Antigen-Induced Somatic Diversification of
Rabbit IgH Genes: Gene Conversion and
Point Mutation
This information is current as
of June 14, 2017.
Candace R. Winstead, Shi-Kang Zhai, Periannan Sethupathi
and Katherine L. Knight
J Immunol 1999; 162:6602-6612; ;
http://www.jimmunol.org/content/162/11/6602
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References
Antigen-Induced Somatic Diversification of Rabbit IgH Genes:
Gene Conversion and Point Mutation1,2
Candace R. Winstead,3 Shi-Kang Zhai, Periannan Sethupathi, and Katherine L. Knight4
A
diverse Ab repertoire is essential for effective humoral
immunity. The mechanisms by which such diversity is
generated and maintained are versatile and differ among
species. In mouse and human, a major contributor to Ab diversity
is combinatorial joining of multiple V, (D), and J gene segments at
the heavy (H) and light (L) chain loci (1–3). Other species instead
rely on postrearrangement somatic diversification mechanisms, somatic gene conversion and somatic hypermutation, to build the
primary Ab repertoire. Somatic gene conversion, first described in
Ig genes of chickens (4, 5), is homologous, nonreciprocal recombination between upstream donor V gene segments and the rearranged V(D)J genes. The result of such gene conversion is that the
rearranged V(D)J gene acquires part of the sequence of the upstream V gene segment, which itself remains unchanged (6, 7). In
this way, the upstream V gene segments serve as a reservoir of
diversity for the rearranged V(D)J genes. Somatic hypermutation
is the targeted accumulation of a high frequency of point mutations
by an as yet unknown mechanism (reviewed in Ref. 8).
Both somatic gene conversion and hypermutation can contribute
to the diversity of the primary Ab repertoire. For example, in
sheep, somatic hypermutation but not somatic gene conversion
contributes to Ab diversity during fetal and neonatal development
(9). Conversely, in chicken, somatic gene conversion is the preDepartment of Microbiology and Immunology, Loyola University of Chicago, Maywood, IL 60153
Received for publication October 19, 1998. Accepted for publication March 10, 1999.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by National Institutes of Health Grant AI16611.
2
Genes discussed here have been deposited in GenBank under accession numbers
AF098225 through AF098241.
3
Current address: Department of Immunology, IMM-25, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037.
4
Address correspondence and reprint requests to Dr. Katherine L. Knight, Department of Microbiology and Immunology, Loyola University of Chicago, 2160 South
First Avenue, Maywood, IL 60153. E-mail address: [email protected]
Copyright © 1999 by The American Association of Immunologists
dominant mechanism that diversifies the single functional VH and
VL gene segment used in H and L chain gene rearrangements (4,
5). Rabbits use both somatic gene conversion (10) and hypermutation (11, 12) to diversify an IgH repertoire that is limited by
preferential rearrangement of the 39-most VH gene segment, VH1,
in 80 –90% of the VDJ gene rearrangements (13–15).
In species that use postrearrangement somatic mechanisms to
generate a primary Ab repertoire, gut-associated lymphoid tissue
(GALT)5 plays a critical role in the diversification process. For
example, diversity by somatic hypermutation in fetal sheep occurs
predominately in the ileal Peyer’s patch (9), diversity by somatic
gene conversion occurs in the bursa of embryonic chicken (16, 17),
and in young rabbits, VDJ genes are diversified in GALT by both
somatic gene conversion and hypermutation. Somatic gene conversion-like mutations and point mutations have both been observed in clonally related VDJ gene sequences obtained from
6-wk-old rabbit appendix follicles (18), and when organized
GALT, including appendix, sacculus rotundus, and Peyer’s
patches, were removed from rabbits shortly after birth (GALTless
rabbits), somatic diversification of Ig genes by gene conversion
and hypermutation were significantly delayed (19).
Somatic diversification also occurs during T cell-dependent immune responses within germinal centers (20 –22). In chicken, somatic gene conversion appears to occur not only in an exogenous
Ag-independent fashion in the embryonic bursa, but also during
immune responses, as demonstrated by the analysis of clonally
related VJ genes obtained from splenic Ag-induced germinal centers (23). In rabbit, we do not know whether somatic gene conversion and/or somatic hypermutation occur during specific immune responses.
To determine which mechanism(s) of somatic diversification is
used in rabbit during Ag-specific immune responses, we examined
5
Abbreviations used in this paper: GALT, gut-associated lymphoid tissue; PLN, popliteal lymph node; PBL, peripheral blood leukocyte; HSA, human serum albumin;
CHO, Chinese hamster ovary; CDR, complementarity-determining region; FR, framework region.
0022-1767/99/$02.00
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During T cell-dependent immune responses in mouse and human, Ig genes diversify by somatic hypermutation within germinal
centers. Rabbits, in addition to using somatic hypermutation to diversify their IgH genes, use a somatic gene conversion-like
mechanism, which involves homologous recombination between upstream VH gene segments and the rearranged VDJ genes.
