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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 8 (October 15), 1998:
pp. 2802-2814
Ig Heavy Chain Third Complementarity Determining Regions (H
CDR3s) After Stem Cell Transplantation Do Not Resemble the
Developing Human Fetal H CDR3s in Size Distribution and Ig Gene
Utilization
By
Erhan Gokmen,
Frank M. Raaphorst,
David H. Boldt, and
Judy M. Teale
From the Department of Medicine, Division of Hematology, and
Department of Microbiology, The University of Texas Health Science
Center at San Antonio, San Antonio, TX.
 |
ABSTRACT |
Previous studies have suggested that the B-cell repertoire after
stem cell transplantation resembles the developing repertoire in the
fetus. Fetal and adult repertoires differ strikingly at the molecular
level in Ig heavy chain third complementarity determining region (H
CDR3) size distribution and Ig gene utilization. Previously, the
posttransplant repertoire has not been studied fully in this regard. In
this study, we analyzed H CDR3s posttransplant using CDR3
fingerprinting, single-strand conformation polymorphism (SSCP), and
random sequencing. Eleven adult patients who received either autologous
(n = 6) or allogeneic adult sibling (n = 5) hematopoietic stem cell
transplants were studied. IgM H CDR3 repertoires demonstrated limited
clonal diversity within the first 6 to 10 weeks posttransplant. By 3 to
4 months, the IgM H CDR3 repertoires were as diverse as those in
healthy adults. Reconstitution of the IgM diversity correlated with the
expansion of the multimember VH3 family. By contrast, the
contribution of the single-member VH6 family was limited in most patients up to 6 to 9 months. No evidence was seen for greater contribution of VH6 posttransplant. IgG repertoires
remained clonally restricted at all times. In all patients, H CDR3
sizes fell within adult limits. Direct nucleotide sequencing of H CDR3s
showed adult-type N-nucleotide insertions and Ig gene utilization.
These results indicate that the emerging repertoire posttransplant does
not resemble the developing fetal repertoire at the molecular level.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
HIGH-DOSE CHEMO-RADIOTHERAPY with
hematopoietic stem cell support has become the standard treatment for
several otherwise incurable malignant and nonmalignant diseases.
However, the success of stem cell transplantation can be hampered by
delayed B-cell immune reconstitution and associated infectious
complications. Several studies have suggested that this delay is due in
part to the time period required for recapitulation of B-cell
ontogeny.1-11 To address this issue at the molecular level,
we have analyzed the emerging B-cell repertoire posttransplant using
fingerprinting and single-strand conformation polymorphism (SSCP) of Ig
heavy chain third complementarity determining regions (H CDR3s) in
combination with direct sequencing. This allowed for a global view of
the repertoire so that the nature and diversity of the emergent
repertoire could be objectively examined.
Ig heavy chain (Ig H) diversity is generated by somatic recombination
of variable (VH), diversity (DH), and joining
(JH) gene segments.12 Further diversification
is accomplished by the addition of nongermline encoded N-nucleotides to
the VH-DH and DH-JH
junctions by the enzyme terminal deoxynucleotidyl transferase (TdT).
Endonuclease activity and the addition of P-nucleotides provide
additional junctional diversity. These mechanisms together generate a
hypervariable segment of Ig H termed the third complementarity
determining region (H CDR3). H CDR3s provide essential residues for
direct interaction with antigens.
Diversification of H CDR3s is differentially regulated during ontogeny
resulting in characteristic differences between fetal and adult
repertoires. Several studies have shown that the single-member family
VH6 is preferentially used in the fetal
repertoire,13-19 comprising up to 10% of Ig H transcripts
in the second trimester. In contrast, 0% to 5% of transcripts in
adults use VH6.19-22 DHQ52, the
shortest DH element, is used in up to 40% of the fetal
transcripts14,15,17,18,23-25 and is rarely seen in the
adult repertoire.20-22,25-27 JH3 and
JH4 genes, among the shortest in nucleotide length, are
used frequently in the fetal repertoire.14,15,17,18,23
Because of limited TdT expression in utero,28 fetal Ig H
transcripts often lack or contain fewer numbers of
N-nucleotides.14,15,17,18,23,25 Together, these mechanisms
result in fetal H CDR3s that are smaller in size than that seen in
adults.
It has been argued that the newly acquired repertoire after bone marrow
transplantation (BMT) resembles a fetal one, suggesting that the full
ontogenetic program must be repeated for successful reconstitution of
the B-cell repertoire.6,7,9,11 Previous studies have
generally focused on morphologic, phenotypic, and functional
characteristics of the posttransplant B-cell repertoire6-8 and VH gene utilization.9,11,29 However, other
important molecular characteristics that typify fetal and adult
repertoires were not examined in detail. In fact, published studies of
H CDR3s after high-dose therapy and stem cell transplantation are
limited to VH6 containing rearrangements in 2 patients.30
The objectives of this study were to enhance our understanding of
B-cell generation posttransplant at the molecular level and to further
characterize the developing repertoire. In this study, the Ig H CDR3
size distribution and diversification posttransplant show that the
emerging repertoire is clonally restricted. However, the repertoire
does not resemble a fetal one, because it displays adult-size H CDR3s,
adult-type Ig gene utilization and no evidence of bias towards
VH6. IgG repertoire diversification lags significantly behind IgM. This suggests impaired memory reconstitution and provides an explanation for the immunodeficiency after stem cell
transplantation.
