ehrlich 9 ignited 

a series of studies with transplantable mouse tumors. 

However the underlying issue turned out to be more 

complex than had first been presumed. More than one 

century was required to elucidate its molecular and 

genetic features. 

The first outcome was not a progress in anti-tumor 

vaccination, but instead the definition of a few rules of 

allograft rejection. Transplanted tumors were rejected by 

immunized host not because they expressed a particular 

TAA but because they were from histoincompatible 

mice and displayed normal allogeneic histocompatibility 

antigens. 10 Later studies with syngeneic mouse 

strains showed the feasibility of immunizing a mouse 

against a subsequent tumor challenge. 11 However, the 

suspicion that residual unnoticed histocompatibility differences 

were involved in these vaccination-rejection 

studies was not ruled out until experiments by George 

Klein in 1960. 12 Carcinoma was induced by methylcholanthrene 

in syngeneic mice. The carcinoma was 

then surgically removed, and its cells were cultured in 

vitro and used to repeatedly immunize the mouse in 

which the tumor had arisen. Finally the immunized 

mouse and a few control syngeneic mice were challenged 

with the carcinoma cells of the original tumor 

maintained in vitro. While these cells gave rise to a carcinoma 

in control mice, they were rejected by the immunized 

mouse in which the carcinoma had arisen originally. 

This evidence of the possibility of immunizing 

against a lethal dose of own tumor was of seminal 

importance and had notable consequences. A very large 

series of subsequent studies established a few basic 

foundations of tumor vaccination. 13 

Looking back at tumor immunology over the last 20- 

30 years, the importance of models in influencing 

immunologic beliefs is strikingly evident. Inappropriate 

use of an experimental model may produce wrong 

beliefs, from which it is then very hard to escape. Virusand 

chemically-induced tumors form highly immunogenic 

models. Since they are easy to handle, these were 

used to establish the rules of tumor vaccination. However, 

it was disputable whether the information from 

these models was relevant to the situation of patients 


158 

M. Bocchia et al. 

with cancer. Using a series of murine spontaneous 

tumors, Hewitt concluded that it was not possible to 

immunize against these tumors. 14 This observation had 

crucial importance in shaping subsequent studies. The 

possibility that the experimental work done with high 

immunogenic transplantable tumors had little relevance 

to human tumors was a dark shadow that hindered the 

progress of tumor immunology. Later, more careful use 

of these spontaneous tumors and more refined immunization 

techniques showed that Hewitt's conclusions 

were wrong. 15 

Boon led the genetic and molecular identification of 

a large series of TAA. Initially his studies were performed 

using conventional transplantable mouse tumors. 16 

Then, tumor antigens were detected on the same spontanous 

tumors that had previously been classified as 

non-immunogenic by Hewitt. 15 Now many antigens 

associated with human tumors have been identified. 17,18 

The targets 

Boon and others 18,19 provided an unambiguous definition 

of TAA, an important finding that definitively laid 

to rest the doubts on the foundations of tumor 

immunology in man. 20 In many cases TAA are peptides 

presented by class I and class II glycoproteins of the 

major histocompatibility complex (MHC). Things that 

may give rise to these tumor-associated peptides are 

enhanced or diminished expression of some normal proteins 

and the new expression of altered or normally 

repressed molecules. Less frequently these antigens are 

tumor-specific as they derive from mutated proteins. 

Lastly, various TAA are shared by tumors with distinct 

histology and origin (Table 1). Telomerase catalytic subunit 

looks like another widely expressed TAA recognized 

by T-lymphocytes. It is markedly activated in most 

human tumors while it is silent in normal tissues. 21 

Why? 

The central tenet of antitumor vaccination is that the 

immune system is able to destroy tumor cells and to 

retain a long-lasting memory provided that TAA are first 

efficiently recognized. While the studies aimed at the 

definition of TAA progressed quickly, investigations of 

lymphocyte receptors and their idiotype network, costimulatory 

molecules, and cytokines were leading to a 

more exact description of the requirements for the 

induction of an immune response. 22 Finally, technical 

refinement of genetic engineering is making the development 

of new cancer vaccines easier . 23 The convergence 

of these issues is once again placing antitumor 

vaccination at the cutting edge of biological research. 

A survey by Science 24 indicates that antitumor vaccination 

is expected soon to become an established therapeutic 

option. 

When? 

Whereas individuals are immunized with microbial 

vaccines prior to encountering the pathogen, cancer 

patients have to be immunized when a tumor has been 

already detected. It is not yet possible to predict which 

combination of gene mutations will give rise to cancer. 

Therefore, the common clinical setting is elicitation of 

an immune response in a tumor-bearing patient, rather 

than prior to tumor development. The very concept of 

vaccine is somewhat distorted since it has moved from 

being preventive to being therapeutic. 23 

The kind of patients who should be considered eligible 

for tumor vaccination is not a minor issue. In many 

trials patients with advanced diseases are enrolled both 

for compassionate reasons and because of the constraints 

imposed by ethical considerations. But, do experimental 

data suggest that vaccination could be effective 

in advanced stages of neoplastic progression? The experimental 

data provide an unambiguous picture of the 

potentials and limits of vaccination. This picture is not, 

however, generally taken into account. Perhaps unconscious 

reasons lead to experimental data being assessed 

with optimistic superficiality. 25 Many experimental studies 

have shown that an antitumor response can be elicited 

by new vaccines. This results in strong resistance to 

a subsequent tumor challenge and inhibition of minimal 

residual disease remaining after convention therapy. The 

pitfall hidden in the evaluation of these vaccine-re-challenge 

experiments is that successful immunization of 

healthy mice against a subsequent re-challenge with 

tumor cells does not demonstrate a true therapeutic 

effect. 26 Examination of more realistic studies of the ability 

of vaccines to cure existing tumors shows that only 

a minority of tumor-bearing mice could be cured. Furthermore, 

the limited therapeutic efficacy of vaccines 

was lost when they were not given in the first few days 

after the implantation of tumor cells. 26 

A similar picture is emerging from phase I studies on 

vaccination of cancer patients. The vaccination is safe, 

but the results suggest that only a minority of patients 

(about 10%) display an objective response. The immunologic 

performance status of these patients is obviously 

suboptimal for this type of therapy. Even so, one would 

have expected a greater number of responders to support 

the promise of new sophisticated vaccines. 23 

Therapeutic vaccination has not had much success in 

the management of infectious diseases. Its use against 

the progression of an established tumor is very challenging, 

since it must secure an effective immune 

response capable of getting the better of a well-established, 

proliferating tumor. Of the several objectives that 

Table 1. Cross-expression of some tumor-associated antigens 

among histologically different human tumors from distinct 

organs. 

bladder BAGE GAGE 

breast BAGE MAGE CEA p53 ras MUC-1 

colon CEA p53 ras 

lung BAGE CEA p53 

melanoma BAGE GAGE MAGE ras 

pancreas CEA p53 ras MUC-1 

sarcomas 

GAGE 



159 

have been made approachable by antitumor vaccines, 

the cure of advanced tumors is both the most difficult 

and at the same time the most common in clinical trials. 

The strong emotions kindled by cancer suffering provide 

the main justification for these attempts. 27 Perhaps, 

major improvements in antitumor vaccination will make 

this goal approachable. However, the data reviewed 

show that this is far from the present reality. 

Nevertheless it should be considered that most tumor 

lethality depends on a few neoplastic cells remaining 

after surgical excision of a tumor mass or after having 

escaped direct killing by chemo- and radiotherapy. Many 

experimental findings 28-30 suggest that a stage of minimal 

residual disease is one in which it is possible to 

foresee a significant cure by immunization. After successful 

conventional management the tumor burden 

may be low, and the tumor may reappear after a long 

dormancy. This is a realistic setting in which vaccination 

could lead to the induction of anti-tumor immunity 

capable of extending the survival of patients. As there 

are grounds for believing that antitumor vaccines could 

be used as an effective anticancer tool, the purpose of 

this review is to describe the types of vaccines that are 

being experimented, emerging clinical results and the 

new perspectives opened by this scientific endeavor. 

Common tools 

Cytokines and cellular signals 

The immunologic attempt of the immune system to 

prevent the development of a neoplastic disease may 

be ineffective due to either a lack of immunogenicity of 

tumor cells, or to a weak reaction unable to contrast the 

neoplastic proliferation. In both cases, it is likely that 

most of the physiologic mechanisms of priming of the 

immunologic effector cells may be impaired or absent. 

In fact, initiation of immune responses requires that 

professional antigen-presenting cells (APC) deliver a first 

signal to T lymphocytes through the binding of the T-cell 

receptor by the peptide enclosed in the HLA molecule, 

that is responsible for the specificity of the immune 

response, and a second or co-stimulatory signal that is 

not antigen-specific but it is required for T-cell activation 

31,32 mainly through CD80 (B7-1) and CD86 (B7-2) 

binding to CD28 receptor, or the CD40:CD40L pathway. 

Moreover, the capacity of dendritic cells (DC) to activate 

natural killer (NK) cells by ligation of the CD40 molecule 

with its counter-receptor has recently been demonstrated. 

33,34 Immunocompetent cells may also determine 

the type of immune response by the expression of 

chemokines and by the release of pro-inflammatory, or 

anti-inflammatory cytokines which drive T-cells to different 

activities or even to suppression. 35 

Therefore, given the complex network of regulatory 

signals by professional APC and naive and memory lymphocytes 

occurring in antigen-specific immune responses, 

it is not surprising that tumor cells may fail to induce 

efficient humoral and cellular immune reactions even 

when a well known TAA is present. In this review, several 

strategies to overcome the immune escape mechanisms 

of tumor cells will be considered, such as the 

direct use of TAA to elicit specific reactions, the use of 

dendritic cells to present TAA in order to enhance the 

immune response, and the use of tumor cells genetically 

modified to function as professional APC or to release 

soluble factors. Animal models have been widely used 

for many years to demonstrate the effect of different 

cytokines, added to or secreted by tumor cell-based vaccines, 

in increasing the in vitro and in vivo cytotoxicity 

against tumor challenge. The role of the main cytokines 

involved in activation of humoral and cellular immune 

responses is represented in Figure 1. On the basis of 

cytokine functions it has been previously shown in 

experimental models that: the number of APC in the 

site of tumor infiltration can be increased by cytokines 

such as granulocyte-macrophage colony-stimulating 

factor (GM-CSF) and interleukin (IL)-4, which should 

allow also the differentiation of DC precursors; B- and 

T-cell responses are potently increased by IL-2, IL-12, or 

GM-CSF; 36 and in particular T-cell cytotoxicity is 

enhanced by GM-CSF, IL-2, IL-12, interferon (IFN-γ), 

tumor necrosis factor (TNF-α), while NK activity is 

enhanced by IL-12 or FLT 3-L. 36,37 However, different 

models and different TAA resulted in controversial findings. 

Furthermore, since these cytokines are likely to be 

more effective when released within the tumor area, 

the transduction of cytokine genes into tumor cells and 

their use as cellular vaccines after irradiation has been 

tested in animal models and in humans. 38-40 

Initial clinical experiences in patients with advanced 

melanoma or renal cell carcinoma suggest that tumor 

cell-based vaccines, either engineered to produce GM- 

CSF or IL-12 or with exogenous GM-CSF, may facilitate 

marked infiltration of DC and CD4 + and CD8 + T-lymphocytes 

into tumor lesions, potentially improving the 

antitumor effect. These data provided evidence of the 

feasibility of the approach but were unlikely to be able 

to address the point of efficacy, due to the large tumor 

burden of these patients. Future immunotherapy 

attempts should, in fact, focus on the possibility of eradicating 

minimal residual disease. More recent data 

demonstrated the role of GM-CSF as a useful adjuvant 

in peptide-based vaccines in ovarian and breast cancer 

and in follicular lymphoma, as will be described later in 

this review. 

Figure 1 also shows that cell-to-cell contact via 

CD40:CD40L plays a pivotal role in activating specific T- 

cell, B-cell and NK-cell responses. On the other hand, T- 

cell tolerance can be obtained by blockade of CD40L 

receptor in non-human primates undergoing solid organ 

allogeneic transplantation, and in mice receiving either 

allogeneic bone marrow or solid organ transplantation. 

41-43 

Recent experiments demonstrated that stimulation, via 

an activating anti-CD40 antibody, resulted in the activation 

of host APC and could convert lymphocytes of 

mice treated with a tolerogenic peptide vaccine into 

cytotoxic T- cells. Moreover, this stimulation induced the 


160 


regression of established tumors that had not been 

affected by previous vaccination alone, 44,45 thus showing 

that triggering the CD40 molecule may both overcome T- 

cell tolerance in a tumor-bearing animal and greatly 

potentiate a peptide-based vaccine. Moreover, gene 

transfer by an adenovirus vector of CD40L in human B- 

cell chronic lymphocytic leukemia (B-CLL) allowed the 

activation of bystander non-infected B-CLL cells that 

upregulated co-stimulatory molecules such as CD80 and 

CD86 and stimulated autologous cytotoxic T-cells. 46 

Another important issue concerns the way T-cells are 

turned off by CTLA-4 receptor following activation via 

CD28. In fact, both CD28 and CTLA-4 bind with high 

affinity to CD80 and CD86 and CTLA-4 physiologically 

blocks T-cell activation. In the case of antitumor T-cell 

response it has been demonstrated that blockade of negative 

regulatory signals by an anti-CTLA-4 monoclonal 

antibody may retard tumor growth in experimental systems. 

47,48 More recent studies in mice suggested that this 

molecule was extremely efficient in causing tumor 

regression when used in combination with subtherapeutic 

doses of melphalan, or with a GM-CSF-expressing 

tumor. 49,50 

Altogether, these studies strongly support the role of 

cytokines or immunomodulatory molecules in anticancer 

vaccine strategies. However, they do not clarify 

whether a strict T-helper (Th1) response is required to 

achieve tumor killing, or whether a humoral response 

induced by anti-inflammatory cytokines should also be 

pursued. Finally, future directions of anticancer vaccine 

research are likely to deal with monoclonal antibodies 

enhancing or blocking specific receptors. 

