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Rearrangement and Selection in the Developing V Repertoire of the Mouse: An Analysis of the Usage of Two V Gene Segments¹

Immunology Program, Sackler School of Graduate Biomedical Sciences, and the Department of Pathology, Tufts University School of Medicine, Boston, MA 02111

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Detailed analysis of the rearrangement and expression of two mouse V genes has been used to examine B cell repertoire development. The V1-A gene is used by a large proportion (9.6%) of splenic B cells in the adult primary repertoire, whereas the V22 gene is used at a much lower frequency (0.16%). Consistent with these results, quantitative PCR (Q-PCR) assays revealed that the number of splenic B cells with rearranged V1-A genes is much greater than the number with rearranged V22 genes. Q-PCR was also performed on both normal bone marrow pre-B cells and transformed pre-B cells induced to rearrange their loci at high frequency. In contrast to splenic B cell rearrangements, the numbers of V1-A and V22 rearrangements in pre-B cells differ by only two- or threefold, suggesting that the intrinsic rearrangement frequencies of these two V genes are not significantly different. Further evidence of disproportionate selection was obtained by comparing the percentages of productive rearrangements amplified from genomic splenic DNA. Sequence analysis showed 84% (37 of 44) of the V1-A rearrangements but only 57% (29 of 51) of the V22 rearrangements to be in-frame. Together these results suggest that B cells expressing V1-A-encoded light chains are preferentially selected either in the periphery or in the transition from pre-B to B cell. Sequence data also reveal a surprisingly restricted diversity of VJ junctions, apparently due to biases introduced by the rearrangement mechanism.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The primary (preimmune) B cell repertoire is comprised of those B cells that have never been activated by Ag to differentiate into Ab-secreting or memory clones. This repertoire includes the majority of resting, Ag-specific B cells in the periphery and is the foundation required to develop humoral immunity against pathogens not previously encountered by an individual. The diversity of the primary repertoire and the mechanisms underlying its development are not yet completely understood. It has been well documented, however, that reproducible biases in the repertoire result in certain V genes being preferentially used.

Differences in the rearrangement frequency among V gene segments possibly contribute to a biased repertoire. Yancopoulos et al. (1) originally observed that transformed mouse pre-B cells preferentially rearrange the more D-proximal mouse V_H genes (V_H7183 family), especially in rearrangements occurring during cell culture. Although the over-representation of rearranged D-proximal V_H genes is most pronounced in fetal pre-B cells (2, 3), there is evidence that adult pre-B cells are also skewed toward the D-proximal genes (4, 5). Similarly, the light chain locus is biased toward gene segments of the V4 family in rearrangements occurring in Abelson virus-transformed pre-B cells (6, 7). In addition to V gene biases associated with cell lines and the fetal repertoire, studies using extrachromosomal rearrangement constructs have shown that both the heptamer/nonamer sequence and the 3' sequence of the V exon can influence the efficiency of rearrangement (8, 9). Thus, features of the Ig loci, including both sequence and organization, are likely to play a role in the preferential rearrangement of particular V gene segments.

The adult repertoire does not display the marked bias toward V_H7183 and V4 family members observed during early development. Instead, V gene families are represented at frequencies that are more consistent with their estimated size. The shift to the adult B cell repertoire has been called normalization (10) and is thought to reflect selective processes at one or more stages of B cell development (11). Despite this normalization, individual V_H genes can be used at markedly different frequencies in both mouse (12) and human repertoires (13). Such skewing of the repertoire is remarkable given the ability of junctional and combinatorial diversification processes to use any given V gene sequence in the context of an enormous number of CDR3⁴ sequences and partner heavy or light chain.

There is substantial experimental support for the argument that heavy chain selection can occur at the pre-B cell stage (14, 15; reviewed in Ref. 16), presumably via the pre-B cell receptor. Evidence of V_H gene selection via B cell clonal expansion is consistent with the finding that the expression of a functional pre-BCR is required for maturation to the pre-B cell stage (17) and that signaling through Ig- is required for generating the peripheral B cell pool (18). In addition to BCR-mediated positive selection during B cell development, negative selection of autoreactive clones by deletion (19), anergy (20), or receptor editing (21) will also impact repertoire development, as will any limitations on the association of particular heavy and light chains.

Although the potential for multiple mechanisms to participate in shaping the primary B cell repertoire is well documented, the relative influence of these mechanisms and the stage at which they operate are still largely unknown. In the present study we examine the rearrangement of two V gene segments. We have determined that the V1-A gene is preferentially used in the adult repertoire and have compared this over-represented V gene segment with the V22 gene, which is represented at a much lower frequency. Consideration of rearrangement frequencies at several stages of B cell development, productive/nonproductive rearrangement ratios, and analysis of VJ junctions from splenic B cells and bone marrow pre-B cells demonstrate that selection is more influential than rearrangement frequency in preferential V1-A gene utilization. Our data also support the possibility that sequence-based biases of the rearrangement mechanism have led to the selection of germline V and J sequences that preferentially form particular, presumably beneficial, CDR3 sequences.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

Female BALB/cByJ mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and maintained in the Tufts University School of Medicine (Boston, MA) animal facility.

