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Distinct Clonal Ig Diversification Patterns in Young Appendix Compared to Antigen-Specific Splenic Clones

Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The young rabbit appendix is a dynamic site for primary B cell repertoire development. To study diversification patterns during clonal expansion, we collected single appendix B cells from 3- to 9-wk-old rabbits and sequenced rearranged H and L chain genes. Single cells obtained by hydraulic micromanipulation or laser capture microdissection were lysed, PCR amplified, and products directly sequenced. Gene conversion-like changes occurred in rearranged H and L chain sequences by 3–4 wk of age. Somatic mutations were found in the D regions that lack known conversion donors and probably also occurred in the V genes. A few small sets of clonally related appendix B cells were found at 3–5 wk; by 5.5 wk, some larger clones were recovered. The diversification patterns in the clones from appendix were strikingly different from those found previously in splenic germinal centers where an immunizing Ag was driving the expansion and selection process toward high affinity. Clonally related appendix B cells developed different amino acid sequences in each complementarity-determining region (CDR) including CDR3, whereas dominant clones from spleen underwent few changes in CDR3. The variety of combining sites generated by diversification within individual clones suggests that at least some clonal expansion and selection, known to require normal gut flora, may be driven through indirect effects of microbial components rather than solely by their recognition as specific foreign Ags. This diversity of combining sites within B cell clones supports the proposed role of appendix in generating the preimmune repertoire.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the majority of rabbit B cells, one predominant V_H gene (V_H1) rearranges to one of several D_H and J_H genes (1, 2). Nontemplated additions of N-region sequences at the V–D and D–J junctions during the rearrangement process generate diverse sequences in H complementarity-determining region (CDR)³ 3. Further diversification of the rearranged H chain sequence occurs by two mechanisms: somatic mutation and gene conversion (3).⁴ Previous studies from this laboratory showed that the rabbit draws upon a diverse set of L chain V sequences to form rearranged VJ (4), but then, during an Ag-specific immune response in germinal centers (GCs) of adult rabbit spleen, gene conversion further alters both rearranged H (5, 6) and L chain (7) sequences.

Gene conversion as a mechanism for diversifying rearranged H and L chain sequences was first described in chickens, where the introduction of homologous sequences from upstream donor genes initiates during embryonic development in a hind-gut organ, the bursa of Fabricius (8, 9). The appendix and other gut-associated lymphoid tissues (GALT) of rabbits have a homologous function. However, whereas diversification starts in chickens before exposure to exogenous Ags, the process in rabbits occurs only after birth and only in the presence of normal gut flora. Moreover, development of a normal complement of preimmune B cells in rabbits is dependent upon the presence of both normal GALT and gut flora. Just as bursectomy leads to deficiencies in numbers of chicken B cells and their repertoire of specificities, neonatal appendectomy and removal of other GALT early in life leads to B cell deficiency (10) and diminished diversity of V_HD_HJ_H gene sequences in B cells of young rabbits (11). Remarkable development and expansion of B cells takes place in rabbit appendix between birth and 6 wk of age (10, 12, 13, 14). It is accompanied by diversification of rearranged V_HD_HJ_H sequences (14, 15) and dependent upon the presence of normal gut flora (16, 17, 18).

Growth and expansion of B cells is also accompanied by apoptotic death, suggesting that both negative and positive selective processes affecting the B cell repertoire are at work (12, 13, 19, 20). Previously, existing technology did not permit determination of rearranged V_HD_HJ_H gene sequences within single cells (15). Only a few clonally related appendix cell sequences from 6-wk-old rabbits could be obtained after cloning products of hemi-nested PCR amplification of a portion of the rearranged V_HD_HJ_H sequences extending from the second framework region through the J_H region. We found that the sequences in clonally related cells were undergoing changes that could be explained by both gene conversion-like mechanisms and somatic hypermutation (15).

