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The Journal of Immunology, 1999, 162: 6602-6612.
Copyright © 1999 by The American Association of Immunologists

Antigen-Induced Somatic Diversification of Rabbit IgH Genes: Gene Conversion and Point Mutation1 ,2

Candace R. Winstead3, Shi-Kang Zhai, Periannan Sethupathi and Katherine L. Knight4

Department of Microbiology and Immunology, Loyola University of Chicago, Maywood, IL 60153


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
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, 2, 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 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 predominant 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 3'-most VH gene segment, VH1, in 80–90% of the VDJ gene rearrangements (13, 14, 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, 21, 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 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.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
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).

Immunizations and generation of rabbit Ag-specific hybridomas

Rabbits were immunized with FITC-HSA (800 µg) (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 µg) in IFA.

The HGPRT- 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 ligand-presenting cells (4 x 106) were irradiated with 5000 rad and incubated with 4 x 107 PLN cells with FITC-HSA (10 µg/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 x 104 cells/well, and medium supplemented with hypoxanthine-aminopterin-thymidine was added 24 h 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 µg/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 IGo. The FITC-specific hybridomas were cloned by limiting dilution.


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  		Table I. Summary of fusions from FITC-HSA-immunized rabbits after primary and secondary responses

 
Cloning and sequencing of hybridoma VDJ genes

RNA was isolated from 1 x 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 µg of FITC-specific hybridoma RNA as described (25). VDJ genes were amplified from the cDNA using Taq polymerase with VH leader-specific primer, VHRPS (5'-AGGAATTCTGCAGCTCTGGCACAGGAGCTC-3'), as the 5' primer and one of two pan JH primers (JHpBR (5'-GTCGAATTCACCTGAGGAGACGGTGACCA-3') or JHR1 (5'-CTCGAGAATTCTGAGGAGACGGTGACCAGGGTGCC-3')) as the 3' primer as described (14). VJ genes were amplified with a VK-specific leader primer (5'-TCGGATATCCACCATGGACACGAGGGCCCCCACTCAGCTGCTG-3') and JK-specific 3' primer (5'-GCAGTCGACTTACCTTTGACCACCACCTCGGTCCCTCCGCCGAA-3'). 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 VH2-a3,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).

PCR amplification of VDJ genes from germinal center DNA

PLN tissues were embedded in OCT (Tissue-Tek, Torrance, CA) and sectioned (7 µm and 14 µm) with a cryostat (2800 Frigocut, Reichert-Jung, Germany). Adjacent sections were placed on the same slide, fixed in ice cold acetone, and stored at -20°C, before staining with Harris hematoxylin and eosin. Germinal center tissue was scraped under a dissecting microscope (Olympus, Tokyo) at x40 magnification using pulled, siliconized 50-µl 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 µl of 1x PBS diluted 1:5.

Germinal center genomic DNA was prepared as described (20). Briefly, the germinal center material was incubated with 5 µl 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-µl reaction consisted of 1 µl of germinal center genomic DNA plus the following: 1x Pfu polymerase buffer (Stratagene, La Jolla, CA), 200 µM mixed dNTPs (equimolar of each dNTP; Pharmacia, Piscataway, NJ), 0.1 or 0.2 µM 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 5' primer was located in the promoter region of VH1 (VHPr) at approximately -250 nucleotides from the ATG start site. The 3' primer used in the first round anneals immediately 3' of the JH4 gene segment (3'JH4), which is rearranged in 80% of VDJ genes. The second round of PCR was either nested on both sides or seminested, in that the 5' primer was the same as in the first round and only the 3' primer was internal. For fully nested PCR, the internal 5' primer was still within the VH1 promoter, at -212 nucleotides from the ATG start site (upVH-H3). The 3' primer for either fully nested or seminested PCR was the pan JH primer, JHpBR, described above. Primer sequences are: VHPr, 5'-TAACAAGCTTAAAAATTCATATGATCTGAATC-3'; upVH-H3, 5'-TCCAAGCTTATCACAGCCATCAC-3'; and 3'JH4, 5'-GTAGGAGCTCGAGTTGGCAAGGACTCAAC-3'.

To specifically amplify VDJ genes from a particular B cell clone, we designed 3' primers that began in complementarity-determining region 3 (CDR3) region and ended within the J region of that clone. The specific 3' primers were used in both rounds of seminested PCR, and the 5' 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 3' CDR3-specific primers and probes were as follows. For clones from germinal center 15: 3' primer (CDR3-4228) 5'-AGGGAATTCATCAAAGCACCAAT-3', and oligo probe (CDR3p-4228) 5'-TGTGCGAGAGGCCTCTAT/CGAT-3'. For clones from germinal center 22: 3' primers (CDR3a-4254) 5'-GGCGAATTCCCACAACTTCCAGGG-3', (CDR3b-4254) 5'-CCAGAATTCGGGCCATAACCAG/ACATAA-3', and oligo probe (CDR3p-4254) 5'-TGTGCGAGAGGTGGTTATGTT-3'.

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 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
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
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 diversification 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. 1Go). All of these sets were derived from hybridomas obtained after a secondary immune response (Table IGo) and all used the germline gene VH1 in their VDJ gene rearrangements, based on nucleotide sequence analysis (Fig. 2Go and data not shown). Five of the sets contained clones with mutations within some, but not all, members of the set (Figs. 1Go and 2Go), and these were analyzed further for evidence of both ongoing somatic point mutation and ongoing somatic gene conversion.



