1. Healthcare

Relationship Between Patterns of Engraftment in Peripheral Blood
and Immune Reconstitution After Allogeneic Bone Marrow
Transplantation for (Severe) Combined Immunodeficiency
By J.E.M. van Leeuwen, M.J.D. van Tol, A.M. Joosten, P.T.A. Schellekens, R . Langlois van den Bergh,
J.L.M. Waaijer, N.J. Oudernan-Gruber, C.P.M. van der Weijden-Ragas, M.T.L. Roos, E.J.A. Gerritsen,
H. van den Berg, A. Haraldsson, P. Meera Khan, and J.M. Vossen
We report the outcome of allogeneic bone marrow transplantation (BMT) as treatment for severe combined immunodeficiency disease (SCID) in 31 patients grafted from
1968
until 1992. The patients received a graft froman HLA-identi(n =
cal related (n = 10). anHLA-haplo-identicalrelated
19). or a closely HLA-matched unrelated (n = 2) donor that
resulted in the long-term survival of 6 of IO, 9 of 19, and
0 of 2 children, respectively. Major complications included
failure of engraftment and early death caused by respiratory
failure. The chimerism pattern and immunologic remnstitution were evaluated in 15 children who survived more than
1 year with sustainedengraftment.
The pattern of engraftment was investigated within flow-sorted peripheral
blood (PB) T- and B-lymphoid, natural killer (NK), and myelomonocytic cell populations using the amplification of variable number of tandem repeats by the polymerase chain
reaction. The immunologic reconstitution was assessed by
various in vitro andin vivo parameters. Although thenum-
ber of PB T cells and thein vitro T-cell proliferative response
was in the lower region of normal in the majority of cases
and even subnormal in some, in all cases donor T-cell engraftment and reconstitution of T-cell immunity was observed. Residual host-type T cells (1% t o 5%) were detected
in eight cases at multiple occasions. All children showed
normal serum IgM and IgG subclass levels and produced
specific IgG antibodies after vaccination, irrespective of donor B-cell engraftment. However, three HLA haplo-identical
cells
graft recipients with host-type B lymphoid and myeloid
have a persistent selective IgA deficiency. NK cells were either of donor, host, or mixed origin.Donor NK cell engraftment restored defective in vitroNK cell function of the
recipient. We conclude that determination
of lineage-specific
engraftment patterns provides valuable information for the
understanding of the immunologic reconstitution
after allogeneic BMT for SCID.
0 1994 by The American Society of ffemato/ogy.
S
graftment of hematopoietic non-T-cell lineages and immunologic reconstitution.
We have evaluated the outcome of 31 consecutive cases
of allogeneic BMT for SCID performed at the Department
of Pediatrics of the Leiden University Hospital. To define
the basis of incomplete immune reconstitution after allogeneic BMT for SCID, we determined (1) the degree of engraftment of different hematopoietic cell lineages using fluorescence-activated cell sorting (FACS) and amplification
of highly polymorphic variable number of tandem repeats
(VNTRs) by the polymerase chain reaction (PCR),8.9and (2)
the immunologic reconstitution by a variety of in vivo and
in vitro parameters in 15 cases who showed sustained engraftment and survived more than 1 year after BMT.
EVERE COMBINED immunodeficiency (SCID) is a
heterogeneous group of congenital disorders, characterized by severe impairment of both cellular and humoral immunity, usually leading to death within 2 years of life.’.*
Patients with SCID have been treated successfully with HLA
identical allogeneic bone marrow transplantation (BMT)
since 19683.4and with HLA haplo-identical T-cell-depleted
(TCD) BMT since 1981.5 A European survey of the period
1968 to 1989 has shown a probability of survival of 76%
and 52% after HLA identical and HLA nonidentical BMT
for SCID, respectively.6 Moreover, SCID patients treated
after 1983 with an HLA-identical graft showed a probability
of survival of 97%: Unfortunately, HLA haplo-identical
BMT was frequently associated with failure of engraftment
or incomplete immune reconstitution. The use of more intensive conditioning regimens has resulted in a higher frequency
of engraftment of B lymphoid and myelomonocytic lineages
and improved immunologic reconstitution after HLA haploidentical BMT,6.7which suggests a relation between en-
From the Department of Pediatrics, Leiden University Hospital;
MGC- Department of Human Genetics, Leiden University, k i d e n ;
and Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands.
Submitted April 11, 1994; accepted July 27, 1994.
Supported by a grant from the Dutch Cancer Society ( I K W 885).
Address reprint requests to J.M. Vossen, PhD, MD, Department
of Pediatrics, Leiden University Hospital, Rijnsburgerweg 10, 2333
AA Leiden, The Netherlands.
The publication costsof this article weredefrayed in part by page
charge payment. This article must therefore behereby marked
“adveaisement” in accordance with 18 U.S.C. section 1734 .solely to
indicate this fact.
0 I994 by The American Society of Hematology.
0006-4971/94/8411-0017$3.00/0
3936
MATERIALS AND METHODS
Patients. From December 1968 until July 1992, 47 infants and
young children suffering from SCID were referred to the Department
of Pediatrics of the Leiden University Hospital for treatment. Eleven
of these patients died before transplantation could be performed.
Five patients, who lacked a suitable donor and were treated with fetal
liver cell transplantation before 1978, died without immunologic
reconstitution.“’ The remaining 31 patients, included in this study,
were treated by allogeneic BMT. Data were collected until July
1993, giving a minimum follow-up of I year for all children. This
study was approved by the Institutional Review Board on Medical
Ethics.
Patients were classified according to criteria of the World Health
Organization” into one of the following categories: SCID with a
low number of T and B cells (T-B-), SCID with a low number of
T cells, but a normal number of B cells (T-B’), SCID caused by
adenosine deaminase deficiency (ADA-), Omenn’s syndrome (OS),
and HLA class I1 deficiency (Table 1). Two patients (unique patient
numbers [UPNs], 81 and 82) presented with atypical T-B- SCID
characterized by a progressive decrease in T-cell function and an
almost complete absence of pre-B and more mature B-cell stages,
whereas Ig heavy- and light-chain genes of Epstein-Barr virus (EBV)
Blood, Vol 84,No 1 1 (December l), 1994: pp 3936-3947
CHIMERISM AND IMMUNITY AFTER BMT FOR SCID
3937
Table 1. Patient Characteristics and Laboratory Findings at Presentation
UPN
Sex/
Age
(mos)
2
5
17
26
50
74
92
105
131
139
4
28
41
53
56
59
81
82
93
94
96
108
112
114
118
126
167
168
183
189
199
M14
MI1
F115
F14
F15
F144
MI6
F182
MI1
F146
MI7
MI9
MI1
MI4
F13
M16
F156
F142
M Pl
F114
F12
MI8
MI4
MI6
MP
M18
F19
F16
F14
MI11
MP
Lymphocytes (96)
SCID
Type
Inheritance
T-B'
T-B'
T-B' (N)
T-BT-B' (N)
XL
Class 11-
AR
T-B'
T-B'
AR
ADAT-B'
T-B'
T-B'
T-BT-B'
T-B+ (N)
T-B'
T-B"
T-B-*
Class IIClass IIADAT-B'
T-B'
T-B'
OSt
T-B'
os
T-B-
?
