1. Art and Diseño

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Clonal Dysregulation of the Antibody Response to Tetanus-Toxoid After
Bone Marrow Transplantation
By E.J.A. Gerritsen, M.J.D. Van Tol, M.B. Van ’t Veer, J.M.A. Wels, I.M.S.L. Khouw, CR. Touw,
CM. Jol-Van Der Zijde, J. Hermans, H.C. Rumke, J. Radl, and J.M. Vossen
After bone marrow transplantation (BMT), a prolonged dysregulation of humoral immunity can be observed. In the
present study, we investigated whether this is reflected in
an abnormal production of specific antibodies (Ab)t o the Tcell-dependent recall antigen tetanus-toxoid (TT). The study
group consisted of children receiving transplants of an unmodified allogeneic graft and of adults receiving eithera Tcell-depleted allogeneic or an unmodified autologous BM
graft. Findings were compared with those in healthy controls. In pediatric graft recipients, who were routinely
revaccinated early after BMT, the Abresponse was quantitatively
superior to thatin adult graftrecipients who did not receive
early revaccination. In the majority of graft recipients, the
time periodafter vaccination required
t o reach the peak level
of antibodieswas prolongedand the number of responding
TT-specific B-cell clones was markedly decreased in comparison withcontrols. In controls, a low frequency of dominant
B-cell clones may produce low quantities of homogeneous
Ab components (H-Ab) against a heterogeneous background. However, in BM graftrecipients, “overshooting“ of
Ab production by separate B-cell clones was observed, resulting in the development of H-Ab at a relatively high concentration. These abnormalities were present up t o 10 years
after BMT, irrespective of eitherthe age of the recipient, the
modulation of the graft, or the vaccination schedule used.
It is hypothesized that thedysregulated Ab production is the
consequence of activation of a restricted number of resting
memory B cells, present in germinal centers, repopulating
gradually after BMT. Our data show that routinerevaccination early after BMT improves the humoral immune response. However, because of a clonally dysregulatedAb production, long-lastingqualitative defects maybe present
even after normalization of Ab titers.
0 1994 by The American Societyof Hematology.
B
ONE MARROW transplant (BMT) recipients may sufanti-TT response, as assessed by the electrophoretic heterofer froman immunologic deficiency up to several years geneity of TT-specific IgG antibodies in sera from children
after transplant.’ In the absence of chronic graft-versus-host
and adults vaccinated after
BMT. The findings were comdisease (GVHD), normalization of B-cell function is considpared with those found in routinely vaccinated healthy in1 to 2 years.’ Although B-cell
eredto takeplacewithin
fants, children, and adults. Although normal Ab titers were
numbers become normal at 4 to 8 months posttransplant,
already reached at 4 months after BMT in the majority of
their phenotype is immature and their function is inadequate
recipients, the number of B-cell clones involved in the rein a way similar to that found during ~ntogeny.’.~ Transient sponse wasmarkedly reduced for a prolonged periodof time.
Ig (sub)class deficiencies may be observed after BMT.’” In
Sometimes the response was largely dominated by a single
clone and seemingly monoclonal. These abnormalities could
addition, Ig synthesis is clonally dysregulated for some time
be found as long as 10 years after BMT.
after transplantation, resulting in monoclonal gammopathies
(MG) and restricted heterogeneity of Ig.7.xAn abnormal huMATERIALS AND METHODS
moral immune response may thus persist despite a normalization of serum Ig (sub)class levels. Specific antibody (Ab)
Controls and Patients
production can be detected from 3 months after BMT onThis study was approved by the Institutional Review Boards on
wards, depending on the vaccines used”” and on whether
Medical Ethics at the University Hospital (BMT center for children),
the donorsorthe
recipientshad been vaccinatedbefore
Leiden, The Netherlands and the
Dr. Daniel den Hoed Cancer Center
BMT.I4-IXHowever, detailed information on qualitative as(BMT center for adults), Rotterdam, The Netherlands.
pects of the Ab response in BMT recipients is still lacking.
In thepresentstudy,
we investigated more extensively
Controls
than hitherto the humoral
immune response to the T-cellSera from 20 healthy infants and children, observed from
birth
dependent (TD) recall antigen tetanus-toxoid (TT). This inup to the age of 4 years, and from 20 healthy adult volunteers with
vestigation was performednotonly
by quantification of
amedianage
of 35years(range,25to
45 years)wereused
as
specific antibodies, but also by qualitative evaluation of the
controls.
