1. No category

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1996 87: 2795-2804
Evidence for oligoclonal diversification of the VH6-containing
immunoglobulin repertoire during reconstitution after bone marrow
transplantation
I Nasman and I Lundkvist
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Copyright 2011 by The American Society of Hematology; all rights reserved.
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Evidence for Oligoclonal Diversification of the VH6- Containing
Immunoglobulin Repertoire During Reconstitution
After Bone Marrow Transplantation
By Ingrid Ngsrnan and lnger Lundkvist
Patients who have undergone bone
marrow transplantation
(BMT) remain immunodeficient for months to years posttransplantation. To evaluate the basic molecular events underlying reconstitution of the humoral immune response,
we have performed a detailed nucleotide sequence analysis
of VH6 containing rearrangements
in circulating B cells
from
two BM donorlrecipient pairs. Our results show that the
third complementarity determining region (CDR3) diversity
is much lower early after transplantation, compared with
that of the donors, and that the clonal variability remains
low for 3 months. Repertoire diversificationfollows an oligoclonal pattern where B lymphopoiesis appears to occur
in waves up to 6 months posttransplantation. The repertoire
among donated marrow cells is not reflected in peripheral
blood lymphocytes fromthe transplanted patients. There is
differential D geneutilization among both donor andpatient
samples, whereas JH gene usage is biased toward Jn4, 5,
and 6. One month after transplantation the vast majority
of the sequenced clones are functional and contain a high
frequency of replacement mutations in the CDRs of the v ~ 6
gene. We conclude that lg gene expression is very restricted
early after B M 1and that development of the B-cell repertoire
appears to follow a wavelike pattern.
0 1996 by The American Society of Hematology.
I
frequently expressed family is vH3. Interestingly, vH3 gene
family usage in the fetal repertoire is not random but three
genes, 2 0 ~ 1 ,30p1, and 5 6 ~ 1 ,are markedly overrepre~ented.’~.’~
The vH3 family also dominates the repertoire
among peripheral B lymphocytes from healthy a d ~ l t s . ~ ~ ~ ’ ~
Among naturally activated lymphocytes, however, the VH6
gene is overrepresented while expression of the vH1 and
vH3 gene families is decreased (Davidkova G, Pettersson S,
Holmberg D, Lundkvist I: Selective usage of VH-genes in
adult human B lymphocyte repertoires. Submitted for publication). Fumoux et al’’ have analyzed VH gene family usage
in BMT patients and found that vH3 utilization is decreased
twofold to threefold, compared with normal adults, and is
compensated for by transient overexpression of vH4, vH5,
and VH6.This bias in VHgene family usage is most significant 60 days after transplantation, although the pattern is
obvious also at 30 and 90 days posttransplantation. It has
also been reported that the vH3 genes that characterize the
fetal repertoire, 56pl and 2 0 ~ 1as
, well as the v H 6gene are
markedly overutilized 2 to 5 months posttransplantation.2n
At 21 months after BMT the percentage of B cells that use
these genes are still twofold to sixfold higher than in healthy
adults.”
The importance of the VH6 gene in fetal and neonatal
VH gene repertoires, together with the similarities between
ontogenic development and the posttransplantation humoral
T IS WELL KNOWN THAT bone marrow (BM) transplant recipients develop a cellular and humoral immune
deficiency that can last for months to years after the transplantation. The time required for recovery is prolonged if
the patient also develops chronic graft-versus-host disease
(GVHD).’ It has been suggested that reconstitution of the
humoral immune system follows ontogenic de~elopment.’.~
The first circulating IgM+ B lymphocytes are detected in
the periphery 2 to 4 months posttransplantation’ and the
phenotype of these B cells resembles cord blood B cell^.^,^
There are few IgG and IgA expressing B cells present after
BM transplantation (BMT).3 The B-cell deficiency in BMT
patient^^.^ has been suggested to be caused by defective B
cells, decreased TH cell function, or a suppressive effect
caused by T cells or natural killer (NK) cells.’ The serum
levels of Igs are normalized 6 months (IgM and IgG) to 1
year (IgA) post BMT.’ Although total IgG levels are normal,
the subclass pattern is biased against y2 and 74,compensated by an increased amount of y 1.
The pattern of reconstitution of cellular subsets and serum
Igs evoked the question whether the posttransplantation immune deficiency could be explained by restriction in utilization of Ig genes similar to that seen in ontogeny. A number
of studies have addressed the question of expression of genes
coding for the variable region of the Ig heavy chain (V,) at
various time points in ontogeny and in central and peripheral
lymphoid tissue. The variable and constant region genes for
the human Ig heavy chain are located on chromosome 14q.
The diverse repertoire of Ig specificities is obtained through
a multi-step process where the initial event is recombination
of one of at least 30 diversity (D) genes’ to one of six joining
(JH) genes9 This D-JH complex is then recombined to one
of roughly 100 VH
In addition to combinatorial
diversity,15junctional diversity (addition of N and P nucleotide~),“.’~
differential association with K or A light chains,
and somatic mutation’’ contribute to the variability of the Ig
repertoire. The human VH genes are divided into seven different families based on nucleotide sequence hom01ogy.l~
The largest family is vH3, which contains 28 gene segments
with open reading frame and the smallest is V,6, with one
functional gene.20
Studies on VH gene usage in the fetal repertoire show an
overrepresentation of the VH6family,21.22
although the most
Blood, Vol 87, No 7 (April I ) , 1996: pp 2795-2804
From the Department of Immunology, Microbiology, Pathology
and Infectious Diseases, the Division of Clinical Immunology, Karolinska Institutet at Huddinge University Hospital, Huddinge, Sweden.