AR
AR
AR
AR
AR
AR
?
XL
AR
?
AR
?
AR
AR
AR
AR
AR
?
?
?
AR
?
AR
AR
os
os
AR
T-B'
XL
AR
Lymphocytes1
PL
600
1,584
1,300
1,650
615
2,888
532
234
96
294
357
1,430
338
248
1,800
532
147
248
1,066
504
300
576
402
1,400
2,886
1,900
1,200
338
5,304
6.31 8
5,375
Serum Ig lmg/dL)
K56211
CD3'
slg+
CD16'
IgM
W
W
1
+
ND
ND
ND
ND
ND
44
28
197
169$
20
716
96*
1286
667*
563*
868
4346
362*
ND
1
+
66
21
11
81
2
18
5
11
23
13
2
13
94
53
5
3
1
<l
<l
6
43
43
40
30
71
54
<l
<l
3
<l
21
<l
71
<l
84
86
<l
+
99
<l
66
2
52
<l
<l
12
38
54
49
89
92
77
78
<l
<l
<l
11
99
<l
6
20
58
66
ND
ND
ND
ND
ND
5
44
66
12
3
48
47
<l
7
1
13
24
56
25
4
<l
<l
127
43
70
97
<l
25
5
59
<l
12
622
130
17
2
1
1
30
153
8
65
47
45
<l
<l
4
109
58
8
185$
9656
129
667
40*
515*
539$
193$
1454
338$
8%
306$
48
740*
56
257*
322$
1616
402$
64*
<l
138
<l
126
13
1
132
<l
932
<l
18*
<l
11*
54
<l
21
13
<l
<l
47
<l
<l
1
54
<l
<l
<l
<l
7
<l
Lysis
-1
ND
-1
ND
Nll
1
N
N
-
ND
ND
-
N
NI
Nn
-
1
1
N
1
ND
ND
-
ND
1
ND
ND
N
-
ND
ND
Abbreviations: N, Nezelof type; XL, X-linked; AR, autosomal recessive; ND, not done; +, present.
* Atypical T-B- SCID.''
t Atypical OS?'
*After intramuscular or intravenous-globulin administration and/or transfusion with plasma.
6 IgG probably of maternal origin.
11 Absent (-1, below (C), or above (N) 10th percentile of normal controls.32
Presence or absence of NK activity confirmed using flow-sorted CD16+ NK cells.
transformed B-lymphoblastoid cell lineages were retained in germline configuration." Three patients with T-B+ SCID showed Xlinked inheritance as evidenced by pedigree analysis (UPN 28 and
199) or nonrandom X-chromosome inactivation of maternal lymphocytes (UPN 2). Three patients presented with normal or increased
serum Ig levels, cytoplasmic-Ig (cIg) containing plasma cells in the
BM, and palpable lymph nodes, which are characteristic of Nezelof
syndrome." Homogeneous Ig components were detected in the serum ofthe latter patients.I4 UPN 105 showed atypical late-onset
T-B' SCID in combination with central deafness. She produced
specific IgG antibodies in response to viral infections and vaccination
before BMT. UPN 139 showed atypical T-B' SCID with absence
of pre-B and B-cell stages, but presence of plasma cells in the BM
and presence of IgA plasmacytosis. Three patients presented with
HLA class I1 deficiency.15-" Three patients presented with diffuse
erythrodermia, blood eosinophilia, hepatosplenomegaly, lymphadenopathy, failure to thrive, increased serum IgE, and recurrent infections, which are all characteristic of OS.'8,'9 Finally, UPN
118 presented with atypical OS; he had diffuse erythrodermia, hepatosplenomegaly, and an oligoclonal expansion of CD8+ T cells, but
he showed no eosinophilia and lacked detectable levels of serum
IgE.*'
The majority of the patients presented with diarrhea, failure to
thrive, and recurrent or persistent infections of the respiratory tract
(eg, with Pneurnocystis carinii), digestive tract (eg, with rotavirus),
mucosa, and/or skin (eg, with C albicans and Staphylococcus
aurew). All patients showed an absent or severely decreased in
vitro T-cell response towards phytohemagglutinin (PHA) (data not
shown).
BMT. Transplant-related variables are listed in Table 2. Patients
received a BM graft from an HLA-identical related (n = lo), an
HLA-haplo-identical related (n = 19) or a closely HLA-matched
unrelated (n = 2) donor. Retransplantations were undertaken in six
cases because of lack of engraftment and immune reconstitution
after a previous attempt. The date ofthe most recent attempt of
BMT is considered day 0. Conditioning ofthe patients was performed according to protocols formulated by the European Group
for Immunodeficiency (EGID) and the European Group for Bone
Marrow Transplantation (EBMT) (Table 2). Measures to prevent
graft rejection included rabbit antithymocyte globulin (ATG), Cam-
3938
Table 2. Transplant-Related Variables and Outcome
UPN
SClD
Type
2
5
17
T-B+
T- B
T-B' (N)
26
50
T-BT-B' (NI
74
92
+
Class 11T- B
+
105
131
139
4
28
41
T-B+
ADAT-B+
T-B+
T- B
T-B-
53
T-B+
56
59
81
+
T-B' (N)
T-6'
T-B-
Age at
BMT
GVHD
Proph.