Somatic gene conversion and somatic hypermutation occur in young rabbit gut-associated lymphoid tissue and are thought to
diversify a primary Ab repertoire that is otherwise limited by preferential VH gene segment utilization. Because somatic gene
conversion is rarely found within Ig genes during immune responses in mouse and human, we investigated whether gene conversion in rabbit also occurs during specific immune responses, in a location other than gut-associated lymphoid tissue. We
analyzed clonally related VDJ genes from popliteal lymph node B cells responding to primary, secondary, and tertiary immunization with the hapten FITC coupled to a protein carrier. Clonally related VDJ gene sequences were derived from FITC-specific
hybridomas, as well as from Ag-induced germinal centers of the popliteal lymph node. By analyzing the nature of mutations within
these clonally related VDJ gene sequences, we found evidence not only of ongoing somatic hypermutation, but also of ongoing
somatic gene conversion. Thus in rabbit, both somatic gene conversion and somatic hypermutation occur during the course of an
immune response. The Journal of Immunology, 1999, 162: 6602– 6612.
The Journal of Immunology
6603
clonal populations of B cells responding to immunization. If
somatic diversification occurs within clonally related VDJ genes,
then we expected to find mutations in some, but not all, of the
related clones. Furthermore, if somatic gene conversion is induced
by immunization, then we expected that some of the differences
found between related clones should match sequences of upstream
donor VH gene segments. We used the GALTless rabbit model for
these studies, because early removal of GALT results in a significantly lower mutation frequency within non-Ag-specific VDJ
genes derived from peripheral blood leukocytes (PBL) compared
with controls (19). We obtained clonally related VDJ gene sequences during immune responses to the hapten FITC conjugated
to human serum albumin (HSA). These sequences were derived
from FITC-specific clonally related hybridomas and from Ag-induced germinal center microenvironments in the popliteal lymph
node (PLN). We report here analyses of ongoing mutations found
within these clonally related VDJ gene sequences.
later. Medium was exchanged every 5–7 days, and hybridomas were observed after 10 –14 days. FITC-specific hybridomas were identified by an
Ag-specific ELISA with FITC-OVA, OVA, or HSA-coated microtiter
plates (1 mg/ml). The secondary Ab, biotinylated goat anti-rabbit L chain,
was detected with an avidin-biotin complex (Vector Laboratories, Burlingame, CA) and developed with the appropriate substrate. A summary of the
percentage of hapten and carrier-specific hybridoma clones obtained for
each fusion is shown in Table I. The FITC-specific hybridomas were
cloned by limiting dilution.
Cloning and sequencing of hybridoma VDJ genes
Materials and Methods
Rabbits
Rabbits of known VHa allotypes were maintained at Loyola University of
Chicago. Surgery to render rabbits GALTless was performed as described
(19). Briefly, in newborn rabbits the appendix and the sacculus rotundus
were surgically excised and at 3 to 6 wk of age the Peyer’s patches were
removed. GALTless rabbits were inspected visually at sacrifice for residual
organized GALT, and, except for rabbit #72L5, in which a single Peyer’s
patch was identified, no organized GALT was observed.
Three GALTless rabbits were used for fusions; rabbits #72L4 (VHa1/a3
allotypes) sacrificed at 12 wk of age (at day 10 of a primary response),
#72L5 (VHa1/a3 allotypes) sacrificed at 14 wk of age (at day 4 of a secondary response), and #323 M2 (VHa2/a2 allotype) sacrificed at 12 wk of
age (at day 3 of a secondary response). GALTless rabbits for germinal
center analyses (VHa3/a3 allotype) were sacrificed at day 14 after primary
immunization (rabbit #347N4, 12 wk of age) and at day 5 after tertiary
immunization (rabbit #347N3 at ;18 wk of age).
PCR amplification of VDJ genes from germinal center DNA
PLN tissues were embedded in OCT (Tissue-Tek, Torrance, CA) and sectioned (7 mm and 14 mm) with a cryostat (2800 Frigocut, Reichert-Jung,
Germany). Adjacent sections were placed on the same slide, fixed in ice
cold acetone, and stored at 220°C, before staining with Harris hematoxylin
and eosin. Germinal center tissue was scraped under a dissecting microscope (Olympus, Tokyo) at 340 magnification using pulled, siliconized
50-ml glass capillary tubes (CMS, Broomall, PA). Germinal center material
was transferred with the glass capillary tube by breaking the tip into microfuge tubes containing 20 ml of 13 PBS diluted 1:5.
