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Monoclonal Antibody T-Cell-Depleted HLA-Haploidentical Bone Marrow
Transplantation for Wiskott-Aldrich Syndrome
By Stephen L. Rumelhart, Michael E. Trigg, Sheldon D. Horowitz, and Richard Hong
Four patients with Wiskott-Aldrich syndrome received
bone marrow transplants (BMT) using monoclonal antibody
T cell-depleted HLA-haploidentical marrow from a family
member donor. The patients did not receive a significantly
larger inoculum of mature T cells than other recipients of T
cell-depleted marrow transplants. All four patients achieved
quick engraftment, and three of the four patients are alive
and well today. The three living patients have all had a
complete return of normal T-cell and 6-cell function.
Infectious complications in the surviving patients were
minimal: however, all three experienced some degree of
graft-versus-host disease (GVHD). Two of these three
patients received GVHD prophylaxis. The patient not
HE WISKOTT-ALDRICH syndrome (WAS) is an
T X-linked recessive, primary immunodeficiency charac-
terized by eczema, thrombocytopenia, recurrent infection,
and increased susceptibility to lymphoreticular malignancies.
Antibody production is deficient, particularly to polysaccharide
antigens, and the serum immunoglobulin pattern is
characterized by a low IgM and elevated IgA. Patients with
this disorder usually succumb to overwhelming infections or
severe hemorrhage before age 10 years.’
A recent review reported that 80% (33/41) of WAS
patients who received a bone marrow transplant (BMT)
from a human leukocyte antigen (HLA)-identical donor
survived, whereas of those whose donors were only haplomatched
at HLA loci, survival was 23% (3/13).* In contrast,
BMT from HLA-haplomatched donors correct other primary
immunodeficiency disorders just as well as BMT from
HLA-identical donor^.^.^ Therefore, Wiskott-Aldrich syndrome
may represent a more difficult disease to correct with
haplomatched donors.
We describe our experience with four WAS patients who
have been transplanted with HLA-haplomatched bone marrow
depleted of mature T cells by treatment with monoclonal
antibody (MoAb) CT-2 and complement. Good engraftment
was attained in all, and three have survived from 300 to 900
days. Analysis of these transplant experiences provides
further insight into the consideration of haplomatched T-
depleted bone marrow as treatment of WAS.
Patients
MATERIALS AND METHODS
The clinical courses and major clinical features of the patients are
shown in Table 1. The history of infectious diseases varied, but was
most severe for patient 4. Patient 1 was transplanted relatively early
in the course of his disease, whereas the remainder were transplanted
at 5 to 10 years of age. All patients showed the small platelets and
lack of isohemmagglutinins characteristic of WAS. Patient 1 was
the first patient with WAS transplanted at The University of Iowa
and patients 2, 3, and 4 are three consecutive patients with WAS
who underwent BMT at the University of Wisconsin.
Preparation and Patient Care
All patients received cyclophosphamide (45 mg/kg body weight
per day for two doses) and cytosine arabinoside (2 gm/m2 body
receiving GVHD prophylaxis experienced severe GVHD and
had a difficult posttransplant course. The patient who did
not survive was chronically ill before BMT. whereas the
other patients were in relatively good health at the time of
BMT. Since the majority of individuals with this disease
lack a matched bone marrow donor, our results using
partially matched donors suggest that a greater number of
patients can be successfully treated for Wiskott-Aldrich
syndrome and that outcome is related to control of GVHD
and state of health before BMT. Marrow transplantation
should be offered earlier in the disease course before the
onset of major infectious problems.
0 1990 by The American Society of Hematology.
surface area twice daily for 3 days). Patients 2, 3, and 4 received
total body irradiation (TBI; 1,320 cGy total dose) given in eight
equal fractions. Patient 1 was given busulfan 4 mg/kg for four daily
doses in lieu of TBI because of the deleterious effects of TBI in young
infants. Previous experience had shown that cytosine arabinoside
added to the preparative regimen was necessary to obtain engraftment
when using T cell-depleted haplomatched bone marrows for
transplant?