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MATERIALS AND METHODS |
Stem cell recipients and control subjects.
Eleven adult patients who underwent hematopoietic stem cell
transplantation between June and December 1996 in the BMT Units of the
University and the Audie L. Murphy Veterans Affairs Hospitals in San
Antonio under Institutional Review Board approved protocols were
studied. The patient characteristics are summarized in
Table 1. Four patients received allogeneic
BMT from HLA-identical siblings. These patients received
graft-versus-host disease (GVHD) prophylaxis consisting of cyclosporin
and a short-course of methotrexate. One patient with acute myeloid
leukemia (AML) who had relapsed after allogeneic BMT received a
leukocyte infusion from the same donor (HLA-identical sibling) after
cytoreduction. Intravenous immune globulin (IVIG) was administered to
allogeneic BMT recipients until day +100 posttransplant. Only 2 patients (patients no. 1 and 5) developed grade II GVHD involving the
skin and the gut at 13 and 9 weeks posttransplant, respectively. GVHD
was treated with cyclosporin and steroids. The remaining 6 patients
received autologous stem cell transplantation (2 peripheral blood stem cell transplantation [PBSCT], 2 BMT, 1 BM plus PBSCT, and 1 CD34 selected PBSCT). None of the autologous stem cell recipients received IVIG posttransplant. Granulocyte colony-stimulating factor (G-CSF) was
administered to all patients until the absolute neutrophil count was
greater than 5,000/µL in 2 consecutive days. Control subjects were
the healthy donors for the allogeneic transplant recipients.
Cell collection, separation, and RNA isolation.
Baseline peripheral blood (PB) samples were collected before transplant
from patients and donors. Posttransplant samples (minimum of 6) were
collected from patients at various times up to 9 months or until the
patients returned to their referring institutions. PB mononuclear cells
(PBMCs) were separated by Ficoll-Hypaque density gradient (Organon
Teknika, Durham, NC) and washed twice with phosphate-buffered saline
(PBS). RNA was isolated from approximately 1 × 107
PBMCs using TRIzol total RNA isolation reagent (Life Technologies, Gaithersburg, MD) according to the protocol supplied with the reagent.
cDNA synthesis and polymerase chain reaction (PCR) amplification.
One microgram of RNA isolated from blood samples was used for
first-strand cDNA synthesis using a combination of oligo(dT) and random
hexamers and SuperScript II Reverse transcriptase in a final volume of
20 µL. The protocol supplied with the cDNA synthesis kit was used
(Life Technologies).
The amount of Ig constant region transcripts was estimated for each
cDNA sample in semiquantitative PCRs to control for variations in
B-cell numbers and transcriptional activity. This method has been
previously used in TCR repertoire studies.31-34 The Ig
constant region transcripts were amplified in PCRs using 0.25, 0.5, 1, and 2 µL of the cDNA together with the Cµ15 and Cµ2RC primers (Table 2). PCR cycles consisted of 30 seconds of denaturation at 92°C, 35 seconds of annealing at
68°C, and 1 minute of extension at 72°C. PCR conditions were
otherwise identical to that described below. Samples were obtained
after 20, 23, 26, 29, and 32 PCR cycles to visualize the products.
Based on the amount of the Ig constant region transcripts estimated for
each sample, the amount of cDNA used in subsequent analyses was
adjusted in a final volume of 5 µL per PCR. In this way, it was
assured that samples with similar amount of constant region transcripts
were being compared.
Ig H CDR3s were amplified in nested PCRs to generate a sufficient
amount of product for fingerprinting and SSCP. Primers for PCR are
shown in Table 2. Ig H panVH (consensus) or family-specific VH3 or VH6 primers were used in combination
with Cµ15 or C 15 in the primary and Cµ5 or C5 in the first
nested PCRs. Primary and first nested PCRs consisted of 20 cycles
composed of 30 seconds of denaturation at 92°C, 35 seconds of
annealing at 68°C, and 1 minute of extension at 72°C (final
cycle, 10 minutes). The panVH-FR3 5 primer was used
in combination with Cµ2 or C2 in the second nested PCRs. This
primer is designed to recognize framework 3 (FR3) regions irrespective
of the VH family. The second nested PCRs consisted of 30 cycles of 30 seconds of denaturation at 92°C, 35 seconds of
annealing at 58°C, and 1 minute of extension at 72°C (final
cycle, 10 minutes). PCRs were performed in a final volume of 25 µL,
including 20 pmol of both 5 and 3 primers, 50 mmol/L KCl,
10 mmol/L Tris-HCl (pH 9.0), 0.1% Triton X-100, 1.5 mmol/L
MgCl2, 0.2 mmol/L of each dNTPs, and 2.5 U of Taq
DNA Polymerase (Promega, Madison, WI).