V cDNA phage libraries

Details of the construction of the V cDNA libraries have been published (22). Briefly, cDNAs were synthesized using poly(A)⁺ RNA isolated from BALB/cByJ spleen cells following 3-day culture with 50 µg/ml LPS. Each library was prepared using spleen cells pooled from four or five 11- to 15-wk-old female mice. First-strand synthesis was conducted using an oligonucleotide primer specific for the constant region (C). Double-stranded cDNA was prepared and treated with EcoRI methylase, ligated to EcoRI linkers, and size fractionated (600–650 bp) by electrophoresis through 5% polyacrylamide. These cDNAs were cloned into gt10. Two libraries, libraries 1 and 2, have been amplified once. The original (nonamplified) libraries contained 240,000 (library 1) and 700,000 (library 2) C⁺ phage. These libraries (both before and after amplification) have been thoroughly characterized (22) and used to examine the use of V gene families, individual V genes, and J gene segments in adult splenocytes.

Preparation of spleen cells

Splenic IgD⁺ cells were prepared from individual 3- to 5-mo-old mice by panning with the IgG fraction of polyclonal sheep anti-mouse IgD (The Binding Site, San Diego, CA). Spleen cell suspensions were prepared in Dulbecco’s PBS (10% FCS; HyClone, Logan, UT). Erythrocytes were removed by lysis with 17 mM Tris-Cl and 140 mM ammonium chloride (pH 7.2), and white blood cells were resuspended in Dulbecco’s PBS (5% FCS). Cells (3 x 10⁷) were gently overlaid onto 100-mm plates previously coated with 0.75 mg/ml anti-IgD in 10 ml 50 mM Tris-Cl, pH 8.5. Cells were allowed to adhere for 1 h at 4°C with occasional swirling. Adherent cells were removed by washing with Dulbecco’s PBS (5% FCS) with the aid of a rubber policeman. Cell purity was determined by flow cytometry following staining with FITC-coupled goat anti-mouse IgM (Fisher Scientific, Pittsburgh, PA) and biotin-conjugated anti-mouse Thy1.2 (Becton Dickinson, Mountain View, CA). The second step reagent for biotin-conjugated Abs was either FITC-conjugated streptavidin (Sigma, St. Louis, MO) or phycoerythrin-conjugated streptavidin (PharMingen, San Diego, CA). Spleen preparations were at least 95% IgM⁺ and were <1% Thy1.2⁺.

Preparation of bone marrow cells

Bone marrow cells were harvested from tibias and femurs of 2- to 5-mo-old mice by flushing with Dulbecco’s PBS (5% FCS), and single cell suspensions were prepared by expressing the cells several times through a 27-gauge syringe needle. Erythrocytes were lysed as described above. For some experiments, surface Ig-depleted bone marrow cells were prepared by panning to remove IgM⁺ cells using goat anti-mouse IgM (Cappel, Westchester, PA). Nonadherent cells were saved and analyzed by staining with FITC-conjugated polyclonal goat anti-mouse Abs (Southern Biotechnology Associates, Birmingham, AL). Unfractionated bone marrow cells contained 8% anti--staining cells, and IgM-depleted populations contained 2% or fewer anti--staining cells. Ig-negative bone marrow cells used for PCR amplification and sequence analysis were prepared by sorting IgM-negative (FITC-conjugated anti-IgM), B220-positive (biotin-conjugated rat anti-B220, clone RA3–6B2, PharMingen) cells pooled from two littermates. Cell sorting was performed using a FACStar Plus, and cells were analyzed on a FACScan flow cytometer using FACScan software (Becton Dickinson, Mountain View, CA).

Cell lines

103/BCL-2/4 (referred to in the text as 103/4) is a pre-B cell line transformed by temperature-sensitive mutant Ab-MLV and transfected with a human BCL-2 gene (23). Cell line 25A was derived by Ab-MLV transformation of NIH-3T3 cells (24). Cell line 103/BCL-2/4 and transformed 3T3 cell line 25A (Ab-MLV) were provided by Dr. Naomi Rosenberg (Tufts University School of Medicine). The V22-expressing plasmacytoma S107 (25) was provided by Dr. Matthew Scharff (Albert Einstein College of Medicine, Bronx, NY).

Preparation of DNA for PCR

Cells were pelleted by centrifugation and resuspended in PCR lysis buffer (50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl₂, 100 µg/ml gelatin, 0.46% Nonidet P-40, and 0.45% Tween-20) at 3 x 10⁶ to 1 x 10⁷ cells/ml. Pronase (type XIV from Streptomyces griseus, Sigma) was added (60 µg/ml), and cells were lysed by heating to 55°C for 60 to 90 min and to 95°C for 10 min.

