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PATIENT: H.O'N. 104L-_L-1~1 __ ~1 __ ~1 __ ~1 __ ~1 ___ ~1_L-1_L-1~ \ 4oM. \ \ 3Or-\ ~ \ f\ 20r \ ~ I \ \ '~' I \s"'\ 10r ~~ I \ \ AI \~ I ~. I ~ _LI. I I OL--2~-4~~6~~8~~1~O~1~2~1~4~1~6~1~8~ WEEKS IN CULTURE Fig. 3. Kinetics of generation of granulocytes and GM-CFUc in vitro over 14 weeks in a representative human continuous marrow culture. Results are the me an of at leat four cultures at each time point. Cultures were established in McCoy's 5A medium supplemented with 10- 7 M hydrocortisone, 12.5% FCS, and 12.5% horse serum, with delayed removal of red blood cells after 5 days corticosteroid-dependent, insulin-resistant lipogenesis in marrow preadipocytes of mutation diabeticobese mice. Nature 275: 752-754 - Greenberger JS (1979a) Corticosteroid-dependent differentiation of human bone marrow preadipocytes. In vitro 15 :823-828 - Greenberger JS (1979b) Phenotypically-distinct target cells for murine sarcoma virus and murine leukemia virus marrow transformation in Vitro. J Nat Cancer Inst 62: 337-348 - Greenberger JS, Davisson PB, Gans PJ (1979a) Murine sarcoma viruses block corticosteroid-dependent differentiation of bone marrow preadipocytes assoeiated with long-term in vitro hemopoiesis. Virology 95:317-333 - Greenberger JS, Donahue D, Sakakeeny MA (1979c) Induction of ecotropic endogenous murine leukemia virus in long-term bone marrow cultures from mouse strains of varying naturalleukernia incidence. J. Reticuloendothel Soc 26:839-853 - Greenberger JS, Gans PJ, Davisson PB, Moloney WC (1979c) In vitro induction of continuous acute promyelocytic cell lines in longterm bone marrow cultures by Friend or Abelson leukemia virus. Blood 53: 987 -1001 - Greenberger JS, Sakakeeny MA, Parker LM (19791) In vitro proliferation of hematopoietic stern cells in longterm marrow cultures: Principals in mouse applied to man. Exp Hematol (Supp15) 7: 135-148 - Greenberger JS, Newburger PE, Lipton JM, Moloney WC, Sakakeeny MA, Jackson PL (1980) Virus and cell requirements for Friend virus granulocytic leukemogenesis in long-term bone marrow cultures of NIH swiss mice. J Natl Canc Inst 64:867-878 - Greenberger JS, Eckner RJ, Ostertag W, Colletta G, Boshetti S, Nagasawa H, Karpas A, Weichselbaum R, Moloney WC (to be published a) Release of spleen focus-forming virus (SFFV) from differentiation indueible promyelocytic leukemia cell lines transformed in vitro by Friend leukemia virus. Virology - Greenberger JS, Newburger P, Sakakeeny MA (to be published b) Phorboi myristate acetate stimulates macrophage proliferation and differentiation and alters granulopoiesis and leukemogenesis in long-term bone marrow cultures. Blood - Greenberger J, Wroble LM, Sakakeeny MA (to be published c) Murine leukemia viruses induce macrophage production of granulocyte-macrophage colony stimulating factor in vitro. J N atl Canc Inst - Mauch P, Greenberger JS, Botnick LE, Hannon EC, Hellman S (1980) Evidence for structured variation in self-renewal capacity within long-term bone marrow cultures. Proc Natl Acad Sei USA 77:2927-2930. 293
Haematology and Blood Transfusion Vol. 26 <strong>Modern</strong> Trends in Human Leukemia IV Edited by Neth, Gallo, Graf, Mannweiler, Winkler © Springer-Verlag Berlin Heidelberg 1981 Cloning Cells of the Immune System N. A. Mitchison The purpose of this introductory note is to explain why the immunological papers in this collection concentrate on cloning. There are three good reasons for choosing cloning as an appropriate subject at the present time. One is that cloning provides a valuable means of acquiring information about the working of the immune system. Another is that several of the technical problems which prevented satisfactory cloning have just been solved, so that rapid progress can and is being made. The third is that application of the new procedures is providing insight into leukaemia. The value of cloning follows from the way in which the immune system is arranged as a loose population of cells which traffic from place to pI ace interacting through transient contacts and soluble factors. In consequence cells are differentiated from one another not by their anatomical position and connections but by their genetic and epigenetic makeup. There is a strong contrast here between the nervous and immune systems, the two most complex and highly integrated systems of the body which otherwise share many features in common. No doubt clones from the nervous system can provide interesting information about such topics as receptor function and metabolic control, but they cannot be expected to tell us directly how neurones work. This is not true of lymphocytes and to a lesser extent, of antigen-presenting cells: here we can expect clones to express all major functions. The major technical problem has been to find ways of keeping cells alive and multiplying outside the body. The first step forward was to maintain clones of B cells under antigenic stimulation in irradiated mice (Askonas and Williamson 1972). For B cells the in vitro problem has now largely been solved by the hybridoma technique (Köhler and Milstein 1976). Monoclonal antibodies produced by this technique turn out to be immensely powerful tools in biochemistry, cell biology, and medicine. Their application is weIl exemplified by Beverley's study of stern cell surface markers described in this volume. The hybridoma revolution is sweeping all before it, leaving only little room for alternatives such as the immortalization of human immunoglobulin-secreting cells by Epstein-Barr virus infection (Steinitz et al. 1977). For T cells, hybridomas have thus for proved less successful. Our own experience has been that immunoregulatory activity can be maintained in this way for a while, but tends to decline in an unpredictable and uncontrollable way (Kontiainen et al. 