Dendritic cells as initiators of immune 

response 

Dendritic cells (DC) represent a heterogeneous population 

of leukocytes defined by morphologic, phenotypic 

and functional criteria which distinguish them from 

monocytes and macrophages. 32 From among the professional 

APC, DC are the most potent stimulators of T- 

cell responses and play a crucial role in the initiation of 

primary immune responses. 32 

The DC system comprises at least three distinct subsets, 

including two within the myeloid or non-lymphoid 

lineage, and a third represented by lymphoid DC. 32,51 

There is also a continuum of differentiation within each 

of these subsets, from precursors circulating through 

blood and lymphatics, to immature DC resident in 

peripheral tissues, to mature or maturing forms in the 

thymus and secondary lymphoid organs. Recent studies 

have focused on the different roles of lymphoid and 

myeloid DC: more resident lymphoid DC induce tolerance 

to self, whereas migratory myeloid DC, including 

Langherans cells, are activated by foreign antigens in the 

periphery and move to lymphoid organs to initiate an 

immune response. 32 

DC have always been described as having two distinct 

functional stages: 1) immature, with high antigen 

uptake and processing ability, and poor T-cell stimulatory 

function; 2) mature, with high stimulatory function 

and poor antigen uptake and processing ability. Bacterial 

products such as lipopolysaccharides, and inflammatory 

cytokines such as IL-1, TNF-α, type I interferons 

(IFN α or β) and prostaglandin E2 (PGE2) stimulate DC 

maturation, whereas IL-10 inhibits it. 52 Interestingly, 

Figure 1. Principal cytokines 

involved in the antitumor immune 

response. 



161 

human and murine DC upregulate the synthesis of HLA 

class I and II molecules, and B7-1, B7-2 and CD40 molecules, 

after ingestion of bacteria or bacterial products, 

such as lipopolysaccharides, can prime naive T-cells. 

An emerging concept is that APC activate T-helper (Th) 

cells not only with antigen and co-stimulatory signals, 

but also with a polarizing signal (signal 3). This signal 

can be mediated by many APC-derived factors, but IL-12 

and PGE2 seem to be of major importance. As for Th cells, 

APC can be functionally polarized. In vitro experiments 

with monocyte-derived DC showed that the presence of 

IFN-γ during activation of immature DC induces mature 

DC with the ability to produce large quantities of IL-12 

and, consequently, a Th1-driving capacity (APC1 or DC1). 

In contrast, PGE2 primes for a low IL-12 production ability 

and Th2-driving capacity (APC2 or DC2). 32,53 DC-stimulated 

CD4 + cells upregulate CD40L/ CD154 that reciprocally 

activates DC via CD40. This renders DC more 

potent stimulators of CD8 + cytotoxic T-cell (CTL). 54 This 

novel concept is in contrast to simultaneous stimulation 

of CD4 + and CD8 + T-cells by DC, whereby the CD4 + T- 

cells secrete helper lymphokines in support of CD8 + CTL 

development. 55 Together with CD40 L and CD40, two different 

groups have discovered another paired member of 

the TNF-TNF receptor family. This factor, termed TNFrelated 

activation induced cytokine (TRANCE) or receptor 

activator of NF-kB ligand (RANK-L), is expressed by T 

cells. 56 Its corresponding receptor, receptor activator of 

NF-kB (RANK7) or TRANCE R, is expressed by mature DCs 

but not on freshly isolated B cells, macrophages, or T 

cells. 57 Ligation to this receptor causes either activation 

of T-lymphocytes or enhancement of DC survival. In addition, 

IL-12 is a critical mediator of DC-supported differentiation 

of naive, but not memory, B-cells, 58 indicating 

that direct interactions occur between DC and B cells, 

apart from those that occur via cognate CD4 + T-cell help. 

Lastly, it should be mentioned that the primary and 

secondary B cell follicles contain another population of 

DC, the follicular DC (FDC). The origin of these cells is 

not clear, and most investigators believe that they are 

not leukocytes. FDC trap and retain intact native antigen 

as immune complexes for long periods of time, present 

it to B cells and are likely to be involved in the affinity 

maturation of antibodies, the generation of immune 

memory and the maintenance of humoral immune 

responses. 59 

In conclusion, there is a general agreement in considering 

DC as very important players in the game of 

immune responses against foreign antigens either of 

infectious agents or of neoplastic cells. Many developing 

immunotherapeutic strategies against danger antigens 

are aimed at exploiting the powerful antigen-presenting 

properties of DC by an in vivo or ex vivo engineering 

of the DC system. In fact, subcutaneous or intramuscular 

injection of antigens relies on the local recruitment 

and activation of DC to capture and present antigens to 

the immune system. Although the techniques for targeting 

tumor antigens to DC in situ might eventually obviate 

the need for ex vivo manipulation of DC, novel methods 

for ex vivo generation and activation of large 

amounts of human DC have been developed. 

Antitumor vaccination: types 

and formulations 

Killing for priming and killing to destroy 

The way cell vaccines die when injected in vivo influences 

DC loading. The initial activity of cytokines transduced 

into the cell vaccine is to select the leukocyte type 

recruited and stimulated at the site of injection. Tumor 

cells are killed by effectors of the innate response; NK 

and polymorphonuclear cells also produce secondary 

cytokines that set up a local inflammation recruiting DC, 

whereas T-cell response is activated later. Of note, gene 

engineering allows the manipulation of the first phase of 

the process through the choice of cytokine and/or cofactor 

and by deciding the level of cytokine to be produced. 

Once the infiltrate leukocytes are activated, their 

response to the triggering cytokine is physiologic and 

independent. They produce other cytokines thus amplifying 

the magnitude and the complexity of response. The 

initial stage is finalized to T- and B-cell activation, killing 

of cell vaccine is to provide the antigen(s) and the 

inflammatory response should provide the right environment 

for such activation. 60 

The final stage is aimed at the destruction of existing 

tumor. Specific immune response is often insufficient 

to fight solid tumor nodules. Among the possible causes, 

immunosuppression, low effector-target ratio, MHC 

downregulation on tumor cells are the most common. 

Animal studies have shown that these problems can be 

overcome by general inflammation associated with neutrophil 

influx. This combination may destroy the tumorassociated 

blood vessels 61 in a way that may resemble 

tissue damage in vasculitis. In this setting a specific 

immune response is not directly responsible for tumor 

elimination but should be strong enough to at least 

begin and direct the inflammatory response to the tumor 

site. In this perspective, tumor vessels are the main target 

of a non-specific immune response, their functional 

impairment increasing the effect of either T- or B- 

cell-mediated specific immune responses. 

An add to DC-common link? 

Although DC have been indicated as central in alerting 

and activating the immune system, it is now clear that 

certain peptides are not and can not be presented by 

mature DC. This observation, made by Van den Eynde and 

colleagues, 62 concerns autoantigens and T lymphocytes 

that recognize them without being deleted in the thymus 

and normally without provoking autoimmunity. In fact, 

APC differ from other cell types by the proteosome that 

digests protein into immunogenic peptides to fit the MHC 

groove. APC immunopreoteosomes have three catalytic 

subunits substituted by those induced by IFN-γ, thus generating 

slightly different peptides from those generated by 

non-APC cells. 

Several self-antigens identified as tumor-associated 

because of CTL recognition may not be processed by 


162 


immunoproteosome. The implication is that such CTL were 

not generated through DC presentation or at least not 

through DC processing unless this happened during transition 

from immature to mature DC. 63 Perhaps free peptides 

can be captured on the surface of DC for presentation, 

or perhaps other as yet unknown mechanisms are 

involved. 

Genetically modified tumor cell vaccines 

Old and recent discoveries confirm the possibility of a 

cancer vaccine made of tumor cells. 

An empirical approach, such as the use of allogeneic 

whole-cell vaccine composed of 3 allogeneic melanoma 

cell lines established in vitro, allowed a 3-fold increase of 

the five-year survival of patients with stage IV melanoma 

as reported by Morton et al. 64 The most active component 

of Morton’s vaccine has been identified by Livingston and 

colleagues 65 to be a ganglioside (GM2). Patients who 

develop antibody response to ganglioside showed a significant 

survival advantage. Whether a CTL response was 

also activated has not been investigated but does probably 

exist. However, this finding prompted a phase III study 

in patients with stage III disease. 

In the autologous setting, irradiated melanoma cells 

were modified with dinitrophenyl and used to treat 

patients with metastatic disease. Clinical evidence of 

inflammatory response to superficial metastases was 

reported. The same treatment administered to phase III 

patients who remain tumor-free after resection of lymph 

node metastases has resulted in 50 and 60% 4-year 

relapse-free and overall survival, respectively. 66 Immunostaining, 

TCR repertoire analysis and functional data of 

node-metastases post-vaccination have shown that treatment 

with autologous dinitrophenyl-modified melanoma 

cells can expand certain T-cell clones at the tumor site. 67 

The above results bring up two issues: autologous versus 

allogeneic tumor cells and chemical or genetic (see 

below) modifications of tumor cells to be used as cancer 

vaccines. 

Before discussing these issues we should, however, 

address a more basic question, that is how to use cancer 

cells as vaccine. Well before identification of tumor antigens, 

their existence was inferred in melanoma on the 

basis of expansion and characterization of cytotoxic T- 

lymphocytes recognizing autologous tumor, 68 whereas 

antibodies against a variety of tumor types were isolated 

from patient sera. Antigens recognized by CTL can be 

tumor-specific and even restricted to the autologous 

tumor or cross-react among different neoplasms of the 

same or different tissue origin depending on the mechanism 

generating such an antigen: point mutation, incorrect 

splicing, over-expression, translocation and other (see 

Table 2). The number of tumor antigens is always increasing 

thus suggesting that our knowledge of the antigenic 

repertoire of tumor cells is partial. In addition, which antigen 

among those already characterized should be used in 

a vaccine? 

These problems can be solved altogether by using tumor 

cells that would represent the entire antigenic repertoire 

with a single drawback, that is immunoselection of certain 

antigens. Tumor cells from different patients may 

undergo different processes of immunoselection and 

therefore a pool of these tumors would be ideal for preparing 

an allogeneic vaccine. The finding that antigens 

belonging to allogeneic cells are processed by host antigen-presenting 

cells, 69 so that they can be recognized by 

host T-lymphocytes (a phenomenon called cross-priming), 

removes any conceptual obstacles to the use of allogeneic 

cells. Allovaccines have several advantages over autologous 

vaccines: cell lines that are extensively characterized 

in vitro can be used to treat all the patients included 

in a clinical study. Genetic modifications of tumor cells 

have been widely studied as a way to increase immunogenicity. 

These modifications can be easily made to a cell 

line, thus avoiding the need to isolate cells from every 

tumor. The rate of success in culturing tumor cells from a 

primary tumor varies according to the type of tumor and 

experience of the operator. Transduced cell lines can be 

selected for production of a certain amount of transgene 

thus assuring that the same vaccine will be given to all 

patients. Which modification is most efficient in inducing 

tumor immunity has not been unequivocally determined 

since variability among different tumors has been 

described. Chemical modification, also called haptenization, 

aims at adding helper determinant to tumor cells, 70 

although the exact mechanism and the downstream pathways 

activated in this way are not clearly understood. 

Genetic modification is now preferred since the effect of 

several cytokines and co-stimulatory molecules are known 

at molecular level. Their genes can be transduced into 

tumor cells that acquire new immunoregulatory functions. 

In this way a desired immune response can be fine-tuned 

through gene dosage, recruitment of certain cell types, 

deflection of Th1 or Th2 type of response and several others 

mechanisms. 60 

To summarize the most recent and promising 

approaches we may consider two strategies aimed at 

favoring vaccine interaction with DC or directly with T 

lymphocytes. The two approaches can be viewed for 

priming or boosting (Figure 2). A combination of 

cytokine and co-stimulatory factors is likely to be synergistic 

as generally occurs when soluble and cell-contact 

signals are given together. Tumor cells transduced 

with both GM-CSF and CD40L have been shown to be 

heavily infiltrated by DC. GM-CSF induces proliferation 

and maturation of hematopoietic cells, and has been 

shown to stimulate DC accessory properties and 

enhance the immune response initiated by these cells. 71 

The CD40/CD40L interaction plays a critical role in 

cell-mediated immunity, and in proliferation and activation 

of APC, as shown for B cells, monocytes and, more 

recently, DC. 72 Ligation of CD40 on monocytes and DC 

results in the secretion of several cytokines, including IL- 

1, IL-6, IL-12, and TNF-α. The murine tumor transduced 

with both GM-CSF and CD40L genes showed that tumor 

infiltrating DC can take up cellular antigen from the 

tumor and can present it to T lymphocytes in vitro. 73 In 

this setting, DC bridges cell vaccine-T-lymphocytes 



163 

interaction and can be envisaged as the way to prime 

patients against weaker antigens otherwise ignored by 

the immune system. A complementary approach would 

consider the possibility of boosting the primed or the 

existing immune response. In this case DC that reencounter 

activated T-cells can be lysed and may not be 

appropriate for boosting. Boosting can be done using 

tumor cells transduced with IL-2 and B7-1 such as to 

provide both cell expansion and co-stimulation from 

the tumor cell vaccine directly to T lymphocytes (Figure 

2). The way cellular antigen can be captured by DC for 

priming of T-lymphocytes depends on how tumor cells 

die after injection. Irradiation of the cell vaccine induces 

apoptotic cell death and apoptotic bodies can be captured 

by DC. 74 Others suggest that peptides of cellular 

protein complexed to heat shock protein (HSP), the natural 

chaperon of peptide from proteosome to membrane-associated 

TAP, leave the cells upon necrosis to 

be taken up by DC. 75,76 DC loading must be followed by 

DC maturation and migration to the lymph node to 

ensure correct antigen presentation. 32 

The way cytokines and co-signals modulate vaccinehost 

interactions may determine the extent and the efficacy 

of treatment. Systemic activation of the immune 

response (T-cell cytotoxicity, antibodies) is easy to measure 

but can not be used as a read-out system to predict 

whether the tumor will be rejected. Tumor nodules might 

be reached by circulating lymphocytes which, however, 

may be neutralized because of either immunosuppression 

or peripheral tolerance. The former is likely dependent 

on tumor size and is less expected when a small 

tumor, minimal residual disease or prevention of recurrence 

is the target to be treated. The latter could be surmounted 

by appropriate co-signals, for example CD40L. 45 

Soluble proteins as immunogens 

Soluble proteins derived from autologous cancer cells 

are not as immunogenic as proteins derived from infectious 

agents. The degree of foreignness, which depends 

on the reciprocal distribution between epitopes subject 

and not subject to self-tolerance, is low in TAA. Moreover, 

TAA do not have the particulate nature of infectious 

agents that is a key factor in increasing their 

immunogenicity. Finally, infectious agents are rich in 

molecules that are able to raise immune responsiveness 

without being themselves immunogenic. A significant 

effort has been made over the last few years to translate 

the immunogenic properties of infectious agents 

into cancer vaccines for clinical use. The most suitable 

TAA should be directly involved in the malignant behavior 

of tumor cells; contain multiple immunodominant B 

cell and T cell epitopes, including both helper and cytotoxic 

T-cell epitopes; contain a very high degree of foreignness; 

be unprotected by self-tolerance mechanisms. 