Now, using both hydraulic micromanipulation and laser capture microdissection (LCM) to collect single B cells, we returned to a more comprehensive analysis of rearranged V_HD_HJ_H sequences in developing appendix. B cells were collected at 0.5- or 1-wk intervals between 3 and 9 wk of age. We analyzed the diversification of rearranged H and L chain gene sequences in appendix tissues of young rabbits after nested PCR amplification and sequencing of V_HD_HJ_H and VJ from single cells. For the first time we have been able to analyze L chain sequence diversification in appendix tissue and find that the diversification patterns parallel those seen for H chains. Our analyses reveal that the diversification of sequences is strikingly different from what we previously found in adult spleen after immunization with the specific hapten-protein conjugate DNP-bovine -globulin (5, 6, 7). There, where a specific Ag was driving the expansion and selection process toward high affinity, we found that dominant clones underwent few changes in the CDR3 while changes in CDR1 and CDR2 continued to occur in both H and L chain sequences. In contrast, in the appendix, we find that clonally related cells develop very different amino acid sequences in each CDR including CDR3. This suggests that a variety of potentially different combining sites exist within a single expanding clone. We hypothesize that rather than being driven by specific Ag, the cells are being driven by less specific mechanisms dependent upon the presence of normal gut flora.

	Materials and Methods

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Animals

Rabbits were from our National Institute of Allergy and Infectious Diseases allotype-defined colony. The rabbits used here were homozygous for H chain V_Ha2 (haplotype F-I) and L chain Cb5 allotypes. All animal experiments were in compliance with institutional guidelines and regulations and were reviewed and approved by the animal care and use committees of the National Institute of Allergy and Infectious Diseases and Spring Valley Laboratories (Woodbine, MD).

Immunohistochemistry

Appendices of 3- to 9-wk-old rabbits were cut into 2-mm slices, submerged in OCT, and quickly frozen in a slurry of dry ice and isopentane. Five-micrometer serial sections were cut at -24°C in an IEC cryostat microtome (Sakura Finetek USA, Torrance, CA), kept overnight in the desiccator, and fixed in acetone for 10 min at room temperature. Tissues were stained for Ig H chain isotypes using goat anti-rabbit IgM (Southern Biotechnology Associates, Birmingham, AL), goat anti-rabbit IgA (Bethyl Laboratories, Montgomery, TX), and mouse anti-rabbit IgG (Spring Valley Laboratories). For the B cell-specific marker CD79a, we used mouse anti-human CD79a that cross-reacts with rabbit ((IgG1 clone HM47; BD PharMingen, San Diego, CA). Proliferating cells within GCs were identified with mouse mAb (Ki-S5) to the human nuclear proliferation Ag Ki-67 which cross-reacts with rabbit (Roche Diagnostics, Mannheim, Germany). Biotinylated rabbit anti-goat IgG (Southern Biotechnology Associates) was used for identifying primary Abs raised in goat and biotinylated goat anti-mouse IgG (Southern Biotechnology Associates) was used for identifying primary Abs raised in mice. Cells were then visualized with avidin-biotin complex (ABC) conjugated to alkaline phosphatase or peroxidase (ABC-peroxidase) and Vector Blue or 3,3'-diaminobenzidine in nickel chloride chromogens (Vector Laboratories, Burlingame, CA). For photography, ABC-alkaline phosphatase-stained slides were coverslipped with aqua mount (Lerner Laboratories, Pittsburgh, PA); 3,3'-diaminobenzidine-nickel chloride-stained slides were dehydrated and coverslipped with permount (Fisher Scientific, Fairlawn, NJ).

Microdissection of frozen tissue sections by hydraulic micromanipulation or LCM

Tissue sections (8 µm) were cut and stained for CD79a or Ki-67 as described above. Sections were dehydrated in five changes of graded alcohol (once in 75% ethanol for 30 s, once in 95% ethanol for 30 s, and three times in 100% ethanol for 2 min each) and then cleared in three changes of xylene for 2 min each (21). Single cells from GCs were collected from sections stained with anti-CD79a or Ki-67 using a hydraulic micromanipulator (Narishige, Greenvale, NY) assembled on an inverted microscope (Olympus, Lake Success, NY) or by LCM with a PixCell II and PixCell II Image Archiving Workstation (Arcturus Engineering, Mountain View, CA) (Ref. 21 ; details of LCM methods can be found on the National Institutes of Health LCM web site at http://dir.nichd.nih.gov/lcm/lcm.htm). Individual GCs were designated by uppercase letters, sections within a GC by lowercase letters, and single cells by numbers. A suffix of -H or -k was added to the designation of a sequence from a single cell to indicate H or L chain sequence. Each individual cell was transferred to a 0.2-ml microfuge tube containing 2.5 µl of alkaline lysing solution (200 mM KOH/50 mM DTT). 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/200 mM HCl) (5, 6, 7).