Pediutric Patients
From the Departments of Pediatrics and Medical Statistics, University Hospital, Leiden; the Department of Hematology, Dr. Daniel
den Hoed Cancer Center,Rotterdam; the National Institute of Public
Health and Environmental Protection, Bilthoven; and the TNO Institute of Ageing and Vascular Research, Leiden. The Netherlands.
Submitted April 6, 1994; accepted August 23, 1994.
Address reprint requests to E.J.A. Gerritsen, MD, Department oj
Pediatrics, Leiden University Hospital, PO Box 9600.2300 RC
Leiden, The Netherlands.
The publication costsof 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
indicute this fact.
0 1994 by The Americun Society of Hematology.
0006-4971/94/8412-0018$3.00/0
4374
ChildrenreceivinggraftsofBMcellsfromanHLA-identical
sibling between 1982 and 1989, who were cured
of their original
disease and who are alive and well, were included
in the study (n
= 38). The characteristics of these patients are given in Table 1. All
receivedanunmodified BM graftwhilestayingintheprotective
environment of a laminar flow isolator and after antimicrobial suppression of theirintestinal micro-flora.” By January I , 1993,the
7.5 years (range, 1.0 to1 1.0 years).
median follow-up after BMT was
As we previously reported, complete chimerism was present in the
majority of patients.20
Adult Patients
=
Recipients treated with either an HLA-identical sibling graft (n
26) or an autologous graft (n = 9) between 1980 and 1992 who
Blood, Vol 84, No 12 (December 151, 1994: pp 4374-4382
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CLONALDYSREGULATION
4375
OF THE ANTIBODYRESPONSE
Table 1. Characteristics of the Patients
Children (n = 381
Age (yr)
Median
Range
16-60
Diagnosis
AML
ALL
NHL
CML
Others
Conditioning regimen
CTX 120 TB1 12
CTX 120 TB1 10
6CTX 120 TB1 7-8
2
BU 16 CTX 120-200
Other
Graft
26
Allogeneic
Autologous
GVHD prophylaxis
TCD
MTX
CSA
2MTX + CSA
Acute GVHD
24
Absent
Grade I
32 Grade II
Chronic GVHD
33
Absent
Limited
Extended
9
Adults (n
=
35)
39
0.5-18
16
14
15
5
1
2
5
4
0
0
33
3
11
14
2
2
38
0
9
0
14
8
16
5
6
26*
2
18
30
2
8
ria toxoid (D)-pertussis (P)-tetanus toxoid (T)-inactivated polio virus
type I, 11, and 111 (IPV) at 3, 4, 5 , and 11 (n = 16) or at 14 (n =
4) months (DPT-IPV I to IV) and with DT-IPV at 4 and 9 years of
age (DT-IPV V and VI; Table 2). In addition, 20 healthy adult
volunteers received a DT-IPV booster (B) vaccination.
Pediatric patients. The patients and their BM donors had been
vaccinated during infancy and early childhood with D(P)T-IPV vaccine, according to the Dutch National Vaccination Program. Twentyseven of 38 patients were immunized with DT-IPV within l month
before BMT (DT-IPV 0; Table 2). In the light of other studies,"."
5 BM donors also received a DT-IPV vaccination within 2 weeks
before graft donation.
In the first 4 months after BMT, the recipients received 3 DTIPV booster injections (I to 111) with an interval of 1 month, starting
from 6 weeks after BMT. A limited number of children received an
additional injection (IV) between 6 and 24 months after BMT. In
1992, 21 children with a follow-up period of at least 3 years (range,
3 to 10 years) after BMT again received a DT-IPV injection (DTIPV V).
Adult patients. As far as could be evaluated, all adult BM graft
recipients and their donors had received routine D(P)T-IPV vaccinations in infancy and childhood. In addition, most male patients had
received a DT-IPV injection at the start of their military service.
None of the adult patients was vaccinated shortly before BMT and
none received revaccination after BMT until the occurrence of an
outbreak of poliomyelitis in The Netherlands (November 1992). At
that time, all patients received three DT-IPV vaccinations at monthly
intervals and one vaccination 6 months later. The response to the
second vaccination (DT-IPV 11) was investigated.