Germinal center genomic DNA was prepared as described (20). Briefly,
the germinal center material was incubated with 5 ml of proteinase K (10
mg/ml) for 2–3 h at 56°C or overnight at 37°C; proteinase K was inactivated at 94°C for 10 min. Germinal center VDJ genes were amplified in a
nested or seminested fashion. Each 50-ml reaction consisted of 1 ml of
germinal center genomic DNA plus the following: 13 Pfu polymerase
buffer (Stratagene, La Jolla, CA), 200 mM mixed dNTPs (equimolar of
each dNTP; Pharmacia, Piscataway, NJ), 0.1 or 0.2 mM of each primer, and
0.5 U of Pfu DNA polymerase (Stratagene). After a hot start of 5 min at
96°C, polymerase was added when the samples had cooled to 80°C. For the
first round, the 59 primer was located in the promoter region of VH1 (VHPr)
at approximately 2250 nucleotides from the ATG start site. The 39 primer
Immunizations and generation of rabbit Ag-specific hybridomas
Rabbits were immunized with FITC-HSA (800 mg) (kindly provided by
Dr. E. Voss, University of Illinois-Urbana-Champaign; hapten substitution
;25 hapten groups per carrier) in CFA, subcutaneously in the lower leg to
induce a response in the PLN. Boosts were given ;30 days later with
FITC-HSA (500 mg) in IFA.
The HGPRT2 rabbit fusion partner 240E-1 was grown in modified
RPMI 1640 plus 15% FCS and fused with PLN cells as described (24).
Briefly, PLN cells and fusion partner cells were fused at a 2:1 ratio immediately after isolation of the PLN cells or after activation in vitro with
CD40 ligand-presenting Chinese hamster ovary (CHO) cells (obtained
from Dr. Melanie Spriggs, Immunex, Seattle, WA) and Ag. In vitro
activation was performed to improve fusion efficiency. The CD40 ligandpresenting cells (4 3 106) were irradiated with 5000 rad and incubated with
4 3 107 PLN cells with FITC-HSA (10 mg/ml) in modified RPMI 1640
plus 15% FCS for 48 –72 h. The cells were washed at room temperature in
serum-free RPMI 1640 and fused in 50% PEG-4000 (EM Science, Cherry
Hill, NJ) at 37°C. Fusions were plated at 5 3 104 cells/well, and medium
supplemented with hypoxanthine-aminopterin-thymidine was added 24 h
Table I. Summary of fusions from FITC-HSA-immunized rabbits after primary and secondary responses
Primary Fusion
Secondary Fusion
Hybridomas
(No.)
Day 10 (rabbit
#72L4)
Day 10 (rabbit
#72L4)
Day 4 (rabbit
#72L5)
Day 3a (rabbit
#323M2)
Total
FITC-specific
HSA-specific
Sequencedb
Related sets
25
1(4%)
0
1
0
103
32 (31%)
2 (1.9%)
7
0
40
7 (17.5%)
3 (7.5%)
7
1c
525
72 (14%)
22 (4.2%)
33
6
a
b
c
a
Lymph node cells were cultured in the presence of CD40 ligand-transfected CHO cells plus 10 mg/ml of Ag for 48 –72 h prior to fusion.
Number of FITC-specific hybridomas from which the nucleotide sequence of the VDJ gene was determined.
Clones 48A2.1 and 20A6.23. All other related sets were obtained from the secondary fusion, day 3.
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RNA was isolated from 1 3 107 cloned, Ag-specific hybridoma cells by the
TRIzol (Life Technologies, Grand Island, NY) method according to the
manufacturer’s directions. First strand cDNA was synthesized from 2 mg of
FITC-specific hybridoma RNA as described (25). VDJ genes were amplified from the cDNA using Taq polymerase with VH leader-specific primer,
VHRPS (59-AGGAATTCTGCAGCTCTGGCACAGGAGCTC-39), as the
59 primer and one of two pan JH primers (JHpBR (59-GTCGAATTCAC
CTGAGGAGACGGTGACCA-39) or JHR1 (59-CTCGAGAATTCTGAG
GAGACGGTGACCAGGGTGCC-39)) as the 39 primer as described (14).
VJ genes were amplified with a VK-specific leader primer (59-TCG
GATATCCACCATGGACACGAGGGCCCCCACTCAGCTGCTG-39) and
JK-specific 39 primer (59-GCAGTCGACTTACCTTTGACCACCACCTCG
GTCCCTCCGCCGAA-39). PCR products were cloned into M13 mp19 for
nucleotide sequencing using the restriction sites indicated by the underlined
sequences in the primers (26). To distinguish the Ag-specific VDJ gene of
lymph node cells from that of the fusion partner, we used differential hybridization with a pan VH probe (VH550) and a probe specific for the VH gene used
by the fusion partner, y33 (VH50) (11). The nucleotide sequence of clones that
were positive for the VH550 probe and negative for the y33 specific probe was
determined (27). The L chain genes were distinguished from the fusion partner
by sequence analysis, which confirmed that none of the clones analyzed were
derived from the fusion partner VDJ gene or VJ gene. The sequences of donor
VH genes are taken from the data of Knight and Becker (13) for VH2a3,VH3-a3 and VH6-a3, Crane et al. (28) for VH6-a2, Raman et al. (29) for
VH8-a3 and VH1-a3, Bernstein et al. (30) for VH34-a3 and VH25-a3, and Currier et al. (31) for P269P2 (a3).