GVHD
Grade
(mod
5
4
26
33
5
13
18
M/F
M/M
F/M
F/M
F/M
FIF
F/F
-, -,-, -, -
43
7
12
94
1
47
F/F
M/M
M/M
F/M
M/F
F/F
M/F
M/M
A,-,
M/M
-, -, -, -, -, -, -,-, -, -, -, -, -, -, A, -, DRt
1
1
1
1
1
1
1
1
2
M/M
M/M
F/F
M/M
F/M
F/F
F/F
F/M
F/M
FIM
M/M
A, -, DR
A, -, DR
A,B,DR
A, B. DR
A,B, DR
A,B, A,B, A,B, -t
A, B, -t
A,B, -t
A,B, DR
2
2
2
2
2
3
3
2
2
2
3
A, DR
B,
3
-
CsA
CsA
CsA
-t
2
2
2
3
3
-t
1
CsA
Died?, +21 d, respiratory failure
Engraftment failure
Died?, +43 d, VOD
AW. +6 yrs
AW, + 4 yrs
Diedll?, + l 2 d, respiratory failure
Diedlln, + l 2 d. respiratory failure
Died?, +71 d, acute GVHD
Diedl, +3 mos, accidental cardiac
tamponade
Engraftment failure
A, + 9 yrs
A, +9 yrs, hypopigmentation
AW, +9 yrs
Engraftment failure
Aplasia
Died, +6 mos, aplasia
Engraftment failure
Aplasia
Died, +6 mos, aplasia
Autologous recovery, died +29
mos, intracranial
lymphoproliferative syndrome
Autologous recovery, died +2
mos, after second BMT at age
7 yrs, intracranial bleeding
AW, +6 yrs, mental retardation
AW, +5 yrs
AW. +5 yrs
Died, +l
yr. GVHD + AdV
Autologous recovery, died +20
mos. e.c.i.
Died, +2 yrs, respiratory failure
2
2
2
2
CsA
CsA
CsA
CsA
Died//l, + l 2 d, respiratory arrest
AW, t2.3 yrs
AW, t1.7 yrs
Diedlln, +l
1 d, cardiac arrest
2
CsA
AW, +1.0 yr
a
28
4
7
12
93
class II-
8
8
57
65
68
43
54
55
71
94
class II-
14
F/M
a
F/M
MF
M/F
A,
M/M
82
T-B-
96
108
112
114
118
ADAT-B+
T- B
T-B+
126
167
T-B+
os
168
183
189
199
T-B+
+
10
5
6
8
HLA Disp
(A. B, DR)
-, -,-
-*
-,
B.A.
-, DR
10
13
M/F
B,
A,
F/M
-,-,
T-B-
7
os
os
12
B,F/F
A,
F/M
MIM
DR
B,
A,
9
MIM
B,
A,
4
-
M/M
DRtt
-
-
-
MTX
-
CsA
CsA
CsA
-
CsA
-
CsA
-
CsA
-
-
Acute
Chronic
Outcome, Follow-up,
Main Cause of Death
A, +24 yrs
AW, +22 yrs, lost to FU
Engraftment failure,
AW. + l 6 yrs
Diedllf, + l 4 d. respiratory failure
Engraftment failure
AW, + l 0 yrs. stunted growth
-*
DR
-,-,
Other
1
1
1
1
1
1
1
-, -, -, -, -,--,-, -
B,
A,
B,
A,
os
Conditioning
TCD§
Regimen
Sex
R/D
DRS
DR
Abbreviations: RID, recipient/donor; HLA disp, disparity for HLA A, B, and DR antigens on the noninherited haplotype; ATG, 2 mg/kg body
weight (BW) x 3 (days -6 through -4); Camp, Campath-IG MoAb at 0.2 mg/kg BW x 2 (days -5 and +3) plus Campath-IM MoAb at 0.2 mg/
kg BW x 1 (day 0); LFA1, 0.2 mg/kg BW x 13 (days -3 through +lo); CD2, 0.2 mglkg BW x 11 (days -2 through +8); VPlGW0, total dose of
etoposide in mg/m2; Buwlsno, total busulfan dose in mg/kg BW; Cy~ow12ww, total
dose of cyclophosphamide in mglkg BW;TBIw, total-body
irradiation dose in Gy; na, not applicable; A(W), alive (and well); FU, follow-up; VOD, veno-occlusive disease; AdV, adenovirus; MTX, methotrexate; CsA, cyclosporin A; Propho, prophylaxis; TCD, T-cell depletion.
1 Ag mismatched (UPN 281 or matched (UPN 167) unrelated donor.
t S Patient is homozygous for A t or DRS antigens shared by the donor.
5 TCD protocol: 1, albumin gradient; 2, albumin gradient E-rosetting; 3, Campath-IM plus human complement.
I/ Engraftment not evaluable.
ll Immune reconstitution not evaluable.
+
CHIMERISM AND IMMUNITY AFTER BMT FOR SCID
3939
and 11 to14 months of age. Data of these infants were kindly
path monoclonal antibody (MoAb) (supplied by the Department of
provided by Dr H. RUmke (NIPHEP). Leukemia patients received
Pathology, University of Cambridge, Cambridge, UK),” antilymDT-IPV vaccine (I through IV) at 2, 3, 4, and 6 to 24 months after
phocyte function-associated antigen-l (LFA1) MoAb?’ or anti-CD2
HLA-identical BMT. Data of leukemic graft recipients were kindly
MoAb (Table 2). HLA-identical graft recipients received albumin
provided by Dr J. Labadie (Department of Pediatrics, Leiden Univergradienvstem cell-enriched l-log TCD BM.23 The majority of
sity Hospital, Leiden, The Netherlands).
HLA-non-identical allograft recipients received more than 2-log
TCD BM cells after albumin gradient and E-rosette ~eparation?~
or
after treatment with Campath-1M MoAb plus human complement.2s
RESULTS
Additional graft-versus-host disease (GVHD) prophylaxis consisted
of cyclosporin A (CsA), 2 m&g/d intravenously (day -5 to +30,
MaternoTfetal T-cell engraftment. Materno-fetal T-cell
followed by 6 mg/kg/d orally through day +l201 and tapered off
engraftment was only looked for in typical SCID patients
until discontinuation at day + 180, or methotrexate (Mtx) intrave(ie, with T-B-, T-B+, or ADA-type SCID) in case the absonously at weeklyintervals up to day + 102.26All patients were nursed
lute PB CD3+ T-cell count was more then 200/pL at admiswithin the environment of a laminar flow isolator and received prosion (n = 4). This was performed by HLA class I serotyping
phylactic antimicrobial medication to suppress their intestinal miof
E-rosette-positive T cells and by FPV analysis of
~roflora.’~
PBMCs. Maternal T cells were detected by both techniques
Chimerism analysis. Chimerism analysis of fractionated periphin two patients (UPN 56 and 59) who also showed clinical
eral blood mononuclear cells (PBMCs), obtained after Ficoll-Isosigns of GVHD at diagnosis. Maternal T cells were absent
paque (Pharmacia, Uppsala, Sweden) density-gradient centrifugain one other patient (UPN 17), and no material was available
tion, was performed after cell sorting followed by amplification of
VNTRs by PCR, so called FACS/PCR-INTR or FPV a n a l y ~ i s . ~ , ~ for analysis in the fourth case (UPN 26). Using FPV chimeCD3+CD4+ and CD3+CD8+ T cells, CD20TD19’ B cells,
rism analysis, we also excluded the presence of circulating
CD16+CD14- natural killer (NK) cells and CD14+CD16- myelomaternal T cells in one patient with OS (UPN 183).
monocytic cell populations were investigated. Two children could
Transplant
outcome.