PCR amplification

The standard PCR reaction contained 50 mM KCl, 10 mM Tris (pH 8.3), 1.5 mM MgCl₂, 100 µg/ml gelatin, 0.25 µM of each primer, 200 µM of each dNTP, and 2.5 U of Taq polymerase (Perkin-Elmer, Branchburg, NJ) per 100-µl reaction. Oligonucleotides used for amplification are as follows: V1-5'-G ATG ACC CAA ACT CCA CTC (codons 3–9, FW1); V1-3'GGA GCT CAA GCC TCC ATC TCT TG (codons 16–23, FW1); V22-5'-G GTC ACC ATT AGT TGC ACG GC (codons 18–25, FW1/CDR1); V22-3'-CA AGC AAA CAC AAG GTG CAC (codons 27d-31, CDR1); and J5'-CCAAGCTTGTACTT ACG TTT CAG CT (J5/intron). Codon positions are numbered according to Kabat (26), and FW/CDR positions are indicated. Restriction sites were engineered into the 5' ends of the V1–3' (SacI) and V22–5' (SalI) primers but were not used.

Amplifications were performed with a Perkin-Elmer GeneAmp PCR System 9600 at 95°C for 45 s, 55°C for 45 s, and 72°C for 90 s for 30 cycles or with a Perkin-Elmer 4600 at 95°C for 1 min, 55°C for 1 min, and 72°C for 2 min for 30 cycles. After cycling there was an additional 72°C incubation for 5 min, and the samples were cooled and stored at 4°C until analysis.

Cloning of PCR products

For all V22 and V1-A rearrangements from bone marrow, the initial amplification of 30 cycles was performed with a 5' V-specific primer (V1–5' or V22–5') and the J5 primer. A sample (5–10%) of the resulting product was subjected to a further 30 cycles of nested amplification with an internal V-specific primer (V1–3' or V22–3') and the J5 primer. The amplification of V22 rearrangements from spleen used the same two-stage, nested primer protocol (amplification using the V22–5'/J5 primer pair followed by amplification using the V22–3'/J5 pair). Amplification of splenic V1-A rearrangements required only one set of 30 cycles using a V1-A-specific primer (V1–5' or V1–3') and the J5 primer. Following amplification, the 5' overhangs of the PCR products were filled in by treatment with 10 U of T4 DNA polymerase I (Life Technologies, Gaithersburg, MD) for 15 min at 37°C. The products corresponding to J5 rearrangements were purified by agarose gel electrophoresis and ligated into SmaI-digested pGEM-3Z (Promega, Madison, WI) as previously described (27).

Q-PCR competitive constructs

Q-PCR competitors were made by amplifying the 5' and 3' ends of full-length V to J5 rearrangements for both the V1-A and V22 genes using the following primers (EcoRI sequences are underlined). A 137-bp portion of the 5' end of the V1-A gene was amplified using the primers: 5'-GAATTCGAGACTGGCCTGGCTTC-3' (forward, codons 38–44) and the V1–5' reverse primer described above (codons 3–9). A 117-bp portion of the 5' end of the V22 gene was amplified using 5'-GAATTCGATGCCCCGTATATCAGCAG-3' (forward, codons 46–52) and the V22–5' reverse primer described above (codons 18–25). The 3' end of both rearrangements consisted of the identical 53 bp of J5-coding and 3'-flanking sequence amplified with the J5/intron primer (forward, see above) and 5'-GAATTCGCTCACGTTCGGTGCTG-3' (reverse, codons 95–101 of V-J5 rearrangement). The purified products were digested with EcoRI and ligated overnight, and ligated product was reamplified with the external primers. The resultant products were purified and cloned as described for PCR products. The full-length amplified product is approximately 300 bp, while the competitors are approximately 200 bp. The V1-A and V22 competitor constructs are denoted V1-A and V22, respectively.

Q-PCR assay

Dilutions of PvuII (Life Technologies)-linearized competitor plasmids were added to otherwise identical PCR reactions. V1-A assays used J5/intron (forward) and V1–5' (reverse) primer pairs. V22 assays used J5/intron (forward) and V22–5' (reverse) primers. The reactions were resolved on an agarose gel, transferred to nitrocellulose and hybridized with an oligonucleotide probe specific for either V1-A or V22 (see below). Hybridization was quantified using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA), and copy numbers were calculated as described by Piatak et al. (28). Copy numbers were determined by plotting log(signal of target/signal of competitor) on the y-axis against log(copy number of competitor) on the x-axis. In our assay, the target refers to endogenous rearrangements of either V1-A or V22 to the J5 segment. Competitor refers to either V1-A or V22, described above. The genomic rearrangement copy number was calculated from the y-intercept of the plotted values, the point at which the signals of the competitor and the target are equivalent.

Southern blots

High m.w. DNA was digested to completion and separated by electrophoresis through 0.8 to 1% agarose gels. PCR products were separated through 2% agarose gels. DNA was transferred to BA-S85 nitrocellulose (Schleicher and Schuell, Keene, NH) by capillary transfer. Hybridization with restriction fragment probes was performed as described previously (29). Hybridization with oligonucleotide probes was conducted in 5x SSC (1x SSC = 0.15 M NaCl and 0.015 M sodium citrate), 2x Denhardt’s solution (0.4% each of Ficoll-400, polyvinylpyrrolidone-360, and BSA), 0.5% SDS, and 100 µg/ml sonicated salmon sperm DNA. Unless otherwise indicated, all Southern blot reagents were obtained from Sigma. The V1-A specific oligonucleotide used for Q-PCR hybridization was 5'- GTCAGAGCCTTGTACACAG-3'. The V22-specific oligonucleotide used for Q-PCR hybridization was 5'-CAAGCAAACACAAGGTGCAC-3'. Both V1-A and V22 oligonucleotide hybridization probes were used at 60°C.