6604
Ag-INDUCED SOMATIC GENE CONVERSION OF RABBIT IgH VDJ GENES
used in the first round anneals immediately 39 of the JH4 gene segment
(39JH4), which is rearranged in 80% of VDJ genes. The second round of
PCR was either nested on both sides or seminested, in that the 59 primer
was the same as in the first round and only the 39 primer was internal. For
fully nested PCR, the internal 59 primer was still within the VH1 promoter,
at 2212 nucleotides from the ATG start site (upVH-H3). The 39 primer for
either fully nested or seminested PCR was the pan JH primer, JHpBR,
described above. Primer sequences are: VHPr, 59-TAACAAGCTTA
AAAATTCATATGATCTGAATC-39; upVH-H3, 59-TCCAAGCTTAT
CACAGCCATCAC-39; and 39JH4, 59-GTAGGAGCTCGAGTTG
GCAAGGACTCAAC-39.
To specifically amplify VDJ genes from a particular B cell clone, we
designed 39 primers that began in complementarity-determining region 3
(CDR3) region and ended within the J region of that clone. The specific 39
primers were used in both rounds of seminested PCR, and the 59 primer
VHpr was used in the first round and upVH-H3 in the second round. The
specific VDJ genes were cloned and screened using an oligomer probe
specific for the CDR3 region of each clone for hybridization. Sequences of
the 39 CDR3-specific primers and probes were as follows. For clones from
germinal center 15: 39 primer (CDR3-4228) 59-AGGGAATTCAT
CAAAGCACCAAT-39, and oligo probe (CDR3p-4228) 59-TGTGC
GAGAGGCCTCTAT/CGAT-39. For clones from germinal center 22: 39
primers (CDR3a-4254) 59-GGCGAATTCCCACAACTTCCAGGG-39,
(CDR3b-4254)
59-CCAGAATTCGGGCCATAACCAG/ACATAA-39,
and oligo probe (CDR3p-4254) 59-TGTGCGAGAGGTGGTTATGTT-39.
Misincorporation frequency of polymerases
Control amplification of cloned DNA samples, using the same conditions
as were used on the germinal center DNA was performed with both Pfu
(Stratagene) and ULTma (Perkin-Elmer, Norwalk, CT) polymerase. Two
plasmids containing an undiversified VH1a3 VDJ gene (41-3) and a diversified VH1a3 VDJ gene (15-23) were mixed at dilutions such that no PCR
product was obtained after 40 cycles of PCR amplification, but equivalent
amplification of each plasmid was obtained after two rounds (80 total cycles). The PCR products were cloned and sequenced and compared with
the known sequences of these two clones (29, 32). The misincorporation
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FIGURE 1. Nucleotide sequence comparisons of CDR3 among seven sets of clonally related FITC-specific hybridomas. Related clones are grouped
together (A through G) and are shown compared with utilized germline DH and JH gene segments. N segments are indicated. The sequence of L chain CDR3
regions was determined for two of the related hybridomas (8.2-2 and 27.2-3), and sequences are shown compared with each other (D); the deduced amino
acids are shown. Dots indicate nucleotide identity. GenBank accession numbers for the complete sequences for the H chain VDJ genes are AF098225
through AF098239 and for the L chain VJ genes are AF098240 and AF098241.
The Journal of Immunology
6605
frequency using this protocol for the two DNA polymerases was 36 mutations of 2662 base pairs for ULTma (8 per VDJ gene clone) and 2 mutations of 2680 base pairs for Pfu (0.4 per VDJ gene clone). Because of the
high misincorporation frequency obtained with ULTma polymerase, all
germinal center sequences were amplified using Pfu polymerase.
Results
Clonally related, FITC-specific hybridoma VDJ genes
We investigated Ag-driven somatic diversification by analyzing
VDJ genes cloned from FITC-specific hybridomas. Rabbits were
immunized with FITC-HSA, and, after primary and secondary immune responses, cells from the draining PLN were fused with the
rabbit hybridoma fusion partner, 240E-1. We searched for sets of
clonally related hybridomas because nucleotide differences within
these sets would provide evidence for Ag-induced somatic diver-
sification that occurred during clonal expansion. By examining the
CDR3 regions of VDJ gene sequences from 48 FITC-specific hybridomas, we found 7 sets of VDJ gene sequences that were
clonally related (Fig. 1). All of these sets were derived from hybridomas obtained after a secondary immune response (Table I)
and all used the germline gene VH1 in their VDJ gene rearrangements, based on nucleotide sequence analysis (Fig. 2 and data not
shown). Five of the sets contained clones with mutations within
some, but not all, members of the set (Figs. 1 and 2), and these
were analyzed further for evidence of both ongoing somatic point
mutation and ongoing somatic gene conversion.