H CDR3 fingerprinting and SSCP.
For fingerprinting, PCR products were purified by phenol extraction,
separated on 5% denaturing polyacrylamide gels containing 7 mol/L
urea, and visualized by silver staining as described
previously.27,35 Briefly, samples were heated at 92°C
for 5 minutes and chilled on ice before loading. The gels were run for
approximately 2 hours at 65 W and fingerprinting profiles were
visualized using Promega silver staining system according to the
supplier's instructions. For SSCP,36,37 7% polyacrylamide
gels containing 5% ultrapure glycerol (J.T. Baker, Phillipsburg, NJ)
were run for approximately 7 hours at 25 W while being cooled with an
electric fan. SSCP images were obtained by silver staining (Promega).
The new technologies of H CDR3 fingerprinting and SSCP were used,
because they have overcome several shortcomings of the other methods
used in B-cell repertoire analysis. Hybridization with VH
family-specific probes, a method used in the previous analysis of
posttransplant repertoire,9 does not show the diversity of
clones within VH families. Flow cytometry has similar
limitations. Cloning and sequencing is technically laborious and,
unless the sample size is very large, the less frequently used genes
are overlooked. Fingerprinting of CDR3s, on the other hand, generates a
snapshot of the total repertoire.27,33,35 It also provides information regarding the clonality within VH families.
There are also limitations of the fingerprinting method. Although
clones within a VH family that differ in CDR3 size are
visualized as separate bands, fingerprinting does not show information
regarding the number of clones within a particular band. SSCP analysis
was incorporated in this study to overcome this limitation and to estimate the number of clones within a family.
Effect of B-cell numbers on H CDR3 fingerprinting.
To determine the influence of B-cell numbers on fingerprinting
profiles, we analyzed normal donor samples using serial dilutions of
PBMCs. B-cell numbers were determined by counting anti-CD19 antibody
(Becton Dickinson, San Jose, CA) labeled cells using fluorescence-activated cell sorting (Becton Dickinson). In these experiments, as few as 2 × 104 normal donor
CD19+ cells exhibited a diverse panVH,
VH3 and in some samples VH6 fingerprinting
pattern. In other samples, 3 to 7 × 104
CD19+ cells were needed for a diverse VH6
fingerprinting pattern. A threshold of 1 × 104 PBMCs
has been described for TCR repertoire analysis using
fingerprinting.33
To determine the influence of B-cell numbers on the VH6
repertoire posttransplant, IgM VH6 H CDR3 fingerprinting
profiles of 3 normal adults and 3 stem cell recipients were compared in a limiting dilution analysis using equal numbers of B cells. Samples from patients no. 1, 9, and 11 obtained at 36, 36, and 26 weeks, respectively, were studied. These samples were chosen for analysis based on the availability of the flow cytometric data. B-cell numbers
were determined by counting anti-CD19 antibody (Becton Dickinson)
labeled cells using fluorescence-activated cell sorting (Becton
Dickinson). PBMCs containing 5 × 105
CD19+ cells were used for each patient and normal adult.
RNA isolation and cDNA synthesis were performed as above. Before the
PCR amplification of the VH6 transcripts, the total amount
of constant region transcripts was estimated for each sample using PCR
amplification with Cµ15 and Cµ2RC primers as described above.
Similar to that described in T-cell receptor repertoire
studies,31-34 potential differences in the transcriptional
activity between samples can be detected in this way. All 6 samples
exhibited similar transcriptional activity in these studies; therefore,
no further adjustment was necessary. PCRs were performed using 5 µL
cDNA and 10-, 100-, and 1,000-fold dilutions in an attempt to simulate
the low B-cell counts of the posttransplant period. PCRs and
fingerprinting were performed as described above. In these experiments,
dilutions of cDNA instead of PBMCs were used, because patient samples
stored in TRIzol reagent were analyzed retrospectively.
Cloning, sequencing, and analysis of H CDR3s.
Nested PCR products were used for random cloning and sequencing as
described elsewhere.27,35 The H CDR3 sequences were analyzed using DNASTAR software (DNASTAR Inc, Madison, WI). Kabat's nomenclature38 was used for identification of H CDR3
regions. V BASE directory and the published germline DH
gene sequences were used to search for homology with known human
germline DH and JH sequences.39-46
Straight and inverted orientation of DH sequences were
analyzed using DNASTAR COMPARE and SEQCOMP programs. For H CDR3s >12
amino acids (aa), a minimum of 9 consecutive base pair (bp) identity
and for H CDR3s  12 aa, a minimum of 6 consecutive bp identity was
required with known germline DH genes for DH
gene assignment. The sequences analyzed in this study can be found in
the GeneBank Database under accession nos. AF028092-AF028121.
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RESULTS |
B-cell repertoire is oligoclonal early posttransplant.