Submitted June 28, 1995; accepted July 27, i995.
Supported by grants from the Children Cancer Foundation of
Sweden, Jeansson’s foundations, King Gustaf the Vth 80-Year Foundation, the Swedish Cancer Society, and the Baxter Novum Research
Laboratory.
Address reprint requests to Ingrid Nasman, BSc, Division of Clinical Immunology, F79, Huddinge University Hospital, S-I41 86 Huddinge, Sweden.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 19% by The American Society of Hematology.
0006-497//96/8707-0006$3.00/0
2795
From www.bloodjournal.org by guest on December 3, 2014. For personal use only.
2796
NASMAN AND LUNDKVIST
immunesystem, haveledus
to study the developmental
program of Ig gene rearrangementpost BMT. To gain better
insight into the molecular mechanismsunderlying repertoire
expressionposttransplantation, we have collectedmaterial
before and at several timepoints after BMT and performed
a detailed nucleotide sequence analysis of VH6-containing
rearrangements. For this purpose we haveused the polymerase chain reaction (PCR) technique with VH6-specific and
JH consensus primers. The first post BMT sample is taken
amount of B
at 4 weeks,ie, before there isasignificant
lymphocytes in the periphery. We have also analyzed samples from the BM donor to be able to evaluate
the importance
of the repertoire of the donated cells versus that
of the microenvironment on reconstitution of the immune system.
MATERIALS ANDMETHODS
Patients and donors. Peripheral blood (PBL) samples were derived from two BM recipients. The first (patient l ) was a 50-yearoldwomanwiththediagnosisacutelymphoblasticleukemiaof
pre-B type (pre-B ALL, LI). TheBM donor (donor 1) was an HLAidentical sibling. Patient 1 was in partial remission with 7% to 8%
lymphoblasts in theBM 1 week beforeBMT.Theprotocolfor
conditioning of patient I was cyclophosphamide (60 mg/kg/d) for
2 consecutive days (days S and 4 before BMT) followed
by total
body irradiation with a dose of 10 Gy. The lungs were shielded and
I wasadministered
receivedadose of 9Gy.AfterBMTpatient
methotrexate
and
cyclosporin
as
prophylaxis
against
GVHD.
3 months. Details reCyclosporin treatment was administered for
garding treatment have previously been published.2" Six days after
BMT patient I developed a mild (grade I) acute GVHD manifested
as a localized skin rash. Five months after BMT patient 1 developed
chronic GVHD that engaged the skin and mouth cavity. Before BMT
patient I was negative for cytomegalovirus (CMV), but 2months
after BMT CMV was detected in blood samples by PCR, the infection was asymptomatic. Nine months after BMT patient I is healthy
with no relapse. The second recipient (patient 2) was a 44-year-old
man with the diagnosis lymphoma (K1 +). The BM donor (donor 2)
was an HLA-identical sibling. Patient 2 was in complete remission
with no detectable tumor cells in the BM I week before BMT. The
protocol for conditioningof patient 2 was as described above. Patient
2 was treated with methotrexate and cyclosporin. No sign of GVHD
was seen and the cyclosporin treatment was stopped 4 months after
BMT. Patient 2 had IgG antibodies against CMV before BMT and
theseantibodieswere
still present 6 months post BMT. No IgM
anti-CMV antibodies were detected in serum. There were no signs
of CMVdisease.Patient2
is healthy with norelapse I 1/2 years
after BMT.
Serum Ig levels and white blood W// count. Values in Table I
were obtained from standardized routine analysisof patient samples.
Cell preparation. Mononuclearcellswereobtainedfrom
both
BM andPBL by gradientseparationonlymphoprep(NycomedPharma AS, Oslo, Norway). The cells were washed twice in RPM1
(GIBCO BRL, Life Technologies Ltd, Paisley,UK) without supplements and the cell pellets from PBL samples were frozen in -70°C
until genomicDNApreparationswereperformed.BMcellswere
used for enrichment of pre-B and mature B lymphocytes.
Monoclonal antibodies (MoAhs). Fluorescein (F1TC)-conjugatedanti-Calla(CD10).cloneW8E7ofmouse
Ig%/K isotype
(Becton Dickinson, Mountain View, CA), was
used to enrich CDIO+,
CD 19' B-cell precursors. R-phycoerythrin (RPE) conjugated antiCD19, clone HD37 of mouseIgG,/K isotype (DAKO A/S, Glostrup,
Denmark), was used for enrichment of mature CDlO-, CD19' B
lymphocytes.
Enrichment of pre-B und mature B 1ymphocyle.s from the BM
sample. Aschematicrepresentation of the purification procedure
is presented in Fig l . The mononuclear cell fraction was incubated
with FITC-conjugated antiCD10 MoAb (10 pL/I0" cells in total 50
p L phosphate-buffered saline [PBSIbovine serum albumin [BSAl)
15 to 30 minutes on ice and washed twice in cold PBS supplemented
with 0.5% BSA, 5 mmol/L EDTA, and 0.01% NaN3 (PBSIBSA).