6
30
4
4
Serum Samples
1
1
Abbreviations: AML, acute myeloblastic leukemia in first ( n = 30)
or second (n = 1) remission; ALL, acute lymphoblastic leukemia in
first ( n = 6 ) or second ( n = 13) remission; NHL, non-Hodgkin's
lymphoma; CML, adult (Ph') chronic myeloblastic leukemia; Others,
for children, severe aplastic anaemia (n = 2).@-thalassemiamaior (n
= 2),and combined immunodeficiency(n = l ) ,for adults, M. Hodgkin
(n = 2), multiple myeloma (n = 2), paroxysmal nocturnal hemoglobinuria (PNH; n = l ) , and mamma carcinoma (n = 1); CTX 120-200,
cyclophosphamide 2 x 60 or 4 x 50 mg/kg; TB1 7-12,total body irradiation in grays (Gy), dose depending on age of recipient administered
in one ( 5 8 Gy) or 2 ( 2 1 0 Gy) fractions with a dose rate of 24 to 48
cGy/min; 61.116,busulphan for 4 days at 4 mg/kg; GVHD, graft-versushost disease; TCD, r2 log T-cell depletion; MTX, methotrexate <50
mg/m2 in only first 2 weeks (n = 20). >l00 mg/m2 until day +l00 (n
= 14; children with MTX only); CSA, cyclosporin A 2 mg/kg intravenously or 6 mg/kg orally for at least 3 months after BMT.
In 13 patients, MTX and/or cyclosporin A was administered in
addition to TCD.
were alive and well were included. The characteristics of these patients are givenin Table l . In case of an allogeneic BMT, the
graft was more than 2 log T-cell-depleted by albumin gradient
centrifugation and E rosette sedimentation?' By January 1, 1993,
the median follow-up of these patients was 2.5 years (range, 0.5 to
13.0 years).
Methods
Vaccination
Controls. Twenty healthy infants and children were vaccinated
according to the Dutch National Vaccination Program with diphthe-
Serum samples were obtained after informed consent before and
at a median of 3 weeks (range, 2 to 4 weeks) after vaccination,
with the following exceptions. From children of the control group
receiving DPT-IPV IV at 14 months of age (n = 4), the postvaccination serum sample was taken 4 weeks later; from those immunized
at 11 months of age (n = 16), serum was taken at 4 months after
vaccination. Cord blood samples were available from all healthy
infants included in the study. Sera from healthy adults were obtained
at 2 and 4 weeks and at 3, 6, and 12 months after the booster. From
pediatric graft recipients, serum samples were also obtained at 3 and
6 months after DT-IPV IV. From adult graft recipients, only one
serum sample, taken 4 weeks after the second DT-IPV vaccination,
was available. All sera were kept frozen at -20°C until analysis.
Serum Analysis
Quantification of IgG anti-7T antibodies. The concentration of
IgG anti-TT antibodies was measured by enzyme-linked immunosorbent assay (ELISA), as has previously been described.**The concentration was expressed in arbitrary units (AU) per milliliter using a
reference serum for standardization. The Ab response in healthy
children after DT-IPV VI at 9 years of age was not investigated in
this study. Data on IgG anti-'M levels after vaccination of 8 pediatric
graft recipients early after BMT werenot included, because the
children were being treated at that time for acute or chronic GVHD
with immunosuppressive therapy.
Dejinition of response to TT. In healthy infants (controls), a
primary Ab response to 'M was scored as positive if the postimmunization IgG titer was at least twice the preimmunization titer and
reached 20.2 AU/mL. In pediatric and adult controls andinthe
pediatric patients a secondary Ab response was considered positive
in case of an increase of the post-immunization IgG titer to 125%
of the pre-immunization titer, reaching at least 1.0 AU/mL. In two
cases, newly arising TT-specific homogeneous Ab components (HAb) were detected in the absence of an increase of the Ab titer. This
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4376
GERRITSENET
AL
Table 2. Vaccination Protocols in BMT Patients and Controls
19
Controls
Vaccination no.
No. of 20
individuals
Age*
Median
Range
Pediatric patients
Vaccination no.
No. of patients
Period post-BMT
Median
Range
Adult patients
Vaccination no.
No. of evaluable cases
Period post-BMT
Median
Range
II
111
IV
V
B
20
4 m0
5 m0
12 mo
11-14 mo
4 yr
35 yr
25-45 yr
111
IV
V
9 mo
6-24 wk
7 yr
3.5-10.5 yr
I
19
20
3 m0
0
27
I
21
-3 wk
11
-(1-4) wk
38
I1
2338
7 wk
6-10
10-14
wk
24
wk
wk
15 wk
wk
14-20
II
35
2.5 yr
0.5-13 yr
Abbreviation: -, before BMT.