Transplant-related variables and
not be evaluated by FPV analysis either because of technical reasons
clinical outcome are listed in Table 2. Engraftment could
(UPN 108) or because none of the 5 VNTR markers yielded patient
not be evaluated in five children who died within 21 days
and/or donor-specific alleles (UPN 131). In these cases, a sex misafter BMT. Successful engraftment after the first BMT was
match between the donor and the recipient permitted analysis by
obtained in 17 of 26 evaluable children. In 4 of 9 cases with
fluorescent in situ hybridization with a Y-chromosome-specific
primary graft failure, sustained engraftment was obtained
probe (Y-FISH) in combination with simultaneous fluorescent labelafter a second transplant (following intensification of the
ing of cell surface antigens.” Ig allotypes were determined in a
hemagglutination inhibition assay?9
conditioning regimen). These cases included three patients
Immunologic teesrs. Immunophenotypic analysis of PBMCs was
who did not receive cytoreductive treatment before the first
performed by two-color immunofluorescencemicroscopy or by flowBMT (UPN 17,50 and 53), and one patient (UPN 92) who
cytometric analysis using a FACStar flow cytometer and commershowed residual alloreactivity and expressed no adverse clincially available MoAb in appropriate dilutions. From 1 year after
ical
symptoms after BCG vaccination and transfusion with
BMT onwards, investigations were performed at irregular time internonirradiated blood products before BMT. The other 5 cases
vals. Age-matched reference values for PB counts of lymphocytes,
of primary graft failure were observed after HLA-haploCD3’ T cells, surface Ig+ (sIg+), or CD20+ B cells and CD16+ NK
identical TCD BMT, despite the use of high-dose myeloablacells (5% to 95% confidence intervals) were kindly provided by
tive conditioning. Strikingly, the children with atypical T-BProfessor Dr J.J.M. van Dongen (Erasmus University Rotterdam,
The Netherlands). Serum Ig isotype and IgG subclass levels were
SCID (UPN 81 and 82) showed repeated graft failure, even
determined by radial immunodiffusion and dot immunobinding
after infusion of Campath MoAbs in vivo and conditioning
assay, respectively, and compared with age-matched reference valwith total-body irradiation. Additional attempts of grafting
ues ( 2 2 SD).30 in vitro T-cell proliferative capacity was assessed
were not undertaken in the remaining cases of graft failure,
either in whole blood or in standard PBMCs using microculture
ie, in two siblings with HLA class I1 deficiency (UPN 93
techniques following mitogenic (PHA and/or antilymphocyte serum
and 94) and in one infant with atypical OS who showed a
[ALS]), allogeneic (mixed lymphocyte culture [MLC]), or pseudonormal
response in MLC before BMT (UPN 118), either
antigenic (anti-CD3) stimuli and compared with normal reference
because of parental refusal (UPN 93 and 94) or because the
values (5% to 95% confidence intervals)?’ In vitro NK cell function
patient had suffered from severe veno-occlusive disease after
was measured by spontaneous cytotoxicity towards the K562 erythBMT (UPN 118). UPN 94 ultimately died after a second
roleukemia cell line in standard 4-hour 5’Crrelease assays and compared with reference values (10% to 90% confidence intervals).”
transplant performed elsewhere.
Specific IgG antibody production was determined in serum taken 2
The actual survival percentage after HLA-identical reto 4 weeks after in vivo immunization with diphtheria toxoid (D),
lated, HLA-haplo-identical related, and closely HLAtetanus toxoid (T), andheat-inactivated poliomyelitis (P) virus type I,
matched unrelated BMT was 60% (6/10), 47% (9/19), and
11, and 111(DT-IPV) vaccine using routine techniques at the National
0% (0/2), respectively. As seen in Table 2, major causes of
Institute of Public Health and Environmental Protection (NIPHEP,
death were primary graft failure (n = 5) and respiratory
Bilthoven, The Netherlands). The children received the DT-IPV infailure
(n = 5). All children who died from respiratory failure
jections at irregular time intervals after BMT. Data obtained within
presented with severe and progressive lung infection (Pcari1 month after discontinuation of daily plasma infusions or biweekly
nii, Aspergillus or cytomegalovirus) before BMT.
intravenous immunoglobulin (IVIG) therapy were excluded from
With the exception of three cases (UPNs 2, 53, and 56),
analysis. Control groups consisted of 20 healthy infants and 20
children who received an HLA-identical BM graft for leukemia.
all long-term survivors are alive and well (Kamofski score,
Healthy infants received DT-IPV vaccine (I through IV) at 3, 4, 5 ,
100%; Table 2). UPN 2 suffered from recurrent skin infec-
VAN LEEUWEN ET AL
3940
Table 3. Cell Lineage-Specific Patterns of Engraftment After BMT
Time
UPN
2
17
50
105
131
53
56
59
96
108
112
126
168
183
199
Diagnosis
T-B+
(N)T-B+
(N)T-B+
T-B'
ADAT-B'
T-B' (N)
T-B+
ADAT-B'
T-B+
T-B'
T-B-
os
T-B+
Conditioning
Regimen
CylzoTBI,
B~16cV200
BUSCYZOO
ATGlCVloo
ATGICVZOO
ATG
ATGlB%Cyzoo
B~lscYzoo
B~sCy,oo
ATGJCymc
BUSCYZOO
LFAl/Bu&mo
BU&ZOO
TCD
1
1
1
l
1
2
2
2
2
Chimerism*
(vs)
Post-BMT
+22.9
+15.4
+8.8
+5.1
+2.2
+8.1
+8.4
+8.1
+5.7
2
+2.7
2
+4.0
+1.0
+1.0
+1.0
+1.0
1
2
2
2
VNTR
r/D
33.6
HRAS
r/D
D
r/D
D
r/D
r/D
APOB
D
YNZ22
Y-FISH*
D
APOB
33.6
D
YNZ22
YNZ22
YNZ22
D
D
D
D
D
rlD
r/D
r/D
YNZ22t
APOBS
Y-FISH*
33.6
B
NK
R/d
RID
RID
D
r/D
RID
R
r/D
RID
D
r/D
D
R
R/d
R/d
RID
D
R
D
D
R/d
R/d
RID
T
APOB
R/d
D
R
D
D
r/D
r/D
R/d
MM
R/d
D
r/D
R
R
R/d
R
R
R/d
R/d
R/d
Abbreviations: TCD, T-cell depletion; MM, myeloid/monocytic cells; TBI. total-body irradiation; Bu, busulfan; Cy, cyclophosphamide; ATG,
antithymocyte globulin; APOB, apolipoprotein B; HRAS, Harvey RAS.