Figure 1A, B, and C show PB IgM H CDR3
fingerprinting profiles of a normal donor (NL) and recipients of
allogeneic BMT (patient no. 1), autologous PBSCT (patient no. 9), and
CD34 selected autologous PBSCT (patient no. 11), respectively. IgM
panVH profiles were generated at various times
posttransplant by PCR amplification of H CDR3s using the Ig H consensus
primer together with the Cµ primers. As shown in lanes NL, a
typically diverse PB repertoire of a normal donor is characterized by
16 to 20 bands separated from each other by 3 bp. Each band corresponds
to a certain H CDR3 length. The bands show a Gaussian distribution in
intensity, with central (average size) bands being darker than the
larger and shorter bands at both ends. Intensity of a band correlates directly with the total amount of H CDR3s present within the
band.33
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| Fig 1.
IgM H CDR3 fingerprinting after (A) allogeneic BMT
(patient no. 1), (B) autologous PBSCT (patient no. 9), and (C) CD34
selected autologous PBSCT (patient no. 11). IgM H CDR3s were amplified
in PCRs using PBMC cDNA as described in Materials and Methods. The Ig H
panVH (consensus) primer, VH3 primer, or
VH6 primer was used in combination with the Cµ primers to
obtain panVH, VH3, or VH6
fingerprinting profiles, respectively. PCR products were run on 5%
polyacrylamide sequencing gels that result in resolution of CDR3s
according to basepair size. Therefore, each band in fingerprinting
corresponds to a certain CDR3 size. Molecular markers (MW) are provided
of known basepair size and the corresponding number of encoded aa.
Bands are separated from each other by 3 bp because of the abundance of
in-frame Ig transcripts in total RNA. Nevertheless, violation of 3-bp
spacing of bands can be seen occasionally. The numbers above the lanes
indicate the number of weeks when the blood samples were collected
posttransplant. Lanes marked NL show the H CDR3 profiles of a normal
donor. The number of bands in each lane correlates directly with the
repertoire diversity. The band intensity reflects the total amount of
CDR3 transcripts that comprise the band. The band intensities are
comparable to each other within a lane. Absolute lymphocyte counts per
microliter at corresponding weeks were as follows: (A) 10 (2 weeks),
136 (3 weeks), 196 (4 weeks), 398 (5 weeks), 1,175 (7 weeks), 1,479 (10 weeks), 1,295 (12 weeks), 627 (14 weeks), 910 (24 weeks), and 1,240 (36 weeks); (B) 200 (2 weeks), 500 (4 weeks), 1,918 (6 weeks), 1,500 (19 weeks), and 1,231 (36 weeks); (C) 19 (2 weeks), 200 (3 weeks), 700 (4 weeks), 400 (6 weeks), 1,000 (7 weeks), and 1,100 (26 weeks). Shown are
IgM fingerprinting profiles representative of 11 patients and normal
donors of allogeneic BMT recipients.
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When patients were analyzed at various times posttransplant, fewer
bands were obtained for up to 6 to 10 weeks after transplantation compared with the normal donor sample. This suggests a more restricted B-cell repertoire during this time (panVHs in Fig 1A, B,
and C). Primary and nested PCRs were performed in triplicates to
ascertain that fewer bands observed early posttransplant were not
simply due to preferential amplification of certain H CDR3s. The same results were obtained in these experiments, consistent with a more
restricted repertoire early posttransplant. A few of the bands in some
samples appeared darker in intensity than expected from the Gaussian
distribution when compared with the other bands in the same sample.
This observation suggests clonal expansions. At around 3 to 4 months
posttransplant, the number of bands reached that seen in the normal
adult repertoire.
Contribution of VH3 and VH6 families to
posttransplant Ig H CDR3 repertoire.
To determine the contribution of individual families to posttransplant
repertoire, fingerprinting analysis was performed at several times
posttransplant using the Cµ primers together with either the
VH3 or VH6 specific primers instead of the
consensus Ig H primer (Table 2). VH3 is the largest
VH family, with approximately 22 functional members that
account for 50% to 60% of rearrangements in the normal adult
repertoire.20-22 Limited number of bands were observed in
IgM VH3 H CDR3 profiles within the first 6 to 10 weeks (Fig
1A, B, and C). This would be predicted from the restricted panVH H CDR3 repertoire seen during the same time period.
However, by 3 to 4 months, the IgM VH3 repertoire in all
recipients was as diverse as in healthy adults.
VH6 is a single-member family that has been reported to be
overly expressed after BMT.9,11,30 The IgM VH6
repertoire showed a limited number of bands for as long as 6 to 9 months posttransplant (Fig 1A, B, and C). This suggests a relatively small contribution of this family and a limited diversity. In light of
the fact that VH6 represents less that 5% of transcripts in normal individuals,19-22 it was important to determine
the influence on these results of overall B-cell numbers, which are
known to be low during this period. To this end, IgM VH6 H
CDR3 repertoires from PB of 3 normal adults and 3 stem cell recipients
were compared using 5 × 105 CD19+ cells.