The children were nursed on bone marrow transplant units that
used filtered air; however, laminar-flow rooms were not used.
Hyperchlorinated water was used for patient 1. Antibiotics were
given empirically with the onset of fever in excess of 38.5OC.
Protocols for treatment of the children and collection of specimens
for research purposes were approved by the respective Human
Subject Committees at the University of Iowa and the University of
Wisconsin.
Marrow Preparation
Bone marrow was obtained from the donors under regional or
general anesthesia. The marrow was depleted of mature T lymphocytes
by the addition of CT-2 (a mouse IgM anti-CD2 antibody) and
rabbit serum as a source of complement.6 The marrow was given
intravenously upon completion of the depletion process; the nucleated
cell dosage is given in Table 1. Estimates of residual T cells were
made as described by Martin and Hansen.’
From the Division of Pediatric Bone Marrow Transplantation,
Department of Pediatrics, University of Iowa, Iowa City, IA; and
the Department of Pediatrics, University of Wisconsin, Madison,
WI.
Submitted May 24,1989: accepted October 25,1989.
Supported in part by the Pediatric Cancer Research Fund of the
University of Iowa.
Address reprint requests to Michael E. Trigg, MD. Associate
Professor of Pediatrics and Director of Pediatric Bone Marrow
Transplantation, The University of Iowa Hospitals and Clinics,
2524 John Colloton Pavilion. Iowa City, IA 52242.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1990 by The American Society of Hematology.
0004-4971/90/7504-0015$3.00/0
Blood, Vol 75, No 4 (February 15). 1990: pp 1031-1035 1031

1032 RUMELHART ET AL
Table 1. Clinical Features and Clinical Courses
Pt 1 Pt 2
Pt 3
Pt 4
Age at diagnosis
Age at BMT
Average platelet
count (x io3)
Small platelets
Major infections
Donor
Significant bleeding
HLA typing (PtIDo)
HLA-A
HLA-8
HLA-DR
MLC reactivity (Pt/Do)*
Pt x Do,
Do x Pt,
Conditioning regiment
Nucleated cell dosage (/kg1
No. T cells givenlkg body weight
GVHDS
Skin
Liver
GI
Other
Outcome
5 wks 6 mos
13 mos 5 Yrs
30 20
Y
Recurrent diarrhea, infected
eczema, otitis
Father
N
Y
Sinus
Father
N
2.2612.26 3.2613.28
8,1418 7.45/7,35
3.7/3,7
6801426 2991244
6,6131727 26,36512,264
BuICYlara
CY/ara/TBI
8.9 x lo8 2.3 x 10'

~ ~
MISMATCHED BMT FOR WISKOlT-ALDRICH SYNDROME 1033
Days post-BMT*
Platelet count ( x io3)
Total lymphocytes
T cells (CD3)t
CD4 i
CD8 i
PHAS
PWM
conA
SKSD
Tet
Cand
B cellst
B1
04
IgG (mg/dL)
IgA (mg/dL)
IgM (mg/dL)
IgE (IUImL)
Isoagglutinins§
anti-A
anti-B
Natural Killer
NKH-1
Table 2. Laboratory Parameters
Pt 1 Pt 2 Pt 3
34 1
33 1
1,840
41
31
8
34
27
28
0.7
0.5
47.0
2
2
7061839
158117
61/61
NDIND
O/ND
O/ND
ND/3 1
895
599
3,600
36/70
28/43
10118
5371993
15011 7 1
19015 1 1
1.014.0
2.011 .o
1.0122.0
1015
NDl8
2,960/807
91/28
34/52
ND/28
018
010
ND/7
332
27 1
1,900
4213 1
37/29
18/16
13011 86
14/43
3319
1.012.0
2.018.0
6.0/ 1 .O
913
NDl5
6941655
204198
4911 17
49/3
010
010
ND114
Abbreviations: Pt, patient; ND, not determined; PWM, pokeweed
mitogen; conA, Concanavalin A; SKSD, streptokinase-streptodornase;
tet, tetanus toxoid; cand, candida.
