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The Journal of Immunology, 1999, 162: 3984-3995.
Copyright © 1999 by The American Association of Immunologists

Gene Conversion and Hypermutation During Diversification of VH Sequences in Developing Splenic Germinal Centers of Immunized Rabbits

Enrico Schiaffella1,*, Devinder Sehgal1,*, Arthur O. Anderson{dagger} and Rose G. Mage2,*

* Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; and {dagger} U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21702


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
The young rabbit appendix and the chicken bursa of Fabricius are primary lymphoid organs where the B cell Ab repertoire develops in germinal centers (GCs) mainly by a gene conversion-like process. In human and mouse, V-gene diversification by somatic hypermutation in GCs of secondary lymphoid organs leads to affinity maturation. We asked whether gene conversion, somatic hypermutation, or both occur in rabbit splenic GCs during responses to the hapten DNP. We determined DNA sequences of rearranged heavy and light chain V region gene segments in single cells from developing DNP-specific GCs after immunization with DNP-bovine {gamma}-globulin and conclude that the changes at the DNA level that may lead to affinity maturation occur by both gene conversion and hypermutation. Selection was suggested by finding some recurrent amino acid replacements that may contribute increased affinity for antigen in the complementarity-determining region sequences of independently evolved clones, and a narrower range of complementarity-determining region 3 lengths at day 15. Some of the alterations of sequence may also lead to new members of the B cell repertoire in adult rabbits comparable with those produced in gut associated lymphoid tissues of young rabbits.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Germinal centers (GCs)3 in secondary lymphoid organs are specialized structures within which somatic diversification of rearranged V genes occurs that lead to affinity maturation of Abs in immune responses to T-dependent Ags. During some stages of GC development, a dark zone and a light zone (LZ) can be defined histologically. B cells in the dark zone (centroblasts) are characterized by intense proliferative activity (1, 2). The rearranged V region genes of the centroblasts undergo somatic hypermutation (3, 4, 5, 6, 7). Centroblasts then move to the LZ, become nondividing centrocytes, contact follicular dendritic cells (FDC) with the Ag in a native conformation on their surface, and undergo selection. B cells with high-affinity receptors for the Ag continue to differentiate toward Ab producing cells or memory B cells (8, 9). Cells that fail to bind Ag or that bind with a lower affinity die by apoptosis, or reexpress the recombination-activating genes 1 and 2 (RAG-1 and RAG-2) gene products and undergo receptor editing (10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20). The presence of RAG-1 and RAG-2 proteins in secondary follicles detected by immunohistochemistry (14), the re-expression of RAGs in activated B cells stimulated by LPS and IL-4 (10) and the presence of intermediate products of VJ recombination at the {kappa} locus, (15, 16) reflect GC B cells in which receptor editing or revision occurs.

Emerging evidence suggests that the description of GCs outlined above oversimplifies the actual events that occur. It was recently shown that proliferating cells are present in the LZ in contact with the FDC network at least until day 10 of the response of BALB/c mice to 2-phenyl oxazolone-BSA (21). Successive cycles of hypermutation and selection are thought to occur (22, 23) involving continued proliferation and selection of cells with surface B cell receptor (BCR) near FDC in the T cell-rich LZ (21) rather than by cells recycling between proliferative and selective compartments of the GC (24, 25). Studies in an adoptive transfer model suggest that Ag-specific CD4 T cells interact with dendritic cells in T cell areas. They subsequently participate in cognate T–B cell interactions at B cell follicular borders where lymphokines and signaling between CD40 and its ligand CD154 drive B cell proliferation and development of peanut agglutinin-positive GCs (26). Interactions of CD40 and its ligand remain crucial during much of the GC reaction. Other interactions such as between OX40 and OX40 ligand (27) and between B7–2 and CD28/CTLA-4 are also important. Interference with B7–2 function by administration of Ab interrupts hypermutation and generation of memory populations (28). Affinity-based selection and receptor editing may also be influenced by cognate T–B cell interactions involving CD40 and lymphokine signaling. B cells with low affinity for Ag may be signaled to up-regulate recombinase expression whereas B cells with high affinity receptors may generate BCR-mediated signals that inhibit recombinase reactivation (19). Very recently, the possibility has been raised that some repertoire development may occur in GCs of mice and humans (14, 17, 29) that resembles the primary repertoire development that occurs in tissues such as young rabbit appendix and the chicken bursa of Fabricius (30, 31, 32, 33, 34).