VH6 profiles showed missing bands in specimens from 2 of 3 normals and 2 of 3 patients (Fig 2). This
would be supportive of the smaller contribution of VH6 to
the repertoire compared with VH3 but fails to support a
greater contribution of VH6 in reconstituted patients. In
addition, fingerprinting profiles for VH6 were generated
using 10-, 100-, and 1,000-fold dilutions of the cDNA from all 6 individuals in an attempt to simulate the low B-cell counts of the
posttransplant period (Fig 2). Under conditions representing limiting
B-cell numbers, the differences in profile complexity between normals
and patients became more obvious, with patients, if anything, being
less complex. Taken together, these results suggest a limited but
variable contribution of VH6 in both normal and patient H
CDR3 repertoires, with no apparent evidence of a greater contribution
of VH6 in the patient population.
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| Fig 2.
Comparison of the IgM VH6 H CDR3
fingerprinting profiles of 3 normal adults and 3 stem cell transplant
recipients using equal numbers of B cells for cDNA synthesis.
Fingerprinting profiles were obtained using 5, 0.5, 0.05, and 0.005 µL of the cDNA. p1, 36-week sample from patient no. 1; p2, 36-week
sample from patient no. 9; p3, 26-week sample from patient no. 11 (patient numbers are according to Table 1). n, normal donor.
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Analysis of H CDR3s by SSCP.
With the technique of fingerprinting, the excessively dark bands could
be interpreted as a clonal population or many different clones with H
CDR3 sequences of the same size.33 To distinguish between
these possibilities, SSCP studies were performed. Using SSCP analysis,
different clones sharing the same H CDR3 size can be separated as
individual bands based on differences in tertiary structures.36-37 IgM H CDR3s from PB of normal adults
showed a smear-like pattern in SSCP analysis, indicating the presence
of polyclonal H CDR3s (lane NL in Fig 3).
In contrast, samples obtained within the first 7 weeks posttransplant
showed several bands with less intense background (Fig 3). SSCP studies
were performed in duplicate to confirm the reproducibility of the
observed patterns. These data indicate that the limited number of bands
early posttransplant in fingerprinting profiles reflect relatively few
clones. Expansion of the IgM H CDR3 repertoire to normal adult levels
by 10 weeks posttransplant was shown by SSCP analyses that showed a
smear-like pattern typical of a polyclonal repertoire (Fig 3).
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| Fig 3.
Analysis of IgM H CDR3s posttransplant by SSCP. IgM
panVH, VH3, and VH6 H CDR3s were
amplified in nested PCRs as described in Materials and Methods. Two
times the amount of PCR product used in fingerprinting experiments was
loaded on to 5% polyacrylamide gels containing 5% glycerol. Gels were
run at room temperature for 7 hours while being cooled by an electrical
fan. Images were obtained by silver staining. In SSCP, CDR3s of the
same basepair size can be separated due to differences in their
mobility based on their tertiary structures. NL, normal donor. Pt,
patient. Patient numbers are according to Table 1. Patient no. 12, who
died of refractory NHL 2 months after an autologous PBSCT,
was not included in other analyses. Bands indicated by the arrows
represent renatured DNA determined by the simultaneously run control
samples that were not denatured before loading (data not shown).
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H CDR3s posttransplant are adult size.
The size of a H CDR3 is determined by the size of DH and
JH elements used in the rearrangement and the number of
nucleotides inserted or deleted at VH-DH and
DH-JH junctions. Adult human H CDR3s range
between 4 and 30 aa in size, with an average of 15 aa (lanes NL in Figs
1 and 4).
Fetal H CDR3s, on the other hand, are significantly shorter in length.
This is depicted in Fig 5, which compares
the size distribution of fetal liver H CDR3s with those of adult PB and
BM. In this study performed by F.M.R. using the same fingerprinting
methodology, the 12-week-old fetal liver H CDR3s ranged in length from
4 to 16 aa in two samples and 6 to 18 aa in the third
sample.27 Shorter sizes of fetal H CDR3s have also been
shown by direct nucleotide sequencing.14,15,17,18,23,25 In
all patients analyzed in this study, the size of H CDR3s posttransplant fell within the adult range. Most H CDR3s were longer than the average
fetal H CDR3 size of 10 aa. This was observed even in the earliest
stages posttransplant when the repertoire was limited in diversity
(Figs 1 and 4). H CDR3s as long as 20 aa were not uncommon. It should
be noted that adult size range includes those seen in the fetal
repertoire; however, H CDR3s longer than 16 to 18 aa are absent in the
fetal repertoire, although they are common in the adult and
posttransplant repertoire. These results suggested extensive
N-nucleotide additions to the recombination joints and/or
absence of a bias towards utilization of shorter DH and
JH elements posttransplant, unlike that seen in the fetal repertoire.
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| Fig 4.
IgG H CDR3 fingerprinting after (A) allogeneic BMT
(patient no. 1), (B) autologous PBSCT (patient no. 9), and (C) CD34
selected autologous PBSCT (patient no. 11). IgG panVH,
VH3, and VH6 H CDR3 fingerprinting profiles
were obtained as described in Fig 1 and Materials and Methods. C
primers were used instead of Cµ primers to amplify H CDR3s in PCRs.