Cloning the germline V22 gene

Approximately 200 ng of size-fractionated (4 kbp range) EcoRI fragments from BALB/c liver DNA were ligated with 1 µg of gt10 EcoRI arms (Promega) and packaged in vitro using Packagene extracts (Promega). Recombinant phage containing the germline V22 gene were identified by plaque lift hybridization using a V22 family probe as described previously (22).

Sequencing

Double-stranded sequencing of purified plasmid preparations was performed with the Sequenase 2.0 kit (United States Biochemical, Cleveland, OH) according to the manufacturer’s protocols.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Previous analyses of both V_H and V utilization suggested that individual V genes are not equally represented in the naive repertoire. To gain insight into the mechanism(s) responsible for the unequal usage of V genes, we compared the rearrangement of two individual V genes, V1-A and V22. These two functional V gene segments were chosen for comprehensive study because, as detailed below, they are used at remarkably different frequencies in the adult primary Ab repertoire. The V gene utilization studies described below involve the analysis of two V cDNA phage libraries, both prepared using RNA isolated from LPS-stimulated BALB/c spleen cells. This molecular cloning approach to repertoire analysis has been described in detail (22, 30) (see Materials and Methods).

Utilization of the V1-A gene

The V1 family consists of three functional genes (V1-A, V1-B, and V1-C) in the BALB/c germ line (31). We and others have shown that, despite its small size, the V1 family is expressed by a large proportion (13–26%) of naive B cells (22, 32, 33). To determine the contribution of a single member of the V1 family, an oligonucleotide probe was designed to be specific for the V1-A gene. The V1-A-specific hybridization temperature was determined empirically by melting experiments using a panel of 96 V cDNA-containing phage isolated based on hybridization with a V1 family probe (22). Based on these experiments, more than one-half (54 of 96) of the V1 cDNAs were V1-A. Three V1-A-hybridizing and one non-V1-A-hybridizing cDNAs were randomly selected from the V1 panel and sequenced. All three V1-A hybridizing phage contained a V gene segment corresponding to the published V1-A germline sequence, whereas the nonhybridizing V cDNA was identical with the published V1-C sequence (31) (data not shown).

Using the V1-A-specific oligonucleotide probe, 9.6% of approximately 19,500 C hybridizing phage were found to contain V1-A sequence (Table I). This figure is consistent with the finding (above) that more than half of the V1-positive phage hybridized with the V1-A-specific probe and that 13% of the C-sequence containing cDNAs in the library have V1 family sequences (22).

View this table: [in this window] [in a new window]   Table I. Utilization of V1-A and V22 gene segments in adult spleen cells1

Utilization of the V22 gene

Based on Southern blot analysis, the V22 family consists of a single gene (34). Previous analysis of two V cDNA libraries, using a digoxigenin-labeled probe, indicated that the V22 gene is used at a relatively low frequency of 0.18% (22). This low frequency of V22 representation was verified by screening approximately 36,000 C-hybridizing phage with a ³²P-labeled V22 probe. Consistent with previous results, only 0.16% (59 phage) hybridized with the V22 probe (Table I). To make certain that the V22 probe recognized only one germline sequence, four of the V22-hybridizing phage were chosen at random, and the cDNA inserts were sequenced. The rearranged V genes of all four cDNAs were identical in sequence and also identical with the V22 gene expressed by the plasmacytoma S107 (25).

RSS of V1-A and V22

It is possible that RSS and/or associated sequences contribute to differences in the utilization of V1-A and V22. To compare the RSS of these two genes, the 4.0-kbp EcoRI fragment containing the germline V22 gene was cloned from BALB/c liver DNA. The sequence of the germline V22 gene was determined and was found to be identical with that of the V gene expressed by S107 and the four V22 cDNAs from our V phage library (Fig. 1).

View larger version (48K): [in this window] [in a new window]   FIGURE 1. Sequence of the BALB/c germline V22 gene, the rearranged and expressed V22 gene of the BALB/c plasmacytoma S107 (25), and four V22 cDNAs isolated from a BALB/c V cDNA phage library (22). The heptamer-nonamer sequence of the germline gene is underlined. The S107 rearrangement and the rearrangements of cDNA clones 1, 2, and 4 are to J5. The cDNA clone 3 is a V22-J2 rearrangement. Dots denote nucleotide identity with germline gene, except for the J region, where they denote identity with the S107 sequence. Codon numbering is according to Kabat et al. (26). V22, germline sequence was submitted to GenBank (accession number AF044198).

Coding sequences immediately flanking the heptamer sequence can also affect the rearrangement efficiency of gene segments, as assessed using extrachromosomal substrates. Gerstein and Lieber (9) found a hierarchy of rearrangement efficiencies correlating with the nucleotide immediately flanking the heptamer signal (C, G > A > T), where a C or G immediate to the heptamer is the most efficient, and an A or a T is less efficient. The V22-coding region ends in a T, while the V1-A gene ends in a C. This T flanking the V22 heptamer sequence could further contribute to a decreased rearrangement efficiency of the V22 gene relative to that of the V1-A gene.