GCs are also present in primary lymphoid organs such as the young rabbit appendix (35) and the chicken bursa of Fabricius where they mainly function to produce a broad B cell repertoire rather than to generate a specific immune response (30, 31, 32, 33, 34, 36). Unlike mice and humans, rabbits rearrange only a few heavy chain V region (VH) genes, so that the diversity generated by combinatorial mechanisms is limited (37, 38). Appendix GCs are seeded by B cells that have already rearranged their Ig genes in sites such as fetal liver, omentum, or bone marrow (36, 39). They colonize the GCs of the appendix during the first weeks of life, proliferate in response to Ags and superantigens (36) and their rearranged VH genes diversify (30) by somatic hypermutation and a gene conversion-like mechanism (38, 40, 41). We refer to the mechanism in rabbits as gene conversion-like because nonreciprocal exchange has not been formally shown in rabbits whereas studies in chickens (42) showed that donor sequences were unaltered.

The objective of this work was to investigate diversification of rearranged VH and light chain V region sequences in splenic GCs during T cell-dependent immune responses to DNP-bovine {gamma}-globulin (DNP-BGG) in adult rabbits, an Ag chosen because of its previous use in documenting affinity maturation in the rabbit (43, 44). We addressed the question of whether VH-gene conversion, somatic hypermutation or both also take place during the GC reaction in response to a T cell-dependent Ag in GCs of the rabbit spleen. We combined the techniques of microdissection of single Ag-specific B cells with a PCR-based sequencing strategy that avoids PCR artifacts. This approach is particularly successful in rabbit because the B cells rearrange only a few VH genes and the majority do not rearrange the second allele (45). Although it was previously reported that VH sequences in adult rabbit B cells were highly diversified (46) we found that cells collected during early stages of the GC reaction (days 7 and 10) from adult rabbit spleen had rearranged VH sequences that were identical or nearly identical with germline sequences (47). This report presents our observations, both at the histological and molecular levels, on the progression of the GC reaction in rabbit spleens during the response to the T cell-dependent hapten DNP. We find changes that may lead to affinity maturation occur by both gene conversion and somatic hypermutation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Ag and immunization

DNP37-BGG was prepared as described (47, 48). Rabbits from 1 to 2 years of age, homozygous for VHa2 (haplotype F-I) and C{kappa}b5 allotypes, were given a priming immunization of 500 µg of BGG in CFA injected s.c. Between 7 and 10 days later, 500 µg of DNP-BGG were given i.v. Control rabbits received BGG at both time points.

Immunohistochemistry

At 7, 10, and 15 days after the second injection, rabbits were killed, and the spleens embedded in OCT and quickly immersed in liquid nitrogen. Serial (7 µm) sections were cut from the spleens in a cryostat microtome, kept for 30 min at room temperature, fixed at 4°C in acetone for 10 min and stored at -70°C. DNP-binding cells from splenic GC were revealed by incubation with DNP-alkaline phosphatase (DNP-AP) followed by incubation with an appropriate substrate as detailed below. The preparation of DNP-AP and staining of tissue sections were as described previously (47). Spleens from BGG-immunized control animals were also stained with DNP-AP to exclude any nonspecific binding. As an additional control, splenic sections from DNP-BGG immunized rabbits were stained with unconjugated AP. To identify proliferating cells within GCs, an adjacent section was stained with mouse anti-human Ki-67 mAb that crossreacts with rabbit (Dako, Carpinteria, CA), followed by biotinylated horse anti-mouse IgG (Vector Laboratories, Burlingame, CA) and avidin-biotin complex conjugated to AP (Vector Laboratories). To obtain information about isotype expression we used biotinylated polyclonal goat anti-rabbit IgM (µ heavy chain-specific) and biotinylated polyclonal goat anti-rabbit IgG ({gamma} heavy chain-specific) Abs (Southern Biotechnology Associates, Birmingham, AL). In some experiments for double staining with DNP-AP and anti-IgG, IgM, or CD4 we used VECTOR Blue Substrate kit (Vector Laboratories) in combination with the VECTOR Red Substrate kit.

Microdissection of frozen tissue sections

The stained sections were incubated with 5 mg/ml collagenase H (Boehringer Mannheim, Indianapolis, IN) as described (49). Single cells from GCs obtained at days 7, 10, and 15 were collected from the sections using a hydraulic micromanipulator (Narishige, Greenvale, NY) assembled on an inverted microscope (Olympus, Lake Success, NY). From days 7 and 10 we collected cells from DNP+ GCs that were either DNP+ (day 7), Ki-67+ (day 10), or both (day 10) (47). From day 15 we collected proliferating cells based on positive staining with anti-Ki-67 from a GC that stained positive for DNP-binding cells.