MWs are provided of known basepair size and the corresponding number of
encoded aa. The numbers above the lanes indicate the number of weeks
when the blood samples were collected posttransplant. Absolute
lymphocyte counts at corresponding weeks are as shown in Fig 1. Lanes
marked NL show the IgG H CDR3 profiles of a normal donor. IgG H CDR3
repertoire reconstitution was significantly delayed posttransplant.
Shown are IgG fingerprinting profiles representative of 11 patients and
normal donors of allogeneic BMT recipients.
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| Fig 5.
IgM panVH H CDR3 fingerprinting on human
fetal liver and adult B cells. Fingerprinting methodology is identical
to that described in the Materials and Methods. Abbreviations: ABM,
adult BM; APBMC, adult peripheral blood mononuclear cells;
CD10 , CD10CD20+ mature
B-cell population sorted from ABM by FACS; CD10+,
CD10+ CD20+ pre-B cells sorted from ABM by
FACS; FL, 12-week-old human fetal liver; MW, molecular markers; nt,
nucleotides; aa, amino acids. Reprinted with permission from
International Immunology, Vol 9, No 10, pp 1503-1515 by permission of
Oxford University Press.
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Direct nucleotide sequencing of H CDR3s confirms an adult repertoire
pattern.
A nucleotide sequence analysis of 32 panVH H CDR3 clones
obtained from patients no. 1, 9, and 11 at 7, 16, and 6 weeks
posttransplant, respectively, was performed to further investigate
whether adult size H CDR3s observed posttransplant contained adult-like
N-nucleotide insertion and DH-JH gene
utilization (Fig 6). These time points were
chosen because they roughly corresponded to the expansion of H CDR3
repertoire in fingerprinting experiments. Two clones each from patients
no. 1 and 11 had identical sequences. All rearrangements were in-frame.
The average H CDR3 size was 13.8 aa (range, 8 to 22 aa). Only four
clones were shorter than the average fetal H CDR3 size of 10 aa. All
clones exhibited N-region additions. Two clones lacked N-nucleotides at
either VH-DH or DH-JH
joint but not at both. The remaining 30 clones contained N-nucleotides
at both recombination sites. The mean numbers of N-or P-nucleotides in
VH-DH and DH-JH joints
were 5.6 (range, 0 to 21) and 8.2 (range, 0 to 26). DH gene
assignment was not possible in 7 clones according to the sequence
homology required by established criteria. Members of the DXP family
were used in 9 rearrangements, whereas the remaining clones used
members of DLR (3 clones), DK (3 clones), DN (5 clones), and DM (1 clone). In 3 clones, there were convincing sequence homologies with DIR
genes alone. One clone was best explained with a DN-DIR fusion.
DHQ52 gene utilization, which is well documented in up to
40% of the fetal transcripts by several
groups14,15,17,18,24,25 and by one of the authors of this
study (F.M.R.)23 was not observed in any of the 32 clones.
Eleven clones used JH3, 15 clones JH4, and 6 clones JH6. These characteristics of the posttransplant PB
H CDR3s are adult-like and similar to that described for adult BM pre-B
and mature B cells.27
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| Fig 6.
Direct nucleotide sequencing of posttransplant H CDR3s.
Shown are the 6 representative panVH CDR3 sequences from
patients no. 1, 9, and 11. JH family assignments are shown
in parentheses following the sequences. Also shown are the germline
(GL) DH sequences that match CDR3s. Asterisks represent
gaps introduced to optimize homology. N- and P-nucleotides are
underlined. Italic letters represent P-nucleotides. CDR3 aa sizes are
shown.
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IgG repertoire reconstitution significantly lags behind IgM
repertoire reconstitution.
To gain insight into the memory compartment of B-cell immunity
posttransplant, IgG H CDR3 fingerprinting profiles were obtained. Shown
in Fig 4A, B, and C are IgG panVH, VH3, and
VH6 H CDR3 fingerprinting profiles of an allogeneic BMT
recipient (patient no. 1), an autologous PBSCT recipient (patient no.
9), and a CD34-selected autologous PBSCT recipient (patient no. 11),
respectively.
The IgG panVH, VH3, and VH6 H CDR3
repertoires in patients were strikingly less complex when compared with
normal donor counterparts (Fig 4A, B, and C). The number of bands in
most patient samples did not reach adult levels as late as 9 months
posttransplant. Nevertheless, an improvement in IgG panVH
repertoire was seen in most patients, as demonstrated by the increasing
number of bands observed over time (panVHs in Fig 4B and
C). The expansion of the IgG panVH repertoire in these
patients was likely due to usage of many VH families,
because the expansion observed could not be attributed solely to the
IgG VH3 and/or VH6 H CDR3 repertoires (Fig 4B and C). The IgG panVH, VH3, and
VH6 H CDR3 repertoires were significantly less complex also
when compared with the IgM counterparts of the same patient (Figs 1 and
4). The IgG panVH H CDR3 repertoire of a representative
patient shown in Fig 4A showed 4 bands at 12 weeks, whereas the IgM
counterpart of the same patient shown in Fig 1A contained 18 bands at
12 weeks.