1978). Other laboratories find the same decline. On the other hand, T cells are proving highly amenable to less drastic cloning procedures. One such procedure is to maintain them on T cell growth factor (TCGF). Another is to restimulate cultures with antigen at intervals. Both of these procedures are discussed and evaluated in detail at the International Congress of Immunology this year, and the latter is weIl exemplified by Hengartner's study described here. Our approach (Czitrom et al. 1980) has been to generate allospecific helper T cells by stimulation in vitro. Our previous work had shown that the adoptive secondary response in mice could be successfully adapted for the study of helper T cells directed at cell surface antigens. We generate helper T cells by alloantigen-induced proliferation in vitro directed at Ik (A.TH anti-A.TL) and test for their ability to help in vive primed B cells directed at D b (A.TH anti-BIO) in an adoptive 294
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Haematology and Blood Transfusion 2
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Dr. Rolf Neth, Universitäts-Kinder
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Participants of the Meeting Aaronso
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Kurth, R, Friedrich-Miescher-Labora
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Wilsede Scholarship Holders (Grante
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Preface Gut ist eine Lehrart, wo ma
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Personal and scientific discussion
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Acknowledgement We should like to t
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Wilsede, June 21 1978 Memorial Trib
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limiting benign lymphoproliferative
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this most unlikely, however (for re
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longer subject to the same control
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Bregula U, Wiener F, Harris H (1971
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fresh lymph node biopsies from ten
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Fig. 3. Binucleate mitosis in an ob
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y Fig. 4. Schematic diagram of post
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than that among patients with Stage
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Tests of binding affinity, agglutin
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N Engl J Med 297: 963-968 - Green I
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Clinical Aspects
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"ring chromosome". "Mar" is marker,
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191 chromosomally abnormal patients
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tumors of both African and non-Afri
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Haematology and Blood Transfusion V
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Retrospective group 26/93 evolved l
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Treatment
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advantage in terms of duration of f
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leukaemia. An analysis of 168 patie
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11. Treatment Program Chemotherapy
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efractory congestive heart failure
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period (Sequence II) was gene rally
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children with a multiple drug proto
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DAYS 1 5 10 15 20 25 30 35 , I I I
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Biologically Drug Outcome fit" sens
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may counterbalance the potential be
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Ht (0--0) 50 40 30 20 10 0 Platelet
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10 7 ,-----------------------------
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11. Haematological and Biochemical
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0\ 0\ INTERFERON ... 10 3 rl E "'
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("pre-B" phenotype) has been descri
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human bone marrow cells exhibit the
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10 9.,0 MBKCC .. .,4 B prot:oool 1'
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demann W, Büehner T, Arlin Z, Gee
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-...l 00 a-tLDRENS CAt«:ER S TU>Y
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prognostic characteristics under in
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significantly higher in HR} (14.5%,
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normal or elevated immunoglobulin l
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References Andreeff M, Darzynkiewic
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R GROUP 1 ( 1970 - 1971 ) 17 PTS. R
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Table 3. Influence of initial featu
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LIJ (f) a.. < -1 LIJ Q: u. 0 >- I-
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me nt indicated that additional the
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42:2123-2134 - Chessells JM, Cornbl
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Baum et al. 1979). This study was b
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1. 0 '--'1--" 0.9 e: o .- VI VI E C
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e: o e: 1. 0 ,.--...-- 1'"'\ ......