Individual cells were each transferred to 0.2-ml microfuge tubes containing 5 µl of an alkaline lysing solution (200 mM KOH and 50 mM DTT). The tubes were incubated at 65°C for 10 min before adding 5 µl of neutralizing solution (900 mM Tris-HCl (pH 8.3), 300 mM KCl, and 200 mM HCl).

DNA amplification and sequencing

We elected to use direct sequencing of PCR products from single cells without a cloning step to avoid two potentially serious artifacts. First, without a cloning step the effect of Taq DNA polymerase errors is minimized (50, 51, 52) because Taq DNA polymerase-based errors are then detected in sequences only if they occur in the first few cycles of amplification. Second, by amplification of VHDHJH sequences from single cells we avoided producing hybrid VHDHJH sequences that were found especially during the somatic hypermutation stage of the GC reaction (6).

The rearranged VHDHJH and V{kappa}J{kappa} sequences were amplified from single cells of DNP+ GCs using a nested PCR strategy. The primers used are shown in Table IGo. In the first round, the rearranged VHDHJH and V{kappa}J{kappa} sequences were amplified simultaneously. Nested PCR was performed separately for the rearranged heavy and {kappa} light chain using internal primers specific for the rearranged VHDHJH and V{kappa}J{kappa} sequences. The temperature conditions for the first and second round of touchdown PCR were the same (53). There was an initial 95°C 2-min denaturation step. This was followed by five cycles of denaturation at 94°C for 1 min; for the first cycle annealing was at 66°C for 30 s that dropped down to 56°C at the end of the fifth cycle at the rate of 2°C per cycle; and an extension step at 72°C for 1 min. The annealing temperature for the remaining 29 cycles was 56°C. The PCR ended with a 5-min extension at 72°C. The PCR was performed on a PTC-100 programmable thermal cycler (96-well model with hot bonnet) (MJ Research, Watertown, MA).


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  		Table I. Primers used for amplifying rearranged VHDHJH and V{kappa}J{kappa} sequences by single cell PCR

 
For the first round, MgCl2, Tris-HCl (pH 8.3), Triton X-100, and external primers (DSH1, DSH7, and DSH8 for the heavy chain; and DS{kappa}1 and DS{kappa}7 for the {kappa} light chain) (see Table IGo) were added to the tube containing the neutralized cell lysate such that the final concentration in a 40 µl volume were 3.75 mM, 10 mM, 0.1% (v/v), and 0.75 µM (of each primer), respectively. The DNA was denatured at 95°C for 10 min. Thereafter, 20 µl of a mix containing 10 mM Tris-HCl (pH 8.3), 0.1% (v/v) Triton X-100, 600 µM of each dNTP, and 5 units of AmpliTaq DNA polymerase (Perkin-Elmer, Branchburg, NJ) was added and the tube setup for PCR. From the first round PCR, 2.5 µl of the product served as the DNA template for the second round touchdown PCRs (53) that were done for the rearranged VHDHJH and V{kappa}J{kappa} sequences separately. The internal primers used for amplifying the rearranged heavy chain were DSH2 and DSH5 (Table IGo). The corresponding set of internal primers used for amplifying the rearranged {kappa} light chain were DS{kappa}2 and DS{kappa}14 (Table IGo). In addition to the template DNA, the other components in the 50 µl of PCR were 1x PCR buffer (containing MgCl2) (Perkin-Elmer), 200 µM of each dNTP, 0.5 µM of each primer (internal to those used in the first round) (DSH2 and DSH5 for the heavy chain; DS{kappa}2 and DS{kappa}14 for the {kappa} light chain) (see Table IGo) and 2 units of AmpliTaq DNA polymerase. The PCR parameters were the same as for the first round.

As a control we also conducted a blind experiment where we collected a group of 23 CD4+ cells from the T cell area along with 69 Ki-67+ cells from within the GC. In the PCRs run at the same time, we amplified 48 of 69 VHDHJH sequences from the cells collected from the GC; none of the 23 T cells gave a PCR product.

Recovery of PCR products for sequencing was done as described (47). The Prism Ready Reaction DyeDeoxy Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) was used following the manufacturer’s instructions to sequence both strands using the second round primers on an Applied Biosystems model 377 automated sequencer (54). The sequences were analyzed using AUTOASSEMBLER version 1.3 (Applied Biosystems) and MACVECTOR versions 5.0 and 6.0 (Kodak Scientific Imaging Systems, Rochester, NY; Oxford Molecular Group, Campbell, CA).