Of course, HLA haplotype remains a major constraint in 

determining whether TAA-derived immunodominant 

peptides can elicit tumor-specific immune responses in 

a given individual (see also below). 

Table 2a. Tumor-associated antigens recognized in class I 

HLA restriction. 

Antigen defined Type of antigen Type of tumor HLA-restriction 

and distribution 

allele 

gp 100 Melanocyte diff. Melanoma A2, A3, A24, Cw8 

Melanocortin receptor 1 Melanocyte diff. Melanoma 

MART-1/MelanA Melanocyte diff. Melanoma A2, B45 

Tyrosinase Melanocyte diff. Melanoma A1, A2, A24, B44 

TRP-1 (gp 75) Melanocyte diff. Melanoma A31 

TRP-2 Melanocyte diff. Melanoma A2, A31, Cw8 

p15 Widely expressed Melanoma A24 

SART-1 Widely expressed Lung carcinoma A2601 

PRAME Widely expressed Melanoma, renal A24 

NAG-V* Melanoma sheared Melanoma A2.1 

β catenin Unique tumor specific Melanoma A24 

CDK4-Kinase Unique tumor specific Melanoma A2 

MUM-1 Unique tumor specific Melanoma B44 

TRP2/INT2 Unique tumor specific Melanoma A6801, Cw8 

CASPASE-8 Unique tumor specific Head/neck cancer B35 

HLA-A*201 mutated Unique tumor specific Renal cancer A2.1 

KIAA0205 Unique tumor specific Bladder cancer B44*03 

BAGE Cancer/ testis Melanoma Cw 1601 

GAGE-1/2 Cancer/ testis Melanoma Cw 6 

MAGE-1 Cancer/ testis Melanoma A1, Cw16 

MAGE-3 Cancer/ testis Melanoma A1, A2, B44 

RAGE Cancer/ testis Renal cancer B7 

NY-ESO-1 Cancer/ testis Melanoma, ovarian, A2 

esophageal cancer 

K-RAS-D13 mutated Shared tumor-specific Colon cancer A2.1 

p53 mutated Shared tumor-specific Colon and lung A2.1 

Bcr/abl Shared tumor-specific CML A2.1, A3, 

A11, B8 

MUC-1 Shared tumor-specific Breast, colon, A11 

pancreatic cancer 

HPV16E7 Viral related Cervical cancer A2.1 

Table 2b. Human tumor antigens recognized by HLA class 

II-restricted CD4 + T cells. 

Antigen Tissue distribution Class II restriction 

Tyrosinase Melanoma/melanocytes DRb1*0401 

Tyrosinase Melanoma/melanocytes DRb1*1501 

Triosephosphate isomerase Melanoma, unique DRb1*0101 

mutated form 

MAGE-3 Melanoma and other tumors, testis DRb1*1301 

MAGE-1, -2 or -6 Melanoma and other tumors, testis DRb1*1301 

MAGE-3 Melanoma and other tumors, testis DRb1*1101 

RAS-D12 mutated Colon and pancreatic cancer DR1 

HER-2/neu Breast and ovarian cancer DR11 

PML/RARα Acute promyelocytic leukemia DR2 

Bcr/abl Chronic myeloid leukemia DR1, DR4, DR11 

CDC27 Melanoma DR4 

*N-acetylglucosaminyl transferaseV. CML: chronic myeloid leukemia. 

Building up immunogenicity of soluble proteins 

Even the most suitable TAA is not immunogenic unless 

it is processed and presented by professional APC to the 

host immune system. To this aim, TAA should interact 

with APC directly. The route of administration and adjuvants 

are key factors in determining the final outcome 


164 


Figure 2. Priming of DC and boosting of T-cell responses by transduced tumor cells as vaccine. 

of immunization. Intravenous injection of soluble antigens 

induces tolerance, whereas subcutaneous or intramuscular 

injection of the same antigens results in immunity 

because of the interaction with epidermal Langerhans 

cells or dermal dendritic cells. Accordingly, delivery 

of antigens via mucosal surfaces may result in immunity 

because antigens may interact with the numerous DC 

located just beneath the epithelium of mucosal lymphoid 

organs. The association between soluble antigens and 

APC is made much more intense by adjuvants. Adjuvants 

act via different principles and pathways, but the common 

goals are to prolong the interaction with APC by 

promoting a slow antigen release, and functionally activate 

APC themselves by delivering danger signals. 

Cytokines have also emerged as potent immunoadjuvants 

since they can influence the immune responses at 

different levels (see above). 

Particulation of soluble proteins 

Precipitation with aluminium hydroxide or aluminium 

phosphate has been used to particulate antigens in diphtheria, 

tetanus, hepatitis B, and other vaccines. These 

types of vaccines induce antibody formation, but very little 

delayed cutaneous hypersensitivity (DTH) or cell-mediated 

cytotoxicity. In a pilot study, five stage I-III patients 

with multiple myeloma were immunized with autologous 

idiotype (Id) precipitated in aluminium phosphate suspension. 

Three patients developed idiotype-specific T- 

and B- cell responses, but these responses were transient 

and their amplitude was low. 77 

The use of immunostaining complexes (ISCOMs) is 

another strategy that has been used to particulate antigens. 

ISCOMs are cagelike structures made of cholesterol, 

saponin, phospholipid, and viral envelope proteins 

to which other proteins can be associated. Saponin is a 

plant derivative that is critical to the efficacy of ISCOMs. 

QS21 is the most effective fraction of saponin and is 

currently under clinical investigation in several trials. 78 

ISCOMs may reach the endocytic pathway and induce 

DTH and CTL responses other than antibody production. 

Liposomes, virosomes, and proteasomes are alternative 

strategies to ISCOMS. Experimental data have recently 

shown in the 38C13 mouse B-cell tumor that liposomal 

formulation of autologous Id converts this weak selfantigen 

into a potent tumor rejection antigen. 79 

Promoting slow release of soluble antigens 

A slow release of antigen is one of the major goals of 

adjuvants. Antigen polymerization and emulsifying agents 

have been put together to achieve this goal. Polymerization 

can be obtained by association with non-ionic block 

polymers or by association with carbohydrate polymers. 

Non-ionic block polymers have been used as components 

of water-in-oil or oil-in-water emulsions. SAF-1 (Syntex 

Adjuvant Formulation-1) is an oil-in-water adjuvant formulation 

containing non-ionic block polymers, squalene, 

and Tween 80. SAF-1 has been used by Kwak et al. in their 

pioneering study on idiotype vaccination in follicular lym- 



165 

phoma patients. 80 Chemical immunomodulators such as 

derivatives of muramyl dipeptide, the smallest subunit of 

the mycobacterial cell wall that retains immunoadjuvant 

activity, can be added to oil-in-water adjuvants. 81 This 

approach was used in the study by Hsu et al. in which idiotype/KLH 

conjugates were delivered to follicular lymphoma 

patients after mixing with SAF-165 that contained 

muramyl dipeptide as immunomodulator. 82 Lipid components 

of bacteria have also been used as adjuvants in 

experimental models. These are portions of the LPS endotoxins 

of Gram-negative bacteria. These molecules are, 

however, too toxic and chemically modified derivatives 

have been developed for clinical use. So far, monophosphoryl 

lipid A is the least toxic derivative capable of promoting 

cell-mediated immunity. 83 

Polysaccharide polymers have also been used as a sustained-release 

vehicle for TAA. Poly-N-acetyl glucosamine 

is a highly purified, biocompatible polysaccharide 

matrix that has recently become available for 

this purpose. 84 

Peptide vaccines 

T lymphocytes recognize small peptides that represent 

the degradation products of a complex intracellular 

process and are presented on the cell surface complexed 

to 1 of 2 types of histocompatibility leukocyte 

antigen molecules (HLA classes I or II). CTLs (CD8 + ) mainly 

recognize peptides of 8 to 10 amino acids derived 

from intracellular or endogenous proteins and complexed 

to HLA class I molecules. 85-89 CD4 + T- lymphocytes 

recognize exogenous proteins which are ingested 

by APC, degraded to peptides of 12-24 amino acids and 

complexed to HLA class II molecules. 90,91 

Class I and Class II peptides that are presented on the 

cell surface, although randomly derived from the original 

protein, must contain specific amino acids in 1 or 2 

critical positions in order to be able to bind the appropriate 

HLA molecules. Thus, peptide binding is HLArestricted. 

The amino acid motifs responsible for the 

specific peptide-binding to HLA class I and class II molecules 

have been determined for the common HLA types 

by analyzing acid-eluted naturally processed peptides 

and by using cell lines defective in intracellular peptide 

loading and processing. 92-94 

Several tumor-specific and some leukemia-specific 

peptides have so far been identified, and studies aimed 

at evaluating the potential clinical benefit of peptide vaccines 

in cancer patients have begun. Among the reasons 

that make a peptide vaccine strategy interesting are several 

unique advantages that peptide immunization offers 

over other vaccine approaches: 1) peptide vaccines permit 

specific targeting of the immune response against 1 

or 2 unique antigens (thus limiting the potential autoimmune 

cross-reactivity or immunosuppressive activity 

often observed with more complex immunogens); 2) 

emerging technology has made it simple, rapid and inexpensive 

to sequence and prepare larger quantities of 

tumor antigen peptides for both laboratory and clinical 

use; 3) use of synthetic peptides greatly reduces the possible 

risk of bacterial or viral contamination that might 

derive from autologous or allogeneic tissue for immunization. 

On the down side, the main disadvantages of 

peptide immunization are: 1) lack of universal applicability 

as each peptide is restricted to a single HLA molecule; 

2) poor immunogenicity of most native peptides; 3) 

risk of inducing antigenic tolerance. Successful attempts 

to enhance HLA binding affinity have been based on synthetically 

generating peptides with amino acid deletions 

or substitutions while maintaining antigen specificity. 95,96 

In initial studies, synthetic substituted peptides appeared 

to enhance immunogenicity and also to overcome the 

host immune tolerance that exists to native peptides. 97,98 

Conversely it has been reported that changes in the fine 

specificity of modified peptide-reactive T-cells following 

vaccination may occur with subsequent loss of tumor cell 

recognition. 99 

A peptide vaccine approach involves several steps 

which are aimed firstly at identifying the appropriate 

peptide, secondly at checking for its immunogenicity 

and relevance as a tumor-associated antigen (TAA) in 

vitro, and thirdly at formulating a safe product to be 

used clinically. 

Identification of the appropriate peptide 

1) From protein to peptide. This approach involves the 

screening of potentially HLA-binding peptides within the 

sequence of a known tumor-specific protein by using HLA 

anchor motifs and epitope selection. Peptides derived by 

mut RAS, 100 melanoma-associated MAGE protein, 101 

prostate specific antigen 102 and chronic myelogenous 

leukemia (CML) specific P210 were identified by this 

approach. 103,104 In CML, for example, a possible total of 76 

peptides, 8 to 11 amino acids in length, spanning the b2a2 

and b3a2 junctional regions of bcr-abl were screened for 

HLA class I- binding motifs and tested for the effective 

binding property to purified HLA molecules. Four of them, 

all derived from the b3a2 breakpoint, were found to be 

able to bind with either intermediate or high affinity to 

purified HLA A3, A11 and B8. 103 A similar approach 

allowed identification of class II b3a2 breakpoint peptides 

capable of binding HLA DR11, 105 DR4 106 and DR1 107 This 

method for identifying tumor-specific peptides is relatively 

simple, fast and suitable for any known intracellular 

protein that may be a potential TAA. Nevertheless, it 

bears the disadvantage that it cannot, by itself, predict 

whether the identified peptide is found on HLA molecules 

of the leukemia or cancer cells that contain the parent 

protein. 

2) From peptide to protein. Another strategy used to 

identify suitable peptides for cancer vaccines involves the 

structural analysis of naturally processed peptides (NPPs) 

bound to HLA class I and class II molecules of cancer 

cells. NPPs were first isolated and sequenced by acidelution 

from immunoaffinity purified HLA molecules and 

subsequently compared with existing protein 

sequences. 108 Alternative approaches are to obtain NPPs 

by mechanically destroying and acid-treating whole 

tumor cells and/or by exposing living tumor cells to rapid 


166 


acid treatment. 109 These procedures should characterize 

tumor-, differentiation stage- and tissue-specific selfantigen 

MHC-bound peptides as well as the naturally 

processed proteins from which they are derived and use 

them as tools for immunotherapy. The main disadvantage 

of this approach is that many tumor cells express low 

levels of HLA molecules and the yield of NPPs can be 

scarce. Nevertheless some immunogenic peptides derived 

from wild-type p53 protein, melanoma associated MART- 

1 and gp100 proteins were identified by these methods, 

110,111 and naturally processed peptides from acute 

myeloblastic leukemia cells and CML blasts are now 

under evaluation. 112,113 Advantages and disadvantages of 

synthetic versus natural tumor peptides have been 

recently reviewed. 114 

3) From tumor infiltrating lymphocytes to peptide. 

Probably the most clinically relevant tumor peptides are 

those identified from the epitope analysis of tumor infiltrating 

lymphocytes (TIL). 115-118 In preliminary experiments 

HLA class I restricted CTL lines were derived by repetitive 

in vitro stimulation of TIL with autologous tumor cells. 

Subsequently, by transfection of a tumor cDNA library 

and in vitro sensitization assays, the peptide sequences 

recognized by the tumor-specific CTLs were identified as 

were the parent proteins (i.e. peptides derived from 

melanoma associated gp100, tyrosinase and the MAGE 

family). Most TIL-derived tumor peptides found in the 

past few years are MHC class I-restricted; however, a 

novel melanoma antigen resulting from a chromosomal 

rearrangement and recognized by a HLA-DR1-restricted 

CD4 + -TIL has recently been identified. 119 

Checking for peptide immunogenicity 

Except for TIL-derived peptides, all other tumor-specific, 

HLA binding, synthetic or naturally expressed peptides 

still need to be tested for immunogenicity. The 

ability of inducing CTL or specific CD4 + proliferation has 

been evaluated for all tumor-specific peptides that were 

subsequently used in clinical trials. P210-derived peptides, 

for example, were able to elicit peptide-specific T- 

cell immunity both in normal donors, 105,120 and CML 

patients. 121 Their relevance as TAAs has been further 

confirmed by observing peptide-specific HLA restricted 

CTLs and CD4 + cells able to mediated killing of b3a2- 

CML cells and proliferation in the presence of b3a2 containing 

cell lysates, respectively. 106,107 The latter findings 

were the indirect proof of natural processing of P210 

and of HLA presentation of breakpoint-derived peptides. 