The Dutch National Vaccination Program also provides vaccination at 9 years of age. The response to this immunization
was not investigated.
0-v, D(P)T-IPVvaccination number in healthy infants and children, and in BMT recipients. B, DT-IpV booster vaccination in adult controls.
finding was interpreted as an indication for a positive TT response.
I n the adult patients. a preimmunization sample was not available.
In thesepatients,an
A b responsewas defined as positive if the
postimmunization titer was 2 1.0 AUlmL.
7T-sprcific itnrnunohlotfing. To investigate the heterogeneity of
IgG anti-TT and the occurrence o f H-Ab, we applied agar gel-elec'T
trophoresis o f serum samples according to Wieme, followed by I
antigen-specific immunoblotting (WABL) onto nitrocellulose membranes coated with 10 pglmL (3 pg/cmZ) o f I T . The patterns were
developed using Ig isotype-specific monoclonal antibodies (MoAbs:
Nordic Immunological Laboratories, Tilburg, The Netherlands), as
described earlier.2'.2' The serum samples (diluted 1:2SO) were investigated for TT-specific IgG patterns together with a positive and a
negative control25 (Fig IA; these human MoAbs were kindly provided by Dr W.P. Zeijlemaker, CLB, Amsterdam, The Netherlands).
In 7 selected patients with H-Ab at a relatively high concentration
(0.2 to 2 pglmL) in the first year after BMT, we investigated whether
the H-Ab present at 3 to IO years after B M T were still produced by
thesame B-cell clones. T h i s investigation was performed by 'ITspecific W A B L and by isoelectric focusing (IEF) followed by IT
antigen-specific immunoblotting (FABL)"." for the characterization
o f the IgG subclass and light chain type o f the H-Ab.
A.sse.s.srnenf of WABL patfems. Overall patterns and presence o f
H-Ab were analyzed independently by two investigators (E.J.A.G.
and M.J.D.v.T.). The patterns were scored as either heterogeneous
or o f restricted heterogeneity (Fig 2). Besides the number of H-Ab,
the concentration o f H - A h was assessed semiquantitatively as high
(>O.S pg/mL), medium, or low ( 4 . 1 pglmL), using the positive
control sample diluted inphosphate-buffered saline (PRS) as a reference (Fig IB). The limit
o f sensitivityfor the detection o f ITspecific H-Ab by WABLranges from S to SO nglmL, depending on
the intensity of the pattern of the heterogeneous antibodies."
Sfatistical evaluation. Responsepercentages and frequencies o f
tests on 2 X 2 tables, and 9S% confiH-Ab were compared with
dence intervals for the difference of the percentages were calculated.
The titer values were analysed using the Student's f-test.
x'
RESULTS
Quantification of IgG Anti-TT Antibodies
Controls. After thefirst vaccination, 45% of the infants
responded to TT (Table 3). The infants who responded had
a lower preimmunization Ab titer in the cord blood andin
the prevaccination sample than did the nonresponders ( P <
.OOl and P < . O l , respectively; data not shown). This finding
indicates that, in the presence of passively acquired maternal
IgG anti-TT antibodies, the actual Ab production by the
infant is delayed. After further vaccinations, the cumulative
response rate reached 95%. Infants at 12 months ofage,
children at 4 years of age, and adults all mounted an Ab
A
kl
H-Ab a TT
H-Ab a HB
l
TT
BSA
1
--
l
-I"
B
pgm
1.0
0.5
0.2
High
Medium
0.1
0.05
Low
J-j
0.02
Fig l. lT-specific immunoblotting. (AI Specificity of TT-specific
immunoblotting (WABL). Abbreviations: H-Ab a TT, human lgGl
MoAb against tetanus toxoid (concentration, 0.2 p g / ~ n L ) ~
H-Ab
~; a
HB, human lgGl MoAb against hepatitis B surface antigen (concentration, 20.0 pglmL1; lg, blotting on noncoated membrane (signal
intensity is related t o protein concentrationl; TT, blotting on TTcoated membrane (only signal ofTT-specific MoAb is obtained with
increased intensity); BSA, blotting onBSA-coated membrane (no signal isobtained). (B) Sensitivity of WABL and semiquantitative determination ofhomogeneous antibodies (H-Ab) at low (01.medium (@),
and high ( W ) concentration. The values from 0.02 t o 1.0 indicate the
concentration rangeof MoAb against TT in micrograms per milliliter.