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Immunohistochemical characterization of splenic GCs in the rabbit

For histological characterization of GCs in the spleens of immunized rabbits, we stained frozen sections with the DNP-AP in combination with other Abs such as monoclonal mouse anti-human Ki-67 that crossreacts with rabbit, mouse anti-rabbit CD4, polyclonal goat anti-rabbit IgM and goat anti-rabbit IgG Abs. In rabbits that were carrier-primed 7–10 days before i.v. immunization with DNP-BGG, GCs were present at day 7 and their appearance and staining were similar at day 10. Fig. 1Go, A–F shows staining of tissue sections with polyclonal goat anti-rabbit IgM and goat anti-rabbit IgG Abs. DNP-AP staining (blue) in combination with anti-IgM (red; Fig. 1Go, A–C) and in combination with anti-IgG (red; Fig. 1Go, D–F), suggests that isotype switching had not yet occurred at day 7 because all the GCs stained positively for IgM, but little or no IgG staining was evident (Fig. 1Go, A and D). By day 15 (Fig. 1Go, B, C, E, and F) the cells in many GCs had undergone isotype switching. The GC shown in Fig. 1Go, B and E, show evidence of recent isotype switching as adjacent sections stained positively with anti-IgM (B) and IgG (E). Fig. 1Go, C and F, show another GC at day 15 that lost most of its IgM expression (C) and stained with anti-IgG (F). Fig. 1Go, G–J, shows staining of adjacent sections at days 10 and 15 with anti-Ki-67 that identifies a nuclear Ag in proliferating cells (G and H) and of DNP-AP in combination with anti-CD4 (I and J). At day 15 (Fig. 1Go, H and J), proliferating cells were more concentrated in the areas of the GC where cells down-regulated BCR expression, and staining with DNP-AP was less intense. CD4+ T cells constituted a major population in the periarteriolar lymphoid sheath area. Within the GC, they were found interspersed with DNP-binding cells.



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  		FIGURE 1. Progression of rabbit splenic GC reactions between days 7 and 15 after i.v. immunization of BGG primed rabbits with DNP-BGG. A–F, Staining of rabbit splenic follicles and GCs for IgM and IgG on days 7 (A and D) and 15 (B, C, E, and F) of the immune response to DNP-BGG. A/D, B/E, and C/F are serial sections. The sections in A–C are double-stained with anti-IgM (red) and DNP-AP (blue). In D–F, anti-IgG (red) in combination with DNP-AP (blue) was used. G–J, Proliferating and DNP-binding cells in rabbit splenic follicles and GCs at days 10 and 15 of the response to DNP-BGG. G/I and H/J are serial sections at days 10 and 15, respectively. (G) and (H), anti Ki-67 (blue), counterstained with nuclear fast red. I and J, Double staining with DNP-AP (blue) and anti-CD4 (red). Bar = 100 µm.

 
Amplification and clonal relationships of rearranged Ig genes from GC B cells

Using PCR, we amplified the rearranged Ig genes in single cells obtained from several DNP-reacting splenic GCs of DNP-BGG immunized rabbits. Table IIGo summarizes the numbers of cells collected from individual GCs at days 7, 10, and 15 and the numbers of sequences that we were able to amplify and analyze. In the seven different GCs studied at days 7 and 10, 55% and 63% of the sequences respectively, had unique complimentarity-determining region 3 (CDR3) and were unrelated. At these time points we found one to four different clones per GC with only between two and seven members (47). The proportions of independent unique sequences decreased from more than half (82/140) at days 7 and 10 to 9.5% (8/85) at day 15. Conversely, groups of clonally related sequences predominated at day 15 and on average contained larger numbers of members. Although it is generally accepted that GCs develop oligoclonally (55, 56, 57), we observed a predominance of unique clonally unrelated sequences at days 7 and 10, many with 10 or fewer changes from the germline sequence of the rearranged VH gene. For the other small groups of clonally related sequences we could also readily identify the germline VH, DH, and JH genes that rearranged in their precursors. At day 10, the population of proliferating cells still showed considerable heterogeneity (47). However, by day 15, most sequences were clonally related, the V region portion of every heavy chain sequence had nine or more differences from germline sequences and there were also on average more changes in the DH and JH sequences. In GCs from mice immunized with the hapten nitrophenyl (NP), clonal diversity also decreased with time but at 4–6 days postimmunization most GCs had only between three and six different CDR3 region sequences and by day 8, when hypermutation had initiated, most GCs had only one or two clones (6). In chickens immunized with FITC-BSA, there were also numerous unique light chain sequences and great clonal complexity in individual GCs at day 7 but complexity markedly diminished by day 11. The data suggest that at the later time, more hypermutations and fewer gene conversions were occurring (58).