In vivo rearrangement frequency

In addition to the RSS and flanking sequences, other features that might influence the rearrangement frequency of individual gene segments include chromosomal position (36, 37), transcriptional orientation (38), and other associated noncoding elements, such as the octamer element (39), which may affect accessibility to the recombinase. To determine rearrangement frequency in vivo, we developed a quantitative competitive PCR assay with specificity for V1-A and V22. Accurate determinations of the number of rearrangements in a population of cells are obtained by adding a precise amount of competitor DNA to a series of PCR reactions. To verify the accuracy of the assay, experiments were performed with the S107 plasmacytoma cell line, which has rearranged the V22 gene to J5 on one allele (25). To maintain a constant number of cells per reaction, dilutions of S107 cells were made in 25A fibroblasts, which have no locus rearrangements. These pilot experiments demonstrated that the assay was linear between 100 and 10,000 copies. These experiments also allowed the number of V22 competitor molecules to be precisely calibrated by comparison with the number of S107 cells added to a reaction (data not shown). After calibration, the signals between the same number of S107 cells and V22 competitor molecules gave signals within twofold in subsequent experiments.

The concentration of the V1-A competitor was calibrated by comparison to V22. Competitor construct dilution series were amplified using a primer pair specific for plasmid sequences common to both competitor molecules and flanking the V sequences in V22 and V1-A. The PCR products were analyzed by Southern blots hybridized with the J5 oligonucleotide probe that contains sequence shared by both V22 and V1-A.

The in vivo rearrangement frequencies were determined in the absence of selection by examining locus rearrangements in the 103/4 cell line (23). These B-lineage cells are transformed with a temperature-sensitive mutant of Ab-MLV and have been shown to undergo a high frequency of locus rearrangement when maintained at the nonpermissive temperature. Since these cells do not express an Ig heavy chain protein, there is no possibility of BCR-mediated growth selection, and the resulting V gene rearrangements should reflect the intrinsic rearrangement frequency of the joined DNA segments.

For all rearrangement frequency determinations, we compared the amplification of the competitor only to J5 rearrangements. This strategy allowed comparison of PCR products of similar size and avoided differences in the efficiencies of amplification and Southern transfer. In addition, the larger PCR products associated with the more 5' J segments (J1 and J2) were not reproducibly detected in all experiments, most likely because of the larger sizes of those products. Figure 2 shows an experiment performed on the cell line 103/4 induced to rearrange by being grown at the nonpermissive temperature for 20 h. The number of V1-A rearrangements in the 103/4 cells was 2.2-fold greater than that of the V22 rearrangement. A second, independent TS Ab-MLV-transformed pre-B cell line, DE/1, was also examined. Although the low number of copies (<100/100,000 cells) of both V1-A and V22 rearrangements to J5 did not permit reliable quantification, both V segments appeared to be rearranged at comparable low levels in the DE/1 cell line.

View larger version (60K): [in this window] [in a new window]   FIGURE 2. Q-PCR analysis of cell line 103/4 grown at the nonpermissive temperature for 20 h. Rearrangements of V1-A (upper panel) and V22 (lower panel) were detected by hybridization with radiolabeled V1-A- or V22-specific oligonucleotides and quantified using a PhosphorImager. Negative controls are reactions without DNA added (lane 1). Cell lysate equivalent to 100,000 cells was added to each reaction (lanes 2–9). Lanes 3 through 9 received the indicated number of molecules of competitor construct (V1-A in upper panel, V22 in lower panel). The numbers of competitor molecules were normalized using a cell line with a single V22-J5 rearrangement (see Results). Copy numbers were calculated from these data points as described by Piatak et al. (28) and detailed in Materials and Methods. The calculated V1-A and V22 copy numbers from this experiment are included in Table II.

View larger version (65K): [in this window] [in a new window]   FIGURE 3. Q-PCR analysis of unfractionated bone marrow (BM) cells, IgM-depleted BM cells, and IgD+ spleen cells. FACS analysis indicated 8% anti--staining cells in the unfractionated BM cells and 2% -positive cells in the IgM-depleted cell population. For details, see Figure 2. The calculated V1-A and V22 copy numbers are included in Table II.

View this table: [in this window] [in a new window]   Table II. V1-A and V22 rearrangements determined by quantitative PCR

The extent of cellular selection for (or against) the product of particular Ig variable regions results in a distinctive ratio of productive to nonproductive rearrangements involving that V gene segment (see Ref. 14 for detailed discussion) If, as suggested above, the difference in the utilization of V1-A and V22 is the result of differences in cellular selection for the product of those V genes, the frequency of productive rearrangements isolated from spleen should be greater for V1-A rearrangements than for V22 rearrangements. We chose to amplify rearrangements only to J5, since rearrangements to the more 5' functional J segments (J1, J2, and J4) could be either deleted or relocated far upstream of the C gene by deletional and inversional secondary V-J rearrangements. The V1-A and V22 rearrangements might be influenced differently by multiple rearrangement events depending on the location of these V genes within the locus.