The cells were resuspended in cold PBSlBSA (80 pL/107 cells) and
a secondary labeling step was performed
with 20 &/IO7 cells of
anti-isotypic MACS magnetic microbeads (Miltenyi Biotec GmbH,
Bergisch Gladbach, Germany). Cells and beads were incubated
together for 15 minutes at +4"C. The cells were separated on a MiniMACS separation column according to the manufacturer's recommendations and theCDlO' and CDlO- fractions were collected. The
negative cell fraction was incubated with RPE conjugated
anti-CDIY
MoAb, washed, and labeled with anti-isotypic MACS magnetic microbeads as described above. The cells were separated
on a MiniMACS separation column and the CD19' and CD I9 fractions were
collected. All cell fractions were analyzedin a fluorescence-activated
cell sorter (Becton Dickinson FACSort) with the software Lysis II
(Becton Dickinson). The number of CDIO', CD19' cells in the preB cell enrichedsample was 10 timeshigher than theamount of
CDIO-, CD19' cells, and the ratio was
reversed in the sample enriched formatureBcells.Thelymphocytefractionsenrichedfor
pre-B (CDIO ') and mature B (CDl9+) lymphocytes were frozen as
cellpellets at -70°C until genomic DNA preparationswere performed.
Prepuration of genomic DNA. DNApreparationswere
performed as described by Miller al.'"
et Briefly, the cells were dissolved
in a TRIS-EDTA buffer and lysed with proteinaseK and I % sodium
dodecyl sulfate (SDS) in 37°C overnight. Proteins were precipitated
with saturated NaCl andchromosomalDNA
was recoveredfrom
the aqueous phase by ethanol precipitation, and dissolved in 1 X TE
(TE: I O mmol/L TRIS-HCI pH 7.6, 1 mmolL EDTA pH 8.0). The
concentration and purity of the preparations was determined by measuring the absorbance at 260 and 280 nm (DU-62 Spectrophotometer: Beckman Instruments Inc. Fullerton, CA).
PCK u r d PCR primers. PCR was performed in atotalvolume
of SO pL AmpliTaq I X PCR buffer containing0.5 to 1 .O pg template
DNA, 0.8 pmollL of the VH6 primer. 0.53 pmol/L of the JH1,2,4.5
primer.0.13pmol/L
of theJJprimer,
0.13 pmol/L of theJl,6
primer, I O % glycerol, and 2.5 UAmpliTaq(PerkinElmer.Roche
Molecular Systems Inc. Branchburg, NJ). The PCR was performed
using a thermal cycler (Techne Ltd, Cambridge, UK) with a cycling
program as follows: preheating 1 minute at 94°C; I minute at 94°C.
I minute at 69"C, and 0.5 minute at 72°C for 15 cycles; I minute
at 94°C. I minute at 65"C,and0.5minute
at 72°Cfor 25 cycles,
and finally an elongation step of 5.5 minutes at 72°C. The following
PCR primerswereused: V,,6: S' CCTCTCACTCACCTGTGCCA
S' AGGAGACGGTGACCAGGGT 3': JH3: S'
3':
JH1,2,4.5:
GAAGAGACGGTGACCATTGT 3': JH6: 5' AGGAGACGGTGACCGTGGT 3'.
Cloning und sequencing.
TheamplifiedPCRproductswere
subcloned into the pT7-Blue(R) vector according to instructions in
the pT7 Blue T-vector Kit (Novagen,Madison,WI)except
that
electroporation(2.5 kV, 25 mF. and200 $2. onaGenePulser;
BioRad,Richmond, CA) was used fortransformation.Doublestranded DNA sequencing was performed with a Sequenase Version
2.0DNASequencing
Kit asrecommended by themanufacturer
(United States Biochemical, Cleveland,OH). The sequence reactions
were separated on a denaturing6% acrylamide gel and detected with
autoradiography.
Seqrrence unalysis. Sequencingdatawereanalyzed
with GCG
package(HGMPResourceCentre,HarrowMiddx,UK)software
Fasta and Lasergene (DNASTAR, Inc. Madison, WI). For identifi-
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2797
lg GENE USAGE DURING RECONSTITUTION AFTER BMT
Table l . Serum Ig Levels and Cellular Content in Peripheral Blood of Patient 1 and Patient 2
6 wk After
4 wk After
BMT
BMT
1
Total WBC
2
1
2
1
2
1
4.6
2.3
0.17
7
4.2
1.6
38
4.8
1.0
21
6.7
0.49
7
-
-
6.5
0.59
9
0.5
9.4
1.0
-
-
-
-
Lymphocytes
% Lymphocytes
4.1*
1.7
41
-*
IgM
IgG
IgA
0.4t
7.0
1.5
0.3
11.4
1.5
7-8 wk After
BMT
BMT
BMT Before BMT
-
-
2
10 wk After
BMT
3 mo After
6 rno After
1
1
2
1
2
Normal
Range
3.1
0.78
25
0.6
4.7
3.8
0.49
13
6.4
0.85
13
4.1
1.3
32
3-10
1.0-4.0
20-40
0.2
0.4-3.1
2
2.8
4.8
5.2
0.73
0.32
1.2
26
7
23
- 0.9 0.6 0.4
0.3-2.0
"
-
-
0.2
"
6.7.0
5
0.5
6.1
0.7
7-15
* x i 0 9 cells/L.
t g/L.
Not determined.