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4377
CLONAL DYSREGULATION OF THE ANTIBODY RESPONSE
CONTROLS
AUImL
A
0.8
IV D
Hetsrogenrour
31.7
Hetaogeneour
1250
with H-Ab
< 0.1 p i m L
A
A
BMT PATIENTS
Rentrioted
heterogenelty (RH)
wfth H-Ab
0.1-0.5
D
,WmL
4.7
ID
5.8
II D
16.0
Restrloted
lllb
heterogenelty (RH)
wlth H-Ab
420
10.5
pimL
k250
Fig 2. Examples of representativeWABL patterns. (A) WABL pattern of sera from a 4-year-old healthy child (control) before and after
DT-IPV booster vaccination (IVD):electrophoretic heterogeneity of
lgG a n t i - l l after vaccination. (B) WABL pattern of serum from a 4year-old healthy child (control)before and after DT-IPV booster vaccination (IVD):electrophoretic heterogeneity of IgG anti-TT and presence of H-Abat a low concentration(<0.1 pg/mL). IC)WABL pattern
of serum from a 2-year-old boy (after an uncomplicatedBMT for acute
myeloblastic leukemia in first remission) after DT-IPV 111 vaccination
post-BMT IIIID): restricted electrophoretic heterogeneity (RH) and
presence of 7 H-Abof medium concentration (0.1 to 0.5 pg/mL).
WABL for IgG subclasses showed H-Ab in all subclasses (data not
shown). (D) WABL pattern of sera from a 4-year-old girl (after an
uncomplicated BMT for acute lymphoblastic leukemia in second remission) before the first (ID) and after the first, second (113).and
third (IIID)post-BMT vaccinations: restricted electrophoreticheterogeneity (RH) and development of H-Ab, of which one was at a high
concentration (>0.5 pg/mLI. The figures represent IgG Ab titers to
TT in AU per milliliter; 1:250 indicatesthe dilution of serum samples.
response after booster vaccination. The geometric mean of
the Ab titers (GMT) was highest in children at 4 years of
age and lowest in infants at 12 months of age.
Pediatric patients. In none of the 4 patients whoreceived a vaccination within 2 days before the start of the
conditioning regimen for BMT was anAb response observed. Seventy percent of the other evaluable patients (n =
23) responded to vaccination before BMT (Table 3). irrespective of the prevaccination titer and even when vaccinated
as little as S days before the start of the conditioning regimen.
The GMT in responders wasmuch lower than in control
infants and children, probably as a result of preceding antileukemic treatment.
After the first vaccination, at 7 weeks after BMT, 30% of
the pediatric recipients responded. There was no statistically
significant difference between responders and nonresponders
withrespect to prevaccination Ab titers, substitution with
gammaglobulins, or treatment with cyclosporin A. Two revaccinations at I 1 and 15 weeks after BMTresulted in a
cumulative response rate of 75% of the graft recipients. Although this finding was not investigated systematically. we
observed that the maximum Ab titer was reached at 4 weeks
or even later after immunization and notat 2 weeks as in
adult controls after booster vaccination.
Revaccination between 6 and 24 months after BMTresulted in a IO0% response rate and a significantly increased
GMT (Table 3). There was no difference between patients
with (n = 6) or without ( n = 17) preceding acute GVHD.
Booster vaccination at 3 to I O years after RMT resulted in
a 100% response rate and a GMT similar to that in controls.
Follow-up investigations showed a half-life of Ab titers comparable to that in adult controls (data not shown).
Addt patients. There were no significant differences
with respect to the percentage of responders between adult
patients who received a TCD allogeneic graft (14 of 26) and
those who received an autologous graft (4 of 9), nor was
there a difference between patients vaccinated 0.5 to 2 years
(6 of 17) and those vaccinated more than 2 years after BMT
(12 of 18). Also, the GMT was not different between these
subgroups. Ten adult patients (about 30%) had an IgG antiTT titer after two DT-IPV immunizations that may not have
been protective. ie, less than 0.1 AU/mL.
Heterogeneity and Oligoclonnlity qf IgC Anti-TT
Controls. TT-specific H-Abwerehardly
detectable in
cord blood and sera of infants after four DPT-IPV vaccinations (Fig 3A). After revaccination at 4 years of age or
in adulthood, either one or two TT-specific H-Ab of low
concentration (<0.1 pg/mL) could be observed in serum
samples of about 35% of the controls, on top of an overall
heterogeneous Ab response (Figs 2B and 3A).
BMT patients. TT-specific H-Ab oflow concentration
could be observed in preimmunization serum samples of
about 25% of the pediatric patients before BMT (Fig 3B).