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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.

 


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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. {psi}, 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. 1Go.

 
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. 1Go). We found as many as eight nucleotide differences between the related clones (clones 30.14-1 and 27.11-1; Fig. 1GoA), 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. 2Go, A–C). However, in two sets of clonally related VDJ genes, there was evidence of ongoing somatic gene conversion because the gene conversion-like 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. 2GoD). 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. 2GoE), 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. 2Go). On the basis of these mutations, we could draw lineage relationships between the clonally related hybridomas (Fig. 3Go). Fig. 3GoA 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. 3GoB. 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 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.



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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 {psi}8.3-2. 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.

 
To rule out PCR or cloning artifacts as the explanation for the gene conversion-like mutations in these clonally related hybridomas (Fig. 2Go, 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. 1GoD), 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. 4Go). The first two related clones (4228 and 4244) were PCR amplified by using pan VDJ primers. Because 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. 4Go). 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 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.



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  		FIGURE 4. Nucleotide sequence comparisons of related VDJ genes cloned from gc15. The location of the specific 3' 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. 2Go. 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. ==, Gap in the sequence where the genes were almost undiversified.

 
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. 5Go). In each case, the shared mutations between related 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.



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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. 3Go.

 
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, and 4254) were amplified using pan VDJ primers, and we obtained eight more clones using CDR3-specific 3' primers and probes (Fig. 6Go). 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 conversion-like mutations in FR1 (4254, 4337, and 4328), are compared with potential donor gene sequences in Fig. 7Go. 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. 7GoA). The mutations in FR1 of the other two clones (4254 and 4328) closely match any one of three upstream VH gene segments (Fig. 7Go, 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. 6Go). 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. 8Go).



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  		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. 7Go. For other details see the legend to Fig. 2Go. Clone 4427 was confirmed in an independent PCR. GenBank accession numbers for these clones are AF098212 through AF098222. ==, Gap in the sequence where the genes were almost undiversified.

 


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  		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.

 


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  		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. 3Go. The asterisk indicates several VH gene segments could have been donors for this gene conversion event. For details, see Fig. 7Go.

 
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. 9Go). 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 5' (leader intron) and 3' (FR3/CDR3) ends of the genes, we suggest that these clones are not PCR-generated chimeric artifacts.



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  		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. 2Go. GenBank accession numbers for these clones are AF098223 and AF098224.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
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 Ag-induced 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-Cµ 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 PCR-generated 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 alone. Therefore, we conclude that most of the point mutations we observed in the DH regions are not PCR artifacts and instead are likely due to in vivo-induced somatic diversification.

The other PCR artifact of concern is PCR-generated chimeric molecules, caused by template "jumping" during PCR amplification between the homologous VH genes (34, 35). PCR chimeras are especially important to exclude because they could appear as gene conversion-like mutations. We attempted to rule out this type of artifact by performing two separate PCR amplification reactions on the same DNA or cDNA template. We reasoned that it would be highly unlikely to obtain identical chimeras in each of two separate PCR reactions. For the clonally related FITC-specific hybridoma VDJ genes, we independently RT-PCR amplified the clones that contained ongoing somatic gene conversion-like mutations (Fig. 3Go) and obtained identical sequences. Therefore, we conclude that the gene conversions found in these hybridomas do not result from PCR or cloning artifacts. For the clonally related VDJ genes from the germinal centers, we were able to reamplify and separately confirm nearly all of the clones from gc15 in which there were ongoing gene conversion-like mutations in CDR1. We were not as successful at separately reamplfying identical clones from gc22 or gc2.3. Despite this, there are two examples of ongoing gene conversion-like mutations from these germinal centers that are most likely not due to PCR chimeras. The first is a codon deletion in CDR1 of 8 of the 11 related clones from gc22 (Fig. 6Go). The presence and absence of this codon have been confirmed in independent PCR amplification. Although this codon deletion is similar to those reported in some human VDJ genes in which a tandem repeat is deleted (36), we think it more likely that this deletion occurred by somatic gene conversion because 9 of 15 sequenced upstream VH donor gene segments contained a deletion at this codon. Another example of ongoing gene conversion-like mutations that is likely not a PCR chimera is in clones 4349 and 4367 from gc2.3 (Fig. 9Go). These clones share identical mutations both 5' and 3' of the ongoing gene conversion-like mutations in FR1. For these clones to be PCR chimeras, template jumping would need to occur twice for each of the clones. Taking these data together, we conclude that PCR artifacts are excluded as the basis for most of the ongoing somatic point mutations and somatic gene conversion-like mutations that we found.

Function of Ag-induced somatic gene conversion

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 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, 40, 41, 42, 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 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.


   Acknowledgments
 
We thank Roauchania Purnyn for expert and dedicated technical assistance with the germinal center studies.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant AI16611. Back

2 Genes discussed here have been deposited in GenBank under accession numbers AF098225 through AF098241. Back

3 Current address: Department of Immunology, IMM-25, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. Back

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: Back

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. Back

Received for publication October 19, 1998. Accepted for publication March 10, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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