*
cation of D and JH genes comparisonwasmadewithpublished
germline genes8.9, 31-36 The criteria for identification
of a D gene
segment was set to be 9-bp overlap with maximum l-bp mismatch
or 10-bpoverlapwithmaximum 2-bp mismatch.Themismatches
werenot allowed tohaveabiastowardtheend
of theidentified
fragment so thatthestretch of homology outsidetheunmatching
regionwasshorterthanthemismatchstretch.If
two possible D
genes overlapped,the one withthe longest stretch of homology
outside the overlapping region was identified or, if this stretch was
of similar length for the two D genes, both genes were identified.
Lymphoprep.
Mononuclear
cells
Staining with mouse a CD10
m A b ( I g G d and MACS rat
a mIg% microbeads.
I
Fractionation over
column.
"Acs
I
l
Negative
fraction;
Positive
cells)
(pre-B
l
CDlO- cells
'-l"---
* Staining with mouse a CD19
mAb (IgG1) and MACS rat
a mIgG1 microbe ad^.
I
Positive fraction;
CD19+ cells
(mature B cells)
Practionationover
fraction;
CD19- cells
Fig 1. Strategy for enrichment of pre-B and mature B lymphocytes
from the B M sample.
The D gene readingframesweredefined as thefirst, second, and
third frame starting from thefirst nucleotide in the germlinesequence
andafunctionalrearrangement
is statedwhenthere
is an open
reading frame through the CDR3.
RESULTS
Reconstitution of lymphocytes and soluble Ig afer BMT.
Both patients 1 and 2 had normal white blood cell and absolute lymphocyte counts before BMT (Table 1). After the
post BMT nadir, the white blood cell count increased to 2.3
X lo9 and 4.2 X lo9 cells/L, respectively, at 4 weeks and
was normal 6 weeks post BMT. Six months after BMT the
lymphocyte count was less than half of normal values for
patient 1. Serum IgM and IgG levels were somewhat low
before, as well as after, BMT for patient l and varied between 0.4 to 0.6 g/L and 4.7 to 9.4 g / L , respectively (Table
1). Patient 2 had also rather low serum levels of IgM and
IgG levels varied between 6.5 and 11.4 g / L . Before BMT
the serum IgA level was well within the normal range for
both patients. After transplantation the IgA level decreased.
Six months post BMT it was within the normal range for
patient 1 but still low for patient 2.
Nucleotide sequence analysis of V&-containing rearrangements. In patient 1 a total of 161 individual clones
were analyzed and the complete sequences of the CDR3
regions are presented in Fig 2. The average CDR3 length
is similar among the different samples (Table
2) except
for the BM-derived pre-B cell population where the CDR3
length is 19.2 5 8.1 tripletcodons (P = .OS). All sequenced clones have N nucleotide additions either in the
VH6-Djunction, D-JH junction, or in both. P nucleotides,
as defined by Lafaille et al,I7 can be identified in some
clones with similar frequency in D-JH junctions as in VH6D junctions (Fig 2). In patient 2 a total of 122 individual
clones were sequenced and the CDR3 regions were analyzed for functionalrearrangements(data
not shown).
None of the rearrangement events identified in patient 2
were found in patient 1, and vice versa.
The frequency of functional VH6-containing rearrangements in the two donors is 29% to 50% in the BM samples
and 39% in the PBL sample. In PBL obtained from patient
1 before BMT 67% of the sequenced clones represent a
functional rearrangement, and 4 weeks post BMT, taking
both patients together, 75% of the rearrangements are func-
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AND
2798
VI4
NandP
D gene
NASMAN
N andP
LUNDKVIST
F/NF
J, gene
Donor:BM DE-B cells
3.
10.
11.
13.
GGAGGG
G
GGGT
GTCOGG
GATCOG
WGGACGCCC
DATA
AGTC
A
G
m
G
A
19.
17.
5.
6.
GGCATT
TTCCTCTCGAG
7.
m
G
lA
?
A
.G
c
CGGAA
CGCCTTTA
QA
8.
12.
14.
16.
GGGACIVLCCCG
TCCCCGAGATA
DACTAC'IciGGccC
ATTACTACTACTACTACGGTA~CGTCTOOOOCC
CTACTACTACTACAlGGACGE'FOOOOCA
TOACTACTDGOGCC
AGA
CGGCGCTMT
18.
TGCAAGA
19.
20.
21.
TGCA
112).
3.
6.
7.
9.
10.
19.
4.
9.
8.
11.
14.
16.
17.
18.
20.
12131
(JH6b) NF
CCTCC
CGATu%GcDGc
GGGACAACCCG
TGGGG
AcGG
TOCAROR
GGA
GAATTCCCTAACCC
TCAGGGAGAA
3
3
GA
3
C
GATC
G
3
2
1
1
m
2
CCOOOC
TAAGAGGGTACGCG
TTAAGCGGG
1
3
1
1
1
3
3
G
A
m
GATA
TCACCGTC
TGC
00AOCCCCCGlQ
GA
2
1
3
m
3.
4.
17.
18,
19.
GTC
C
GAGGC
3
1
CAOWLCGA
AOOOAT
GAGCl'ACG
(JH6b) F
(JH6b) F
(JH4bl F
IJH4bl F
AGGGAG
lJH4bl
(JH4bl
(JHSbI
(JH4bl
lJH4bl
IJH4b)
NF
NF
TIAGTCGCGTCA
(JH6b)
(JH4al
(JH5b)
(JHPbI
lJH6b)
lJH6b)
NF
NF
NF
NF
NF
NF
C
(JHSbl NF
GT
23.