To analyze specifically for somatic point mutations, we examined the D regions because there are no known upstream germline
sequences to serve as donors for gene conversion (Fig. 1). We
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FIGURE 2. Partial nucleotide sequences of five sets of clonally related FITC-specific hybridoma VDJ genes compared with sequences of germline
VH1a2 or VH1a3. Dots indicate nucleotide identity; forward slashes indicate nucleotide deletion. Back slashes were included in the germline genes for the
purpose of spacing. Gene conversion-like mutations are shown boxed (D and E) with the likely upstream donor VH gene segment indicated below. Beyond
the boxed region, the clone resumes similarity to germline VH1 and is significantly different from the sequence of the proposed donor VH gene segment.
c, Indicates that this clone is nonfunctional, it has a two nucleotide deletion causing a reading frame shift. Amino acid numbering is according to Kabat
et al. (46). GenBank accession numbers for these clones are listed in Fig. 1.
6606
Ag-INDUCED SOMATIC GENE CONVERSION OF RABBIT IgH VDJ GENES
found as many as eight nucleotide differences between the related
clones (clones 30.14-1 and 27.11-1; Fig. 1A), suggesting that somatic point mutations were introduced in the D regions during
clonal expansion. We suspect that somatic point mutation also occurred within the V regions, but this cannot be distinguished from
somatic gene conversion in which a highly similar VH gene segment was used as a donor.
We searched for evidence of somatic gene conversion within the
clonally related VDJ genes by analyzing the nucleotide sequences
for mutations that matched the sequence of upstream donor VH
gene segments. In three of the sets of clonally related genes, the
changes between the related clones were mostly in the form of
scattered point mutations that did not match the sequence of
known upstream VH gene segments (Fig. 2, A–C). However, in
two sets of clonally related VDJ genes, there was evidence of
ongoing somatic gene conversion because the gene conversionlike mutations were present in some, but not all, of the clonally
related VDJ genes. One striking example is found in clones 27.2-3
and 8.2-2 (Fig. 2D). In clone 8.2-2, there is a cluster of 18 mutations, including a codon insertion, in framework region 1 (FR1)
that closely matches the upstream VH gene segment VH6, although
the nucleotide sequence both upstream and downstream of this
cluster closely matches VH1. Because these mutations are not
found in the related clone 27.2-3, they were presumably introduced
during clonal expansion. In another set of related clones (Fig. 2E),
we found three nucleotides changes at the end of FR1 in two related clones 1.12-1 and 14.2-1, but not in the other related clone
8.3-2. These three nucleotides are identical to the upstream VH
gene segment, VH6.
In addition to somatic gene conversion-like mutations, the
clonally related hybridoma VDJ gene sequences have many other
shared and unique mutations (Fig. 2). On the basis of these mutations, we could draw lineage relationships between the clonally
related hybridomas (Fig. 3). Fig. 3A depicts the proposed lineage
relationship between clones 8.2-2 and 27.2-3, highlighting the
gene conversion-like mutations in FR1 of clone 8.2-2. This lineage
includes putative precursors to the related clones, and it begins
with a germline VDJ gene precursor and then diversifies into a
precursor A with three mutations shared by both clones. Clone
8.2-2 differs from precursor A by 7 nucleotides, in addition to the
18 mutations that were presumably introduced by gene conversion
using VH6 as a donor. The putative lineage relationship between
another set of related clones, 1.12-1, 14.2-1, and 8.3-2 is diagrammed in Fig. 3B. Clones 14.2-1 and 1.12-1 share many mutations with one another, including the putative gene conversion-like
mutation of three nucleotides in FR1. The VDJ gene from clone
8.3-2 contains none of the mutations in common with clones
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FIGURE 3. Proposed lineage relationship between FITC-specific hybridoma VDJ gene clones. A, Clones 8.2-2 and 27.2-3. B, Clones 1.12-1, 14.2-1, and
c8.3-2. Germline precursor (G.L.) and a putative precursor A are shown as circles with dashed lines; hybridoma clones are indicated by their numbers within
solid circles. The predicted gene conversion events are designated by the name of a putative donor gene above the arrows, followed by the number of
additional mutations that do not match an upstream VH gene segment. Sequence comparisons of proposed somatic gene conversion are shown enlarged
above the clone(s) containing the gene conversion.
The Journal of Immunology
6607
1.12-1 and 14.2-1, implying that 8.3-2 must have diverged from
1.12-1 and 14.2-1 early in the immune response.