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  		Table II. Summary of PCR amplification, sequence analyses, and clones in splenic DNP+ GCs during immune responses of adult rabbits to DNP-BGG

 
Table IIIGo summarizes the results of analyses of heavy and light chain sequences from cells of GC L and N collected on day 15. All but 7 of the 85 successfully sequenced heavy chains were grouped into clones, 3 in GC L and 6 in GC N. Identification of clonally related light chain sequences showed correspondences with the heavy chain clones. In addition, there was one group of light chain sequences from cells in GC N where we obtained no heavy chain sequences and another pair of cells with related light chain sequences one of which also yielded a heavy chain sequence. Thus GC N had at least eight different clones and GC L had at least three. The emergence of expanding, diversifying dominant clones by day 15 correlates with the switching from IgM to IgG (Fig. 1Go, A–F). Analyses of clonally related sequences from day 15 show that gene conversion as well as hypermutation was occurring during clonal expansion.


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  		Table III. Summary of PCR amplification, sequence analyses of rearranged VH and V{kappa}, and clonally related groups of cells from DNP+ GCs at day 15 of the immune response of adult rabbits to DNP-BGG

 
Gene conversion and hypermutation during clonal expansion of B cells in GCs

Fig. 1GoH shows GC L stained for Ki-67 and Fig. 1GoJ shows its adjacent section stained for DNP. Proliferating cells in GC L were collected from two serial sections (La and Lb) and rearranged VHDHJH and V{kappa}J{kappa} genes were amplified. GC L had a predominant clone (L1) with 29 heavy chain members and an additional 13 cells from which we recovered only light chain sequences related to those in cells with heavy chains. A summary of this largest clone is shown in Fig. 2Go. Fig. 2GoA shows the approximate locations of the cells collected on a diagram of GC L based on the stained sections shown in Fig. 1Go, H and J. Cells belonging to clones L1, L2, and L3 are shown as green, red, and black dots, respectively. Unique members are shown as black squares. We generated genealogical trees based on the changes that occurred in clonally related sequences. For L1, this is shown in Fig. 2GoB and the corresponding sequences in Fig. 2GoC. The cells of clone L1, recovered from the two serial sections appeared to share a common precursor sequence derived from rearrangement of VH1a2, Df and JH4 that had already undergone several sequence alterations involving blocks of sequence that are best explained as arising by gene conversion from known (VH4, shaded orange) and unknown donors (UD, black boxes). The sequence of the rearranged VHDHJH of cell La 55 appears closest to the precursor sequence from which the cells arose. A single point mutation in the sequence of cell La 01, replaced Thr with Asn at amino acid position 79 (Kabat numbering; 59) and the remaining sequences acquired a silent g in the Lys81 codon. Cells with identical sequences were found in serial sections and at some distance from each other in a given section (see Fig. 2GoA). Lb 92 and four other cells (La 02, La 21, La 39, and La 58) had the same VHDHJH sequence (indicated by a plus after the cell name in Fig. 2GoA). The remaining cells diverged from this sequence by acquiring additional point mutations and gene conversions, but only two sequences (La 11 and Lb 22) had replacement changes in CDR3. The rearranged germline sequence of this CDR3 may have already been well suited for binding to DNP. Cell La 40 acquired a stretch of sequence in FR1 and CDR1 that matches a likely gene-conversion-donor gene sequence present in VH6 (boxed in dark blue); in La 04, a smaller tract only affected framework 1 (FR1). Both La 40 and Lb 83 underwent replacements of Gly16 with Asp that is found in many potential donor sequences including VH2, VH4, VH8, and VH9 (pink shading). Cells Lb 29 and Lb 30 exhibited the commonly observed replacements of Leu75 with Glu that are likely to have occurred through gene conversion from donor sequences present in both VH7 and VH9 (red and green box). This tract of changes extended at least through the silent g in Lys81. Lb 82 acquired a Ser codon TCT at position 50 that involved a two-base change plus acquisition of an adjacent silent base change. Another sequence, La 71 also acquired a Ser AGC at position 50 but it was via a single-base change from the Thr codon ACC. Ser61 was changed to Thr independently in several branches of the genealogical tree. This might reflect some selective advantage (e.g., for improved affinity). We also found two different Thr61 codons, ACA in Lb 17 and La 49 and ACC in five other sequences (Fig. 2GoC). Of 24 successful V{kappa}J{kappa} sequences, 11 were from the group of 29 cells with successfully sequenced VHDHJH. This is indicated in Fig. 2GoB by underlining the cell name. As regards the V{kappa} sequences, deduced V{kappa}J{kappa} protein sequences from four cells (La 55, La 64, Lb 22, and Lb 29) were identical; the others had between one and eight replacements. Definition of the complete database of germline V{kappa} sequences (unpublished manuscript) will allow us to reconstruct the events leading to the sequence changes with confidence. Now we can only conclude that the light chain sequences generally complement the heavy chain data (data not shown).