24.
5.
GATCACTACC
1
3
2
1
1
1
3
C
AGGACCACCAXAT
GGA
2
Ac
G
A
m
T
c
G
G
1
1
1
1
aAGGGC
2
AA
l
GG
6.
7.
8.
9.
10.
11.
12.
13.
14.
20.
C'IGGTACCCAGGGT
TOT
GACCAGGCCWC
GAT
G
CGcGA'IGGTTK
CGAOC
GGGTGG
E
F
F
NF
(JH5bl NF
Fig 2. The complete CDR3 region of all sequenced clones in donorlpatient pair 1. Each sequence starts with the end of FR3of the VH6
gene. The number in frontof each sequence indicates clone number and,if several clones had identical sequences, the number of repeatsis
given in parentheses after the clone number. Identified D andJH genes ara specified in parentheses andthe D gene reading frameis given in
front of each D gene. Abbreviations and symbols: ND, not determined; boldface letter, P nucleotides; *, deletion compared with germline
sequence; underlined letter, insertion compared with germline sequence; lowercase letter, nucleotide differing from the germline sequence;
-C, D gene rearrangedin reversed orientation.
tional (Fig 2). At later time points the frequency decreases,
but still 6 months post BMT 56% of the clones contain
functional rearrangements.
In general, the number of mutations in the VH6gene are
low (Table 3) and the pattern of base substitutions indicate
that they are of the intrinsic type."In the CDRs the BMderived mature B cells have a replacement mutational frequency of 1.5 mutations per 100 sequenced bp, resulting in
an €US (replacement to silent mutations) ratio of 7.9. Four
and 6 weeks post BMT the R/S ratios are 12.0 and 7.0,
From www.bloodjournal.org by guest on December 3, 2014. For personal use only.
lg GENEUSAGE
2799
DURING RECONSTITUTION AFTER BMT
Patient PBL
2614). XULA
33.
TGCAAGA
13121. T a m
GAC
TCAAOTrCT
1113). Toc
C
3131.
TGC
5 0 ) .
2
2
1
-
lDIR2-Cl
lD3)
(DIR1-Cl
(ND)
2 (DIR1)
3 lDIR2-C)
2 (DXPIJ
3 (DIRZ)
3 IDxP4)
noopac-ta:
0000tcmCcGG
GAMcI\cG
GAGFXCAT
COOOO
CCCtCCUOOtGcCCCC
C
GTATT"
CTAC
Slx Weeks
2 lDIR2-Cl
1171. TGCAAGA
2111). TGC
OACGCCT
C
2 IDIR1)
2815). TGCAAGA
GATOOO
3 lDIR2-C)
3 lW1)
Seven weeks after BMT
15.
19.
l(4).
3.
4.
1717).
XULA
612).
912).
Toc
T G T m
GATCKC
TGCAAG
TGCAAGA
TGCAAG
T
GGCCTCT
GTCTOOGMTTGG
'ECAAGA
AUA
-
(ND)
1
2
3
2
3
(DK4-C)
(DHO52)
(DLR1)
lDLR1)
lD2-C)
CCGAWLGCC
3 lDK4-Cl
TGCAAGA
11.
TGCAAGA
1512).
22.
TGUVV;A
319). TOCAAG
2 1 4 ) . TGCAAGA
TGCAA
4.
GATA
2
1
1
3
2
12.
TGCAAGA
GGTCCCATAAT
312).
TGCAAGA
AA
1.
TGCAAGA
TGCAAGA
GATAGGG
GAGTCwryiAIL
3 (DXPI)
2 (m4)
1 (DZl/lO)
10.
11.
TGCAAGA
GATCCG
(NDI
T
o
c
m
-
GCGCGC
6.
'ECAAGA
OAocOT
l.
ToCA
1
1
1
3
3
2
2
1
2
(DIR2-C)
(D23/7-C)
(DZ-C)
(D3)
(D3-C)
1
2
1
2
2
2
lDK41
(m4)
(W4)
lDK1)
(DXP'1)
5.
8.
TouuLo
2.
3.
6.
McAAopl
9.
5(2).
1.
4.
7.
E.
TACCGCCGCGTTGGA
GAQXG
T
GGGGATCCGT
T
OCYH30C
(03)
(DKl)
(DXP'11
(DXP'lI
lDHQ521
IND)
1 IDIRZ)
2 lDK1)
1 (Dm)
-
( n u )
ouocc
TGCAAGA
MOG
'ECAAGA
rOcM0
'ECAAGA
M
-
T
AGACAC
2 (DK4)
co1TcToo(rr
3 (m11
2 (DK1)
l (D21
'ECAAGA
GATcGmm
AG
(D21/10-C)
(DIR1)
(DHQ521
"OA
GGAA
m
TA
(m1
:*mx:&C
Fig 2. (Cont'd)
respectively. At later time points after BMT the mutational
frequency and R/S ratio decrease considerably.
Clonal variability. To estimate the degree of diversity in
the antibody repertoire we calculated the fraction of unique
sequences out of all sequences obtained from each sample,
ie, if all individual clones represent unique rearrangement
events the clonal variability will be 100%. As shown in Fig
3, the donors have a varied repertoire of rearrangements
among BM-derived pre-B and mature B cells, as well as
among PBL, with a clonal variability close to or equal to
100%.The clonal variability in patient l before BMT is 43%
and the variability decreases to 13% after transplantation. In
patient 2 the clonal variability is as low as 4% 4 and 6 weeks
post BMT. Seven to 10 weeks post BMT the variability is
20% to 40% and at 3 and 6 months the repertoire in patient
1 is almost as diverse as among the donated cells. However,
the variability remains low in patient 2.