DNA amplification and sequencing

Direct sequencing of PCR products from single cells without cloning was used to minimize two known and potentially serious artifacts: Taq DNA polymerase errors and in vitro recombination during PCR (22, 23, 24, 25). Even with single-cell PCR, there could theoretically be some artifactual recombinants between a germline V gene sequence and the rearranged V_HD_HJ_H or V_LJ_L sequence.

For L chains, we chose to only amplify the rearranged VJ sequences because in the homozygous b5 rabbits which were used here, 85% of B cells have surface Ig bearing L chain. Rearranged V_HD_HJ_H and VJ sequences were amplified from single cells from GCs using primers and nested PCR strategies described previously (5, 6, 7). Briefly, in the first round of PCR, sequences were amplified using a mixture of external primers: one set specific for the rearranged V_HD_HJ_H and one set for VJ. A 2.5-µl aliquot of the first-round PCR product served as the template for the second round of nested PCR, which was performed separately for the rearranged V_HD_HJ_H and VJ sequences using internal primers (5, 6, 7). The temperature conditions for the first and second round of touchdown PCR were the same as described previously (5, 6, 7). PCR was performed on a PTC-100 programmable thermal cycler (96-well model with hot bonnet; MJ Research, Watertown, MA). Throughout this report the amino acid positions are numbered according to the Kabat numbering system (26).

Recovery of PCR products for sequencing was done as described previously (5, 6, 7). 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 (Applied Biosystems). The sequences were analyzed using Autoassembler version 1.4.0 (Applied Biosystems) and MacVector software version 7.0 (Accelrys, San Diego, CA).

Sequence analyses

In the rabbits of V_Ha2 (F-I) haplotype used in this study, the majority of B cells rearrange the V_H1a2 gene and a few cells rearrange V_Hx or V_Hy genes. Thus, we were able to determine the V_H, D_H, and J_H genes used in each V_HD_HJ_H rearrangement and most gene conversion blocks by aligning the sequences of the known rabbit V_H, D_H, and J_H genes in the GenBank database and our laboratory databases. Each nucleotide addition, deletion, or mismatch was scored to determine the numbers of single base differences from the rearranged germline V_H gene. Mismatches at the last codon of V_H that could be attributable to exonuclease activity during the joining process were not scored as single base changes in the V_H gene. Many of the observed nucleotide changes could be accounted for by one or more gene conversion-like events in which the donor for the block was a known germline V_H gene. After scoring total base changes from the germline sequence, we also scored events. The introduction of a gene conversion block was counted as one event. In a few instances, a tight cluster of nucleotide substitutions that appeared likely to be due to a block of gene conversion was also scored as a single event despite the fact that we could not find a known germline V_H gene donor. An initial gene conversion block was identified if there were no more than two superimposed point mutations. Such point mutations were counted as additional events. We cannot rule out the possibility that we may have missed identifying a few gene conversion blocks and in a few instances may have over estimated the number of events because not all rabbit germline V_H genes have been identified. Replacement and silent base changes were counted in all sequences that were not members of clones and ratios of replacement to silent changes were tabulated.

The criteria for identification of D_H gene segments were that at least six consecutive nucleotides were identical to a known D_H gene segment or at least seven nucleotides of identity were interrupted by no more than a single nucleotide substitution. Most D_H gene sequences had 10 or more base pairs identical to a known germline gene. A clone was counted only once in tabulating the D_H gene usage. Both functional and nonfunctional VDJ sequences were included in the analyses.

In general, the approach adopted for identifying the germline VJ, point mutations and gene conversion events was the same as that used for the H chain. The recovered VJ sequence was first aligned to the germline V database (4). The best match was identified as the rearranging germline V gene. The rearranging germline V gene thus identified was used as the reference to identify point mutations and gene conversion events by searching the same germline V database. In rabbits of b5 allotype, all rearrangements involve the only functional J gene J2.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Immunohistochemical staining of developing rabbit appendix