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In conclusion, we can state that th
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Haematology and Blood Transfnsion V
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INDUCTION VCR + PRED + ASNASE (wk 3
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SCHED.INT 1 MTX • ! Adria MTX •
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~ looh---------ooooc>----o---c>----
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Consoll- Irra .tlon t) .tlon Indu
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Highrisk Standard risk Table 1. Cli
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Cumulo/ive comp/el9 remission 1.0 .
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10° 8 = 6 ö 4 Non T Cell .~ :ö
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% SURVIVAL 100 ~ ~II 0 80 60 40 - -
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a) Percentage of donor cell mitoses
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Table 5. Results of bone marrow tra
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...... w N Table 7. Clinical result
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agent had to be added and that in t
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C. Chronic Myelogenous Leukemia The
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Patients with a suitable donor rece
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No. Recurrent Leukaemia Other death
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followed by dialysis against isoton
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HS, HL- Heme >_ 40 0 HS I ;:. =====
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knowing at present. These genes may
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esistance to antifolates. In Vitro
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a DNA probe specific for the gene t
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translocation between chromosomes 9
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Table 1. Subdivision of 1827 Myelo-
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NORMAL CELL ®
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some change of the primary type, in
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F. Transformation of Mouse Bone Mar
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were carried out with 3H-MTX in the
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the lysozyme cDNA prepared from ovi
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et al. 1976). The BJAB cellline in
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dalton bands corresponding to light
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1979). The EBV-carrying cell lines
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producer lines (DAUDI and P3HR-1),
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G, Giovanella B, Westman A, Stehlin
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Dc;bt CT + i J.I9 Raji; 1. i .. 100
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c. Discussion During infectious mon
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C. Nonepisomal Viral DNA We have us
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A 260 3.0 A B 1670 70 N2 2.0 c: E .
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vivo. Nature 260:302-306 - Kirk JTO
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Fig. 1. a Polyacrylamide gelelectro
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1979). The lymphocyte subpopulation
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lysin 0 antigens are found, and mix
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Pope JH (1978) Long-term T cell-med
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Charaeteristie Morphologie maturati
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after TPA-induction (Table 3). The
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PA (1978) Reactivity of a rabbit an
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Blood Monocyte. B lood Monocytes 1-
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and nonhistone proteins can serve a
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The findings of the evaluation of s
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Fig. 3. Tumor grawth of L 428 cells
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Cell biological and Immunological A
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consideration. Sanel (1973) obtaine
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endotoxin serum, were used. A parti
Page 253 and 254: monocytic leukemia cell line in cul
Page 255 and 256: B. Isolation, Characterization and
Page 257 and 258: Haematology and Blood Transfusion V
Page 259 and 260: don al hemopathies generally displa
Page 261 and 262: nitors. Blood Cells 6:595-607 - Min
Page 263 and 264: \I) ....I ....I IU Jl 200 r 100 90
Page 265 and 266: their haemopoietic cells' ability t
Page 267 and 268: normal normal ALL ALL ALL persons p
Page 269 and 270: 64.5 { V)
Page 271 and 272: References Biermann E, Neth R, Gros
Page 273 and 274: ehambers, the growth pattern observ
Page 275 and 276: References Anderssen LC, 10kinen M,
Page 277 and 278: REH CELL UNE TO CALLb~Ö ~q~ V.M. c
Page 281 and 282: the macrophage series in which the
Page 285 and 286: Table 1. Production of factor depen
Page 289 and 290: Table 1. Characteristics of the adh
Page 291 and 292: Fig. 3. A human marrow culture at 6
Page 293 and 294: 300 250 • HYPERPROLIFERATIVE PATT
Page 295 and 296: Fig. 9. Nonadherent population from
Page 297 and 298: The cobblestone areas were often no
Page 299 and 300: L (1976) Induction of colony format
Page 301 and 302: B. Materials and Methods J. Tissue
Page 303: Fig. 2. Ultrastructural appearance
Page 309 and 310: Table 1. Monoclonal antibody reacti
Page 311 and 312: al. 1979; Janossy et al. 1979) is e
Page 313 and 314: to which this is an exact replica o
Page 315 and 316: inger JL (1976) Distribution of la-
Page 317 and 318: I region antigens were found to be
Page 319 and 320: have been reported to be present on
Page 321 and 322: V. FunctionalAssays Assays for PHA
Page 323 and 324: Functional studies of UCHT1 separat
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Page 329 and 330: with immunohistochemieal methods (L
Page 333 and 334: can be dissected by cloning. Utiliz
Page 335 and 336: Table 1. Origin and marker profile
Page 339 and 340: complete identity of gp 100 from no
Page 341 and 342: Table 1. Reaction of single anti se
Page 345 and 346: media. Following 24-h incubation, c
Page 347 and 348: nonleukemic eells (Greaves 1979). L
Page 351 and 352: Fig. 2. Cytocentrifuged smears of b
Page 353 and 354: Modification of amino-groups of hum
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disturb this balance in the directi
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The majority of human blood lymphoc
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of the EBV infected B cells. Howeve
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and immunoglobulins. J Immunol 120:
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" K 10 • j ~ ALL RNlL AL wtfh les
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tion akuter Leukämiezellen in "spo
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D. Results and Discussion J. Acute
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selectively responsive to different
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l1J ~ 100 ~ Q. ::) l1J Z 0 ~ z >- .