To rule out PCR or cloning artifacts as the explanation for the
gene conversion-like mutations in these clonally related hybridomas (Fig. 2, D and E) we performed a second RT-PCR of the
hybridoma cDNA, and in each case obtained identical sequences
from the independent clones. We further confirmed the clonally
related nature of two of the hybridomas (27.2-3 and 8.2-2) by
cloning and sequencing the L chain genes. The CDR3 regions of
the VJ genes from these clones are almost identical (Fig. 1D), and
are very different from that of the fusion partner (data not shown).
We conclude that the changes between clones 27.2-3 and 8.2-2 as
well as among 1.12-1, 14.2-1, and 8.3-2 most likely result from
gene conversion and are not due to cloning or PCR artifact.
In addition to ruling out artifacts, it is important to determine
that the putative donor gene for gene conversion was not used in
the VDJ gene rearrangement. For the mutations in clone 8.8-2, the
putative donor gene, VH6, is nonfunctional because of a translational stop codon in the leader exon (GenBank accession number
U51026). On this basis, as well as the fact that the entire sequence
of the related clones is more similar to VH1 than VH6, we conclude
that VH1 rather than VH6 was the rearranged gene.
Clonally related, germinal center VDJ genes
To obtain larger clonally related lineages, we cloned VDJ genes
directly from the germinal center microenvironment. We obtained
lineages of VDJ genes from three germinal centers (gc15, gc22,
and gc2.3) in which ongoing diversification had occurred.
Germinal center 15 (gc15). A large lineage of clonally related
VDJ gene sequences was obtained from gc15, at day 14 of a primary response (Fig. 4). The first two related clones (4228 and
4244) were PCR amplified by using pan VDJ primers. Because
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FIGURE 4. Nucleotide sequence comparisons of related VDJ genes cloned from gc15. The location of the specific 39 primer and CDR3 oligo probe used
for specific PCR and screening purposes are indicated beneath the sequences. Nucleotide sequences are shown compared with germline VH1a3, D1, and
JH2 used in these VDJ gene rearrangements. For details, see Fig. 2. Clones that have been confirmed in independent PCR amplifications are numbers 4228,
4589, 4498, 4509, 4496, and 4451. GenBank accession numbers for these clones are AF098203 through AF098211. 55, Gap in the sequence where the
genes were almost undiversified.
6608
Ag-INDUCED SOMATIC GENE CONVERSION OF RABBIT IgH VDJ GENES
their nucleotide sequences showed evidence of ongoing somatic
mutation, we searched for additional members of this lineage by
PCR amplification using primers and probes specific for CDR3 of
these related clones (Fig. 4). In this way, we found seven additional clonally related sequences. When the sequences of the nine
clones were compared, we found a cluster of mutations in CDR1
that was present in eight of the nine clones. Because these mutations are highly clustered, we propose that these changes are
caused by ongoing somatic gene conversion rather than hypermutation. However, we have not identified a sequence from an upstream donor VH gene segment that matches these mutations,
which is not surprising because the nucleotide sequences of only
15 of an estimated 100 upstream VH genes (31) are known for this
allotype. In addition to this putative ongoing somatic gene conversion, we found a gene conversion-like mutation in CDR2 and
FR3, with VH3 as the donor that was shared between all of the
FIGURE 6. Nucleotide sequence comparison of related VDJ genes obtained from gc22. Nucleotide sequences are shown compared with germline
VH1a2, D2b, and JH4 used in these VDJ gene rearrangements. Potential donor VH gene sequences are shown in Fig. 7. For other details see the legend to
Fig. 2. Clone 4427 was confirmed in an independent PCR. GenBank accession numbers for these clones are AF098212 through AF098222. 55, Gap in
the sequence where the genes were almost undiversified.
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FIGURE 5. Proposed lineage relationship between clonally related VDJ genes obtained from gc15. Germline (G.L.) and putative precursors (letters in
dashed circles) and mutations are as described in Fig. 3.
The Journal of Immunology
6609
FIGURE 7. Nucleotide sequence comparisons illustrating potential donor VH gene segments for gene conversion-like events in gc22
VDJ genes. All sequences are shown compared
with germline VH1a3. Dots indicate nucleotide
identity. A, Clone 4337, with potential upstream
donor gene sequence shown beneath. VH2,
VH6, VH8, and VH10 are identical in this region. B, Clone 4254 shown with three potential
donor VH genes from the a3 haplotype. C,
Clone 4328 shown with three similar potential
donor gene sequences from the a3 haplotype.
clones are depicted as common precursors A through E. As described above, the key points of the lineage include shared gene
conversion between all clones within CDR2 and FR3 using VH3 as
a donor (germline to precursor A) and a cluster of changes in
CDR1 (aa 25–32) that is shared among all of the clones (precursor
B) except clone 4228. Much of the ongoing diversification appears
to result from point mutations more characteristic of somatic hypermutation than somatic gene conversion.