Typical IgG panVH and VH3 H CDR3 repertoires of
a normal donor (NL in Fig 4) are characterized by 16 to 20 bands,
similar to that seen in the normal IgM counterparts. However, the
distribution of band intensities in IgG repertoires did not show a
clear pattern. In contrast to the average size bands (11 to 16 aa)
being darker in IgM panVH and VH3 H CDR3
repertoires, the darker bands in the IgG panVH and
VH3 H CDR3 repertoires of the normal donor in Fig 4 were 21 and 31 aa in size. IgG VH6 H CDR3 repertoire of the donor
showed approximately 15 bands, 4 of which were significantly darker
than others. The darker bands were not clustered according to size.
These results suggest clonal expansions. Violation of the 3-bp spacing
of the bands was not uncommon in the IgG VH6 H CDR3
repertoire. This may reflect PCR artifacts resulting from limiting
amounts of template. These observations suggest that there is low
abundance of functional IgG VH6 transcripts in the normal
adult repertoire in general, but that expansion of a few clones is
likely.
| |
DISCUSSION |
In this study, B-cell repertoire reconstitution after hematopoietic
stem cell transplantation was analyzed by monitoring Ig H CDR3
diversification using fingerprinting and SSCP in combination with
sequencing. In contrast to previous reports,6,7,9,11,30 it
is shown that the emerging repertoire posttransplant is not fetal-like
when assessed at the molecular level. This conclusion is based on the
demonstration of adult-size H CDR3s and absence of a fetal-like Ig gene
utilization posttransplant.
Several studies have examined B-cell reconstitution posttransplant in
terms of quantitative recovery, morphology, phenotype, and function,
whereby both similarities and differences with fetal B cells were
observed (reviewed in Storek and Saxon7 and Storek et
al8). In the few studies in which posttransplant B-cell repertoire was examined at the molecular level, 9,11,29,30
the characteristics that typify fetal and adult repertoires were partially addressed. Despite only modest similarities between fetal and
posttransplant repertoires, it has been concluded and generally
accepted that a fetal-like pathway is followed in B-cell recovery
posttransplant recapitulating ontogeny.
One of the most striking characteristics of the fetal repertoire is the
relatively small size of H CDR3 regions.14,15,17,18,23,25 This is in part due to N-nucleotide insertions either being absent or
limited in the fetus. Sequencing the expressed H CDR3s was particularly
informative relative to the question of an adult versus fetal-like
N-region diversification. Consistent with the adult-like size of the H
CDR3s detected by fingerprinting, all of the sequences analyzed (n = 32) exhibited N-insertions indicative of an adult repertoire.
Interestingly, in a study in which VH6 sequences from 2 patients were examined posttransplant, all clones also showed N-region
additions in either VH-DH or
DH-JH joints or in both.30 This
contrasts a fetal pattern of diversity in which a significant portion
of VH-DH or DH-JH
joints lack N-regions.14,15,17,18,23,25 Because the
VH6 H CDR3 sizes were found to be close to the length of
the relatively small sample size of fetal H CDR3,47 it was concluded that the posttransplant sequences were fetal-like despite adult-like N-nucleotide insertions.30
We found no evidence posttransplant for a biased expression of Ig gene
segments that typify the fetal repertoire. The DH genes expressed are characteristic of an adult repertoire. The
DHQ52 gene, which is expressed in nearly half of fetal Ig H
chain transcripts,14,15,17,18,23-25 was not found in any of
the sequenced transcripts from patients. JH genes
identified in sequenced clones in this study (JH3,
JH4, and JH6) are common in both fetal and
adult repertoires.14,15,17,18,23-25 Several studies have
suggested preferred expression of VH6 in the fetal
repertoire,13-19 although this is
controversial.48 Our results suggest that the contribution
of VH6 is not increased posttransplant relative to that
seen in normal adults. This is in contrast to that shown by Storek et
al,11 which indicated up to 10-fold higher utilization of
VH6 at 6 to 12 months posttransplant. In another study by
Fumoux et al,9 increased utilization of VH6 was
shown at 60 days but not at 30 or 90 days after allogeneic BMT. In the
same study, VH6 utilization was not found to be different after autologous BMT than in normal adults. Because we examined 26- and
36-week samples in the limiting dilution analysis, we cannot exclude an
increased contribution of VH6 during earlier time points
posttransplant, as previously suggested.9 However, it is
important to note that in the previous studies the largest proportion
of B cells expressing VH6 was 7% and was usually in the
0.1% to 2% range. VH6-containing rearrangements were
shown previously to have a relatively high frequency of replacement mutations, suggesting positive selection of these clones
posttransplant.30 Moreover, identical
VH6-containing rearrangements were identified several
times, suggesting clonal expansion. Therefore, it is possible that the
marginal increases in VH6 usage reported in previous studies of posttransplant patients9,11,30 represents
oligoclonal proliferations rather than preferred usage of
VH6 in clonally diverse B cells.