Although strong peptide-specific CTL and CD4 + responses 

have been shown in vitro for most tumor peptides so 

far identified, few data on T-cell induced immunity after 

peptide vaccination in patients have been generated. 

100,122,123 Thus, strategies to improve peptide immunogenicity, 

by using different adjuvants and delivery systems, 

are currently under evaluation. 

Peptide vaccine formulation 

The goal of experimental clinical protocols using peptide 

antigens for active vaccination is to induce a strong 

CTL response against the immunizing antigen and thereby 

against tumor cells expressing the antigen. The mode 

of peptide-based cancer vaccination critically affects the 

clinical outcome. The synthesis of a peptide on a large 

scale, its purification and testing for common Quality 

Control/Quality Assurance compliance are simple and 

fast procedures but the choice of an effective delivery 

system for the peptide is crucial. Peptide vaccination 

strategies currently being evaluated include: 1) direct 

peptide vaccination with immunologic adjuvants and/or 

cytokines; 124 2) lipopeptide conjugates; 125 3) peptide 

loading onto splenocytes or DC; 126 4) lysosomal complexes. 

127 Recently, a specific formulation of the polysaccharide 

poly-N-acetyl glucosamine has been found to 

be an effective vehicle for sustained peptide delivery in 

a murine vaccine model able to generate a primary CTL 

response with a minimal peptide dose. 84 Finally, triggering 

CD40 in vivo with an activating antibody considerably 

improved the efficacy of peptide-based anti-tumor 

vaccines in mice, converting a peptide with minimal 

immunogenicity into a strong CTL inducer. 44 

Recombinant viruses 

The molecular identification of the antigens on human 

tumors recognized by T- and B-lymphocytes offers the 

opportunity to design novel cancer vaccines based on 

recombinant forms of TAA. Genes coding for TAA can be 

inserted into the genome of attenuated micro-organisms 

such as bacteria and viruses. Viruses are among 

the most interesting vectors since they are able to 

induce antibody, Th, and CTL responses in the absence 

of co-stimulation. 128 Their long-lasting cohabitation 

with human beings has likely favored the evolution of 

specific patterns recognized by the innate immune system 

which create an immunostimulatory environment 

for optimal immune responses. The vector choice, however, 

is limited by the possible disadvantages of recombinant 

virus utilization, such as recombination with 

wild-type viruses, oncogenic potential, or virus-induced 

immunosuppression. 

Vaccinia virus (VV) belongs to the Poxviridae family, 

and its worldwide use in the smallpox eradication campaign 

demonstrated that it was safe and very effective. 

To date, no other large-scale vaccination program has 

had such an impact on human diseases, because smallpox 

has been virtually eliminated from the world population. 

Large amounts of foreign DNA can be stably 

inserted into the VV genome by homologous recombination. 

129 VV employs a built-in transcriptional and posttranslational 

apparatus to produce large amounts of the 

protein encoded by the inserted gene. VV sojourns within 

the host cell cytoplasm and does not integrate nor is 

it oncogenic. 129 The induction of potent cellular and 

humoral immune responses with recombinant (r)VV was 

observed in several tumor systems. 130-132 Preclinical studies 

in models of pulmonary metastatization caused by 

tumors bearing a prototype TAA have revealed some features 

of rVV that are important in determining successful 

therapy. In particular, TAA gene must be expressed 



167 

under the control of a strong, early promoter which 

allows its expression in professional APC such as DC. 133 

Moreover, while prevention from tumor challenge 

requires only the synthesis of the TAA by the recombinant 

virus, genes encoding immunostimulatory molecules 

inserted in the recombinant poxvirus, 134 exogenous 

cytokines, 131,135 or their combination 136 are required to 

induce eradication of established tumors. 

Encouraging results have also been obtained in mouse 

models more relevant to the therapy of human cancer. 

Immunization of mice transgenic for the human HLA- 

A*0201 allele with an rVV encoding a form of the 

melanoma antigen gp100, which had been modified to 

increase epitope binding to the restricting class I molecule, 

elicited CD8 + T-lymphocytes specific for the epitope 

that is naturally presented on the surface of an HLA- 

A*0201-expressing mouse melanoma. 137 Repeated inoculations 

of an rVV encoding the mouse tyrosinase-related 

protein-1 (TRP-1/gp75) caused autoimmune attack of 

normal melanocytes manifested by hair depigmentation 

(vitiligo) and CD4 + -mediated melanoma destruction in 

mice. 138 In a clinical trial, administration of VV encoding 

human carcinoembryonic antigen (CEA) proved effective 

in inducing both humoral and cellular immune responses 

in patients with colorectal cancer. 139 

Poxviruses are not the only choice for tumor immunologists. 

Adenoviruses in which critical genes that 

enable viral replication have been deleted and replaced 

by genes encoding heterologous antigens, have been 

generally used in gene therapy studies, but have also 

provided antitumor activity when employed as immunogens. 

140 Liver toxicity was described following systemic 

administration of high titers of first generation E1- 

deleted Adenovirus vectors optimized for gene therapy. 

141 These side effects, which certainly raise some concerns 

about Adenovirus administration in patients, 

might not restrain their use in cancer immunotherapy 

since systemic delivery of high viral titers would certainly 

not be the favored immunization route. 

Initial clinical trials have unveiled some of the intrinsic 

limitations of recombinant viruses. Many patients, in 

fact, have high neutralizing antibodies against VV as a 

consequence of its use as a vaccine for smallpox prevention, 

and against Adenoviruses, which cause upper respiratory 

tract infections throughout life. High doses of 

recombinant adenoviruses expressing the human 

melanoma antigens MART-1 and gp100 could be safely 

administered to cancer patients, but the high levels of 

neutralizing antibodies present in their sera likely impair 

the ability of these viruses to immunize against the 

melanoma antigens. 142 

These results, largely expected, have not put an end 

to the clinical use of recombinant viruses, as various 

strategies have been exploited to overcome pre-existing 

immunity. Several groups have engineered non-replicating 

viruses which normally do not infect human 

beings. The Avipoxviridae family comprises viruses, such 

as fowlpox and canarypox, which can productively infect 

avian but not mammalian cells, and are not cross-reactive 

with VV. 143 A recombinant fowlpox virus expressing 

a model TAA was able to cure established tumors in 

mice; 144 an important aspect of this study was the observation 

that prior exposure to VV did not abrogate the 

immune responses induced by the recombinant fowlpox 

virus. A different non-replicating virus, canarypox virus 

(ALVAC), has also been employed to elicit immune 

responses against a variety of antigens. 145,146 

A highly attenuated strain of VV, known as modified VV 

Ankara (MVA), has been inoculated as smallpox vaccine 

into more than 120,000 recipients without causing any 

significant side effect. 147 Replication of MVA is blocked at 

the step of virion assembly and for this reason the MVA 

vectors produce recombinant proteins expressed under 

the control of both early and late viral promoters, thus 

mimicking the expression in wild-type virus. An MVA vector, 

and a fowlpox virus vector expressing a model TAA 

showed better therapeutic effects on pulmonary metastasis 

than a VV encoding the same TAA. 148 MVA is a very 

promising vector for the development of recombinant 

vaccines for cancer, and can be efficiently used in combination 

with DNA vaccines. 149 

As an alternative approach, the mucosal route of 

administration was recently shown to overcome preexisting 

immunity to VV. Intrarectal immunization of 

vaccinia-immune mice with rVV expressing HIV gp160 

induced specific serum antibody and strong HIV-specific 

CTL responses in both mucosal and systemic lymphoid 

tissue, whereas systemic immunization was ineffective 

under these circumstances. 150 

Direct immunization of mice with recombinant adenoviruses 

resulted in the induction of high titers of neutralizing 

antibodies, which precluded a boost of CTL 

responses after repeated inoculations. The presence of 

neutralizing antibodies did not, however, affect the 

immunogenicity of infected DC, as repeated administration 

of virus-infected DC boosted the CTL response even 

in mice previously infected with the recombinant vector. 

151 It was also shown that protective immunity against 

mouse melanoma “self” antigens, gp100 and TRP-2, could 

be obtained by DC transduced with Adenovirus vector 

encoding the antigen. Importantly, immunization with 

Adenovirus-transduced DC was not impaired in mice that 

had been pre-immunized with Adenovirus. 152 With the 

help of these novel strategies, recombinant vectors could 

be used in the general population, including those individuals 

previously exposed to the viruses. Gene transfer 

to different human DC subpopulations by vaccinia and 

adenovirus vectors is a conceivable strategy for TAA loading. 

153 

DNA vaccines 

Following the first and somewhat shocking demonstration 

that the intramuscular injection of naked DNA 

(i.e., DNA devoid of a viral coat) encoding the influenza 

A nucleoprotein could induce nuceloprotein-specific 

CTL, and protect mice from challenge with heterologous 

influenza strains, 154 DNA immunization has become a 

rapidly developing technology. This vaccination method 


168 


provides a stable and long-lasting source of antigen, 

and elicits both antibody- and cell-mediated immune 

responses. Compared to recombinant viruses, DNA vaccines 

offer a number of potential advantages because 

they are cheap, easy to produce, and do not require special 

storage or handling. DNA vaccines express virtually 

only the heterologous gene, therefore, they should 

induce an immune response selective for the antigen 

and not the vector, thus supplying a source of antigen 

suitable for repeated boosting. DNA vaccination has 

proven to be a generally applicable approach to various 

preclinical animal models of infectious and non-infectious 

diseases, 155 and several DNA vaccines have now 

entered phase I/II human clinical trials. Although the 

clinical application of DNA vaccines is a very young 

practice, some trials have already demonstrated that is 

possible to elicit a specific CTL response against malaria 

and HIV proteins in human volunteers. 156,157 

This novel vaccination approach involves different 

steps: 1) cloning of a heterologous gene under the control 

of a viral promoter (ordinarily derived from the CMV 

immediate early region); 2) purification of the endotoxin-free 

DNA plasmid from bacteria factories; 3) administration 

of the expression vectors by direct intramuscular 

or intradermal injection with a hypodermic needle 

or using a helium-driven, gene gun to shoot the skin 

with DNA-coated gold beads. Heterologous DNA can 

also be introduced into recombinant Salmonella, 158,159 

or Listeria strains 160 that can be thus administered by a 

mucosal route (Figure 3). In addition to these classic 

routes of DNA delivery, plasmid-based gene expression 

vectors have also been admixed with polymers and 

administered with a needle-free injection device, 

achieving high and sustained levels of antigen-specific 

antibodies. 161 The route of DNA delivery can profoundly 

influence the type of immune response by preferentially 

activating different Th populations: gene gun bombardment 

elicits a Th2 response, while intramuscular 

inoculation induces Th1 activation, even though the 

antigen form (i.e., membrane-bound vs secreted) can 

also exert some effect. 162,163 

The immunostimulatory activity of DNA vaccines has 

been associated with the prokaryotic-derived portion of 

the plasmid, which contains a central CpG motif in the 

sequence PuPuCpGPyPy. 164,165 In their unmethylated 

form, these hexamers stimulate monocytes and macrophages 

to produce different cytokines with a Th1 promoting 

activity including IL-12, TNF-α, and IFN-γ. 166,167 

A plasmid that incorporated several CpG islands in the 

prokaryotic ampicillin-resistance gene induced a 

stronger immune response when compared to a second 

plasmid carrying the kanamycin-resistance gene which 

possesses none. 168 To date, it is not known whether the 

CpG motifs will have the same immunostimulatory 

properties when applied to vaccination of human beings. 

However, it was recently reported that CpG motifs can 

activate in vitro subsets of freshly isolated human DC to 

promote Th1 immune responses. 169 

The mechanism of DNA-induced immunization has not 

yet been fully elucidated. An exclusive role for the direct 

transfection of normal tissue cells, such as myocytes or 

keratinocytes, has been debated because surgical ablation 

of the injected muscle within 1 minute of DNA inoculation 

did not affect the magnitude and longevity of DNAinduced 

antibodies. 170 Moreover, studies with bone-marrow 

chimeras clearly indicated that bone-marrowderived 

APC, either transfected by the DNA plasmid or 

able to capture the antigen expressed by other transfected 

cells, were necessary to prime T- and B-lymphocyte 

responses. 171 Indeed, more recent evidence suggests 

that Th and CTL are activated by DC directly transfected 

in vivo following DNA immunization. 172,173 

The first applications of DNA immunization to preclinical 

models of tumor growth revealed some interesting 

aspects. In general, the potency of naked DNA does not 

equal that of recombinant viruses, probably because DNA 

does not undergo a replicative amplification in the transfected 

cells, which in turn limits the amount of heterologous 

antigen produced. Inflammatory responses caused 

by DNA inoculation are more contained than those occurring 

during infection with viruses; for this reason, repeated 

inoculations of plasmid DNA, or the use of adjuvants 

such as cardiotoxin are generally required for the induction 

of an optimal response. Another emerging issue is 

that the efficacy of the vaccination approach depends 

more on the type of antigen than on the route of administration 

(Table 3). Vaccines based on shared viral antigens, 

or model TAA artificially introduced in the experimental 

tumors can be used to induce a strong, and often 

therapeutic immune response. Using a gene gun for DNA 

immunization, Irvine et al. 174 observed effective treatment 

of established pulmonary metastases, but recombinant 

cytokines were necessary to enhance the therapeutic 

effects. Unlike viral and model TAA, self TAA fail to induce 

sterilizing immunity since therapy of established tumors 

has been rarely reported, and prevention from challenge 

is often partial. 175-178 Central and peripheral tolerance to 

self antigen has thus emerged as the main limitation to 

the successful application of DNA vaccines to the therapy 

of cancer. This conclusion seems to apply to several 

mouse melanocyte differentiation antigens, a class of 

molecules that is expressed in both melanomas and 

melanocytes and includes tyrosinase, TRP-1/gp75, TRP-2, 

and gp100/pmel 17. However, tolerance can be broken by 

the use of a xenogeneic source of TAA. While immunization 

with mouse TRP-1/gp75 or TRP-2 antigens failed to 

induce a detectable immune response, vaccination with 

a plasmid DNA encoding the human homologous antigens 

elicited autoantibodies and CTL in C56BL/6 

mice: 178,179 immunized mice rejected metastatic melanoma 

and developed patchy depigmentation of their coats 

(vitiligo). This “obligatory” association of vitiligo and antitumor 

response was recently questioned by a study showing 

that immunization with mouse TRP-2-encoding plasmid 

could protect CB6 F1 mice in the absence of overt 

vitiligo, suggesting a role for the genetic background in 

controlling both the extent and the consequences of the 

immune activation against self TAA. 180 



169 

A novel vaccine that combines the properties of viruses 

and DNA is based on antigen production in the context 

of an alphavirus replicon. These new vectors rely on 

the ability of the alphavirus RNA replicase to drive the 

replication of its own gene, as well as of subgenomic 

RNA encoding the heterologous antigen. This loop of 

self-replication results in a several-fold amplification of 

protein production in infected cells. 181 Compared with 

traditional DNA vaccine strategies, in which vectors are 

persistent and the expression constitutive, the expression 

mediated by the alphaviral vector is transient and 

lytic, resulting in a decrease of biosafety risks as well as 

the risk of inducing immunologic tolerance due to longlasting 

antigen expression. A single intramuscular injection 

of a self-replicating RNA immunogen at doses lower 

than those required for standard DNA-based vaccines 

elicited antibodies, CD8 + T-cell responses, and prolonged 

the survival of mice with established tumors. 182 Interestingly, 

the enhanced immunogenicity of these vectors 

correlated with the apoptotic death of transfected cells, 

which facilitated their uptake and presentation by DC. 