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  		FIGURE 2. Cells collected from GC L, the clonal relationships of members of the predominant clone L1 and their VHDHJH sequences. Individual GCs were designated by capital letters, sections within a GC by lowercase letters, and cells were numbered. A, The approximate locations from which cells were collected from GC L for sequencing. Fig. 1Go, H and J, show sections serial to those used for collection. The members of clones L1, L2, and L3 that yielded VHDHJH sequences are color coded as green, red, and black dots, respectively. Unique members are shown as black squares. The five cells that yielded identical VHDHJH sequences have a plus (+) after the cell name. La and Lb in the cell names indicate cells collected from two serial sections. B, Diagrammatic representation of the clonal expansion and sequence changes (codon position number: amino acid > new amino acid or silent change (lower case "s")). Codon positions are numbered according to Kabat et al. (59). The hypothetical rearranged precursor is in a rectangular box. Df is a germline DH gene described in Chen et al. (60). Another hypothetical precursor (P) is shown as a shaded circle. Lb 92+ indicates that in addition to Lb 92, four other cells (La 02, La 21, La 39, and La 58) had the same VHDHJH sequence. Underlined cell names indicate that a V{kappa}J{kappa} sequence was obtained from the same cell. Known germline VH genes that can account for blocks of gene conversion are shown as colored boxes. RVH indicates five different potential germline donor sequences (RVH700, 701, 714, 720, and 755) found in an a2 homozygous rabbit by Roux et al. (41). UD, unknown VH donor. (C) Heavy chain sequence alignment showing clonal relationships and a summary of the base changes, replacements and gene conversions (horizontal boxes) that occurred during clonal expansion of these cells. Amino acid replacements are shown above the sequences, silent base changes are in lowercase. The numbers above the reference sequence indicate codon positions according to Kabat et al. (59). Most framework region positions that were invariant are not shown. The VHDHJH sequences of members of clone L1 have been assigned GenBank accession numbers AF087712–AF087740.

 
Although most of the VHDHJH sequences found in cells collected from GC L belonged to a single clone, two other smaller sets of clonally related sequences (clones L2 and L3; Fig. 3Go) and five independent unique sequences were found. Clone L2, with five members provided additional examples of gene conversions that appear to have occurred during expansion of the clone within the GC. The initial precursor appears to have rearranged VH1 to Df and JH4. A common precursor (P1) underwent a large gene conversion that extended from FR1 through the first codon of CDR2 with the likely donor being VH7 (green) and a change involving VH9 (red) extending from CDR2 through position 75 of FR3. Precursor P1 also acquired a silent change in Ile102 of the J region and two amino acid replacements in CDR3. Cells Lb 24 and Lb 31 then underwent additional changes that may have been due to point mutations. Cells Lb 12, La 20, and Lb 35 share a common precursor P3 that arose after a second gene conversion involving VH9 and a silent change in Ser30 that may have been due to VH6.



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  		FIGURE 3. The sequences and clonal relationships of two additional clones from GC L (see also Fig. 2Go). Underlined cell names indicate that a V{kappa}J{kappa} sequence was obtained from the same cell. The approximate locations from which cells were collected for sequencing from GC L were shown in Fig. 2GoA and Fig. 1Go, H and J, show sections serial to those used for collection. The VHDHJH sequences of members of clones L2 and L3 have been assigned GenBank accession numbers AF087741–AF087745 and AF087746–AF087749, respectively.

 
Although clone L3 had only four heavy chain members, we obtained rearranged heavy and light chain sequences from all four cells and there were 10 additional light chain members. The light chain data complemented the heavy chain data and strongly favored the clonal tree we show compared with an alternative tree (data not shown). The presence of a replacement of Ser97 by Gly on two branches of the tree may indicate a selective advantage for improved affinity.