Fig. 1A shows appendix tissue sections from 3-, 6-, and 9-wk-old rabbits stained with Abs to the nuclear proliferation Ag Ki-67, the B cell receptor-specific marker CD79a (Ig), and three Ig H chain classes. There is some variability among animals in the degree of appendix development at a given age. For example, we reported earlier that absolute numbers of lymphoid cells in appendix increased sooner in litters with four or fewer pups (27). The first three rows in Fig. 1 show stained appendix tissue sections from three different animals collected at 3 wk of age. The sections from two animals in the same litter show some difference in appendix follicle development. The larger 3-wk GC shown in the top row is referred to as GC RX in Table I and Fig. 2 and is analyzed separately. At each time point, Ki-67-positive proliferating cells are evident. Staining for CD79a confirms previous reports that the majority of cells in young rabbit appendix are of the B lineage. IgM- and IgA-positive cells are seen at each time point and IgG-positive cells appear by 6 wk of age.

View larger version (66K): [in this window] [in a new window]   FIGURE 1. A, Progression of GC formation in rabbit appendix between 3 and 9 wk of age. Serial sections of the rabbit appendix were stained with Abs to Ki-67, CD79a, IgM, IgA, and IgG to reveal follicles and GCs. Three different animals are shown at 3 wk of age to illustrate variation between different animals, including two littermates. All sections were counterstained with nuclear fast red. Magnification, x100. B, Serial sections a and b from 5.5-wk appendix showing GC RU stained with anti-CD79a. Identified are the light zone (LZ), dark zone (DZ), and approximate locations of the cells collected that were members of clones RU3 (black letters and shown in Fig. 4, A and B) and RU4 (red letters and shown in Fig. 5).

View this table: [in this window] [in a new window]   Table I. Summary of Ig H and L chain sequences recovered from appendices of rabbits of different ages1

View larger version (27K): [in this window] [in a new window]   FIGURE 2. Summary of the number of sequences with a given number of base changes from the rearranged germline VH in rabbit appendix at 3–9 wk of age. , All base changes from the rearranged VH portion of the germline gene sequence. Gene conversion events introduce blocks of replacement changes from germline sequences in rearranged VH. , Numbers of events; a gene conversion that may have introduced several base changes is counted as a single event. R:S indicates the ratio of replacement to silent changes in all sequences that were not members of a clone. Only the R:S for GC RX is shown at 3 wk because there were only replacement changes in the other sequences. Total base changes scored were 3 wk, 48; 4 wk, 186; 4.5 wk, 84; 5 wk, 71; 5.5 wk, 633; 6 wk, 127; and 9 wk, 326. See Materials and Methods for further details.

Increasing diversification of rearranged Ig H chain sequences during development of the young rabbit appendix

In developing appendix from 3 to 5 wk of age, we found small clones but most of the sequences were unique and unrelated (Table I). We collected a large number of single cells from appendix tissue taken at 5.5 wk because sequence diversification was becoming much more extensive. One hundred fourteen H chain sequences were obtained from four serial sections of one GC (GC RU, Table I). Sixty-eight were unique and unrelated to others (59.6%, Table I). The remaining recovered sequences fell into nine groups with 2–21 members that were clonally related. Twenty-six L chain sequences were also analyzed from the same GC. Eighteen were members of clones with 2–10 members (69.2%).

Fig. 2 shows the number of sequences at each time point with a given number of total base changes from the germline V_H gene that had rearranged (hatched bars). Gene conversion introduces multiple base changes during a single conversion event. The black bars represent the total number of events counting gene conversions as one event. The shift of black bars to the left graphically emphasizes that conversions introduce multiple changes in one event.

At 3 weeks, the sequences of V_HD_HJ_H from three of four GC differed by 1–3 bp from the rearranged germline gene (V_H1a2). One example of a possible gene conversion was observed (hatched bar that shifted left to black bar). The D and J region sequences were also close to those of the respective rearranged germline genes with 0–1 bp mismatches in 17–23 bp from the germline D_H genes. The base changes from the germline sequence all led to amino acid replacements occurring mainly in CDRs 1, 2, and 3 (a total of seven replacements in five sequences). The fourth GC appeared to be larger and more developed, (GC RX, Fig. 1, top row). It yielded V_HD_HJ_H sequences that were more diversified and the results are shown in a separate panel (GC RX, Fig. 2). The shift of black bars to the left again indicates that in several sequences, one gene conversion led to multiple base changes. As previously described (28), most gene conversion blocks introduce replacement changes into the V_H portion of the VDJ at most or all positions where a base change is introduced. In GC RX, seven sequences contained 44 replacements (R) and only 4 silent (S) base changes, leading to a ratio (R:S) of 11. This differs from the situation in mouse and man where high R:S ratios found after somatic hypermutation are thought to reflect selection events. The panels showing total base changes or events in sequences from 4- to 9-wk appendix tissue illustrate that this trend continued. Although replacement changes may occur by somatic hypermutation, the remarkable predominance of R vs S changes seen in the unique V_H sequences at all ages (R:S ranges from 5.5 to 11) raises the possibility that even some single base changes were introduced as gene conversions from germline donor sequences. Thus, the numbers of gene conversion events are probably underestimated.