‘Day on which last evaluation was performed.
tFirst number in column is value pre-BMT; second number is last value
determined. Lymphocyte number is given as percent.
*Response to mitogen or antigen is a stimulation index (stimulated
cpmlnonstimulated cpm). All mitogen responses are normal. Stimulation
by antigen should be greater than 3.0.
§Expressed as reciprocal of titer, except absent titers, which are
expressed as 0 (pre-BMT).
marrow at the University of Wisconsin. Steroid therapy was
initiated at 2 mg/kg because of a worsening of the skin on
day 24 posttransplant. At this time, there was slight elevation
of serum gamma glutamyl transferase (GGT) to 172 1U. He
showed gradual worsening of his GVHD despite steroids, and
approximately 6 weeks posttransplant, his pulmonary status
acutely worsened with the picture of adult respiratory
distress syndrome. He required intubation. High dose intravenous
pulses of methylprednisolone (1 gm) were given and
azathioprine was started. His eyes stopped tearing and
corneal ulcers developed. Local prednisone ophthalmic ointment
was added to the treatment regimen.
He gradually improved and ventilatory support was
stopped. His liver remained involved (maximal values: GGT
4359, aspartyl aminotransferase (AST) 554, and bilirubin
8). Approximately 14 weeks after transplant, cyclosporine
was added to the regimen. His skin by now was atrophic with
a prominent livedo reticularis pattern. He was discharged
119 days after transplant. At that time he had chronic
GVHD with skin and liver involvement and a prominent
sicca syndrome requiring artificial tears. He received cyclosporin,
azathioprine, and prednisone for 12 months with
return of his liver enzymes to normal and marked improvement
in his skin. When last seen in September, 1988, two
years after the transplant, his skin was normal and there were
no contractures.
Clinical course of patient 4. Patient 4 had a stormy
posttransplant course and died 37 days after transplant.
Before the transplant, he had experienced repeated bouts of
severe Herpes simplex infections and pneumonia. Early in
the transplant course, he showed evidence of liver disease.
Ten days after the transplant, his creatinine rose to 1.2
(baseline 0.6) and intravenous cyclosporine (5 mg/kg continuous
infusion) was stopped. His renal failure progressively
worsened and hemodialysis was required. He responded, and
over a period of 1 week, his creatinine gradually returned to
baseline levels. During this interval his serum bilirubin
slowly increased and on the ninth posttransplant day was 2.2.
At that time, his alkaline phosphatase was 168, GGT 185,
and AST 255. From this time until his death, his bilirubin
continued to rise and was disproportionately elevated compared
with GGT and AST levels. Terminally, the total
bilirubin was 29.0 (direct reading 19.7), alkaline phosphatase
1127, GGT 554, and AST 144.
He showed definite neutrophil engraftment on day 15
post-BMT (1,400 total leukocyte count), and this level was
sustained until his death. Skin GVHD appeared coincident
with neutrophil engraftment and consisted of a generalized
maculopapular eruption without bullae formation.
He developed severe hemorrhagic gastroenteritis; a colon
biopsy showed only atrophic mucosa without evidence of
GVHD or cytomegalovirus infection. However, Herpes simplex
type I was cultured from the biopsy and adenovirus from
Table 3. Tempo of Engraftment
Pt 1 Pt 2 Pt 3
Neutrophils >500
for 3 consecutive days 15 15 16
Last day of platelet transfusion 17 9 12
Total lymphocytes >750 24 355-393 120-180
CD3f > 675/pL 152 355-393 120-180
CD4i > 45014 152 355-393 120-180
CD8f > 225/pL 152 668-895 120-180
B1+ > 75/pL 341 393-411 120-180
Specific antibody
IgA anti-E Coli ND >895* NAt
IgM anti-€ Coli ND 807 NA
IgA anti-tetanus ND NA NA
IgM anti-tetanus ND 510 NA
IgA anti-diphtheria ND >E95 NA
IgM anti-diphtheria ND 510 NA
Tempo reported as number of days after BMT for values to attain
criterion shown in leftmost column; where a range is shown, the value
was attained during that interval between examinations.