*The predominant origin, either recipient (R) or donor (D), of the cell populations is given uppercase,
in
whereas a minor population( ~ 1 0 % )
is indicated in lowercase (r or d. respectively); If donor and recipient derived cells were present in near equal amounts, both populations are
given in uppercase (ie. R/D).
t The presence of recipient-derived cells cannot be excluded.
Determined by Y-FISH analysis.
*
tions caused by S aureus in association with acne and warts,
and experienced a severe S aureus sepsis and pneumonia
once, at the age of 7 years; he is now under flucloxacillin
prophylaxis. UPN 53 suffered from recurrent upper respiratory tract infections (URTI) with encapsulated bacteria until
the age of about 10 years; he is now scheduled for tympanoplasty of both ears; he still suffers from warts on feet and
hands. UPN 56 recovered from her extensive chronic GVHD
of the skin, except for persisting hypopigmentation of skin
and hair.
Chimerism patterns. Lineage-specific patterns of engraftment and immune reconstitution were evaluated in 15
of 16 children who survived more than 1 year after BMT
with sustained engraftment. UPN 5, who showed complete
immune reconstitution, but lacked engraftment in B
lymphoid and erythroid lineages,33was lost to follow-up
and could not be evaluated. PB T cells were exclusively or
predominantly of donor origin in all children, whereas B -,
NK- and myeloid cell populations were either of recipient,
donor, or mixed origin (Table 3 and Fig l, A through C,
last follow-up). Surprisingly, residual (1% to 5 % ) host T
cells were detected in eight cases up to 23 years after BMT.
Except for UPN 2 and 105, these residual host-type T cells
were detected at multiple occasions. In three cases who received no conditioning before BMT, engraftment of the myeloid cell lineage was absent or extremely limited. In addition, myeloid cells remained predominantly of host origin in
7 of 12 children who received prior cytoreductive treatment.
Five children showed (complete) engraftment in all cell lineages.
The origin of serum Igs after BMT was evaluated by Ig
allotyping. Unfortunately, a discriminating Ig allotype
marker was not available in 12 of 15 donorhecipient pairs.
Both donor and recipient Ig allotypes were detected in the
serum of UPN 17, and recipient Ig allotypes were found in
the serum of UPN 53 and 59 (data not shown), in agreement
with the results obtained by FPV analysis.
PB lymphocyte numbers. Longitudinal analysis of the
absolute numbers of various PB lymphocyte subsets showed
considerable fluctuations of cell counts in the children who
were frequently investigated. Results of the last follow-up
are summarized in Table 4. We repeatedly observed subnormal absolute lymphocyte and CD3' T-cell counts in five
cases (UPN2, 53, 56, 131, and 168), although they were
incidentally within normal range for UPN 2 and 56 at the
last follow-up. In general, decreased counts were observed
in both CD4' and CD8' T-cell subsets in graft recipients
with a diminished number of CD3' T cells. Three children
(UPN 2, 56, and 105) showed an increased percentage of
y6 T lymphocytes at the last evaluation (40%, 17%, and
19%, respectively). Although UPN 168 and 183 repeatedly
showed a decreased number of circulating B cells, the absolute numbers of CD16+ NK and CD20' and/or sIg+ B cells
were within the normal range in the majority of cases.
In vitroT-cellproliferation.
Analyses were performed
at irregular time intervals after BMT. The results of the
last investigation are summarized in Table 4. The in vitro
proliferative T-cell response towards PHA, ALS, and antiCD3 MoAb in whole blood cultures and the response towards PHA in standard PBMC cultures was repeatedly decreased in three children (UPN 2, 53, and 131). The in vitro
T-cell response towards alloantigens, as determined in the
MLC, wasdecreased at multiple occasions in one case (UPN
53).
B-cell immune reconstitution. Longitudinal analysis of
serum Ig classes and IgG subclasses was performed from 1
CHIMERISM AND IMMUNITY AFTER BMT FOR SCID
3941
All evaluable children (n = 14) who survived more than
1 year after BMT with sustained engraftment showed a posi-
LC,
872 bp
-603 bp
I
Fig 1. Cell lineage-specific patterns of engraftment after allogeneic BMT for SCID.(A) UPN
2,23 years after HLA-identicalBMT (APOB
VNTR); (B) UPN 53, 8 years after HLA-haplo-identical BMT (33.6
VNTR); and (C) UPN 168, 1 year after HLA-haplo-identical BMT
(YNZ22 VNTR). Pre-BMT patient (PI- and donor (D)-specificbands are
indicated. PCR-VNTR analysiswas performed on DNA obtained from
the cell populationsindicatedabove each lane. Negative control samples were included in each amplification experiment and found to be
negative (not shown in Fig 1A). Although a faint (+Yo) recipient-typespecific band was visible in the CD3'CD4' and CD3TD8' T-cell fractions on the autoradiogram of Fig 1C. this could not be visualized on
the photograph.
year after BMT onwards. Three children (UPN 53, 59 and
199) showed a persistent selective IgA deficiency (IgA-D)
after BMT. Some secretory IgA was detected in the tears of
UPN 53, but not in those of UPN 59. In several of the other
cases, serum levels of total IgG,IgG subclasses and IgA
occasionally dropped below the lower confidence limit (-2
SD) of age-matched controls, but none of them showed a
persistent deficiency in these subclasses (Table 5).
tive IgG antibody response towards at least one T-cell-dependent recall antigen after repeated DT-IPV immunizations
posttransplant (Table 5). UPN 199 was considered nonevaluable because he received only two injections of DT-IPV
vaccine and has notyetproduced specific IgG antibodies
towards any of the DT-IPV antigens. Although UPN 59 did
not respond to D or IPVI.IIIantigens, even after 6 DT-IPV
injections, he produced specific IgG antibodies toward tetanus, and toward rubella after natural infection (data not
shown).