Conclusions drawn in previous studies regarding a fetal-like emergence
of the lymphocyte repertoire after transplantation of adult stem cells
to patients seem counterintuitive based on the results of the previous
preclinical studies addressing this issue. There is considerable
evidence, particularly in mice, that fetal lymphoid precursors are
distinct from adult precursors in terms of morphological
characteristics, surface marker expression, TdT expression, and
antigen-specific receptor repertoires.49-51 Studies have
been performed to address the question of whether the source of stem
cells (fetal v adult) or the host microenvironment (fetal
v adult) is more important in shaping the characteristics of
the emerging lymphocyte repertoires. In our own laboratory, it was
found that VH utilization in developing fetal B cells
remained fetal-like when propagated in the presence of an adult BM
stromal layer, arguing against the fetal phenotype resulting from
microenvironmental differences.52 Similarly, in mouse fetal
thymic lobe repopulation studies using T-cell precursors from either
fetal liver or adult BM, it was found that the extent of TCR
N-nucleotide insertions was determined by the progenitor source rather
than the microenvironment.53-54 However, in another study,
reconstitution of adult mice with either fetal liver or adult BM
precursors gave rise to lymphocytes with adult
characteristics.55 In any event, adult- but not fetal-like lymphocyte characteristics at the molecular level were observed in
these studies when either adult progenitor cells or adult hosts were
used. Based on these studies, an adult-type repertoire would be
predicted after transplantation of adult progenitors to adult hosts.
In the study presented here, an adult-like repertoire was evident in
all patients analyzed, including the patient receiving CD34+ selected stem cells, whereby CD34
B-cell precursors are largely eliminated. This observation provides evidence against the possibility that adult-like repertoire
posttransplant could be simply due to passive transfer of mature B
cells with established adult characteristics. The patient population in
this study was diverse in terms of stem cell source (allogeneic [n = 5], autologous [n = 6], BMT [n = 6], PBSCT [n = 4], and BM + PBSC [n = 1]), preparative regimen, and disease type.
Adult-like B-cell repertoires observed in all patients suggest that
results are not likely to be biased by any one of these variables.
Our results show a significant lag in diversification of IgG H CDR3s
posttransplant, suggesting limitations in secondary B-cell responses
and memory formation. Previous studies indicate that serum IgM levels
return to normal within 3 to 6 months, whereas recovery of serum IgG
levels can be delayed up to 1 year or longer (reviewed in
Lum,2 Atkinson,5 and Storek and
Saxon7). Other criteria for secondary B-cell responses and
memory development include loss of membrane IgD on antigen-specific B
cells after stimulation and increasing somatic mutation of the CDRs. In
an analysis of 59 BMT recipients, Storek et al56 showed
significantly fewer IgD B cells at 1 year
posttransplant than in normal controls. In addition, Suzuki et
al29 showed that VH genes exhibited fewer somatic mutations posttransplant in 4 patients. This is consistent with
the data shown here indicating restricted diversity in the IgG H CDR3s
being expressed. Taken together, these studies show a significant
impairment in the memory compartment posttransplant. Given that IgG and
memory responses are dependent on germinal center formation, the
kinetics and quality of restructuring the architecture of the secondary
lymphoid organs after transplantation will have a significant impact on
reconstitution of the primary B-cell repertoire. In this regard,
histological analyses of spleen and lymph nodes from nonsurviving
patients of allogeneic BMT showed reduced cellularity and an absence of
lymphoid follicules and germinal centers.57-60 Another
important factor in Ig class switch and memory response is the help
provided by T cells. Failure of T cells has been shown
posttransplant.61-66 Therefore, successful recovery of
secondary B-cell responses will also be dependent on the recovery of
the T-cell compartment.
The data presented here expand the knowledge of B-cell immune
reconstitution posttransplant and demonstrate that it does not recapitulate fetal ontogeny at the molecular level, as previously suggested. Therefore, the delay in B-cell reconstitution is unlikely due to the need for fetal reprogramming. Posttransplant immune deficiency appears to be rather due to functional B-cell defects described in several studies (reviewed in Lum,2
Atkinson,5 and Storek and Saxon7) and an
impairment in the memory compartment that is dependent on germinal
center formation and proper T-cell help. Recent studies have
demonstrated improved numerical and functional recovery of B cells
after allogeneic PBSCT.67,68 One technique to improve
memory compartment reconstitution posttransplant may be adoptive
transfer of donor memory cells. To that end, we are currently
investigating the H CDR3 repertoire reconstitution in correlation with
numerical and functional B-cell recovery after PBSCT and also exploring
the expansion of the B-cell compartment in ex vivo expansion systems
for potential posttransplant use.
| |
FOOTNOTES |
Submitted February 4, 1998;
accepted June 10, 1998.
Supported by Grants No. A I 19896, A I 33221, and N S 35974 from the
National Institutes of Health. E.G. is supported by a fellowship grant
by Amgen.
Address reprint requests to Judy M. Teale, PhD, Department of
Microbiology, The University of Texas Health Science Center at San
Antonio, 7703 Floyd Curl Dr, San Antonio, TX 78284; e-mail: Teale{at}uthscsa.edu.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Dr Manuel Santiago for his help in preparation of the
figures and for his support of fellow research training. We also thank
Oxford University Press for the permission granted to reprint Fig 5.
| |
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Abstract
Introduction
Abstract