Fig. 3 summarizes D_H gene usage at 3–9 wk of age. We previously reported that 41% of V_HD_HJ_H in cDNA from 15- to 28-day rabbit fetal liver and bone marrow utilized the Df gene, with the D2 and D5 genes accounting for another 21 and 14%, respectively (29). These were also the most commonly rearranged D_H genes in appendix B cells through 5.5 wk of age. At 6 and 9 wk, a broader representation of D_H genes was evident. Over the 3- to 9-wk period, J_H2 was found in 10.7% and J_H4 in 89.3% of the 234 independently rearranged sequences (data not shown).

View larger version (22K): [in this window] [in a new window]   FIGURE 3. DH gene usage expressed as the percentage of sequences using any given DH gene (y-axis, left) and in absolute numbers (y-axis, right). UNKN-D, the DH gene used in the rearrangement could not be identified with confidence. DH gene usage converts from a fetal to a broad and diverse representation by 6 wk.

View larger version (45K): [in this window] [in a new window]   FIGURE 4. Examples of clones found among sequences from cells collected from appendix GCs at 4.0, 4.5, 5.0, and 5.5 wk of age. A and C, H chain sequence alignments 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 in italics and single-letter code below the sequences; silent base changes are in lowercase. The numbers above the reference sequence indicate codon positions according to Kabat et al. (26 ). Most framework region positions that were invariant are not shown. Individual GCs are designated by uppercase letters, sections within a GC by lowercase letters, and cells by numbers. B and D, Diagrammatic representations of the clonal relationships between different clone members and sequence changes. Known germline VH genes with sequences that could have acted as donors of blocks of gene conversion are shown as boxes. UD indicates an unknown VH donor. The numbers near the arrows in B and D indicate the number of point mutations. The numbers in parentheses (italicized) placed adjacent to the block of gene conversion (boxed in B and D) represent the minimal size (bp) of the gene conversion block. The hypothetical rearranged precursor is indicated. Additional hypothetical precursors (P) are shown as circles. C, The sequences of clone RU7 are grouped according to their relationship to hypothetical precursors (precursor groups P1-P5). The VHDHJH sequences of members of clones have been given GenBank accession numbers AF434315–316, AF434359–360, AF434384–385, and AF434412–416.

View larger version (47K): [in this window] [in a new window]   FIGURE 5. Sequence alignment and clonal relationships of the largest clone RU4 from GC U. A block of gene conversion superimposed on an earlier gene conversion is indicated by a double underline in sequences RUb-025-H and RUa-131-H. As in Fig. 4C, the sequences of the members of clone RU4 are grouped according to their relationship to hypothetical precursors (precursor groups P1-P15). See legend to Fig. 4. for other details. The VHDHJH sequences of clone RU4 were assigned GenBank accession numbers AF434417–AF434437.

Evidence for gene conversion in the rearranged L chain during clonal expansion of B cells in young rabbit appendix

Fig. 6 shows examples of clones found among VJ sequences obtained from 3-, 4-, 5.5-, and 6-wk appendices. The sequences obtained from the cells of the 6-wk clone RZ-1 are shown to illustrate the gene conversion blocks observed. In each clonal group, examples of one or more gene conversion blocks as well as single base changes were observed. Amino acid replacement changes predominated and are shown beneath each sequence. Silent base changes are shown by lowercase letters.