Based on heavy chain sequences, GC N yielded six groups of clonally related cells. One group of eight cells (N3) all had a frameshift mutation at the VH to DH junction and will not be considered in detail (sequences deposited in GenBank and given accession numbers AF087778–AF087785). Analyses of the other sequences allowed construction of clonal trees shown in Figs. 4Go and 5. In clone N1 (Fig. 4Go), rearrangement of VH1-D2a-JH4 then led to a common precursor (P1) with a gene conversion that was likely due to VH4, four replacement changes in CDR3, and a silent change in J4. Cell Na 73 branched off and 11 other cells shared a set of gene conversions that could have involved VH9 (red box) and either VH2, VH5, or VH8 (pink shading). Na 16, Nb 49, and Nb 56 from two serial sections had the same heavy chain sequence and two additional cells Nb 37 and Nb 72 had only a silent change in Ser31. Nb 09 acquired replacements in FR1, CDR1, and CDR3. The change of Val34 to Met and the reversions of Tyr32, Gly33, and Ile35 to Asn, Ala, and Ser can all be accounted for by a block derived from VH6 (dark blue box). Similarly, the loss of the silent base change in Ser3 (red box) in cells Na 11 and Nb 58 can be explained by a gene conversion block from VH2 or VH7 (green box). The remaining few silent and replacement changes may have been due to point mutations. Fig. 4Go also shows diagrammatically the three small clones N4, N5, and N6. Clones N5 and N6 both had precursors with changes in CDR1 that can be explained by two different gene conversions from the VH4 sequence. All three sequences from clone N5 also had the frequently observed replacements of Leu 75 with Glu that can be explained by conversion involving either VH7 or VH9 (red and green box). In N6 this replacement occurred in the sequence of Na 65 after divergence from Na 77.



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  		FIGURE 4. The sequences and clonal relationships of clone N1 from GC N and diagrammatic summaries of three smaller clones from the same GC (see also Fig. 2Go). Underlined cell names indicate that a V{kappa}J{kappa} sequence was obtained from the same cell. Nb 16+ indicates that two other cells, Nb 49 and Na 16, yielded identical sequences. Nb 37++ indicates that Nb 72 yielded an identical sequence. The VHDHJH sequences of members of clones N1, N4, N5, and N6 have been assigned GenBank accession numbers AF087755–AF087766, AF087786–AF087788, AF087789–AF087791, and AF087792–AF087793, respectively.

 
The sequences and clonal tree derived for clone N2 are shown in Fig. 5Go. During expansion of clone N2 from precursor P1, several additional gene conversion events occurred involving a donor that could have been VH6 (dark blue boxes), and an unknown donor (UD, boxed in black) to generate the 10 functional heavy chain sequences that we recovered. An eleventh sequence (Nb 69) acquired a frameshift at position 6. The common precursor, P1, already showed evidence for gene conversions involving VH3 or VH4 (orange), VH2, VH5, or VH8 (pink), and VH7 or VH9 (red and green). Although studies of VH and/or VL sequences in B cells of splenic GCs from chickens immunized with NP-BSA (61) or FITC-BSA (58) also found somatic gene conversion had occurred during clonal expansion in spleen, the precursor cells with between three and five gene conversion blocks identified 7 days after immunization, were presumed to have initiated the clones in splenic GCs and to have been bursal derived (61). In the rabbit, we cannot presume that they were derived from cells that developed the primary repertoire in appendix or other gut-associated lymphoid tissue (30, 31) because we found splenic precursors with germline or near-germline sequences at days 7 and 10 that underwent gene conversions in the spleen (47).



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  		FIGURE 5. The sequences and clonal relationships of clone N2 from GC N (see also Fig. 2Go). Underlined cell names indicate that a V{kappa}J{kappa} sequence was obtained from the same cell. UD, unknown donor. The VHDHJH sequences of members of clone N2 have been assigned GenBank accession numbers AF087767–AF087777.

 
Evidence for selection of CDR amino acid replacements and heavy chain CDR3 (HCDR3) lengths

In the clonal anti-DNP populations present on day 15 we observed slightly shorter CDR3 lengths. Although differences in the mean lengths were not statistically significant, there appeared to be selective narrowing of the range of CDR3 lengths compared with the earlier time points. These results are summarized in Table IVGo. By day 15 the range of HCDR3 lengths narrowed to 8–14 codons from 4–18 and 4–19 codons at days 7 and 10, respectively. Our results are consonant with those of Brezinschek et al. (62) who recently reported that human memory B lymphocytes have slightly shorter (11.9 ± 3.4 codons vs 14.3 ± 4.3 codons) and a more limited range of HCDR3 lengths than naive B cells and suggested this may facilitate more effective Ag binding. Significantly shorter HCDR3 were also found in sequences that also had affinity-increasing mutations at day 12 of the response to NP in C57BL/6 mice (63).


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  		Table IV. CDR3 lengths of potentially functional sequences obtained from days 7, 10, and 15

 
By day 15, some DHJH regions had and were continuing to accumulate mutations. Because in rabbit there are no known donor sequences for DH regions, it is likely that the replacements in the DH regions were the result of point mutations and selection. Replacement changes in the body of the V region are more complex to analyze because many changes appear to have resulted from gene conversion events. The donors that are the proposed sources of the altered sequences themselves contain base changes that mainly result in replacements. Thus the strikingly low proportions of silent changes in the V regions may reflect both the highly efficient gene conversion mechanism for introducing templated replacements (64) and selection for favorable replacements during the clonal expansion and selection stages of the GC reaction.