V1-A and V22 rearrangements were amplified and cloned from IgD⁺ spleen cells. This naive B cell population was chosen to exclude isotype-switched memory cells from the analysis. Each rearrangement was obtained from an independent PCR reaction to ensure that each clone represents a unique rearrangement event. This is important, since, in contrast to the nearly infinite D segment- and N insertion-enhanced diversity of heavy chain CDR3 sequences, VJ junctions are somewhat limited in junctional diversity. Thus, rearrangements have no CDR3 fingerprint with which to determine clonal independence. A total of 44 V1-A and 51 V22 rearrangements were sequenced (Fig. 4). The V1-A rearrangements were predominantly productive (84%, 37 of 44). In contrast, only 57% of the V22 rearrangements were productive (29 of 51). More than 95% of the sequenced V genes were identical with the corresponding germline sequence, and the occasional single base changes observed most likely resulted from errors during PCR amplification.

View larger version (32K): [in this window] [in a new window]   FIGURE 4. Nucleotide sequence analysis from IgD+ spleen cells. A, V1-A sequences were isolated for three DNA preparations, with 30 cycles of amplification. Twenty-nine of the V1-A sequences were amplified with the 3' V1 primer from two different mice. An additional 15 sequences were amplified with the 5' V1 primer with DNA isolated from another mouse. B, V22 sequences amplified from two different mice using 60 cycles of amplification with nested primers. Nucleotides that cannot be unambiguously assigned to the V or J gene segments are highlighted in bold type. The number to the right of each junctional sequence indicates the number of times that sequence was obtained from an independent PCR amplification. The prefix DP1 denotes a V1-A sequence obtained from spleen cells. The prefix DP22 denotes a V22 sequence obtained from spleen cells. Each of the nine different V1-A junctional sequences is assigned a letter (A–I). Similarly, each of the 16 different V22 junctional sequences is assigned a letter (A–P).

View larger version (21K): [in this window] [in a new window]   FIGURE 5. Sequence analysis from Ig-negative bone marrow. Amplifications of all sequences were from sorted bone marrow (BM; from two mice). Both the V1-A and V22 sequences were subjected to 60 cycles of amplification with nested primers. The prefix BM1 denotes a V 1-A sequence obtained from Ig-negative BM cells. The prefix BM22 denotes a V22 sequence obtained from Ig-negative BM cells. Each distinct sequence is assigned a letter that corresponds to those letters assigned to the spleen cell-derived sequences (DP1 and DP22) in Figure 4. Only one junctional sequence is unique to the set obtained from BM (BM22.Q).

Our experiments provided an opportunity to examine the diversity created by the use of alternative rearrangement sites. The most striking feature of the productive rearrangements of both V1-A and V22 is the very pronounced bias for one or two particular junctions (Fig. 4). Of the 37 V1-A productive rearrangements obtained from spleen cells, 23 have identical junctions (DP1.A). The remaining 14 junctions include a set of eight sequences (DP1.B), a set of two sequences (DP1.C), and only four unique junctions (DP1.D, DP1.E, DP1.F, and DP1.G). Interestingly, the CDR3 encoded by the predominant DP1.A junction, found in 23 independent rearrangements, is identical with the CDR3 encoded by the DP1.C junction, which is represented by only two of the 37 V1-A in-frame rearrangements. Since any selective forces would influence DP1.A and DP.1C rearrangements equally, the large difference in the representation of these two rearrangements must reflect a bias of the rearrangement mechanism.

Great care was taken to avoid contaminating PCR reactions with previously amplified products. For example, all amplification reactions were set up in the laboratory of a colleague not working with Ig genes. Before amplification, all PCR work used dedicated supplies and equipment never exposed in our laboratory. No contamination of V gene products was observed in multiple "no DNA" controls included in all experiments. To further rule out any possibility that the high frequency of repeated productive and nonproductive V1-A to J5 junctions was the result of contamination, the last 15 V1-A products obtained from adult spleen were amplified using a more 5' primer (V1-A 5') that would not be capable of priming previously amplified products. This last set of amplifications continued to yield the dominant productive and nonproductive junctions observed for V1-A to J5 rearrangements.

The bias for certain junctions in the set of productive V22 rearrangements is also noteworthy. Twenty-four of the 29 in-frame V22 junctions obtained from spleen cells (Fig. 4) are identical with one of two repeated junctional sequences, 15 with the 22.A sequence and 9 with the 22.B sequence. The 22.A and 22.B sequences encode the identical amino acid sequence. The remaining five junctions (22.C through 22.G) are each unique. Three of the four productive V22 rearrangements obtained from bone marrow (Fig. 5) also have the 22.A junction. Considering both spleen- and bone marrow-derived productive V22-J5 rearrangements, 27 of 33 (82%) encode the same CDR3 (22.A or 22.B).