Germinal center 22 (gc22). The next large lineage of germinal
center VDJ genes we obtained was from gc22, also obtained on
day 14 of a primary immune response. Three clones (4269, 4268,
FIGURE 8. Proposed lineage relationship between clonally related VDJ genes obtained from gc22. Germline (G.L.) and putative precursors and
mutations are shown as in Fig. 3. The asterisk indicates several VH gene segments could have been donors for this gene conversion event. For details, see
Fig. 7.
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
clones. Because this gene conversion is found in all nine sequences, it must have occurred earlier in the immune response, or
before immunization. However, we do not believe that VH3 was
used in this rearrangement because sequences upstream of the
VH3-like mutations are more similar to VH1 than to VH3. Further,
the sequence of clone 4228 is identical to VH1 except for the mutations due to gene conversion by VH3.
We drew a lineage relationship between the related clones from
gc15, in which we attempted to minimize the number of branches
by maintaining the least number of identical, independent mutations (Fig. 5). In each case, the shared mutations between related
6610
Ag-INDUCED SOMATIC GENE CONVERSION OF RABBIT IgH VDJ GENES
FIGURE 9. Nucleotide sequences and putative lineage relationship of gc2.3-related VDJ gene clones 4349 and 4367. Comparison to germline VH1a3,
D2b ,and JH4. For details. see Fig. 2. GenBank accession numbers for these clones are AF098223 and AF098224.
Discussion
Rabbits use both somatic gene conversion and somatic hypermutation to diversify an IgH repertoire limited by preferential VH
gene usage (10, 13). This somatic diversification occurs at a young
age beginning at ;4 wk (28), preferentially in GALT (18, 19).
Until now, we did not know whether somatic gene conversion in
rabbit could also occur during peripheral immune responses. In the
present study, we obtained clonally related VDJ gene sequences
from FITC-specific hybridomas and from Ag-induced germinal
centers scraped from the PLN. We found evidence of ongoing
somatic hypermutation, as assessed by point mutations in the D
regions of related clones. We also found evidence for ongoing
somatic gene conversion based on mutations that match the sequence of upstream donor gene segments within the V regions of
clonally related VDJ genes. We conclude that both somatic gene
conversion and somatic point mutation were induced during the
course of an immune response in the PLN and, further, that somatic gene conversion in rabbit can occur outside GALT as part of
peripheral immune responses.
We believe that the somatic gene conversion-like mutations as
well as hypermutations were introduced during the course of Aginduced clonal expansion in the PLN. Alternatively, the mutations
might have occurred before the immune response, either within the
PLN or in another secondary lymphoid tissue. Because naive PLN
contains very few germinal centers, it is unlikely that the VDJ
clones diversified here before immunization. If these clones first
diversified in another location, then the related clones would need
to migrate separately into the PLN, where they would be selected
by Ag to expand without further somatic diversification. We consider this an unlikely possibility in any circumstance, but it is even
more unlikely because we used GALTless rabbits in which VDJ
genes from non-Ag-selected peripheral B cells had undergone only
limited somatic diversification. We confirmed this previous observation by PCR amplifying VDJ-Cm genes from PBL of the rabbits
used at the time of the fusions, and indeed, the genes were almost
undiversified, with between 0 and 3 nucleotide changes per VDJ
gene clone (data not shown). Therefore, we believe that somatic
gene conversion and hypermutation occurred during clonal expansion caused by the immune response within the PLN.
Excluding PCR artifacts
Studies that rely heavily on PCR amplification are subject to PCR
artifacts, including misincorporation of nucleotides by the polymerase, which would appear as single base mutations, and PCRgenerated chimeric molecules, which could masquerade as somatic
gene conversion-like mutations (33). We analyzed primarily somatic hypermutation within the DH region of the hybridoma VDJ
genes, which were amplified with Taq polymerase. In our experience, we consistently obtain at most one error per VDJ gene (12,
14). Extrapolating this error frequency to an average-length CDR3
region (55 base pairs), we would expect at most 0.1 mutation per
CDR3. In fact, we found the frequency of mutations in CDR3s is
much higher (1–7 base pairs) than anticipated by the PCR error
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and 4254) were amplified using pan VDJ primers, and we obtained
eight more clones using CDR3-specific 39 primers and probes (Fig.
6). The VDJ gene sequences from these 11 related clones have
evidence of ongoing somatic diversification both in the form of
single nucleotide changes, which do not match known upstream
donor genes, and several somatic gene conversion-like mutations
that match or closely match upstream VH genes. The nucleotide
sequences of clones in the region containing the gene conversionlike mutations in FR1 (4254, 4337, and 4328), are compared with
potential donor gene sequences in Fig. 7. For example, clone 4337
has four nucleotide changes in FR1 that match each of four upstream VH gene segments VH2, VH6, VH8, and VH10 (Fig. 7A).