In an extensive analysis of serum anti-DNP Abs produced by rabbits in our National Institutes of Health allotype-defined breeding colony (44), anti-DNP affinities and concentrations were found to increase most between days 14 and 28. Analyses of affinities of serum Abs indicated that some molecules of the highest affinity species were present in all rabbits by day 42. It is likely that in rabbits as in mice, high-affinity precursors of anti-hapten Ab-producing cells exit GCs and undergo further selection for high-affinity Ab forming cells in the bone marrow (65, 66). The selection that may occur in bone marrow would involve competition between independently evolved cells with high affinity. In addition to narrowing of the HCDR3 lengths in the dominant clones found at day 15, comparisons of the CDR sequences in the separately evolved clones revealed certain recurrent amino acid replacements in the dominant clones that may contribute to favorable increases in affinity for Ag, or on rates of binding to hapten (67). The recurrences shown in Table VGo are particularly striking in view of the variations occurring in parallel in the light chain CDRs and the likelihood that many CDR structures can yield high affinity anti-DNP Abs in rabbits. Although only seen in 2 of 29 sequences from L1 (Lb 81 and Lb 84; Fig. 2Go) the replacement of Asn32 by Tyr was seen in 30 of the 31 sequences from clones L2, L3, N1, N2, and N5. The largest clone from day 10, M1 (47), had two sequences with this replacement (Ma 28 and Ma 34). None of the five sequences in the other two small clones N4 and N6 had this change. In CDR2, there were recurrent replacements of Ala50 by Tyr (or Phe, Ile, and Val) and of Tyr58 by Asp (or Asn in clone N2). Interestingly, the smaller clones (N4, N5, and N6) did not have the replacements in CDR2 seen in the larger clones. In CDR3 we observed a recurrence of Tyr-Ile at positions 99 and 100 (or 100 and 100A) in all L1 and three of four L3 sequences. Whereas in L1, Tyr-Ile was retained from the germline sequence, L3 acquired Ile by a 2-bp change from the germline sequence. Clone L2 maintained Tyr99 from the germline sequence and had a two-base change to yield Trp at position 100. Clone N1 maintained germline Tyr100 and replaced Ala110A with Ser (8/9) or Phe (1/9).


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  		Table V. Recurrent amino acids in the HCDRs of different clones

 
The overall pattern that is documented by this and the previous study ( Figs. 2–5GoGoGoGo and 47) is one of splenic precursor cells whose germline or near germline sequences changed both by gene conversion and point mutations during early divisions and largely by point mutations during later divisions. Because our preliminary data show that within the same expanding clonal populations, considerable diversification of light chain sequences occurred in parallel with the changes in the VH sequences, these events may produce the diverse sequences that serve as substrates for further affinity maturation by selection either within GCs or later among emigrant cells in sites such as bone marrow. The reappearance of gene conversion in rabbit splenic GCs provides an important example of re-expression of immature functions (neoteny) (14, 17, 29). It remains to be determined whether "fine-tuning" of anti-DNP combining site structures occurs by both gene conversions and point mutations or whether the gene conversions are largely an alternative means of receptor editing. Future studies of expressed heavy and light chain pairs should allow us to evaluate this as well as the contributions of observed recurrent replacements in CDRs (Table VGo) to affinities for DNP.


   Acknowledgments
 
We thank Drs. S. Bauer, J. Dasso, M. G. Mage, N. A. Mitchison, and M. Shapiro for helpful comments about this paper. We thank Ms. Shirley Starnes for expert editorial assistance, and Ms. G. O. Young-Cooper and Mr. C. Alexander for outstanding technical assistance.


   Footnotes
 
1 E.S. and D.S. contributed equally to this paper. Back

2 Address correspondence and reprint requests to Dr. Rose G. Mage, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Building 10, Room 11N311, 10 Center Drive-MSC 1892, Bethesda, MD 20892-1892. E-mail: Back

3 Abbreviations used in this paper: GC, germinal center; AP, alkaline phosphatase; BCR, B cell receptor; BGG, bovine {gamma}-globulin; CDR, complementarity-determining region; FDC, follicular dendritic cell; HCDR3, heavy chain complementarity-determining region 3; LZ, light zone; NP, nitrophenyl; RAG, recombination-activating gene; VH, heavy chain V region. Back

Received for publication October 21, 1998. Accepted for publication December 28, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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