Methodology for ex vivo generation of DC 

Investigators working in human and murine systems 

have discovered culture conditions that use hematopoietic 

cytokines to support the growth, differentiation, and 

maturation of large amounts of DC. Therefore, DC can 

also be purified from peripheral blood after removal of 

other defined T, B, NK and monocyte populations by using 

antibodies and magnetic beads or a cell-sorter. 183 However, 

the very low frequencies of DC in accessible body 

samples, especially blood, limits the use of these DC for 

vaccination protocol. The ex vivo differentiation of DC 

progenitors can be traced easily by monitoring changes 

in some key surface molecules such as CD1a (acquired by 

DC) and CD14 (expressed by monocytes and lost by DC). 

Furthermore expression of co-stimulatory molecules such 

as CD40, CD80, CD86, as well as HLA antigens, can be 

used to evaluate the stage of differentiation and the 

degree of maturation of DC during in vitro culture. In 

addition, two new markers, CD83 and p55, have been 

shown to be selectively expressed by a small subset of 

mature DC differentiated in in vitro culture. 184,185 According 

to the knowledge of DC ontogeny, two major strategies 

are used. The first is based on the ability of CD34 + 

progenitors isolated from bone marrow, 186 peripheral 

blood, 187 or neonatal cord blood 188 to differentiate ex vivo 

within 12-14 days into mature CD1a + /CD83± HLA-DR + 

DC in the presence GM-CSF and TNF-α. Both stem cell 

factor (SCF) and FLT3 ligand are able to augment the DC 

yield if these key factors are present in the culture. 189 

The maturation of DC from progenitors is influenced 

not only by cytokines, but also by extracellular matrix 

(ECM) proteins, such as fibronectin which has been 

reported to enhance DC maturation by mediating a specific 

adhesion through the a5b1 integrin receptor. 190 The 

choice of culture conditions, especially the cytokine combination, 

will influence DC purity, maturation and function, 

and this is a consideration of prime importance 

before starting a DC-based immunotherapy strategy. A 

more practical approach is the production of DC from 

CD14 + monocytes, in the presence of GM-CSF and IL-4. 

A future strategy for easy achievement of large 

amounts of DC is the in vivo injection of the same 

cytokines utilized for ex vivo DC generation. In fact, the 

administration of FLT3 ligand either in animals 191 or in 

humans 192 results in a reversible accumulation of functionally 

active DC in both lymphoid and non-lymphoid 

tissues. Therefore, in murine models it has been demonstrated 

that FLT3 ligand caused the regression of various 

tumors supporting the suggestion that DC may be directly 

involved in the antitumor effect of FLT3 ligand. 193,194 

Strategies for delivery of TAA into DC 

Several approaches for delivery of TAA into DC have 

been utilized. To date, in the clinical protocol of vaccination 

by DC both synthetic peptides corresponding to 

known tumor antigens and tumor-eluted peptides have 

been used for DC-mediated antigen presentation. 195 

While synthetic peptides represent only the limited antigenic 

repertoire of the presently known tumor antigens, 

tumor-eluted peptides, though originating from 

unknown proteins, may reflect a wider antigenic spectrum. 

Another potential disadvantage of using defined 

synthetic peptides to activate tumor-reactive T-cells is 

that the generated peptide-specific T-cells may not recognize 

autologous tumor cells expressing the antigen of 

interest. Loading DC with cocktails of different synthetic 

peptides, corresponding to different tumor antigens 

expressed by the same tumor, has been demonstrated to 

be a clinically effective procedure. Nevertheless it is possible 

that the synthetic peptide-approach will limit 

patient selection, on the basis of the HLA phenotype, 

and will prevent the possibility of activating both CD4 

and CD8 T-cells directed to different epitopes of the 

same antigen. To by-pass these disadvantages, several 

alternative methodologies using a mix of TAA have been 

developed. DC are able to internalize complete tumor 

lysates or apoptotic cells and to present derived antigen 

in an HLA I-restricted manner. 196 In addition, DC secrete 

antigen-presenting vesicles, called exosomes, which 

express functional HLA class I and class II, and T-cell 

co-stimulatory molecules. Tumor peptide-pulsed DCderived 

exosomes prime specific cytotoxic T-lymphocytes 

in vivo and eradicate or suppress growth of established 

murine tumors in a T-cell-dependent manner. 197 

However, a possible limitation of these approaches is 

the need for large numbers of primary samples or tumor 

cell lines and the complete lack of control of the nature 

of the antigens that are being presented by the DC. The 

use of RNA instead of protein could constitute a good 

alternative since it could be amplified in vitro. In addition, 

substractive hybridization could allow the enrichment 

of tumor-specific RNA, thus limiting immune 

response against self antigen. 198 A further possibility is 

the engineering of DC with expression vectors carrying 

TAA genes. Among the viral vectors, retroviral, adenoviral, 

and vaccinia vectors have been widely utilized to 


170 


transduce either monocyte or CD34 + cell-derived DC. 

Many authors have chosen retroviral vectors because 

retroviral transduced-DC should be able to constitutively 

express and process TAA to produce long-term 

antigen presentation in vivo. Specific CTLs against transduced-TAA 

are elicited by retrovirus-engineered DC. 199 

However, their low efficiency of transduction limits the 

clinical use of retroviruses. 

In contrast, adenoviral vectors infect replicating and 

non-replicating cells, are easy to handle, and supernatant 

with clinical grade high titer is readily achievable. Both 

monocyte and CD34 + cell-derived DC can be transduced 

with high efficiency by adenovirus combined with polycations. 

200,201 Moreover, DC transduced by adenovirus 

maintain their APC functions. 202 However, the use of adenovirus 

vectors is hampered by their immunogenicity 

which causes the rapid development of a CTL response 

that eliminates virus-infected cells and generation of 

neutralizing antibodies in recipients. Moreover, vaccinia 

virus which is a member of the poxvirus family, is not 

oncogenic, does not integrate into the host genome, is 

easy to manipulate genetically and is capable of accepting 

large fragments of heterologous DNA. 203 The transduction 

of CD34 + cell-derived DC is feasible but their 

use is limited by the narrow therapeutic index between 

optimal transduction and target cell viability. 153 The presence 

of acquired genetic abnormalities in myeloid hematologic 

malignancies might represent the basis for innovative 

immune-based anti-leukemic strategies. In fact, 

intracellular proteins can be processed and presented on 

the cell surface by HLA molecules indicating the possibility 

that leukemia-specific genetic abnormalities may 

be targets for cytotoxic T-cells. The generation of functional 

monocyte-derived DC carrying the specific genetic 

lesion has been reported for both acute myeloid and 

lymphoid leukemia (AML), 204,205 as well as chronic myelogenous 

leukemia (CML). 206 Current protocols for ex vivo 

DC generation from CD34 + cells does not allow large 

numbers of leukemic DC to be produced, probably 

because of a defective proliferative and/or maturative 

capacity of transformed CD34 + cells. 

Recently, a protocol which allows the optimal generation 

of BCR/ABL-positive DC from CML-derived CD34 + 

cells has been reported. 207 

Antitumor vaccination: 

emerging clinical results 

Clinical trials in solid tumors 

Despite the fact that the large number of ongoing clinical 

trials which can be derived from the Physician Data 

Query (PDQ) of NCI (Figure 4) suggests a diffuse interest 

in immunotherapy, there is still a strong need to define 

the clinical impact of immunotherapy in the treatment 

of solid tumors. Table 4 summarizes the already published 

vaccination trials carried out using a) autologous 

or allogeneic neoplastic cells, b) synthetic peptides corresponding 

to defined TAA, alone or pulsed on autologous 

monocytes or DC. 

Melanoma 

Melanoma is the most striking example of a non-virusinduced 

immunogenic tumor in man that is able to elic- 

Figure 3. DNA immunization protocols. 



171 

it T-cell-mediated antitumor immunity. The majority of 

tumor antigens defined by T-cells have been identified 

utilizing patients’ T-lymphocytes as effector cells and 

tumor cells obtained from autologous (metastatic) tumor 

deposits as the target. 68 Several investigators have isolated 

cross-reactive tumor-specific CTLs from peripheral 

blood, lymphocytes, or tumor-infiltrating lymphocytes of 

melanoma patients, and these CTLs are able to recognize 

common tumor antigen expressed in melanomas that 

share the restricting HLA class I allele. 19 Thus, large numbers 

of ongoing or published clinical trials have been carried 

out on patients with metastatic melanoma. Experimental 

strategies are encouraged since with current 

standard therapies the prognosis of patients with 

metastatic melanoma is poor with a median survival of 

about 6 months. 208 Several approaches to induce antitumor 

immune response have been reported. 

Irradiated tumor cells. The initial anti-melanoma vaccines 

were similar in their preparation to the vaccines 

against infectious diseases. Crude preparations of 

homogenized tumor cells were mixed with immunestimulating 

adjuvants such as viral or bacterial particles. 

Some of these early vaccines were tested in clinical trials 

and induced objective clinical responses in about 

25% of the cancer patients. 209 The most impressive trial 

was reported by Morton et al. 64 who administered, to 

136 stage IIIA and IV (American Joint Commitee on Cancer, 

AJCC) melanoma patients, a polyvalent melanoma 

cell vaccine (MVC) comprising 3 allogeneic melanoma 

cell lines. Of 40 patients with evaluable disease, 9 (23%) 

had regressions (3 complete). Induction of cell-mediated 

and humoral immune responses to common 

melanoma-associated antigens present on autologous 

melanoma cells was observed in patients receiving the 

vaccine. Survival correlated significantly with DTH and 

antibody response to MCV and there was a 3-fold 

increase in 5-year survival of patients with stage IV 

melanoma. Livingston et al. 65 randomized 122 stage III 

melanoma patients free of disease after surgery to 

receive treatment with the ganglioside GM2/BCG vaccine 

or with BCG alone. All patients were pretreated with 

low-dose cyclophosphamide. In most patients vaccinated 

with GM2/BCG, an antibody production against ganglioside 

was demonstrated and this was associated with 

a prolonged disease-free interval and survival, although 

the improvement did not reach statistical significance. 

Gene-modified tumor cell vaccine. Autologous or allogeneic 

tumor or fibroblast cells have been modified to 

express cytokines and/or co-stimulatory molecules and/or 

a suicide compound. This genetic engineering is mainly 

performed ex vivo using retroviral vectors. In the published 

human trials, tumor cells have been transduced to 

express several cytokines. Arienti et al. 210 described 12 

stage IV melanoma patients who underwent vaccination 

with HLA-A2-compatible allogeneic human melanoma 

cells (5x10 7 or 15x10 7 cells) engineered to release IL-2. 

Little toxicity with three mixed clinical responses was 

recorded. Among the nine patients immunologically evaluated, 

peripheral blood lymphocytes from three patients 

displayed enhanced non-HLA-restricted cytotoxicity, and 

two of those individuals had an increased reactivity 

against tyrosinase peptide or gp-100 peptide after immu- 

Table 3. DNA vaccines in active immunotherapy of experimental mouse tumors. 

TAA Experimental tumor Route of inoculation Adjuvant Vaccine effects Reference 

Beta-galactosidase adenocarcinoma gene gun bombardment cytokines treatment of 2-day-old (47) 

(model TAA) followed by cytokine i.p. (IL-2, IL-12) pulmonary metastases 

gag from M-MuLV leukemia i.m., 3 inoculations every 10 days none complete protection (56) 

from challenge 

HPV-E7 sarcoma 3 gene gun bombardments, none complete protection (48) 

every 2 weeks 


Neu spontaneous mammalian i.m., 4 weekly inoculations IL-2-encoding plasmid partial protection (50) 

tumor 


P1A mastocytoma i.m., 3 inoculations every 10 days none partial protection (49) 


Idiotype/GM-CSF B-cell lymphoma i.m., 3 inoculation every 3 weeks none partial protection (57) 

fusion protein 


mouse TRP-2 melanoma 3 gene gun bombardments, IL-12-encoding plasmid partial protection (48) 

every 2 weeks 


human TRP-1 melanoma 5 gene gun bombardments, weekly none reduction of pulmonary (52) 

metastases upon challenge; 

vitiligo 

human TRP-2 melanoma 1-4 gene gun bombardments GM-CSF (in therapy setting) reduction of pulmonary (51) 

metastases (prevention 

and therapy); vitiligo 

mouse TRP-2 melanoma i.m. cardio-toxin protection from challenge; (53) 

no vitiligo 


172 


nization. In the only patient for whom the autologous 

melanoma line was available, and following in vitro stimulation 

of the PBLs after vaccination, the frequency of 

CTL precursor (CTLp) was significantly enhanced. Other 

groups utilized a similar approach transducing different 

cytokines, i.e. IL-12, 40 IL-7, 211 and GM-CSF. 39 Despite documented 

specific antitumor reactivity with an increased 

frequency of anti-melanoma cytolytic precursor cells, 

negligible clinical results were demonstrated. To summarize, 

the advantage of a genetically modified autologous 

cell vaccine is that it contains the whole collection of 

tumor proteins and therefore has the greatest chance of 

inducing an immune response against relevant tumor 

antigens. However, growing autologous tumor cells in vitro 

to establish tumor cell lines is time-consuming and 

often unsuccessful. These studies are relevant since they 

show that injection of gene-modified cells into a patient 

a) is safe; b) is followed by efficient and variable transduction 

rate of host tissues; c) is associated with transgene 

expression in the patient; d) is associated with biological 

activity of the transgene product in most instances. 