Reconstitution of the VH6-containing lg repertoire in patient l appears to follow a wavelike pattern in that one of
the sequences, represented in 13 of the 19 clones isolated 4
weeks after BMT, is also present in 11 of 23 clones 6 weeks
after BMT (Fig 4A). In the sample obtained 7 weeks after
BMT none of the rearrangements found at 4 and 6 weeks
after BMT are present. However, 4 clones at 7 weeks after
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2800
NASMAN ANDLUNDKVIST
Table 2. C M 3 Length in Samples Derived From DonorlPatient Pair 1
Before and at Different Time Points After BMT
I
100
-
75
-
50
-
25
-
I
CDR3 Length'
Donor
BM pre-B cells
BM mature B cells
PEL
Patient
PEL before BMT
4 wk after BMT
6 wk after BMT
7 wk after BMT
10 wk after BMT
3 mo after BMT
6 mo after BMT
19.2 f 8.1
14.2 f 3.6
13.2 2 3.6
12.0 f 6.1
12.7 t 4.0
14.7 t 2.5
13.8 ? 4.4
14.6 ? 5.4
14.8 t 8.6
13.8 t 3.9
* CDR3 length is expressed as the average number of triplet nucleotide codons between residues 93 and 102 according to the terminology of Kabat et aI5O t SD.
BMT contain a rearrangement also present in 9 clones 10
weeks after BMT and in 1 clone 3 months after BMT. Two
clones obtained 3 months post BMT share the same rearrangement as well as 2 clones 6 months after BMT, but these
clones are not identical. None of the 8 sequences shown in
Fig 4A are present in the donor-derived samples. A similar
pattern is seen in patient 2, although none of the rearrangements are found at more than one time point (Fig 4B). Also
in patient 2 there is no overlap between donor and recipient
derived sequences.
J, gene usage. In all samples from patient 1 the Cproximal J H genes, JH4, JH5, and JH6, are most frequently
used (Fig 5). In pre-B cells all rearrangements containing
Table 3. Mutational frequency and Replacementto Silent
Mutations Ratio @?/S)in Functional Rearrangements
From DonorlPatient Pair 1
CDR
FR
R
S
R/S
R
S
RIS
0
0.24
0.24
0
0.16
0
1.5
>0.24
0
1.5
0
0
0.19
0
7.9
4.2
1.7
0.79
1.4
2.5
2.5
1.2
>0.56
>0.11
1.0
>0.23
2.0
5.3
4.7
0
0.27
0.27
0.27
1.3
0.44
0.67
0.67
Donor
BM pre-B cells
BM mature Bcells
PBL
Patient
Before BMT
4 wk after BMT
6 wk after BMT
7 wk after BMT
10 wk after BMT
3 mo after BMT
6 mo after BMT
2.0
1.7
0.56
0.11
0.11
0
0
0.23
0
0.11
0
0
0
-
1.5
12.0
7.0
0
>0.27
>0.27
>0.27
Mutational frequency is calculated as total number of nucleotide
changes in the V& sequences at each sample time divided by total
number of sequenced bp, multiplied with 100. Replacement (R) mutations lead to amino acid substitutions, compared to VH6 germline
sequence, whereas silent (SI mutations do not affect the amino acid
sequence. Framework regions (FRI are located between residues 1 to
30, 36 to 49, and 66 to 92. CDRs are located between residues 31 to
35 and 50 to 65 according to the terminology of Kabat et al.50
Fig 3. Clonal variability of the different samples. Clonalvariability
is calcul8t.d as percentage of different clones in total number of
sequenced clones
in each sample. (A) and (B) show the clonal variability fromdonorlpatient p a i o 1 and 2, respectively.
J H are
~ nonfunctional. J H 1 and JH2 are used mainly in BMderived pre-Bandmature
B cells and only 1 of 161 sequences express the J H gene.
~
D gene usage. In general, D gene usage varies more
among pre-B cells compared with mature B lymphocytes in
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lg GENE USAGE DURING RECONSTITUTION AFTER BMT
-1
L
c
c
c
4
4
c
c)
I
*
E
I
2801
gene. D2119 is used by 17.6% of mature B cells from BM,
but is not present in any other sample. DM1 and D21/10
represent two D genes that are rearranged inall samples
from the donor and that reappear in the patient at 3 months
after BMT. DHQ52 is represented in clones isolated 7 weeks,
10 weeks, and 3 months posttransplantation, but all those
rearrangements are nonfunctional. D2, DNI, and DIR2 are
frequently used in clones isolated from BM and PBL from
the donor as well as in PBL from the recipient. All DNIcontaining rearrangements except one are nonfunctional. The
DNI gene is frequently used in pre-B leukemia, adult PBL,
and in the neonatal repert~ire.~"'~
The DXP gene family,
Donor: BM preBcells
1-1
75%
Donor: PBL
75%
Donor: BM mature B cells
-1
75%
m
I
Patient: PBL before BMT
Rap l
0%
Four weeks after BMT
75%
Fig 4. Diversification ofthe lgrepertoire. The frequency of certain
abundant rearrangements at different time points before and after
BMT in patient 1 (AI and patient2 (B). In (A) sequence 1 ( 0 1 represent
13 clones from 4 weeks after BMT and
11 clones from 6 weeks after
BMT. Sequences 2 (01 and 3 (A1 represent 3 clones each from 4
weeks afterBMT, sequences 4 ( 0 1 and 5 (W) represent 7 and 5 clones,
respectively, from 6 weeks after BMT. Sequence 6 ( + l represent 4
clones from 7 weeks, 9 clones from 10 weeks, and 1 clone from 3
months afterBMT. Sequence 7 (01 represent 7 clones from 7 weeks
and sequence 8 (AI 4 clones from 10 weeks afterBMT. In (B] sequence
1 (01represent 24 clones from 4 weeks after BMT, sequence 2 (01
23 clones from 6 weeks after BMT, and sequence 3 (A1 represent 7
clones from 8 weeks after BMT. Sequence 4 (0)
and 5 (W) represent
8 clones each from 3 months after BMT and
sequence 6 ( 1 represent
5 clones from thesame timepoint.