Abbreviations: Pt, patient; ND, not determined MA, not applicable.
’”>” means that value was greater than value as of last day of
examination shown in column.
tThe value did not decrease appreciably after conditioning, remaining
within normal limits. Note patient 1 was never immunized pre-BMT.
Values at 18 months post-BMT were indicative of nonimmunity, and the
child was scheduled to undergo a primary series of immunizations.

1034 RUMELHART ET AL
his stool at this time. His condition progressively deteriorated,
and he died of overwhelming staphylococcal sepsis 37
days after the transplant.
Patient 4 had moderate skin GVHD. Other severe systemic
disease involving the liver and kidneys, followed by
fatal sepsis, make the full extent of GVHD involvement
(other than the moderate skin GVHD) difficult to assess in
his case.
DISCUSSION
Bone marrow transplantation has proven to be an effective
treatment for Wiskott-Aldrich syndrome and other immunodeficiencies
when a matched sibling donor is a ~ailable.~‘~~~~~~~
The problems of thrombocytopenia, eczema, and recurrent
infections have been eliminated after successful engraftment
has been established. However, more extended evaluation is
needed to determine whether successful engraftment also
eliminates the risk for lymphoreticular malignancies characteristic
of the disease. Due to the many complications
associated with this disease, a bone marrow transplant should
be done early in the disease course whenever possible, or at
least before the onset of major infectious complications. In
our experience with other forms of primary immunodeficiency,
delay has resulted in the acquisition of a complicating
infection that greatly increases the morbidity after transplant
and usually results in an unsuccessful outcome! We
believe that our experience with WAS is also consistent with
this view.
In the three children who were in relatively good health at
the time of transplant, the engraftment was rapid and
durable in two. Patient 2, who did not receive GVHD
prophylaxis, experienced chronic GVHD and therefore, a
lengthy recovery. We believe that had GVHD prophylaxis
been instituted, he too would have had a more benign course
and rapid recovery. Patient 4, however, who had a long
history of severe chronic infections and was in poor health at
the time of transplant, had a stormy posttransplant course
and died. He may have suffered from hyperacute GVHD.
If possible, an HLA-matched bone marrow donor is used;
however, the majority of individuals will lack an HLAidentical
donor. In this situation a partially matched family
member or closely matched unrelated individual can be used
as the donor, and the marrow is depleted of T lymphocytes to
prevent or diminish the severity of GVHD.16
Our results reveal that successful engraftment can be
achieved in patients suffering from Wiskott-Aldrich syndrome
who do not have an HLA-identical, mixed lymphocyte
culture nonreactive sibling donor. This has been accomplished
with an ablative regimen consisting of TBI (or
busulfan), cytosine arabinoside, and cyclophosphamide early
in the disease course, followed by the administration of T
lymphocyte-depleted bone marrow.s,8.16 B-cell and T-cell
reconstitution were achieved within 12 months after transplant
in two patients and have been ultimately attained in the
third, after achieving control of chronic GVHD. Except for
patient 4, infectious complications were minimal.
These data suggest that results comparable with those of
Wiskott-Aldrich patients transplanted with HLA-identical
donor marrow might be attained; however, one must be
particularly watchful for complications of graft-versus-host
disease. We believe prophylaxis is mandatory, contrary to
the usual practice for combined immune deficiency disease.
2.4.10-1 s
In contrast to recipients of matched non-Tdepleted
marrow, WAS patients will also require more
pretransplant conditioning (cytosine arabinoside) and may
require more posttransplant immunosuppression for GVHD
control. Although the long term impact on the child of these
additional therapies remains to be determined, three of four
children described in this report are alive and well, having
previously had a disease for which there was no other
treatment yet available with a curative intent. Furthermore,
early transplant in growing children may prevent delayed
growth and development secondary to chronic eczema, recurrent
overwhelming infections, and lymphadenopathy.