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Table 2. Growth inhibition of S49 e
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Reverse Infectious ASC/2X1Q6 transc
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munized animals to 1316 tumor cells
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Fig. 3. Expression of retroviral gp
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pg70. J Exp Med 143: 151-166 - McGr
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Table 1. Expression of tumor antige
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Virological and Molecularbiological
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Table 1. Oncogenic properties of re
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also Fig. 1). It followed that the
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whether this sequence is related to
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zed in infected cells argue that th
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a specific viral transforming prote
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detect viral RNA as the only charac
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Eisen H (1980) Myeloproliferate vir
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p,60- -34K - 1 2 3 4 Fig. 1. In vit
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.. p - • a'T ... I .t'l Fig. 4. D
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Fig. 2. Photomicrographs of NY68 in
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DEAE (o(lJmn 'liI GI -2 2 o e s 1
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vity was deterrnined. For endogenou
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pr 180 - - - P 12/15 - A B c D E F
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85,- 66- -27 - -66 40- 27- -1'9 19-
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= 0.5 o E Q. o ILI 0.4 (J) « ILI .
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Table 1. Oncogenic potential of avi
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100 III GI c: o "8 80 > w
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Expt. No. Infecting virus Proportio
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Haematology and Blood 'fransfusion
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sequences, provided its gag portion
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77:3009-3013 - Hu SSF, Moscoviei C,
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RI 2.2 md and Hind III 2.6 md leuke
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RELATIONSHIP OF ENDOGENOUS AND EXOG
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E ........ E Cl. U E :::l .... Q) (
Page 447 and 448:
References Astrin S (1978) Endogeno
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the same animal were examined (e.g.
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I) 'IQ a \0 - CL -- Fig. 2. Restrie
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~ ..................... ~ Cell 3' 5
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a :Sa,C! + . ~ 111 i' ( " '~ .J. b
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Table 1 Sampie Tissue TS fragment"
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a b Sac I / rep ._-_.-_ K J nl rep
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Haematology and Blood 'fiansfusion
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Osteopetrosis and osteosarcomas occ
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Lymphoma cellline Table 1. T and B
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Table 2. In vivo growth and oneogen
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Table 1. Transforming aetivity of c
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Table 3. Transformation aetivity of
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- Shoemaker C, Goff S, Gilboa E, Pa
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indieate a shifting of target eell
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Table 2. Expression of Jl and Sourc
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~ i ,..1---------,,':::::::::::::::
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fragment of pBR322 DNA, a 3.0-kbp S
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Mak TW (1979) Proc Natl Acad Sci US
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Table 1. Transformation of 3T3 cell
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inserts in the genome of rapidly tr
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pulations, it seemed likely that th
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contains antigens which react with
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le of reacting in sensitive radioim
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gic approach. In: Hiatt HH, Watson
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12 Eco R [ Fig. 1. Hybridization pa
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10 - '1~ )( -~ S:! E A U I o 1 2
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Haematology and Blood 'fransfusion
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cultures. These cells did not conta
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indication of malignant T-cells in
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Fig. 3. Thin-section electron micro
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Table 3. Lack of detectable related
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J. HTLV Was Present in the Primary
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specific signals into nonspecific s
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Table 2. Distribution of HTLV-relat
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u 10 ~ 20 ~ :c 30 a 40 o Fig. 1. Ki
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SSV TrS-gp labeled effectively with
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60 HFF/SSV cDNA CELL RNAs: 0 HF/SSV
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Antisera a Table 1. Immunofluoresce
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Haematology and Blood 'ftansfusion
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y competition RIA yielded virtually
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SiS'V' gp70: ANTIGEN, ANTI BODY, IM
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type-C virus p30 antigen in cells f
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c. Results The fractionated whole-b
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induced antibodies as well as exper
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- Fig. la-f. Retravirus particles p
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eplication of infecting murine leuk
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immunodeficiency, multic10nal lymph
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Bovine leukemia --, proteins of 499
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Human T- cell - -, characterization
Page 563 and 564:
RNA -, of tumor virus 439 Rous sarc
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Haematology andBlood Transfusion Su
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