View larger version (29K): [in this window] [in a new window]   FIGURE 6. Evidence for gene conversion of rearranged L chains during clonal expansion of B cells in young rabbit appendix. A, Clones from 3-, 4-, 5.5-, and 6-wk appendices (GC RC, RG, RU, and RZ, respectively) showing rearranged germline genes used, gene conversion donors, and minimum lengths of conversion tracts (italicized). Numbers below arrows show additional changes from germline sequence. B, The sequence of rearranged germline genes 1–096 and J2 are compared with those of four members of the clone from a 6-wk appendix. The numbers above the reference sequence indicate codon positions according to Kabat et al. (26 ). Parts of framework 1 and 3 sequences with no changes from germline have been omitted. Identical bases (... .) are shown, codon deletions (—) are shown, and silent base changes are lowercase. Encoded sequences are shown beneath each sequence in italics (only replacement changes are shown for the four appendix cell sequences). Gene conversion blocks are enclosed in boxes. The VJ sequences of members of clones have been given GenBank accession numbers: 3 wk, AF434590-591; 4 wk, AF434599-601, AF434602-603; 5.5 wk, AF434627-628, AF434632-634, AF434629-631; and 6 wk, AF434653-656.

At days 7 and 10 during Ag-driven expansion and selection in splenic GCs specific for the hapten DNP, we found small clones and the majority of recovered sequences were unique and unrelated (6). However, by day 15 when switching to IgG was evident by immunohistochemistry, only 10% of sequences from anti-DNP-specific splenic GCs were unrelated and clones with up to 42 members were found (5, 6, 7). In 3- to 5-wk-old rabbit appendix, we also found mainly unique unrelated sequences, but by 5.5 wk we found more clonally related sequences (Table I). Whereas few replacement changes were found in the HCDR3 and LCDR3 of dominant clones from splenic GCs, the clones from appendix had striking diversity in CDR3. Fig. 7 shows the contrast between the HCDR3 sequences in different members of large clones recovered from 5.5-wk appendix (Fig. 4, C and D, and Ref. 5) and the HCDR3 sequences of members of large clones from anti-DNP splenic GCs collected on day 15 (5). The marked contrast between the sequences from cells in expanding Ag-specific clones from spleens and those from young appendix suggests that the latter are developing a diverse set of combining sites consistent with their proposed role as a source of the rabbit’s preimmune repertoire (13, 14, 18).

View larger version (69K): [in this window] [in a new window]   FIGURE 7. Contrast between the level of diversification in the HCDR3 sequences recovered from 5.5-wk-old rabbit appendix and day 15 immunized adult rabbit spleen (5 ). A, left, 5.5-wk appendix clone RU4 (Fig. 5); right, splenic GC clone L1 (GenBank accession numbers AF087712–087740. aSeven other cells had a protein sequence identical to La-55-H) (5 ). B, left, Appendix clone RU7 (Fig. 4, C and D); right, day 15 GC clone N1 (GenBank accession numbers AF087755-AF087766. bTwo other cells had sequences identical to Nb-16-H. cAnother cell had a sequence identical to Nb-37-H (5 ).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

It has been known for many years that GALT of rabbits play a key role in development of B lymphocytes (10). More recently, the importance of GALT as a site for diversification of the rearranged genes that encode Ab H chains was demonstrated (11, 14, 15). The present study considerably extends these earlier observations by presenting an extensive analysis of sequence diversification during the development of rabbit appendix follicles and GCs between 3 and 9 wk after birth. These results were obtained by LCM or manual microdissection of single B cells from frozen appendix tissue sections, cell lysis, PCR amplification of rearranged H and L chains, and direct sequencing. We demonstrate here that diversification of both rearranged H and L chain gene sequences occurs during the period when B cells in rabbit appendix are expanding in numbers to generate the large follicles found by 6 wk of age (Fig. 1 and Refs. 12, 13, 14, 15). These cells are the hypothesized source of the primary repertoire, i.e., B cell precursors generated to exit to the periphery and respond to specific Ags (14). The sequence changes that we find during clonal expansion are accounted for by gene conversions as well as likely superimposed point mutations. We find a remarkably high proportion of replacement changes (Fig. 2). This may be because the majority of base changes introduced by gene conversion lead to amino acid replacements (28). The possibility that some single base changes may actually be due to gene conversions is raised by the fact that we find an excess of replacement changes even where single base changes occur. However, the extensive single base changes from germline D region sequences observed in HCDR3, are more likely due to somatic hypermutation because there are no known donor genes that could donate the changes.

What drives B cell expansion in appendix GCs?