As in this report, a number of investigators have examined nonproductively rearranged Ig loci to study rearrangement biases in the absence of BCR-mediated cellular selection (13, 14, 43). Since there is no selection for or against nonproductive rearrangements, out-of-frame rearrangements from both spleen and bone marrow were pooled for analysis (Figs. 4 and 5). In the case of nonproductive V1-A rearrangements, a bias for the DP1.I/BM1.I sequence was observed in both spleen and bone marrow, with this one junction accounting for 11 of the 14 junctions obtained. The nonproductive V22 rearrangements also included frequent repeats of particular junctions. Of the 28 nonproductive V22 sequences, 11 have the 22.I junction, and 8 have the 22.H junction. Of the remaining rearrangements, one was isolated twice (22.J), and seven are unique (22.K, 22.L, 22.M, 22.N, 22.O, 22.P, 22.Q).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The contribution of an individual V gene segment to the B cell repertoire is determined by the intrinsic frequency of rearrangement, the diversity of junctional sequences formed, and cellular selection mediated via the expressed -chain. To examine these facets of Ig repertoire formation, we have examined and compared two functional V gene segments, V1-A and V22, which are represented at very different frequencies in the naive adult repertoire.

Earlier studies on IgV gene usage in adult mice focused largely on the determination of V gene family usage (7, 10, 22, 30, 32, 33, 44, 45, 46, 47). These studies demonstrated that V gene use is generally correlated with estimated family size and led to the suggestion that individual V genes might contribute equally to the preimmune repertoire. However, more recent studies of both mouse and human V_H genes usage have revealed that individual V genes segments can be used at very different frequencies (11, 12, 13, 48). We have found that 9.6% of C-positive cDNAs from a V cDNA library contain the V1-A sequence. Given this extraordinarily high representation, it is possible that V1-A is the most frequently expressed V segment within the mouse preimmune repertoire. This finding is consistent with studies using hybridization probes, which demonstrated that the small, three-gene V1 family is over-represented in mature B cell populations (22, 32, 33). In contrast, the V22 gene sequence was present in only 0.16% of the V cDNAs. This large disparity between individual V gene segments is not unique, since individual mouse V_H genes can vary in utilization from 0.1% (12) to over 6% (unpublished) in the splenic B cell population.

Sequences within a V gene can influence that gene’s intrinsic rearrangement. For example, variations within the RSS affect the frequency at which an individual V gene will be targeted as a rearrangement substrate. RSSs display considerable variation from the consensus sequence, and it has been demonstrated, using both extrachromosomal (8) and integrated rearrangement constructs (35), that certain positions within the heptamer and nonamer are more critical than others. Coding region sequences adjacent to the RSS have also been shown to influence rearrangement efficiency (9, 49), can potentially influence the relative contribution of a particular V gene (or D or J) segment to the preimmune repertoire, and can be fine-tuned during evolution to optimize the composition of the naive repertoire.

To examine the intrinsic rearrangement potential of V1-A and V22 gene segments in the absence of BCR-mediated selection, we have examined a cell line capable of being induced to rearrange loci. The number of V1-A rearrangements was routinely measured to be about 2-fold greater than that of V22 rearrangements in the cell line 103/4 (2.2-fold in the experiment presented in Table II). The numbers of copies of V1-A and V22 rearrangements are consistent with the number of total rearranged loci recently reported for the 103/4 line by Liu et al. (50). Using a similar Q-PCR assay, these investigators calculated that 103/4 had approximately 37,000 rearranged loci/100,000 cells 16 h following induction of rearrangement (Liu et al. present their data as copies per 20,000 cells). Our calculated value for V1-A and V22 rearrangements to J5 is approximately 200 copies/100,000 cells 20 h following induction (Table II), which, based on the results of Liu et al. (50), represents roughly 0.5% (200 of 37,000) of the total rearrangements. Since the mouse locus consists of about 140 V exons (51), an unknown number of which are pseudogenes, 0.5% is within the expected order of magnitude for a relatively unbiased use of individual V segments.

In contrast to rearrangements in pre-B cells, V1-A rearrangements in mature splenic B cells were present at a 13-fold greater number than V22 rearrangements based on Q-PCR. If these results are adjusted to reflect only productive rearrangements (57% of V22 and 84% of V1-A), there are 20-fold more V1-A productive rearrangements than V22 productive rearrangements in IgD⁺ splenic B cells. It is possible that the full 60-fold difference measured using the V cDNA libraries was not observed by the Q-PCR assay, because V22 may use J5 more frequently than does V1-A. Only 14 of 108 analyzed V1-A cDNAs (13%) were rearranged to J5 (our unpublished observations), whereas the frequency of J5 rearrangements in the total V cDNA library is 20% (30). On the other hand, three of the four sequenced V22 cDNAs from our V phage library (Fig. 1) and the expressed V genes of both S107 (25) and TEPC15 (52) plasmacytomas are V22 to J5 rearrangements, suggesting that V22 may be preferentially associated with J5.