As seen in Table 6, the cumulative response rates of the
grafted SCID patients towards DT-IPV antigens, as observed
within 6 months after BMT, are inferior to those of healthy
infants, but comparable to those observed after BMT for
leukemia. In this context, it should be noted that the majority
of graft recipients received immunosuppressive therapy up
to 6 months posttransplant, which may explain the poorer
specific antibody responses when compared with healthy
infants. Additional immunization with DT-IPV vaccine more
than 6 months after BMT for SCID further increased the
cumulative response rate: graft recipients who received at
least a total of four injections of DT-IPV vaccine or showed
a positive antibody response after previous vaccination
showed a cumulative response rate towards D, T, IPV,, IPVII,
and IPVlllof 75% (9/12), 100% (l3/13), 92% (l2/13), 85%
(1 1/13), and 75% (9/12), respectively (Table 5 ) . In comparison, healthy infants exhibit a 100% cumulative response
rate toward DT-IPV antigens from DT-IPV IV onward, and
children grafted for leukemia exhibit a 100% cumulative
response rate toward tetanus toxoid from DT-IPVIV onward.23a
In vitro NK cell function. The in vitro NK cell function
of the children after BMT is summarized in Table 4 and
Fig 2. Engraftment of donor NK cells was associated with
restoration of NK cell activity in two graft recipients with a
defective NK cell function before BMT (UPN 1 12 and 131).
Although NK cells were completely of donor origin, UPN
126 showed decreased in vitro NK cell activity. The in vitro
NK cell function of this patient before BMT was not determined. The N K cell function was only partially restored in
three graft recipients (UPN 2, 17, and 59), perhaps because
in these children, a mixture of functionally defective recipient NK cells and normal donor NK cells was present after
BMT. None of the latter cases received cytoreductive treatment before BMT. We also observed normal NK cell activity
in transplant recipients without or with only marginal NK
cell engraftment (UPN 53, 56, 168, and 183). This finding
is in agreement with the normal NK cell activity observed
before BMT in three of these cases (UPN 53, 56, and 168).
For unknown reasons, NK cell activity could not be detected
in cryopreserved samples obtained before BMT in the fourth
case (UPN 183).
DISCUSSION
From December 1968 until July 1992,31infantsand
young children with SCID were treated by allogeneic BMT
at the Department of Pediatrics in Leiden, The Netherlands.
3942
ET
LEEUWEN
VAN
Table 4. Immunologic Reconsitution After BMT for SClD
Proliferation
T-cellIn Vitro
PBMC Counts
UPN
2
Diagnosis
Time
(yrsl
PostBMT
T-B+
+22.9
Standard
Lymphocytes/ CD3'1
/JL
@L
pL
2,604
CD20+/
pL
CD%+/
K562
PHA
ALS
Infections z1 yr
Post-BMT
a-CD3
Lysis11MLCO PHA
i
103
5,120t
2,422
6,630
10,900t
5,400t
130 130
HPV (warts),
S aureus
(sepsis.
pneumonia)
17
50
105
131
53
T-B'(N)
T-B' (N)
T-B'
ADAT-B'
+15.4
+8.8
+5.1
+2.7
+8.1
1,846
3,280
2,100
1,280'
1,100'
1,403
2,296
1,176*
755'
495'
158
222
22,415
10,900
37,900
11,500
203
426
459
357
357
90'
358
242
297
56
59
96
108
112
126
168
183
199
T-B+ (N)
T-B'
ADAT-B'
T-B'
T-BT-B-
+8.4
f8.1
+5.7
+4.7
+4.0
+1.8
+2.5
+1.0
+1.0
2,262
1,260*
2,130
1,911'
2,508
884'
504*
2,000
7,410
1,312
1,134
1,065
975*
1,956
339
88*
490
363
426
OS
T-B+
ND
ND
151*
1,500
4,668
45*
20'
2,742
452
25'
554
554
125
ND
111
280
222
53,700
15,100
1
N
N
N
4,810 146
34,689
~
15,000
1,400t
300t
27,700
9,700t
5,300t
11,200
1,060t
150t
14,330
4.592$
287t
59
132
29
2,900t
8,900
19,500
16,400
14,000
11,300
1,900t
26,500
24,900
6,800t
37,100
33.700
29,100
45,800
14,500
10,100t
36,000
49,200
890t
1,760
20,553
12,619
29,590
19,214
28,436
11,965
7,578t
33,469
51,194
166
24
N
l
-
ND
NE
ND
N
-
ND
ND
9,360
2,140
3,800
12,100
6,220
~
-
N
166
117
151
59
252
84
HPV (warts),
URTl
~
-
l
~
N
N
ND
~
Abbreviations: HPV, human papilloma virus; ND, not done; NE, not evaluable.
* Below fifth percentile of age-matched controls.
t Below fifth percentile of control values, ie <6,000 counts per minute (cpm) for PHA, <14,000 cpm for ALS, and <l600 cpm for a-CD3."
Below 2.5 percentile of control values, ie, <8,400 c p ~ n . ~ '
§ Response capacity, percent of two controls (250% is considered normal).
/I Above (N) cq below (1)10th percentile of controls32(see also Fig 2, A through D).
*
The actual survival is 60% after HLA-identical BMT (n =
lo), 47% after HLA-haplo-identical BMT (n = 19) and 0%
after closely HLA-matched unrelated BMT (n = 2). If patients with HLA class I1 deficiency or OS, disorders associated with a poor prognosis,34are excluded from our report,
the actual survival after HLA identical and HLA non-identical BMT for SCID is 67% and 53%, respectively. Similar
probabilities of survival have been reported by the EBMT
in the 1968-1989 survey (ie, 76%and 52% after HLA-identical and HLA-nonidentical BMT, respectively).6Major obsta-
Table 5. Humoral Immune Reconstitution After BMT for SClD
Serum
Ig
UPN
Time (yrs)
Post-BMT
IgM
IgG
No. of
DT-IPV
Vaccinationst
Levels (mg/dL)
Total
IgA
lgGl
lgG2
lgG3
71*
421
167
262
25
29
73
lgG4
Specific IgG Antibody
Productiont
D+
TS
IPV,,,§
IPV,,§
IPV,§
111
II
I
111
111
Ill
___~
_
~
2
1,190 173 +15.4 17
50
105
131
53
56
+8.1 59
085 109 +5.7 96
108
97
112
663
126
04 101 +1.6 168
f1.5 183
199
+22.9
667 110
+8.8
+3.1
+2.7
+8.3
+8.4
194
134
119
398
128
73
1,311
56
57 88 294 1,106
635
162
651
186
877
1,110
917
4'
974
909
44'
1,011
507*
<l*
327*
+4.7
78 933 163
89
+4.0 571 137
+2.2 4,028 369
50 71136 402
350
f 1 . 0 1,769 136
158
80*
87
302
106
82
620
141
2,395
51274 746
125
< l59
* 1,558
417
28
124
71
100
46
14
VI
31
15
11
16
4*
86
50
20
59
127
47
33
20
3
11
9
37
XI
VI
IV
111
IV
VI
Vlll
VI
V
V IV
V
VI
Ill
Ill
I1
Humoral Immune
Reconstitution
-
IVIV
~
111
~
V
IV
~
V
111
IV
I
II
II
IV
I
Ill
"
-
-
VI1
II
111
-
IV
IV
II
-
V
111
IV
I
111
111
-
_
IX
I1
111
111
_
~
~
VI1
II
It1
-
-
-
IV
II
IV
Ill
~
V
v
-
V
-
V
I
Ill
V
I
111
-
-
-
-
_
~
* Below mean -2 SD of age-matched controls."
t The number of the DT-IPV immunization after which the positive response occurred is indicated (-, no positive response).