After pre-BMT vaccination, this percentage was SO%, which
was not significantly different from healthy children at the
age of 4 years and from adults. However, after the first and
second post-BMT vaccination, it increased significantly to
75% and 10096, respectively. In addition, the number of HAb per serum sample and the quantity of some H-Abincreased (Figs 2C and D and 3B).
After booster vaccination in thesecondhalf
year after
BMT, H-Ab ata medium and high concentration were found
in about SO% of the patients. At follow-up investigation, 3
to I O years after BMT, 7S% of the patients still had H-Ab
in their serum. In some cases, these H-Ab were of relatively
high concentration, ie, greater than 1.O p d m L (Figs 2D and
3B).
In about 30% of the adult graft recipients who responded
to TT vaccination, the postimmunization serum showed HAb. These H-Ab were also frequently at a relatively high
concentration. In general, there was a strong correlation between the presence and concentration of H-Ab and the quan-
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GERRITSEN ET
4378
Table 3. Quantification of IgG Anti-TT Antibodies After Vaccination
Before Vaccination
Origin of Serum Samples
Response to D(P)T-IPV I
Healthy infants9 (3 mo)
Pediatric patients (7 wk)
Response to D(P)T-IPV II (cumulative)
Healthy infants (4 mo)
Pediatric patients (1 1 wk)
Adult patients ( 18
S mo)
Response to D(P)T-IPV 111 (cumulative)
Healthy infants (5mo)
Pediatric patients (15 wk)
Response to booster vaccination
A. Controls
Infants (12 mo;
19 IV)
Children (4 yr;
17 V)
Adults (B) 20
B. Pediatric patients
Before BMT (-3 wk; 0)
6-24 mo after BMT (IV)
>36 mo after BMT (V)
Individuals
Investigated
20
30*
Individuals
With Response
9
Responders
GMT
15
17
0.44
79 (54-941 0.03-12.3
0.3-9.8
2.6
59 (39-76)
NE
NE
51 (34-67)
20
24
19
18
95 (75-99)
75 (53-90)
231
23
21
100 (82-100)
100 (80-100)
0.3-9.1
100 (83-100)
16
23
21
Range
(47-87)#70
0.1-21.9
100 (88-100)
0.1-14.0
100 (84-100)
GMT
0.3-12.30.94
4.5
0.01-0.96
0.03
45 (23-68)
0.5-6.6
2.1
30 (15-49)
19
29**
35
19
17
20
Afier Vacclnatlon
% (+2 S D )
3.5
1.8-31
5.5
1.3-128
8.2
Range
2.8-14.0t
0.7-52.9
.O
2.5
4.4
0.7-53.3
0.3-52.9
0.4-31.0
1.7-57.0
5.1
8.8
1.5
0.8
3.2
0.1-5.9
0.4-4.5
9.0
34.9
15.4
1.3-77.5511
14.2-83.211
4.6-81.411
1.4
2.3
1.6
0.2-6.4
4.9
79.7
27.5
1.0-17.1/1
1.0-184.011
2.4-159.01[
Abbreviations: GMT, geometric mean of lgG anti-lT antibody levels (AU/mL) in serum of responders before and one month after vaccination;
NE, not evaluated.
* Patients with acute GVHD (n = 8 ) were excluded.
t P < .05.
One serum sample after vaccination was not available.
5 Values are extrapolated from 4 children investigated 1 month after DPT-IPV IV at 14 months and from 15 children 3 months after DPT-IPV
IV at 11 months of age, respectively.
/I P < ,001. using analysis of variance; the difference of the GMT of antibody titers after booster vaccination was significant between all
subgroups.
1 Patients vaccinated less than 8 days before BMT (n = 4) were excluded.
# P < 0.001; using analysis of variance, the response percentage to booster vaccination in pediatric patients before BMT was significantly
less than after BMT, and in comparison with controls.
*
tity of IgG anti-TT in postvaccination sera of BMT recipients. Combination of the quantitative and qualitative data
indicates that 40% of the sera with IgG anti-TT levels less
than 20 AU/mL contained H-Ab, 20% of whichwere of
medium or high concentration. H-Ab were present in 70%
of the sera with IgG anti-TT levels greater than 20 AU/mL;
60% of these H-Ab were at a medium or high concentration.
In 7 pediatric graft recipients with H-Ab at a high concentration in the first yearafter BMT, WABL and FABL showed
that, at 3 to 10 years after BMT, 2 graft recipients had a
heterogeneous Ab response and 3 had a restricted pattern
with H-Ab produced by the original B-cell clone(s) (Fig 4).