Synthetic and natural peptides. Phase I clinical trials 

have been carried out using synthetic peptides corresponding 

to defined TAA. The clinical trial using 

melanoma differentiation antigen (MAGE-3.A1) by 

Marchand et al., 123 enrolling 39 chemoresistant stage IV 

melanoma patients, was encouraging because monthly 

injection of 100-300 µg peptide alone was associated 

with tumor regression in 7 out of 26 patients who 

received the complete treatment. All but one of these 

regressions involved cutaneous metastases. No evidence 

for CTL response was found in the blood of the 4 patients 

who were analyzed, including 2 who displayed complete 

tumor regression. In contrast, Jager et al. 212 immunized 

similar patients with gp100 peptide along with GM-CSF 

and, in some patients, were able to document an 

increase in the specific CTL activity against the immunizing 

peptide. Rosenberg et al. 213 reported that 31 

metastatic melanoma patients were immunized with 

the modified g209-2M peptide in incomplete Freund’s 

adjuvant (IFA) along with IL-2 obtaining tumor regression 

in 42% of patients. Peripheral blood mononuclear 

cells harvested from these patients after, but not before, 

immunization exhibited a high degree of reactivity 

against the native g209-217 peptide, as well as against 

HLA-A2+ melanoma cells. 

These studies indicate that vaccination with synthetic 

peptides is well tolerated, with occasional occurrence 

of mild fever and inflammation at the site of injection. 

Nonetheless, it should be pointed out that there is usually 

a poor correlation between induction of specific T- 

cells and the clinical response. The reasons for this discrepancy 

might be the selection of the patients enrolled 

in the trials since the majority of patients were in stage 

IV with large amounts of disease. 

Dendritic cells. Autologous DC generated from peripheral 

blood monocytes have been utilized as antigen-presenting 

cells after their loading with specific melanoma 

antigens. Chakraborty et al. 214 found that intradermal 

administration of DC pulsed with a MAGE-1 HLA class 

I-restricted peptide could elicit peptide and autologous 

melanoma reactive-CTLs in patients with advanced 

melanoma. However, despite the presence of these CTLp 

in the vaccination site, peripheral blood, and distant 

tumor sites, no significant therapeutic responses were 

seen. 

Nestle et al. 195 recently described the immunization of 

16 melanoma patients using DC loaded with melanoma 

peptides or tumor lysates. DC were pulsed with a cocktail 

of gp100, MART-1, tyrosinase, MAGE-1, or MAGE-3 

peptides chosen to suit the individual patient class I HLA 

molecules. Four patients whose HLA haplotype was inappropriate 

for peptide pulsing received DC pulsed with 

autologous tumor lysate. Keyhole limpet hemocyanin 

(KLH) was included during antigen pulsing. DC were 

administered by direct injection into uninvolved lymph 

nodes via ultrasound guidance to facilitate entry into the 

lymphatics and to minimize DC loss. Patients received 6- 

10 injections of 1x10 6 cells every 1-4 weeks. Toxicity was 

limited to mild local reactions at the injection sites. 

Immunologic monitoring revealed DTH skin reactions to 

peptides in 11 cases, and peptide-specific CTLs could be 

recovered from the skin biopsies of some patients. 

Regression of tumor was seen in 5 out 16 patients, 

including 2 complete responses lasting over 15 months. 

Responding tumor sites included skin, lung, soft tissue, 

bone, and pancreas. Importantly, two of the responding 

patients received only tumor lysate-pulsed DC, suggesting 

an approach applicable to cancers lacking defined 

tumor antigens. 

In a similar but smaller trial at the University of Pittsburgh, 

215 6 HLA-A2 + patients with metastatic melanoma 

received four weekly intravenous injections of 1-3x10 6 

monocyte-derived DC pulsed with HLA-A2-restricted 

peptides derived from MART-1, gp100, and tyrosinase. 

Complete regression of a subcutaneous mass lasting 

more than one year has been observed in one patient. 

Colon cancer 

Colon cancer is potentially curable by surgery; the 

cure rate is, however, moderate to poor depending on 

the extent of disease. Adjuvant chemotherapy with 5- 

fluorouracil plus levamisole or folinic acid is the standard 

treatment for stage III colon cancer based on the 

results of numerous co-operative and intergroup clinical 

studies. In contrast, adjuvant chemotherapy for stage 

II disease has no benefit. 216 Despite several immunotherapeutic 

approaches having been tested for colon cancer 

patients, only one study has reported clinical results. 

In a prospective randomized study, 217 254 patients with 

stage II or III post-surgery colon cancer were randomly 

assigned to receive active specific immunotherapy, 

namely autologous tumor cell-bacille Calmette-Guèrin 

(BCG) or no adjuvant treatment. The immunotherapy 

program comprised three weekly intradermal injections 

starting 4 weeks after surgery, with a booster vaccination 

at 6 months with 10 7 irradiated autologous tumor 

cells. The first vaccination contained 10 7 BCG organ- 



173 

isms. The 5-year median follow-up showed a 44% 

reduction of risk of recurrence in all patients receiving 

the vaccinations. The major impact of immunotherapy 

was evident in patients with stage II disease, who had 

a significantly longer disease-free period and 61% risk 

reduction. In addition, no patient discontinued treatment 

early because of side effects. 

Recently, Foon et al. 218 generated anti-idiotype antibody, 

designated CeaVac, that is an internal image of 

CEA. Thirty-two patients with resected Dukes’ B, C, and 

D, and incompletely resected Dukes’ D disease were 

treated with 2 mg of CeaVac every other week for four 

injections and then monthly until tumor recurrence or 

progression. Fourteen patients were treated concurrently 

with a 5-FU chemotherapy regimen. All 32 

patients entered into this trial generated a potent anti- 

CEA humoral and cellular immune response. Interesting, 

the 5-FU regimen did not affect the immune response. 

A phase III trial for patients with resected colon cancer 

is ongoing. 

Prostate cancer 

Several prostate-tissue-associated antigens, including 

prostatic alkaline phosphatase (PAP), prostate-specific 

membrane antigen (PSMA), and prostate-specific antigen 

(PSA), are now being explored as targets for prostate 

cancer immunotherapy. 219-221 Valone et al. 221 have carried 

out a dose-escalation trial of peripheral blood DC pulsed 

with recombinant PAP protein in 12 patients with 

advanced prostate cancer. Intravenous administration 

of 0.3, 0.6, and 1.2x10 9 pulsed cells/m 2 monthly for three 

months resulted in T-cell proliferative responses against 

PAP in all patients, the magnitude of which was related 

to cell dosage. Toxicity was limited to myalgias in 

three patients. Clinical outcomes have not been reported. 

Salgallar et al. 222 have recently updated the results of 

another trial 223 in which monocyte-derived DC pulsed 

with HLA-A2-binding peptides derived from PSMA were 

administered to 82 patients with advanced, hormonerefractory 

prostate cancer. Six infusions of up to 2x10 7 

DC were administered every six weeks, with half of the 

patients also receiving systemic GM-CSF. The treatment 

was well tolerated. However, only two patients mounted 

T-cell responses against the PSMA peptides as measured 

by enzyme-linked immunospot (ELISPOT) and DTH 

skin testing. Although four patients had reductions in 

serum tumor markers following vaccination, the concurrent 

administration of radiotherapy and hormonal 

therapy makes interpretation of the vaccine’s effects 

difficult. 

Renal cell carcinoma 

Renal cell carcinoma accounts for 2% of all malignancies 

and many patients have metastatic disease at 

diagnosis and the prognosis is unfavorable. At present, 

neither chemotherapy nor radiation therapy has any significant 

influence on the course of disease or the survival 

time. Immunotherapy using recombinant IL-2 

alone 224 or combined with interferon-α 225 is currently 

the standard therapy for metatastic renal cell carcinoma. 

Cellular immunotherapy includes the adoptive 

transfer of in vitro expanded tumor infiltrating lymphocytes 

226 as well as active immunotherapy with an autologous 

tumor cell vaccine engineered to secrete GM- 

CSF. 38 Although each of these attempts generated 

promising results neither attempt met the expectations. 

Recently, Holtl et al. 227 have administered, to 4 metastatic 

renal cell carcinoma patients, autologous monocytederived 

DC pulsed with autologous tumor cell lysate. 

Each patient received 3 monthly intravenous infusions 

with the immunogeneic KLH. Initial results have shown 

Figure 4. NCI-registered immunotherapeutic trials according to diseases and strategies. 


174 


Table 4. Phase I/II trials of vaccination in patients with solid tumor. 

Disease Stage Pts Vaccine 

Melanoma IIIA/IV 136 

IV 12 

Melanoma Cell 

Vaccine (MCV) 

IL-2 Transduced- 

Melanoma Cell Line 

Route of 

Admin. TA Ads Side Effects Clinical Results 

ID 

SC 

Human 

Melanoma Cell 

Lines 

Melanoma Cell 

Line 

Yes/No 

No 

Mild (Erithema, 

Fever) 


Fever) 

9/40 evaluable pts 

(3 CR, 6 PR) 

3 MR; 1 SD 

Immunol 

Results 

↑Cell mediated and 

humoral responses to 

MCV ; ↑ Activation of 

TIL 

↑ Melanoma-Specific 

CTLp 

Authors 

Morton, 1992 

Arienti, 1996 

IV 10 

IL-7 Transduced- 

Autologous 

Melanoma Cells 

SC 

Autologous 

Melanoma 

Cells 

No Mild (Fever) 2 MR 5 SD 


CTLp 

Moller, 1998 

IV 21 

GM-CSF Transduced- 

Autologous 

Melanoma Cells 

ID and SC 

Autologous 

Melanoma 

Cells 

No 


Induration, Itching) 

1 PR; 1 MR; 3 Minor Res 


CTLp; 80% Tumor 

Destruction into 

Metastases 

Soiffer,1998 

IV 31 

Peptide+IL-2 

SC 

modified g209- 

2M 

IL-2 and 

IFA 

Mild (Erithema) 6 PR, 3 MR, 3 SD 


CTLp 

Rosenberg, 1998 

IV 39 

Peptide 

SC and ID 

MAGE-3 (HLA- 

A1) 

No Mild 3 CR; 4 PR 

No Increase of anti 

MAGE-3 CTLs even 

in Responders 

Marchand,1999 

IV 16 

Ag Pulsed Autologous 

Monocyte- derived 

Lymph nodes 

DCs 

Peptide 

Cocktail or 

Autologous 

Tumor Lysate 

KLH Mild (Fever) 2 CR; 3 PR;1 MR 

DTH to KLH in 16; 

DTH to peptidepulsed 

DCs in 11 

Nestle, 1998 

IV 17 



DCs 

ID 

Autologous 

Tumor Lysate 

No No 1 PR 

DTH to Vaccine in 

9/17; CD8 Cells in 

expanded-VIL 

Chakraborty, 1998 

Colon 

Cancer 

Prostate 

Cancer 

Renal Cell 

Cancer 

II and III 254 

Locally 

Advanced 

82 

IV 12 

Irradiated Autologous 

Tumor Cells 

ID 



Lymph nodes 

DCs 

Ag Pulsed Monocyte 

derived-DCs 

IV 

Autologous 

Tumor Cells 

Peptide 

Cocktail or 

Autologous 

Tumor Lysate 

Autologous 

Tumor Lysate 

BCG 

Mild 

Stage II: 61% Risk 

Reduction for Recurrences; 

Stage III: no Significant 

Benefits 

KLH Mild (Fever) 2 CR; 3 PR; 1 MR 

KLH Mild (Fever) 1 PR 

DTH+: ≥90% Pts 

DTH to KLH in 16; 

DTH to peptidepulsed 

DCs in 11 

DTH to KLH after 1° 

and 2° Vaccination 

Vermorken,1999 

Salgallar, 1998 

Holt, 1999 

Pts: Patients; TA: Tumor Antigens; Ads: Adjuvants; SC: Subcutaneous; MR: Mixed Response; SD: Stable Disease; CTLp: Cytotoxic T Lymphocyte precursor; ID: Intradermic; PR: Partial Response; 

TIL: Tumor-Infiltrating LymphocytesIFA: Incomplte Freund's Adjuvant; KLH: Keyhole Lympocianin; DTH: Delayed Type Hypersensitivity; IV: Intravenous 

that a potent immunologic response to KLH and, most 

importantly, against cell lysate could be measured in 

vitro after the vaccinations. In addition, the treatment 

was well tolerated with moderate fever as the only side 

effect. In contrast, only one partial response after 2 vaccinations 

was observed. 

Recently, Kugler et al. 228 vaccinated 17 patients with 

metastatic renal cell carcinoma using hybrids of autologous 

tumor and allogeneic DC generated by an electrofusion 

technique. After vaccination, and with a mean 

follow-up time of 13 months, four patients completely 

rejected all metastatic tumor lesions, one presented a 

mixed response, and two had a tumor mass reduction of 

greater 50%. These promising data indicate that hybrid 

cell vaccination is a safe and effective therapy for renal 

cell carcinoma and may provide a broadly applicable 

strategy for other malignancies with unknown antigens. 

Hematologic malignancies 

Tumor vaccines in B-cell lymphoproliferative 

disorders 

Multiple myeloma (MM) and low-grade non-Hodgkin’s 

lymphomas (NHL) are clonal expansions of lymphoid cells 

that have rearranged immunoglobulin (Ig) genes. Early 

during development, pre-B-cells become committed to 

the expression of a heavy and light chain Ig variable 

region. The heavy chain derives from the recombination 

of variable (V) with diversity (D) and joining (J) region 

genes with a constant region (C). The V-D-J joins occur 

with a variable number of nucleotide insertions or deletions 

resulting in a unique sequence which creates the 

third hypervariable region (CDR III) and contributes to 

the antigen-binding site. These antigenic regions (idiotype; 

Id) are characteristic for any given Ig-producing 

tumor (e.g. MM and NHL) and can be recognized by an 

immune response consisting of anti-Id antibodies and/or 

by Id reactive T-cells. 229-235 The tumor-derived Id is a self 

protein which, in most circumstances, is poorly immunogenic. 

However, haptens and adjuvants, including 

cytokines, have been used in several animal models to 

increase Id immunogenicity and establish protective anti- 

Id-immunity. 236 Lately, Id vaccines have come into medical 

use in patients with lymphoma and MM. 