The reason for the severity of GVHD in WAS is unclear.
We have examined the degree of T-cell contamination for the
last 21 consecutive T cell-depleted haploidentical BMT
performed at the University of Wisconsin, using the method
described by Martin and Hansen.’ The degree of depletion
by our protocol is of the order of 1.5 to 2 logs. After depletion,
the frequency of phytohemagglutinin (PHA)-responsive T
cells in the bone marrow inoculum averages 1/51 to 50,000,
and no patient has received more than 1.6 x 10’ mature T
cells per kilogram body weight. Kernan et all7 suggest that
lo5 clonable T cells per kilogram body weight will produce
GVHD. Patients 2, 3, and 4, for whom measurements are
available, received 5 x lo4, 1.2 x lo4, and 1.2 x los,
responding T cells per kilogram body weight. One cannot
directly equate the number of PHA-responsive cells as
determined by us to the clonable T cell number described by
Kernan et al,” but the severity of GVHD seen in other
primary immunodeficiency patients previously transplanted
here suggests that our numbers are of the same order of
magnitude as those in their studies. In any event, the WAS
patients did not receive a significantly larger inoculum of
mature T cells than other recipients of T cell-depleted BMT.
We believe that the severity of GVHD is in part determined
by the degree of immunity originally present in the
host, and for this reason, patients with hematologic malignancy
or aplastic anemia have traditionally required GVHD
prophylaxis even with matched BMT, whereas those with
primary immunodeficiency do not. Other factors, such as
leukemia antigen and infection are also factors involved in
the severity of the clinical picture.18 When the marrow is
exhaustively depleted of mature T cells (3 or more log
depletion), GVHD is essentially nil, but poor engraftment
and leukemia recurrence are major problem^."*'^.^^ We have,
therefore, used a less rigorous depletion protocol. In all cases
of primary immunodeficiency other than Wiskott-Aldrich
syndrome, this strategy has worked well without GVHD
prophylaxis!
Although all marrows were T cell-depleted, successful
engraftment was not a problem, as has been previously
recorded.8.” This confirms our experience with this conditioning
regimen, which is used in the preparation of patients with
hematologic malignancy as well as immunodeficien~y.~*~.’~
On the basis of these results, we believe that Wiskott-
Aldrich syndrome can be effectively treated with haploiden-

MISMATCHED BMT FOR WISKOTFALDRICH SYNDROME 1035
cal T cell-depleted bone marrow transplants. Our data
indicate that outcome is related to the control of graft-versushost
disease and the Overall state Of health before the
transplant, and Our results argue for earlier, rather than
later, bone marrow transplantation therapy even in the
absence of an HLA-identical donor.
ACKNOWLEDGMENT
We thank Joan Smith for manuscript preparation, Tom Riley for
preparation of tables and graphics for this report, and the dedicated
nursing staffs at The University of Iowa and The University of
Wisconsin, who provide care to the children undergoing marrow
transplantation.
1. Ammann AJ, Hong R Disorders of the T-cell system, in
Immunologic Disorders of Infants and Children. Philadelphia, PA,
WB Saunders, 1989, p 276
2. Buckley RH: Advances in the correction of immunodeficiency
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3. OReilly RJ, Brochstein J, Collins N, Keever C, Kapoor N,
Kirkpatrick D, Kernan N, Dupont B, Burns J, Reisner Y: Evaluation
of HLA-haplotype disparate parental marrow grafts depleted of T
lymphocytes by differential agglutination with a soybean lectin and
E-rosette depletion for the treatment of severe combined immunodeficiency.
VoxSang 51:81,1986 (suppl)
4. Moen RC, Horowitz SD, Sondel PM, Borcherding WR, Trigg
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