* Positive if the postimmunization titer wasat least twice thepreimmunization titer.
§
Positive if the postimmunization titer was at least 4 times the preimmunization titer.
_
_
~
Complete
Complete
Complete
Complete
Complete
Selective IgA-D
Complete
Selective IgA-D
Complete
Complete
Complete
Complete
Complete
Complete
Selective IgA-D
~
AND
CHIMERISM
FOR SCID
3943
Table 6. Cumulative Response RatesToward DT-IPV Antigens After Three Vaccinations of Healthy Infants.
and of Pediatric Graft Recipients Within 6 Months After BMT
D
60%
Healthy infants (n = 20)
Pediatric BMT recipients
Leukemia (n = 20)
60%
SCID
IWm
IPV,,
IPV,
T
~PVI-III*
83%
89%
90%
90%
90%
25% (2/8)t
64% (7/11)
56% (5/9)
44% (4/9)
25% (2/8)
100%
67% (6/9)
Response to either IPV,, IPV,,, or IPV,,,.
t Number of patients who showed a positive response among those who either received three DT-IPV injections or showed a positive response
after previous vaccination within 6 months after BMT.
cles for short-term success were early death caused by respiratory failure (n = 5), and primary graft failure (n = 9)
ultimately leading to death in five cases. The children who
died from respiratory failure all presented with progressive
lung infection before BMT, an important adverse risk factor
in the EBMT survey: Graft failure was observed after unconditioned BMT (n = 3) and, despite prior cytoreductive
treatment, in patients with T-B+ SCID (n = l), atypical OS
(n = 1)'; atypical T-B- SCID (n = 2),12and HLA class I1
deficiency (n = 2). Successful engraftment was obtained in
four of six patients who received a second transplant.
Sixteen graft recipients were alive for more than 1 year
after BMT, and data on these cases are included in this
evaluation (except for UPN 5, who was lost to follow-up).
With the exception of an episode of severe S aureus infection
in UPN 2, recurrent URTI in UPN 53, and persistenthecurrent warts in both cases, no signs or symptoms of a subnormal immunologic defense capacity were present in the long-
A.
B.
'"1
0
5
10
15
20
0
25
5
10
15
20
25
EK mtlo
ratio
D.
C.
ap
10
/
l
0
15
5
20
10
ratio
25
,
,
n
0
5
10
1s
20
26
E/T mtlo
Fig 2. in vitro NK c e l l activity after BMT for SCID.PBMCs of the children were collected M o r e (pre) or after (post) BMT. Testa were
lines represent 10% and 90% confidence intervals of fresh
performed using either fresh (A andB) or cryopreserved IC and D) material. Dashed
(n = 66;A and B) or cryopreserved In = 33; C and D) control samples. Posltransplant NK cells of the children were either of rmlpient origin
(A and C), donor and recipient origin (B), or donor origin (D).
3944
VAN LEEUWEN ET AL
term survivors at a median (range) follow-up of 6 (1 to 24)
sponses for many years after BMT, in contrast to the obseryears after BMT. Noneof the childrenreceived Ig suppletion
vations of Wijnaendts et al.37
more than 6 months after BMT. Except for UPN 2 , who is
Defective in vitro NK cell function in SCID patients before BMT32.38.39 and nonrandom X-chromosomeinactivation
on flucloxacillin prophylaxis for his tendency to develop S
aureus skin infection, none of the long-term survivors rein NK cells of female obligate XSCID carriers4" has been
ceived antimicrobial prophylaxis more than 6 months after
described, suggesting that NK cells are
intrinsically defective
BMT.
in a subset of patients with SCID. Here, we show that NK
In 15 children and young adults, weinvestigated the relacells can be either
of recipient, donor, ormixed origin. Donor
tionship between engraftment of different cell lineages and
T- and B-cell engraftment was not always associated with
immune reconstitution. In the majority of cases, FPV chimedonor NK cell engraftment,
supporting the ideathat NK cells
rism analysis showed the presence of complex, cell lineage
belong to a separate lymphoidlineage. All evaluable children
specific patterns of engraftment, which are not observedafter
showed in vitro NK cell activityafter BMT. Importantly,
high-dose conditioning andallogeneic BMT for le~kemia.'.'~ engraftment of NK cells was associated with increased
in
Interestingly, conditioning withbusulfan (8 to 16 mgikg
vitro NK cell function in children who showed defective in
BW) plus cyclophosphamide was not
sufficiently myeloablavitro NK cell activity before BMT. Accordingly, incomplete
NK cell engraftmentresulted in partial restoration of in vitro
tive inthe majority of children to eradicate host-type hematopoiesis. In a previous study, we noted a high incidence of
NK cell function in such cases.
All children with sustained engraftment showed evidence
persistent host-type hematopoiesisinchildren
less than2
of humoral immune reconstitution after BMT. None of the
yearsof age grafted for leukemia
afterconditioningwith
busulfan (16 to24mgikgBW)plus
cy~lophosphamide.~~ children showed apersistentIg isotype deficiency, except
for three cases (UPN 53,
59, and 199) who lacked B-cell
These findings may be explained by altered pharmacokinetengraftmentand displayedalong-lastingselective
IgA-D
ics of busulfan in children as compared with adults leading
after BMT for T-B' SCID. Selective IgA-D after HLAto decreased drug exposure in children? Thus, the use
of
haplo-identical BMT for SCID hasbeenreported also by
either no or arelativelymildpreparative
regimenbefore
other investigator^."^^' Importantly, specific IgG antibodies
BMT for SCID leads to the persistence of host-type stem
were detected in all evaluable children after repeated DTcells. In turn, these may occupy the space in the BM necesIPV immunization (and after natural infection), irrespective
sary for engraftmentof donor stem cells. Under
these circumstances, donor cells may be detectable only at the
maturaof donor B-cell engraftment. However, the recovery of specificantibodysynthesisafter
BMT for SCID may
not be
tional stage of thehematopoieticcelllineages
inwhich
complete in all cases as suggested by holes in the antibody
recipient cells have defectsin their development andor funcresponses after repeated vaccination (eg, in the case of UPN
tion as a result of a selective growth advantage.