Idiotype vaccination in human lymphoma 

A pioneering study was carried out in 9 lymphoma 

patients in CR or partial remission. They were immunized 

with subcutaneous injections of autologous Id, conjugated 

to KLH and emulsified in an oil-in-water emulsion 

containing non-ionic block polymers. 80 Specific anti-Id 

humoral and/or cellular responses were observed in 7/9 

patients. Two patients with measurable disease showed 

a clinical improvement. These results have been con- 



175 

firmed in a larger series of patients. 82 Following standard 

chemotherapy, 41 patients with B-cell lymphoma 

received subcutaneous injections of autologous Id-KLH 

conjugates mixed with an oil-in-water emulsion containing 

non-ionic block polymers and threonyl-muramyl 

dipeptide. Approximately 50% of the patients generated 

specific anti-Id responses and isolated tumor regressions 

were observed. In particular, 11/16 patients had a significant 

increase in the frequency of tumor-specific cytotoxic 

T-lymphocytes precursor (CTLp). 237 The median 

duration of freedom from cancer-progression was significantly 

prolonged and this resulted in a survival advantage, 

especially in patients who generated cell-mediated 

anti-Id immunity. 

These pioneering studies did not prospectively investigate 

the effect of Id vaccines on tumor burden, since 

most patients were already in clinical remission, and 

standard tumor regression criteria could not be used. A 

recent study has directly evaluated the ability of Id vaccines 

to eradicate residual t(14;18) + lymphoma cells in 

20 patients in first remission after ProMACE-based 

chemotherapy. 238 These patients received multiple injections 

of Id /KLH conjugates in the presence of GM-CSF. 

Eight of eleven patients with detectable translocations 

in the peripheral blood converted to a PCR negative status 

after vaccination. Tumor-specific cytotoxic CD8+ and 

CD4 + T-cells were uniformly seen in most patients. Antibodies 

to autologous Id were also detected, but they 

were apparently not required for molecular remission 

since the latter was achieved in some patients without 

a detectable antibody response. Clinical monitoring indicates 

a 90% disease-free survival after a median followup 

of 3 years. This is encouraging compared with the 

44% disease-free survival (after a median follow-up of 

3 years) in another series of patients treated at the same 

Institution with anti-B4-blocked ricin in first remission 

after ProMACE-based chemotherapy. The encouraging 

results obtained in lymphoma patients have provided the 

rationale for exploring the use of Id vaccination in MM. 

Id vaccination in human MM 

MM has several biological features that can advantageously 

be exploited in the setting of active specific 

immunotherapy. Among others, pre-existing tumor-specific 

T-cell immunity, 229-232 the possibility of using clonal 

markers to track the fate of residual tumor cells, 239 

and the preserved susceptibility of chemoresistant 

myeloma cells to the effector mechanisms of cytolytic 

T-cells 240 may, together, represent a favorable basis for 

the efficacy of Id vaccines. 

In a pilot study, five MM patients were repeatedly 

immunized with autologous Id precipitated in aluminium 

phosphate suspension. 77 Four patients were previously 

untreated and one patient was in stable-partial 

remission following chemotherapy. Three patients developed 

specific anti-Id T- and B-cell responses, but these 

responses were transient and their magnitude was low. 

A more effective immunization schedule was developed 

with the goal of achieving long-lasting T-cell anti-Id 

immunity. This effort was focused on early stage MM, 

based on the assumption that Id-specific T-cells are present 

at higher frequency mainly in patients with early 

stage MM or MGUS. 231 Most of these Id-reactive T-cells 

are Th1-type cells in early stage MM, whereas they are 

predominantly Th2-type in patients with advanced disease. 

Thus, a more effective antitumor T-cell immune 

response may be expected if vaccines are delivered when 

the frequency of T-cells with the potential to develop 

cytotoxic activity is higher. In this series, patients 

received subcutaneous injections of autologous Id precipitated 

in aluminium phosphate suspension, together 

with free GM-CSF. 241 Long-lasting Id-specific T-cell 

responses were induced in all five immunized patients. 

Moreover, one patient showed a decrease of circulating 

Id upon immunization. 

A vaccination trial in which Id/KLH conjugates and 

GM-CSF were administered subcutaneously as a maintenance 

treatment after high-dose chemotherapy and 

PBPC infusion has recently been published. 242 Most 

patients generated Id-specific DTH reactions. DTH specificity 

was confirmed in one patient by investigating the 

reactivity to synthetic peptides derived from the VDJ 

sequence of the tumor-specific Ig heavy chain. In 3 

patients with minimal residual disease, the DTH skin 

tests remained positive up to two years after the last 

immunization, but residual tumor cells were not eliminated 

by these long-lasting immune responses. Nevertheless, 

these patients remained in clinical remission 

without any further maintenance treatment. Thus, it is 

possible to generate anti-Id immune responses that are 

not potent enough to eliminate residual tumor cells, but 

are sufficient to hold the disease in check for extended 

periods. 

These results have been confirmed in a preliminary 

report of 18 patients with MM receiving Id/KLH conjugates 

and GM-CSF in first remission after high-dose 

chemotherapy and tandem transplantation followed by 

PBPC infusions. In particular, 50% of the patients generated 

positive DTH reactions and 2 patients, in partial 

remission at the time of vaccination, achieved a complete 

remission following vaccination. A retrospective 

pair-mate analysis has shown a trend for a better clinical 

outcome in MM patients receiving Id vaccines compared 

to those receiving IFN-α alone. This tendency is 

particularly evident in patients who generated positive 

Id-specific T-cell responses. Although preliminary and 

retrospective, this is the first study providing clinical evidence 

that the generation of T-cell immune response 

against tumor cells may positively influence the clinical 

outcome of MM patients in the remission phase (N. 

Munshi and L. Kwak , personal communications). 

DC-based anti-Id vaccination 

As previously discussed, a proportion of lymphoma 

and MM patients enrolled in clinical trials mounted an 

Id-restricted antibody and T-cell response and some of 

them showed tumor regression. However, there are 

important differences between lymphoma cells and MM 


176 


plasma cells. In the case of lymphomas, the cells are 

characterized by high surface expression and little antibody 

secretion whereas myeloma cells have very low 

levels of cell surface Ig with high levels of antibody 

secretion. Thus, it is unlikely that the sole generation of 

an antibody-based anti-Id immune response will be 

beneficial for MM patients. In fact, anti-Id antibodies 

may be blocked from reaching the tumor cells by the 

high levels of circulating Id. Moreover, despite the existence 

of a pre-plasma cell stem cell compartment in 

MM with a higher expression of surface Ig, it is possible 

that tumor cells would not express enough target 

protein for the antibodies to be effective. Conversely, 

an Id specific T-cell response would not need to bind to 

cell surface Ig to be active. T-cells do not recognize 

intact protein, but are specific for processed peptide 

fragments of the Id expressed on class I or II molecules. 

The advantage of a cytotoxic T-cell response is that it 

would not be blocked by free circulating paraprotein 

and would not depend on the expression of the native 

protein on the surface of tumor cells. Moreover, B-cells, 

including putative myeloma stem cells, are known to 

process and present peptides of the Ig on their membrane 

associated with class I and II molecules. Therefore, 

optimal strategies for Id vaccination may require the 

induction of a T-cell-mediated immune response which 

is best achieved by the use of APC. In this view, the rapid 

generation of a T-cell immunity in healthy volunteers 

after a single injection of mature DC has recently been 

described. 243 Nine normal subjects were given subcutaneous 

injections of monocyte-derived mature DC 

unpulsed or pulsed with KLH, tetanus toxoid (TT) or HLA- 

A*0201-positive restricted influenza matrix peptide. 

Four other individuals received these antigens without 

DC. Of note, administration of unpulsed DC or antigens 

alone failed to induce any T-cell response. Conversely, a 

CD4 + T-cell response was observed in 9/9 and 5/6 subjects 

injected with KLH or TT-pulsed DC, respectively. 

Moreover, a significant stimulation of effector and 

memory CD8 + CTLs was also reported. This feasibility trial 

provides the first controlled evidence of the capacity 

of DC to stimulate T-cell immunity. 

DC-based anti-Id vaccination has been reported In B- 

cell malignancies in a few papers. Hsu et al. have 244 

described 4 low-grade non-Hodgkin’s lymphoma (NHL) 

patients resistant to conventional chemotherapy or 

relapsed who were injected intravenously with Id-pulsed 

DC freshly isolated from PB by subsequent enrichment 

steps. A tumor-specific T-cell response was observed in 

all cases associated, in one case, with tumor regression. 

Sixteen patients have been treated so far and an anti- 

Id restricted cellular response has been observed in 8 

subjects (R. Levy, personal communication). The same 

strategy of targeting the Id has been applied by the same 

group to induce a T-cell immune response in MM 

patients. 245 Twelve patients were injected, 3 to 7 months 

after autologous stem cell transplantation, with Idpulsed 

DC followed by 5 subcutaneous boosts of Id/KLH 

administered with adjuvant. Whereas 11/12 patients 

developed a strong KLH-specific cellular proliferative 

response, thus suggesting immunocompetence after 

high-dose chemotherapy, only 2 individuals generated 

an anti-Id restricted T-cell proliferation and only 1/3 

patients showed a transient Id-specific CTLs response. 

This approach raises concerns about the efficacy of 

uncultured blood DC of stimulating efficiently T-cells, 

the capacity of Id-loaded DC to reach secondary lymphoid 

tissues to prime T-cells escaping the entrapment 

of the lungs and the role of Id-KLH boosts after DC 

administration. 

Wen et al. 246 reported immunization of one MM patient 

injected with Id-pulsed DC derived from adherent 

mononuclear cells in the presence of appropriate 

cytokines. In this paper, Id-specific T- cell proliferation 

and secretion of IFN-γ were reported, as were the production 

of anti-Id antibodies. Similar results have recently 

been reported by Cull et al. who treated two patients 

with advanced refractory myeloma with a series of four 

vaccinations using autologous Id-protein pulsed DC combined 

with adjuvant GM-CSF. 247 DC were derived from 

adherent mononuclear cells. Both patients generated a 

specific T-cell proliferative response that was associated 

with the production of IFN-γ, indicating a Th-1-like 

response. However, no Id-specific cytotoxic T-cell 

response could be demonstrated. Lim and Bailey-Wood 

have also treated 6 MM with DC generated from adherent 

mononuclear cells. 248 DC were pulsed with the autologous 

Id and KLH as a control vaccine. All patients developed 

both B- and T- cell responses to KLH, suggesting the 

integrity of the host immune system. Id-specific responses 

were also observed. In one patient, a modest but consistent 

drop in the serum Id level was observed. Lastly, a 

vaccine formulation based on CD34 stem cell-derived DC 

pulsed with Id-derived peptides has recently been used in 

11 MM patients with advanced disease. 249 Five patients 

generated Id-specific immune responses and one patient 

showed a decreased plasma cell infiltration in the bone 

marrow. 

New strategies in Id vaccination 

The generation of an effective antitumor response 

greatly depends on the final activation of tumor-specific 

cytolytic CD8 cells. This is the final event resulting from a 

series of cognate and non-cognate interactions occurring 

among tumor cells, professional APC, CD4, and CD8 cells. 

Each of these cell populations plays a unique role, and 

may represent a possible target of immune intervention to 

improve the efficacy of vaccination. Malignant B-cells can 

be modified to become efficient APCs themselves and present 

peptides from their own tumor-specific antigens to 

autologous T cells. To this end, a number of strategies are 

currently under preclinical and clinical evaluation. One 

possibility is to fuse tumor cells with dendritic cells. The 

fusion product will combine the functional properties of 

DC with the full antigenic repertoire of tumor cells. 250 As 

an alternative, malignant B-cells can be turned into effective 

APC by stimulating cell surface CD40 with its specific 

counter-receptor CD40 ligand. 251,252 Genetic engineering 



177 

is another approach that may turn malignant B-cells into 

effective APC. Transfection with immunologically relevant 

DNA sequences coding for cytokines or co-stimulatory 

molecules greatly enhances the ability of malignant B- 

cells to activate antitumor immune responses. This 

approach has recently been used in MM taking advantage 

of the selective expression of functional adenoviral receptors 

on the cell surface of myeloma cells. 253 

Interestingly, there has been a description 254 of the 

immunization of a matched related donor of allogeneic 

bone marrow with the myeloma derived Id (conjugated 

with KLH) isolated pretransplant from patients. After 

transplantation, a CD4 + T-cell line was established from 

the peripheral blood of the recipient and found to be of 

donor origin and proliferate specifically in response to 

the myeloma Id. Thus, this experience demonstrates the 

principle of transfer of donor immunity with the advantage 

of immunizing a tumor naive donor who may be 

more likely to be able to generate an immune response 

against the tumor Ig without interference or suppression 

by the malignant cells. However, the lack of an anti-Id 

CTL response and ethical concerns on the immunization 

of healthy donors with tumor-derived products, suggest 

that in the future an alternative strategy based upon the 

generation, ex vivo, of Id-reactive CTL clones by means of 

APC, will be needed. 

The prerequisite for any vaccine-based strategy is the 

possibility of differentiating tumor cells from normal cells. 

Id is absolutely tumor-specific, but is not directly related 

to the malignant phenotype of myeloma cells, and is 

self-Ag. As such, it is protected by self-tolerance mechanisms. 

There is a growing list of alternative tumor-specific 

antigens that can be exploited as targets for active 

specific immunotherapy. Among others, the core protein 

of Muc-1, 255,256 antigens encoded by MAGE-type genes, 257 

overexpressed or fusion proteins resulting from chromosomal 

abnormalities may all represent alternative 

immunogens. 258,259 Compared to Id, some of these antigens 

may be more intrinsically related to the malignant 

phenotype of tumor cells. 

Polymorphism of the HLA molecules is a major obstacle 

in the outcome of vaccines aimed at triggering 

cytolytic T-cell responses. By combining amino acid 

sequencing of tumor-specific antigens and HLA typing, it 

is now possible to predict whether the HLA alleles of a 

given individual can bind tumor-derived peptides. Several 

groups are planning to use Id vaccination only in those 

patients for whom preliminary sequencing demonstrates 

a compatible restriction between HLA and peptides. 

Finally, a new generation of immunogens has been 

developed using DNA-based technologies. The whole 

tumor-specific immunoglobulin or the variable region 

sequences of both heavy- and light-chains have been used 

as immunogens or to produce recombinant proteins in 

bacteria. However, it has soon become clear that naked 

DNA is not immunogenic per se and additional sequences 

are required to elicit protective immune responses. 260 

Sequences coding for cytokines, chemokines or xenogeneic 

proteins have been included in these constructs 

and used as adjuvants. 261-264 Experimental data indicate 

that these fusion genes have indeed the potential to raise 

protective immunity in both NHL and MM. 