The other 2 cases showed, in addition to H-Ab produced
by the original B-cell clone, H-Ab at a high concentration
produced by new clones, obviously triggered by revaccination.
DISCUSSION
Our previous study on the recovery of serum Ig isotypes
and the occurrence ofMG after BMT’ was extended here
to investigate the quantity and quality, in terms of electrophoretic heterogeneity, of the specific IgG Ab formation
after vaccination with the T-cell-dependent TT antigen.
In accordance with previous reports,”1owe found a quanti-
tatively decreased Ab production in recipients of a BM graft
early after BMT in comparison with the response on a
booster vaccination in healthy controls (but not in comparison with infants who received the first course of 3 vaccinations). IgG antibodies to TT are producedbymemory
B
cells with the help of T cells. Early after BMT, the number
of memory B cells may be low. It has also been shown that
the number of T-helper cells and precursors is low.26 In
addition, the production of and response to interleukin-2 (IL2) is de~reased~’.’~
and this has a detrimental effect on the
proliferative response after stimulation of T cells via the Tcell receptor-CD3 complex by antigens andanti-CD3.28,2’
Therefore, it seems paradoxical that specific Ab production
to recall antigens in BMT recipients precedes normalization
ofin vitro T-cell responses by a number of months.‘ This
finding is in contrast to the finding of a relatively high number of CD4’ CD45RO’ T lymphocytes in peripheral blood
after BMT,30unless this population belongs to the TH2 subset.31”2
Alternatively, the majority of T cells in the germinal
centers of BMT recipients may be activated by other as
yet unknown mechanisms. It is attractive to speculate that
alloantigens may be involved in this activation. B cells may
interact with activated T cells in a major histocompatibility
complex (MHC)-unrestricted and antigen nonspecific way
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CLONAL DYSREGULATION OF THE ANTIBODY RESPONSE
CONTROLS
A
I
% V
II
v
IV
111
v
v
V
B
V
V
100.
B
PATIENTS
v
6-12111
adults
>36m
v
>6m
children
before BMT
after BMT
Fig 3. Percentage (and 95% confidence intervals) ofresponders
with H-Ab in IgG anti-TT. (A) Healthy pediatric and adult controls
before (b) and after (a1 the indicated D(P)T-IPV vaccination (V).(B)
Pediatric patients before and after BMT and adult patients after BMT.
The sequential DT-IPV vaccinations are indicated (V,see also Table
2). Sera were investigated by WABL before (b) and after (a) vaccination. Note themarked difference in frequency ( * P < .01, **P < .001)
and concentration of H-Ab in patients compared with children after
DT-IPV V and adults after boostervaccination (see also Fig 2). H-Ab
at a (0)low, (W) medium, and (W) high concentration, respectively.
The numbersindicate the number of individualsinvestigated all individuals were analyzed before (b) vaccination; only samples of responders were investigated after (a) vaccination.
and proliferate and mature to Ig-producing cells in the presence of interleukins (ie, IL-4 and IL-10).31”4Interestingly, Ig
levels in graft recipients with acute GVHD are significantly
higher than in recipients witho~t.~’
In the present study, we showed that the occurrence of HAb at a low concentration within an otherwise heterogeneous
response is a normal phenomenon, because this type of
clonal dominance was also found in healthy children after
DT-IPV V and in adult controls after booster vaccination.