59; Table 5). Interestingly,the cumulative responserates
Surprisingly, we detected residual (1% to 5%) host-type
towards DT-IPV antigens of the group of grafted SCID paT cells in eight of the graft recipients, even more than 23
tients were similar to those observed in children after HLAyears after BMT. Although we cannot formally exclude the
identical BMT for leukemia (Table 6).
possibility that purified T cells might have been contamiAEuropeansurveyhasshownthatincompletehumoral
nated with recipientnon-T cells, we believe that
this explanaimmune reconstitution (ie, lack of specific antibody production is unlikely for several reasons. First, all T-cell population or Ig isotype deficiency) after HLA nonidentical BMT
tionsthat were purified by cell sorting showed aclearly
for SCID is stronglyassociatedwith failure of B-cell endistinct stainingpattern in fluorescence diagrams (results not
graftment.'j In agreement with these data, we only observed
shown). Second, residual host-type T cells were observed in
incompletehumoral
immune reconstitution(ie,selective
some, but not all children withcelllineage-specific
enIgA-D) inchildren who showedno or onlymarginalBgraftment patterns; these host-type T cells were detected on
cell engraftment after HLA-haplo-identical BMT for T-B'
several occasions in all six children who were investigated
SCID. Several mechanisms, which are not mutually exclumore than once. Moreover, residual host-type T cells were
sive,may explain the above findings.First,lack of B-cell
detected in one childwith predominantly donor cells innonengraftment may lead to incomplete humoral immunereconT-cell lineages (UPN 105). It seems unlikely that the persisstitution after BMT for T-B' SCID, because subtle defects
tence of residual host-type T cells can beconfirmed by stanin in vitro B-cell function have been described in a subset
dardmetaphasecytogeneticsbecause,
in ourexperience,
of T-B+ SCIDpatients42and because nonrandom X-chromoFPVchimerismanalysis
ismoresensitive
andbecause
some inactivation has been observed in B lymphocytes obthese host-type T cells most likely do not respond to polytained from female obligate XSCID carriers, suggesting an
clonal activators (eg, PHA) used to obtain metaphases for
intrinsic B-cell defect.47 More recently, it has been shown
karyotyping. In addition,informative cytogenetic markers
that XSCID is caused by mutations in the common y ( y J
were not available in several of these cases.
chain,44 a functionally important component of the IL-2, ILAll children who showed sustained engraftment and sur4, and IL-7receptors.ds-4RThus, we cannot excludethe possivived for more than 1 yearafter BMT showed significant
bility
that the persistent host-type B cells of children with
improvement of T-cell immunity as evidenced by several in
incomplete humoral immune reconstitution (UPN 53, 59,
vivo andin vitro parameters. However,wefound thata
and 199) are intrinsically defective for some, but not all, Bnumber of long-term survivors continued to have low absocellfunctions. However, thehypothesisdescribed
above
lute T cell counts and low in vitro proliferative T-cell re-
CHIMERISM AND IMMUNITY AFTER BMT FOR
3945
SCID
does not explain the general finding that incomplete humoral
immune reconstitution is observed almost exclusively after
HLA nonidentical BMT.6.7.37,49
Second, incomplete humoral immune reconstitution after
HLA-nonidentical BMT may be caused by the inability of
donor T cells to cooperate with host B cells across major
histocompatability complex (MHC) barriers. This model
seems unlikely for several reasons: (1) postthymic T cells
of murine radiation BM chimeras display MHC restriction
patterns towards host MHC antigens5's5' because positive
selection is mediated by thymic epithelial cells of host MHC
(2) T-B-cell cooperation across MHC barriers
gen~type~'-'~;
has been shown in several SCID patients after HLA-nonidentical BMT, even at the clonal l e ~ e l ~ ~and
. ~ '(3)
; T-B-cell
cooperation after HLA-haplo-identical BMT can always occur via the shared HLA haplotype.
Third, lack of self-tolerance towards donor-specific HLA
class I1 antigens has been observed in SCID patients with
incomplete humoral immune reconstitution after HLAhaplo-identical BMT.62" Various studies in mice have indicated that clonal deletion of self-reactive thymocytes is mediated by BM-derived antigen presenting cells in the thymus.65-68
Thus, graft recipients with split chimerism may fail
to delete donor thymocytes reactive towards the nonshared
donor-specific HLA antigens. Indeed, T-cell-mediated donor-antidonor reactivity towards donor-specific HLA class
I1 antigens has been observed in split ~hirneras,6*".~~."but
not in complete chimera^.^' In our study, 7 of 10 HLA haploidentical allograft recipients showed split chimerism. Complete humoral immune reconstitution was observed in split
chimeras with B-cell engraftment (UPN 56, 168, and 183).
Interestingly, studies performed by De Villartay et a16' and
Keever et a163 suggest a relation between lack of B-cell engraftment and the presence of donor-antidonor HLA class I1
reactivity. It is tempting to speculate that resting donor B
cells, which constitutively express HLA class I1 antigens,
but lack costimulatory activity (ie, B7 expression):'may
play a role in the functional inactivation of naive donor
T cells that recognize donor-specific HLA class Wpeptide
complexes. The remaining children with split chimerism
(UPN 53, 59, 108, and 199) lacked B lymphoid and myeloid
engraftment. Strikingly, LJPN 108 received BM from an
HLA class I1 phenotypically identical, MLC-negative, related donor (which was also identical at the HLA DR and
DP loci as determined by oligotyping) and showed complete
humoral immune reconstitution, whereas the other three graft
recipients showed selective IgA-D after HLA class I1 haplomismatched BMT. In these children, expression of donor
HLA class I1 antigens is restricted to activated T cells. Although there is no direct evidence showing a causal relationship between the presence of donor-antidonor class I1 reactivityand incomplete humoral immune reconstitution, it
seems possible that the presence of donor-antidonor HLA
class 11 reactivity in these cases may interfere with T-cell
activation, leading to a reduced efficiency of T-B-cell cooperation and incomplete humoral immune reconstitution?*
Importantly, such a model provides an explanation for the
finding that incomplete humoral immune reconstitution is
found almost exclusively after HLA-nonidentical BMT and
is restricted to graft recipients who lack B lymphoid and
myeloid engraftment.
ACKNOWLEDGMENT
We thank M. van der Keur and J. Slats for technical assistance
in cell sorting experiments and Professor Dr D.W. van Bekkum for
critical review of the manuscript.
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