We observed a striking difference in the proportions of productive and nonproductive rearrangements involving V1-A vs V22 gene segments in the adult spleen. Rearrangements of V22 to J5 were found to be in-frame 57% of the time. The ratios of productive to nonproductive rearrangements for both V4 family members (53) and V 21 family members (40) have also been reported to approach 1.0 in adult spleen. In contrast, 84% of the V1-A rearrangements were found to be in-frame. Based on this high proportion of productive rearrangements for V1-A, we suggest that V1-A-expressing B cells are preferentially expanded in the naive repertoire. Although the feature of V1-A chains being selected is not known, one possibility is that a BCR-ligand interaction, perhaps with self Ag, is a mandatory checkpoint in B cell development. Recent evidence suggests that the naive repertoire contains self-reactive clones that are expanded in the primary response (54). Another possibility is that V1-A can form functional associations with many heavy chains, whereas other V genes can effectively pair with a more restricted set of V_H regions. Although there are not yet sufficient data to test this idea, Kaushik et al. (55) found that V and V_H families appear to associate with each other in a stochastic fashion.

The junctional diversity of rearrangements is more limited than heavy chain rearrangements due to the absence of D segments. In addition, mouse rearrangements generally have few N nucleotide additions (56, 57), presumably because terminal deoxynucleotidyl transferase expression is down-regulated as a result of µ-chain expression before most locus rearrangements (58, 59). Junctional variability is further restricted by a bias to form particular junctions between particular V and J segments. For example, both Milstein et al. (53) and Victor et al. (43) examined large sets of V rearrangements involving members of the V4 or V21 gene families, respectively, and reported that the nonrandom deletion of nucleotides from V and J results in biased junctions. Similarly, we found preferences for one or two junctional sequences for productive and nonproductive rearrangements of both V1-A and V22.

The finding that 23 of 37 productive splenic V1-A/J5 rearrangements have the DP1.A rearrangement cannot be explained solely by selection, since the identical amino acid sequence is encoded by the DP1.C rearrangement, which accounted for only 2 of the 37 productive V1-A rearrangements (Fig. 4). These data provide compelling evidence for a strong mechanistic bias in the formation of V/J junctions. Interestingly, the frequently formed productive junction, DP1.A, is not mediated by short regions of homology (56), suggesting that other features of the recombinase are responsible for the preference for the DP1.A junction (40). The V22 junctions amplified from adult spleen also show biases. The DP22.A junction occurs in a region of sequence homology between the V and Jk5 gene segments. This sequence is also observed at a high frequency in the bone marrow-derived sequences (three of four in-frame junctions are BM22.A), indicating that this junction is favored by the rearrangement process. DP22.A and DP22.B encode identical CDR3 sequences and are expressed in both S107 and TEPC15 anti-phosphocholine light chains (25, 52). Since these junctions make up 24 of 29 (83%) of the splenic junctional sequences, it is likely that this CDR3 sequence is positively selected in the repertoire.

Other biases noted are the high percentage of nonproductive junctions occurring within regions of sequence homology (32 of 42). Since nonproductive junctions cannot be selected by expression of BCRs, these must represent independent rearrangement events and are the result of a biased rearrangement mechanism.

Analysis of 115 V1-A and V22 rearrangements revealed only a single nucleotide that is unambiguously an untemplated N addition. Rearrangements of other mouse V genes have had a slightly greater frequency of N-containing junctions: 5% for V4 (53) and 10% for V21 (43). It must be noted that given that N insertions in are often a single nucleotide, there is a significant possibility for an N nucleotide to be incorrectly assigned to either V or J segments. Despite this uncertainty regarding the precise frequency of N insertions in light chains, the tight regulation of terminal deoxynucleotidyl transferase expression during B lymphocyte development appears to severely limit nontemplated additions. The paucity of N insertions in fetal heavy chain junctions has previously been postulated to restrict the early repertoire by facilitating homology-directed junction formation (60, 61). Similarly, minimizing N additions during light chain rearrangement would facilitate the coevolution of V genes capable of favoring the formation of useful junctional sequences.

It has become increasingly accepted that the preimmune repertoire does not consist of a random assortment of V, D, and J segments linked by arbitrary junctions. Rather, the content of the primary repertoire can be determined by the frequency of rearrangement of individual gene segments, bias in the formation of junctions, and cellular selection based on Ig chain amino acid sequence (62). Thus, the Ig loci and V(D)J rearrangement mechanism have probably evolved together to provide the species with a preimmune Ab repertoire that represents a compromise between sufficient diversity and the expression of particularly useful heavy and light chains.

	Acknowledgments

 
We thank Drs. Naomi Rosenberg, Erik Selsing, and Henry Wortis for advice during the course of these studies. We also thank Naomi Rosenberg for providing cell lines, and Naomi Rosenberg and Brian Haines for reviewing this manuscript. We thank Gerry Parker for expert photography.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grant GM36064.

² Current address: Department of Molecular Microbiology and Immunology, Oregon Health Sciences University, 3181 S.W. Sam Jackson Park Rd., Portland, OR 97201.

³ Address correspondence and reprint requests to Dr. Peter H. Brodeur, Department of Pathology, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111.

⁴ Abbreviations used in this paper: CDR3, complementarity determining region 3; BCR, B cell receptor for antigen; C, constant region; Ab-MLV, Abelson murine leukemia virus; Q-PCR, quantitative polymerase chain reaction; RSS, recombination signal sequence; TS, temperature-sensitive.

Received for publication August 1, 1997. Accepted for publication January 9, 1998.

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