Vaccine trials in chronic myelogenous 

leukemia 

Chronic myelogenous leukemia (CML) is a biphasic 

neoplastic disorder with a prolonged indolent phase lasting 

an average of 4 years followed by an acute phase of 

blastic transformation which inevitably leads rapidly to 

death. There is no curative therapy for CML other than 

allogeneic bone marrow transplantation, an option that 

is available only to a small fraction of patients who have 

both a matched donor and are young enough to tolerate 

the procedure. 

Recently, IFN-α has been shown to induce hematologic 

remissions in most CML patients, with a relevant portion 

of them also experiencing several degrees of cytogenetic 

response and ultimately a statistically significant prolongation 

of their chronic phase. However, still too few CML 

patients are long survivors if not cured regardless of the 

treatment option they received. Because of the unique features 

of this disease, the hallmark translocation that characterizes 

all neoplastic cells, a therapeutic targeting 

approach only the Ph+ clone could be a powerful tool in 

the treatment of CML. 

The first direct evidence of the immune system’s crucial 

role in recognizing and eliminating Ph+ CML cells 

came from the demonstration that infusion of large doses 

of peripheral blood leukocytes from the marrow donor 

induced durable remission in patients with CML who had 

relapsed following a T cell depleted marrow allograft. 265,266 

This latter finding proved that in CML the graft-versustumor 

effect is mediated by the cellular arm of the 

immune system. While the nature of the response is likely 

to be largely allogeneic a possible role for specific anti- 

CML responses is suggested by the lack of this observation 

in patients with other myeloid leukemias undergoing 

the same treatment. 267 

The hypothesis that CML cells could be recognized by 

the immune system through the presentation of P210, 

the tumor-specific product of the bcr/abl hybrid gene, 

was first tested. The evidence that P210 b3a2-breakpoint 

peptides were able to bind HLA class I and HLA class II 

molecules and to elicit specific T cell responses in normal 

donors provided the rationale for a peptide vaccine in 

CML patients. 103-106 Pinilla-Ibarz et al. 124 have recently 

completed a phase I dose escalation trial (5 doses over 10 

weeks) of a multivalent peptide vaccine (5 peptides) plus 

QS21 in patients with CML and b3a2 breakpoint. Patient 

characteristics included hematologic remission, IFN-α 

therapy and no HLA restriction. In a preliminary report the 

peptide vaccine appeared safe with patients experiencing 

only minimal discomfort at the site of injection. 

With regards to the immune response, peptide-specific 

delayed hypersensitivity (DTH) in vivo and peptide-specific 

proliferation in vitro were shown but no peptidespecific 

CTL response was induced. Bocchia et al. are currently 

conducting a multicenter phase I/II trial of a pen- 


178 


tavalent peptide vaccine plus QS21 and GM-CSF in b3a2- 

CML patients expressing any of HLA A3, A11 B8 or DR11. 

Patient characteristics also include major or complete 

cytogenetic response with or without IFN-α maintaining 

therapy. The protocol comprises 6 s.c. vaccinations with 

peptides + QS21 at 2 weekly intervals, with GM-CSF 

injected at the vaccine site for 4 consecutive days starting 

the day before each vaccination. Goals of the study 

are the evaluation of the induction of peptide-specific T- 

cell response, of the role of GM-CSF as immunologic 

adjuvant in CML patients and the impact of the peptide 

vaccine on minimal residual disease. 

As in other cancers, the use of DC as powerful inducers 

of an active specific immune response in CML is now 

under in vitro evaluation. 107,268 Interestingly, most CMLderived 

DC carry the t(9;22) translocation and therefore 

could naturally present P210 derived peptides. In fact, 

CML derived Ph + DC were able to strongly stimulate 

autologous T-cells that displayed vigorous cytotoxicity 

activity against autologous CML cells but low reactivity 

to HLA-matched normal bone marrow cells or autologous 

remission state bone marrow mononuclear cells. 206 

P210-derived peptides could have a role in inducing this 

leukemia-specific response and a vaccine strategy which 

combines Ph + DC and breakpoint peptides will be investigated. 

Conclusions 

This review shows that there are many prospects of 

curing cancer through the active induction of a specific 

immune response to TAA. The terms of the matter are 

now defined with molecular and genetic details for 

melanomas. Ongoing research is aimed at defining TAA 

on other forms of tumors. Indeed, experimental data and 

very recent clinical evidence suggest that antitumor vaccines 

will soon be a new form of tumor treatment that 

will be able to be adopted for the management of 

defined stages of neoplastic disease, in sequential association 

with conventional treatments. 269 

Prediction of when the efficacy of antitumor vaccination 

will be assessed and will become a routine procedure 

is beyond a simple scientific evaluation. While preclinical 

research has identified several possible targets 

and strategies for tumor vaccination, the clinical scenario 

is far more complex and as yet no specific clue has 

emerged to clearly envisage a clinical development strategy 

which could make biotechnology investments in this 

area attractive enough to pharmaceutical companies. 

Patent issue complexity further contributes to slowing 

down the development of expensive clinical trial programs. 

A cautious, yet attentive attitude seems, at the 

moment, to be the general behavior of the pharmaceutical 

industry. 

At present peptide vaccination may appear of more 

immediate application. Several phase I clinical trials have 

already been carried out using synthetic peptides from 

defined TAA. These peptides have been administered 

alone 123 or combined with adjuvants, or presented by 

monocytes or DC. 269 Nearly all studies indicate that this 

form of vaccination is well tolerated, mild fever and 

inflammation at the site of injection being the only occasional 

side effects observed. Nonetheless it should be 

pointed out that there is usually a poor correlation 

between peptide ability to induce a T-cell response and 

clinical response. Among the several reasons that may 

account for this discrepancy, the choice of the peptides 

may have a critical importance. Most peptide-based vaccines 

have considered HLA class I restricted peptides 

only, whereas there is increasing evidence that tumorspecific 

CD4 + T-cells may be important in inducing an 

effective antitumor immunity. The addition of peptides 

that bind class II HLA glycoproteins to peptide vaccines 

could lead to an amplification of the immune response 

as well as to better clinical effect. 

A survey of the outcomes of vaccination trials shows 

that the poor correlation between induction of immunologic 

responses and the clinical results is a consistent 

finding, independently of the immunizing strategy 

adopted. Many factors may contribute to this poor correlation, 

e.g.: 

a) the selection of the patients enrolled in the trial: 

tumor burden, stage of disease, and others, as discussed 

above; 

b) the techniques used for the immune monitoring in 

vitro: most of the current studies evaluate T-cell induction 

through in vitro peptide stimulation of PBMC, while 

the use of tetrameric soluble class I-peptide complexes 

270 or reverse solid phase ELISPOT analysis 271 may provide 

complementary information; 

c) the assays for immune monitoring in vivo: often a 

positive DTH test does not correlate with evident tumor 

regression. 

Perhaps, fine needle biopsies at the site of regressing 

and non-regressing tumors could provide a more direct 

insight into the events associated with the clinical outcome. 

The immune pattern within the tumor should be 

compared to the one found in the skin. This should allow 

evaluation of the exact role of vaccine-induced T-cells. 

Which of the existing in vitro and in vivo assays correlates 

most accurately with clinical responses remains to 

be established. 

Several recent studies showing that the immune system 

recognizes TAA during tumor growth did not clarify 

whether such recognition was indeed associated with 

subsequent tumor cell destruction. The development of 

reliable assays for efficient monitoring of the state of 

immunization of cancer patients against TAA is as an 

important goal that will markedly affect the progress of 

antitumor vaccines. A major problem in testing the efficacy 

of antitumor vaccination in adjuvant settings 

depends on both the long period of time and large number 

of patients required. The possibility of effectively 

monitoring the immune response induced acquires critical 

importance since it may provide a much earlier surrogate 

end-point, predictive of the clinical outcome. 

The downregulation of the expression of TAA represents 

another crucial issue for vaccination therapies, 

since it may lead to the immunoselection of tumor cell 



179 

clones that hide the target TAA. An ideal TAA is a protein 

that is essential for sustaining the malignant phenotype, 

and that is not stripped or downmodulated by 

the immune reaction. Mutations that give rise to TAA of 

this kind have been described. 272 However, they will be 

an appropriate target only for the tumors expressing 

these particular mutations and will not be suitable for 

more general cancer vaccines. Improvements in the 

identification of tumor-associated mutations that may 

be potentially recognized by the immune system may 

also open up the possibility of tailoring individual cancer 

vaccines. The recent report of the construction of 

fusion cells composed of autologous tumor cells and DC 

or TAA pulsed-DC represents a step forward towards the 

quick manufacture of tumor-specific and individualized 

vaccines. In contrast, the characterization of the telomerase 

catalytic subunit (hTERT) expressed in more than 

85% of human cancers appears to open the way to a 

novel strategy for a general anticancer vaccination targeting 

a widely distributed TAA. 273 

The in vivo or ex vivo introduction of TAA genes into DC 

through recombinant viral vectors is still hampered by 

the lack of an ideal viral vector and by the induction of 

an immune response against the viral proteins. Nonetheless, 

many viral vectors successfully used in animal models 

and currently tested in clinical trials appear to be safe 

vehicles for gene transfer, without any major toxicity. To 

control transgene expression levels better, investigators 

are exploiting tissue- or cell-specific regulatory elements 

such as cytomegalovirus promoters and enhancers that 

are preferentially expressed in tumor cells. 

Certainly the new prospects opened by antitumor vaccines 

are fascinating. When compared with conventional 

cancer management, vaccination is a soft, noninvasive 

treatment free from particular distress and 

iatrogenic side effects. Antitumor vaccines can be 

expected to have a considerable social impact, but a 

few large clinical trials enrolling the appropriate patients 

are now necessary to assess their efficacy. 

This review will end considering their use not in the 

treatment of cancer patients but to prevent cancer in 

healthy persons, a so far neglected prospect. Current 

studies are leading to the detection of gene mutations 

that predispose to cancer. 274 Identification of the gene 

at risk and its mutated or amplified products would provide 

the opportunity to vaccinate susceptible subjects 

against their foreseeable cancer. Molecular characterization 

of altered gene products predictably destined to 

become a tumor antigen will be the first step towards 

the engineering of effective vaccines to be used for this 

purpose. 25 

An unrestrained imagination may picture an even 

broader application of antitumor vaccines, i.e. their use 

to prevent tumors in the general population. Molecular 

and genetic data suggest that the number of TAA is not 

endless. Several of the TAA detected so far are shared by 

histologically distinct tumors arising in different organs 

(Table 1). The possibility of vaccinating against most 

common human cancers by using not many more than 

twenty TAA may perhaps be conceivable. Experimental 

data suggest that the immunity elicited by specific vaccination 

is much more effective in the inhibition of 

incipient tumors than in the cure of those that have 

already progressed. 275 The risk of inducing an autoimmune 

disease would be a major worry since not rarely 

antigens acting as TAA are expressed by normal tissues. 

276 This risk would be much harder to accept when 

treating healthy individuals than in the vaccination of 

cancer patients, where the scales of risk-benefit are 

biased by a short life-expectancy. On the other hand 

failure to intervene when a disease so diffuse and dramatic 

as cancer can be prevented could also be viewed 

as harmful. 277 Lastly, it should be considered that the 

same or even a higher risk of inducing autoimmune 

reactions is associated with many antimicrobial vaccines. 

Fortunately, they started to be used when this risk 

was not yet perceived. 

In conclusion, even if cancer vaccines are an old 

dream, 278 only recently has their design become a rational 

enterprise. There are now many ways of constructing 

vaccines able to elicit a strong protective immunity. 

This progress is offering ground for optimism. 


The authors were a group of experts and representatives 

of two pharmaceutical companies, Amgen Italia 

Spa and Dompé Biotech Spa, both from Milan, Italy. This 

co-operation between a medical journal and pharmaceutical 

companies is based on the common aim of 

achieving optimal use of new therapeutic procedures in 

medical practice. 

Funding 

The preparation of this manuscript was supported by 

educational grants from Dompé Biotech Spa and Amgen 

Italia Spa. 

Disclosures 

Conflict on interest: Dompé Biotec Spa sells G-CSF and 

rHuEpo in Italy, and Amgen Italia Spa has a stake in Dompé 

Biotec Spa. 

Redundant publications: no substantial overlapping 

with previous papers. 


Manuscript received August 7, 2000; accepted October 

24, 2000. 

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Index of authors 

Aglietta Massimo, 19, 92 

Arcese William, 69 

Aversa Franco, 69 

Bandini Giuseppe, 69 

Bertolini Francesco, 49, 92 

Bocchia Monica, 156 

Bordignon Claudio, 117 

Bronte Vincenzo, 156 

Caligaris Cappio Federico, 32 

Carlo-Stella Carmelo, 1, 92, 117 

Cavo Michele, 32 

Cazzola Mario, 1 

Colombo Mario P., 117, 156 

De Fabritiis Paolo, 1 

De Vincentiis Armando, 1, 19, 32, 49, 69, 92, 117, 156 

Di Nicola Massimo, 156 

Falda Michele, 69 

Forni Guido, 156 

Gianni Alessandro Massimo 1 

Lanata Luigi , 19, 32, 49, 69, 92, 117, 156 

Lanza Francesco, 1, 19 

Lauria Francesco, 1 

Lemoli Roberto M., 1, 19, 32, 49, 69, 92, 117, 156 

Locatelli Franco , 69, 117 

Maccario Rita, 49 

Majolino Ignazio, 32, 49, 69 

Massaia Massimo, 156 

Menichella Giacomo, 19 

Olivieri Attilio, 92, 117 

Ponchio Luisa, 49 

Rondelli Damiano, 49, 117, 156 

Siena Salvatore, 92 

Tabilio Antonio, 49 

Tafuri Agostino, 19 

Tarella Corrado, 1, 32 

Tura Sante, 1, 19, 32, 49, 69, 92, 117, 156 

Zanon Paola, 1, 19, 32, 49, 69, 92, 117, 156 

Direttore responsabile: Prof. Edoardo Ascari 

Autorizzazione del Tribunale di Pavia n. 63 del 5 marzo 1955 

Composizione: = Medit – via gen. C.A. Dalla Chiesa, 22 – Voghera, Italy 

Stampa: Tipografia PI-ME – via Vigentina 136 – Pavia, Italy 

 

Printed in December 2000

In collaborazione con Dompé Biotec s.p.a.

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