I
after
weeks
Seven
BMT
S i x weeks after BMT
75%
Ten weeks after BMT
50%
1
+
months
after
Three
BM and peripheral blood (Fig 2). Before BMT, patient 1 uses
only three different D gene segments in the VH6-containing
rearrangements. With time after BMT the D gene usage
becomes more diverse and is comparable with that of the
donor cells 3 months post BMT.
Twenty-two different germline D gene segments were represented in the samples from patient 1. Seven of those were
only used in BM-derived cells and out of those Dl, DLR5,
DXPI/D5, and DAI/DA4 were found only in pre-B cells.
D4 is only used in the BM samples where 23.5% of the preB cells and 17.6% of the mature B lymphocytes use this
BMT
S i xBMT
months
after
1-1
75%
l
RI
5n%1
A
4l"J
25%
0%
Fig 5. JHgene usage in VH6containing rearrangements in donor/
patient pairl.JH gene usage is expressed as percentage of the number of unique rearrangements
in each sample. Functional rearrangements are represented by shaded bars (RI] and nonfunctional rearrangements by open bars (01.
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2802
NASMANAND
which has been reported to be the most frequently used D
gene family in the adult PBL repertoire, is also repre~ented.’*.~~
DIR genes can be identified in 2 rearrangements before
BMT, 2 rearrangements 4 weeks after BMT, and2 rearrangements 6 weeks post BMT. This high frequency of DIR gene
family utilization could be due to either the low variability
at 4 and 6 weeks post BMT (Fig 3A) or a selection of Bcell clones using the DIR gene in combination with VH6.In
a recent report, Moore and Meekw could not demonstrate
significant rearrangement to the DIR recombination signal
sequences, and they suggest that these GC-rich sequences
may be explained by N nucleotide additions rather than DIR
utilization. We do not think this can explain our results because DIR sequences are the only D genes possible to identify in the more than 30-bp long CDR3 region isolated at 4
and 6 weeks post BMT, even though the match with reported
germline sequences is not absolute.
There are some clones that contain more than one D gene
(Fig 2). In the pre-B cell population S of 17 clones have this
type of rearrangement. Two of these clones are functionally
rearrangedand three clones are nonfunctional. Four to 6
weeks after transplantation three unique rearrangements contain D-D fusions, which all are functional. D-D rearrangements are not common among the other samples and where
they are present the majority are nonfunctional.
D gene reading frame (RF). The junctional regions of
the functional BM-derived VH6-D-JHrearrangements from
patient 1 displayed a bias against D genes translated in RFl
(Fig 2). In pre-B cells none of the D genes were in RF1,
but among mature B cells RFI was found in 28.6% of the
population. This was accompanied by a decrease in RF2
utilization in mature cells. Interestingly, this pattern is indicated also in the early posttransplantation period. Four and
6 weeks post BMT none of the rearrangements use RFI,
but during reconstitution of the lymphocyte repertoire the
frequency of D genes in RFI increases. Because of the limited number of unique rearrangements early after BMT, the
differential utilization of D gene RFs cannot be statistically
confirmed.
In adult PBL, cord blood, and fetal repertoires all three
D gene reading frames are usedin similar frequency, but
independently of VH gene family usage, certain D genes
preferentially use a specific RF.”.”Varade et a14’ found that
58% of the VH6-containing rearrangements in spleen from
young individuals use RF1.
DISCUSSION
The well-documented overrepresentation of the v ~ gene
6
in the fetal Ig repertoire2’.22as well as in the post BMT
s i t ~ a t i o n ~led
~.~
us’to perform a more detailed analysis of
VH6-containing rearrangements before and at several time
points after BMT. Our results show that the repertoire 4 to
10 weeks post BMT displays a limited set of rearrangements
(Fig 3). This is in contrast to results derived from the BM
donor samples and 3 to 6 months after BMT where there is a
higher degree of variability. The high frequency of functional
rearrangements and the high R/S ratios among clones isolated 4 and 6 weeks post BMT (Fig 2 and Table 3) indicate
LUNDKVIST
that there is a positive selection of VH6-expressing B lymphocytes during early reconstitution of the immune system
after BMT.