Clonal dominance has also been described in adults after
influenza vaccination.36However, in BM graft recipients, the
restricted heterogeneity of TT-specific IgG and the development of H-Ab at a relatively high concentration has to be
considered as an abnormal response. This oligoclonal Ab
response was found both in children receiving grafts of unmodifiedBMand vaccinated before and early after BMT
and in adults receiving grafts of either autologous or T-cell-
4379
depleted allogeneic BM who did not receive revaccination
early after BMT. Previous studies in immune-deficient mice
and in Rhesus monkeys after BMT also showed a similar
r e s p o n ~ e . ~There
~ . ~ ’ are at least two possible explanations for
this restricted response of prolonged duration. First, it may
be the result of the transfer of a limited number of memory
B-cell clones from the donor. This hypothesis is in accordance with the finding of a skewed Ig VH repertoire of B
cells early after BMT.39 Under certain conditions, booster
vaccination of the BM donor 2 weeks before donation has
been shown to increase the quantitative Ab response in the
recipient^.'^"'"^ On careful observation of the data presented
in some of those
it can be concluded that this
response was also oligoclonal. Second, it may be that only
a limited number of memory B cells become activated in
view of the restricted possibilities for cell-cell contact with
T-helper cells, resulting from underrepresentation of the latter population in germinal centers after BMT. Results of
animal experiments are in favor of the latter hypothesis because restricted heterogeneity of Ig and H-Ig develop after
thymectomy and disappear again after infusion of peripheral
T ~ e l l s . 4However,
’ ~ ~ ~ the persistence of a restricted Ab response and of H-Ab of high concentration in a number of
graft recipients many years after BMT, when numbers and
functions of specific T cells have become normal,’ indicates
that, in addition to abnormalities at the T-cell level, abnormalities at the B-cell level may persist. In murine graft recipients, germinal center reconstitution after BMT has been
shown to be oligoclonal after antigenic stimulation? In human BMT, a delayed reconstitution of germinal centers has
been described in cases with GVHD.44
Our findings of an impaired specific Ab response postBMT have clinical implications with respect to vaccination
policy of BMT recipients. Previous studies showed longlasting but transient persistence of donor immunity in nonvaccinated BMT recipient^.^^.^"^^ Vaccination of the donor
before graft donation, together with vaccination of the recipient early after BMT, may contribute to a quantitatively good
Ab response. BM graft recipients respond to vaccinations in
the following sequence: TD-recall antigens (at about 3+
months), TD-primary antigens (at about 6+ months), and Tcell-independent antigens (atabout l + year).’ This sequence can be observed after BMT with either an unmodified
graft,’ a T-cell-depleted graft,I5.l6 or a B-cell depleted
graft.47,48
However, under the latter condition, all responses
are primary because of the lack of transferred memory Bcell clones.47In the present study, we showed that repeated
vaccinations of children early after BMT resulted ina quantitatively enhanced specific humoral immunity at 6 to 24
months after BMT, whereas this response in adults was not
yet normal at2 years post-BMT after two vaccinations. However, we do not know whether the qualitative abnormalities,
present as a clonally restricted response, will provide adequate protection. It may be that increased quantity compensates for decreased quality of antibodies in the first years
after BMT.
With our very sensitive technique we have clearly shown
long-lasting oligoclonality attheB-cell
level after BMT.
Preliminary data on the T-cell receptor repertoire, which is
of relevance for antigen-specific help for B cells, do not
indicate a long-lasting oligoclonality at the T-cell level4’
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GERRITSENET
4380
WABL
A
1984
P
I
P
1992
B
t
1986
1992
C
AU/mL
1:250
20.0
t250
1:25
29.0
1:250
150
8.0
1:500
1:400
112.0
1150
10.0
,
1985
1992
FA6 L
1:250
-t
AL
l
~
1500
Fig 4. Follow-up investigation of H-Ab in IgG a n t i - n in 3 patients after BMT. WABL (antigen-specific immunoblotting after agar gel
electrophoresis) and FABL (antigen-specific immunoblotting afterisoelectric focusing) patterns ofsera from 3 children early after BMT (obtained
after DT-IPV 111) and 6 t o 8 years later (obtained after
DT-IPV V).(A) Sera of 1984 and of1992 showed an almost identical Ab pattern of restricted
electrophoretic heterogeneity and the presence of a l"-specific H-Ab at a high concentration. WABL combined with IgG subclass and light
chain type immunoblotting identified this
H-Ab as lgGl K (data not shown). FABL patterns showed identityof the dominantclone. The pattern
of the latter serum also shows some changes in the expression level of other clones. (B) The serum of 1986 showed a restricted patternand
an H-Ab at a high concentration. The serum of 1992 showed a more heterogeneous pattern with other dominant clones in addition t o the
original clone. (C) The serum of 1985 showed a restricted pattern with an H-Ab at a high concentration. The serum of 1992 showed a
heterogeneous pattern. The values from 8.0 t o 112.0 represent the lgG Ab titer toTT in AU per milliliter. The values from 1:25 t o 1:500 indicate
the dilution of the serum samples.
ACKNOWLEDGMENT
The authors thank D. Paddenburg for skilful technical assistance.
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1994 84: 4374-4382
Clonal dysregulation of the antibody response to tetanus-toxoid after
bone marrow transplantation
EJ Gerritsen, MJ Van Tol, MB Van 't Veer, JM Wels, IM Khouw, CR Touw, CM Jol-Van Der Zijde,
J Hermans, HC Rumke and J Radl
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