A key role for gut flora in driving expansion of appendix B cells was suggested by early studies which showed that surgical ligation of newborn appendix to interrupt normal contact with intestinal contents interfered with normal lymphoid development (30). Later studies of germfree rabbits (16, 17) also demonstrated that lymphoid development in GALT required gut flora. More recent studies indicate that specific species of gut flora are necessary to obtain development of a normally diversified primary repertoire (18, 31).

How similar are the biochemical mechanisms of somatic hypermutation and gene conversion?

A recent report (32) supports the idea that gene conversion and somatic hypermutation constitute distinct pathways for processing or resolving a common molecular lesion (e.g., single- or double-stranded breaks) in rearranged Ig V_H and V_L gene segments (33, 34, 35). We found that on a B cell population basis, the machinery for both gene conversion and somatic hypermutation is active in rabbit appendix lymphocytes as early as 3 wk of age, although somatic hypermutation may predominate when judged on a cell-cell basis. However, as discussed earlier, we cannot rule out that some of the single base changes we observed did not result from gene conversions. Therefore, base changes in the D regions are a more reliable indicator of somatic hypermutation. Examples of gene conversions with no changes in germline D were observed as well as base changes from germline D region sequences with no other changes in the rearranged VDJ. Based on our limited data, the gene conversion and somatic hypermutation machineries appear to be acting on the rearranged V_H and V_L genes simultaneously. Both continue to be active at least as late as the ninth week, the last time point assessed in this study. We observed that the odd GC that was more developed than its neighbors tended to have sequences that were more diversified (Fig. 1 and GC-RX in Fig. 2). Thus, our data only allow us to conclude that in the young rabbit appendix these two mechanisms of V gene diversification appear to be occurring in parallel and that the factors required for gene conversion and somatic hypermutation can coexist in the progeny of a single B cell.

The patterns of sequence changes in expanding appendix clones contrast with patterns of sequence changes in splenic GCs during Ag-specific responses

As we analyzed the sequences recovered from appendix GCs, we were struck by the difference between the patterns of sequence changes that occurred in clonally related appendix B cells and in dominant clones from adult DNP-specific splenic GCs (Fig. 7 and Ref. 5). In spleen, where a specific Ag was driving the expansion and selection process toward high affinity, we found that dominant clones underwent few changes in the CDR3, while changes in CDR1 and CDR2 continued to occur in both H and L chain sequences. In contrast, clonally related appendix B cells developed very different amino acid sequences in each CDR including CDR3. Such diversification results in a variety of potentially different combining sites within a single expanding clone. Although it remains possible that some IgA- or IgG-positive appendix B cells may have developed specificities for commensal gut flora, in the context of classical (10) and recent (11, 12, 13, 14, 15) studies of the young rabbit appendix that demonstrate a major role in primary repertoire development and seeding of peripheral sites such as spleen, lymph nodes, and Peyer’s patches, we consider it likely that the clonal sequences we observed reflect preimmune repertoire development. We believe it is unlikely (as suggested by one reviewer) that the variety of different combining sites we find within a single expanding clone are specific for different epitopes of intestinal flora. We favor the hypothesis that rather than being driven by specific Ag, these cells are being stimulated by less specific mechanisms. These could include interactions between B cell receptor framework regions and endogenous superantigens such as CD5 (13, 19, 20, 27) or superantigens from components of the gut flora. In addition, or alternatively, more indirect mechanisms such as via interactions between components of the gut flora and receptors of the innate immune system could lead to release of cytokines or chemokines and stimulation of B cells.

	Acknowledgments

 
We thank C. B. Alexander, M. Mage, B. Newman, R. Pospisil, and R. Sinha for helpful comments about this manuscript, Shirley Starnes for expert editorial assistance, and C. B. Alexander and Glendowlyn O. Young-Cooper for outstanding technical assistance.

	Footnotes

 
¹ Current address: National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India.

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 address: rm3z{at}nih.gov

³ Abbreviations used in this paper: CDR, complementarity-determining region; GC, germinal center; GALT, gut-associated lymphoid tissue; LCM, laser capture microdissection; ABC, avidin-biotin complex; R, replacement; S, silent.

⁴ For simplicity, we refer to the mechanism in rabbits as gene conversion although it should be termed gene conversion-like because nonreciprocal exchange has not been formally shown in rabbits.

Received for publication November 1, 2001. Accepted for publication March 28, 2002.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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