Another similarity between development of the fetal and
the post BMT B-cell repertoire is the frequent use of the
DN and DK gene families (Fig 2).19 In contrast, the DHQ52
gene andthe JH3 gene frequently used in the fetal repert0ire21.24.3Y are only sparsely rearranged in our material. Instead, JH gene usage in all our samples is biased toward the
C-proximal genes, JH4, JHS, and JH6,(Fig 5 ) , which is the
case also in both cord blood and adult peripheral blood repertoires,31.39,42.4~
Yamada et al” have described a DNl variant gene, with
a CA to TG substitution, that could represent either a polymorphism or a new gene in the DN family. This gene has
also been described by Mortari et a14’ and they proposed the
tentative name DN2. We have also identified this variant of
DNl among our samples, but since the DN 1 variant gene
always is found in association with the JH6c allele and the
DN1 gene in association with the JH6b allele (Fig 2), we
believe that this variant represents a polymorphism rather
than a new gene in the DN family.
The CDR3 lengths, as shown in Table 2, do not vary much
between the different sample points and are quite similar to
the 12.3 t- 4.0 triplet nucleotide codons amongfetal C,
transcripts reported by Mortari et
A deviation is seen
in the BM pre-B cell sample where the CDR3 length is 19.2
5 8.1 codons (P = .05). This long CDR3 region is partly
due to frequent N nucleotide additions in both the VH6-Dand
the D-JH junctions and partly torearrangement of multiple D
genes. The possibility to circumvent the 12/23-bp spacer
recombination rule4’maylead
toincreased combinatorial
diversity by D-D gene fusion. This type of rearrangement.
originally suggested by Kurosawa and Tonegawa,Is is most
frequent in the BM pre-B cell sample. The presence of DD fusions has previously been reported in the fetal, cord
blood, and adult Ig repertoire^^'.^^.^ and evidence for the
occurrence of D-D fusions as well as inverted D gene rearrangements has been p r e ~ e n t e d . ~ ” . ~ ~
Interestingly, some clones contain more than two D gene
segments, for example in clone 16 and clone 18 from BM
pre-B cells we can identify four D genes in each rearrangement (Fig 2). Furthermore, some clones contain more than
two P nucleotides. In the suggested mechanism for P nucleotide addition a maximum of two nucleotides are added.”
Our results maybe due to N nucleotides in a palindromic
sequence or represent a new mechanism for Ig gene repertoire diversification.
To exclude the possibility that the low variability found
4 and 6 weeks post BMT in patient 1 (Fig 3A) was caused
by a bias in the PCR amplification process, we performed a
second PCR on the sample material from 6 weeks post BMT
followed by nucleotide sequence analysis. In Fig 2, 7 clones
represented by clone 1 and 3 of the 8 clones represented by
clone 2 origin from the first PCR, whereas the remaining
clones were isolated in the second experiment. These 23
clones only represent three unique rearrangement events, one
of which is represented in clones originating from both PCR
amplifications. These results support the notion that the low
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lg GENEUSAGE DURING RECONSTITUTION AFTER BM1
variability is the result of a restricted repertoire in vivo,
although we cannot formally exclude the presence of other,
nonamplified, rearrangements. It could be argued that the
low clonal variability observed at 4 and 6 weeks post BMT
in patient 1 was caused by a contaminating sequence that
would be preferentially amplified when the number of B cells
is verylow. However, these sequences have never previously
been isolated in our laboratory and, furthermore, none of the
other sequences present at 4 (clone 3 and 5) or 6 (clone 1
and 28) weeks post BMT are found at more than one time
point. Therefore, we believe that the rearrangement isolated
both 4 and 6 weeks post BMT represents an important clone
in the early reconstitution of the Ig repertoire. To further
confirm thepattern of oligoclonal diversification of the VH6containing Ig repertoire we analyzed a second BM recipient.
In patient 2 the clonal variability was even lower at 4 and
6 weeks after BMT (Fig 3B) because only one unique rearrangement was found at each time point.
The limited diversity of the antibody repertoire demonstrated by the low clonal variability early after transplantation (Fig 3) is in concordance with the low Ig concentrations
in serum, the reduced numbers of peripheral lymphocytes
and impaired polyclonai as well as specific immune responses found in BMT patients (Table l).’ Interestingly, the
diversification of the repertoire is obviously not just simple
additions of new clones to the existing pool of specificities.
Instead it looks more like a dynamic system of appearance
and disappearance of consecutive clones (Fig 4A and B). To
our knowledge this type of wavelike diversification of the
Ig repertoire has not been described, though there is evidence
for a sequential rearrangement of human T-cell receptor V,
and V+ genes during ~ntogeny.~’
It is not likely that these
consecutive clones are derived from activated donor B cells
or plasma cells transferred with the BM graft because we
do not detect any of these rearrangements in the BM or PBL
samples from the donor. It has been shown that secretory Ig
levels in saliva peak 2 to 3 weeks after BMT and that these
Igs are derived from activated cells from the BM donor.48
The Ig levels thereafter gradually decrease and remain low
for around 3 months. When the levels increase again the Igs
originate from the reconstituted B-cell compartment. The
same pattern was found for Ig levels in serum even though
the origin of the antibodies was not ~ h o w n . 4These
~
findings
further support our conclusion that the pattern ofIg gene
expression during reconstitution of the B-cell repertoire after
BMT is a property of the new immune system developing
from graft-derived hematopoetic precursors.
ACKNOWLEDGMENT
We are grateful to Dr 0. Ringdkn, L. Markling, M. Remberger,
and C. Tammik for providing us with sample material. We also
thank Drs D. Holmberg, S . Pettersson, and 0. Ringden for critical
reading of the manuscript.
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