The mutations in FR1 of the other two clones (4254 and 4328)
closely match any one of three upstream VH gene segments (Fig.
7, B and C). In addition to these three examples of gene conversion-like mutations in FR1, 8 of the 11 clones have a codon deletion in CDR1 (Fig. 6). In general, codon deletions are rare events
during hypermutation and are more characteristic of somatic gene
conversion. In fact, 9 of the 15 cloned upstream donor genes also
contain a deletion at this codon, although none exactly matches
any of the clones in this lineage. The codon deletion in CDR1
provided a useful mutation with which to begin developing a lineage relationship between the clones (Fig. 8).
Germinal center 2.3 (gc 2.3). We obtained one lineage from
gc2.3, obtained on day 5 of a tertiary immune response, that
showed evidence of ongoing diversification by both somatic gene
conversion and hypermutation (Fig. 9). Mutations in FR1 of clone
4349 appear to have been donated by VH8, whereas in FR1 of
clone 4367, any of several upstream VH segments (VH2, VH6,
VH8, or VH10) could have donated the four observed nucleotide
changes. In addition to this ongoing gene conversion, there are
shared gene conversion-like mutations in CDR2, in which VH3
was a potential donor. Several other mutations, mostly point mutations, were shared between the two clones, and because they
spanned the 59 (leader intron) and 39 (FR3/CDR3) ends of the
genes, we suggest that these clones are not PCR-generated chimeric artifacts.
The Journal of Immunology
variant B cell receptors. In the context of an Ag-specific immune
response, somatic gene conversion, especially within the CDRs,
might change the specificity of the Ag receptor rather than increase
its affinity for Ag. As such, somatic gene conversion may rescue
cells destined to die and thereby be more similar to Ag-induced
peripheral receptor editing than to somatic hypermutation (39 –
43). Alternatively, both Ag-induced receptor editing and somatic
gene conversion may serve to increase the overall diversity of the
Ab repertoire, similar to the function of somatic gene conversion
occurring in GALT of young rabbit or bursa of embryonic chicken.
This idea is intriguing especially in rabbit, because B lymphopoiesis is limited in adults (28) and therefore new specificities are not
continuously generated.
Correlation between the occurrence of somatic gene conversion
and hypermutation
From our experiments, it is clear that both somatic gene conversion
and hypermutation can operate on the same VDJ gene sequences.
A link between the occurrence of somatic gene conversion and
hypermutation has also been described in several other studies
(discussed in Ref. 44). In young rabbits, somatic gene conversion
within the VH region and hypermutation both occur within a similar time frame (11, 12, 28) and in a similar location (18). Chickens
show a similar correlation in diversification of Ig genes in both
adult splenic germinal centers (23), and in the embryonic bursa,
although, somatic gene conversion predominates in Ig gene diversification in the bursa (4, 5). In mouse, although gene conversion
is rarely, if ever, found during normal immune responses, in a
transgenic mouse model in which two Ag-specific VDJ genes were
arranged in tandem, gene conversion-like mutations and nontemplated point mutations were always found on the same sequence
(45). Together, these data show a correlation between the occurrence of somatic gene conversion and hypermutation, suggesting
that both can be induced under similar circumstances and, in fact,
may share some mechanistic features. By exploring the different
environments in which we see one or both types of somatic diversification, we may begin to unravel the relationship between the
mechanisms and requirements of both somatic gene conversion
and hypermutation.
Function of Ag-induced somatic gene conversion
Acknowledgments
Somatic diversification by hypermutation during immune
responses in mouse and human is important for affinity maturation
(37), and Ag-induced somatic gene conversion may serve a similar
purpose. In chicken, somatic gene conversion occurs within
splenic germinal centers (23) and occurs early (day 7) during the
primary immune response, with point mutations accumulating at
later stages during the same immune response (38). This finding
suggests that somatic gene conversion may first generate a large
variety of B cell receptors, and hypermutation fine tunes the immune response later. In this study, we demonstrate that Ag-induced somatic gene conversion likely occurs in a mammalian species, although we do not know what the contribution of somatic
gene conversion is to affinity maturation. Because rabbit uses both
somatic gene conversion and hypermutation to diversify its IgH
genes during an immune response, we suggest that rabbit is an
excellent model in which to study the relative contributions of
gene conversion and hypermutation to the quality of the humoral
immune response.
With regard to affinity maturation, somatic gene conversion is
quite different from hypermutation in that a single recombination
(mutation) event can alter many amino acids. Therefore, diversification by somatic gene conversion could quickly generate many
We thank Roauchania Purnyn for expert and dedicated technical assistance
with the germinal center studies.
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