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The Journal of Immunology, 1999, 162: 1541-1550.
Copyright © 1999 by The American Association of Immunologists

Germline Structure and Differential Utilization of Igha and Ighb VH10 Genes1

Elizabeth A. Whitcomb2, Brian B. Haines, Allen P. Parmelee, Adam M. Pearlman and Peter H. Brodeur3

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Ab heavy chains encoded by mouse VH10 genes have been of particular interest due to their frequent association with DNA binding. We reported previously that VH10 sequences are over-represented in the preimmune repertoire considering the apparent number of germline-encoded VH10 gene segments. In this report, we show that the VH10 family consists of three and two germline genes in the Igha and Ighb haplotypes, respectively. The complete nucleotide sequences of these five genes, including promoters and recombination signal sequences, were determined and allow unambiguous assignment of allelic relationships. The usage of individual VH10 genes varied significantly and ranged from 0.2% to an extraordinary 7.2% of the VH genes expressed by splenic B cells. Since the promoter and recombination signal sequence elements of all five VH10 genes are identical, we suggest that the few amino acid differences encoded by these five germline VH10 genes determine their representation in the preimmune repertoire. Rearrangements of the most frequently used VH10 gene have an apparent bias for histidine at position 95 of complementarity-determining region-3 (CDR3). These CDR3s are also biased for asparagine, an amino acid associated with the CDRs of DNA binding Abs. Together, these results suggest that high VH10 gene use is the result of B cell receptor-mediated selection and may involve DNA and/or ligands that share antigenic features with DNA.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The first member of the VH10 gene family was identified as the expressed VH gene in an anti-DNA autoantibody-producing hybridoma derived from MRL-lpr/lpr mice (1). Southern blot analysis revealed that the VH10 family in various Igh haplotypes consisted of two to four homologous VH genes (2). Curiously, this small set of V genes appears to make an inordinately large contribution to the naive, or preimmune, B cell repertoire. For example, although the VH10 family of C57BL mice includes only two VH10 hybridizing restriction fragments on Southern blots (2, 3), 8.8% of µ transcripts from LPS-stimulated C57BL/6 spleen cells are encoded by VH10 family genes (4). BALB/c mice, which have three VH10 germline genes based on Southern blot analysis (2, 3), also possess an unexpectedly high representation of VH10 sequences among LPS-induced splenic µ transcripts, ranging from 4.7–8.4% (5). Consistent with the analysis of cDNA libraries, in situ hybridization studies have shown that approximately 8% of LPS-stimulated, Ig-secreting, BALB/c and C57BL/6 splenocytes express a VH10 family gene (6). Among BALB/c peritoneal B cells examined by single-cell PCR, a substantial fraction (10%) of the B-1b subset has also been shown to use VH10 genes (7).

We and others have reported previously that individual VH (5, 8, 9) and V{kappa} (10) genes can be represented at different levels in the mouse primary repertoire. Such differences might, in some cases, be explained by the efficiency of an individual V gene to act as a substrate for V(D)J recombination. For example, RSS4 (11, 12), promoter activity (13), and the distance between rearranging elements (14) have all been implicated in determining rearrangement efficiency. Other studies have suggested that differences between individual V gene usage in the preimmune repertoire are influenced by BCR-mediated B cell clonal selection (8, 10, 15, 16).

The VH10 family was chosen for a detailed analysis of individual VH gene utilization for several reasons. First, its small size permitted the genomic cloning and sequencing of all germline VH10 genes, the establishment of allelic relationships, and the design of gene-specific hybridization probes. Second, the association of VH10-encoded heavy chains with DNA binding autoantibodies suggests a possible relationship between the proclivity to encode autoantibodies and the unusually high expression of VH10 genes in the preimmune B cell repertoire. To explore the basis of this high VH10 family usage, we have conducted a comprehensive examination of the VH10 family in two widely studied Igh haplotypes, including the copy number, germline structure, and frequency of individual VH10 gene use. These studies have permitted us to ask whether all VH10 genes are highly expressed and whether unusual coding or noncoding features of the germline genes might account for their overexpression.

We also considered the possibility that VH10-expressing B cells might be expanded by a relatively homogeneous set of Ags, possibly DNA. We have, therefore, examined the CDR3 sequences of VH10 gene rearrangements for evidence of pauciclonal expansion (restricted diversity) and also for the biased use of certain basic and polar amino acids that are commonly associated with DNA-binding Abs (17, 18). The evolution of this highly homologous set of genes and possible explanations for their differential utilization are discussed.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cloning germline VH10 genes

Mice of the Igha haplotype (prototype strain, BALB/c) have three EcoRI fragments (9.5, 8, and 6 kb) that hybridize strongly to a VH10 gene family probe (2). These three VH10 sequence-containing fragments were cloned from BALB/cByJ liver DNA into {lambda}gt10 (6-kb fragment) or {lambda}gtWES (8- and 9.5-kb fragments). The Ighb haplotype includes two EcoRI fragments (12 and 4 kb) that bear germline VH10 genes (2). The two Ighb VH10 genes were cloned from C57BL/10J liver DNA. The VH10 gene containing the 4-kb EcoRI fragment was cloned in {lambda}gt10. The VH10 gene located on the 12-kb EcoRI fragment resides on a 6.5-kb BamHI fragment (data not shown). This BamHI fragment was cloned into the Charon 27 {lambda} vector. Restriction analysis demonstrated that each of the five cloned fragments contains a single VH10 segment.

Sequencing

DNA sequencing was performed with the Sequenase kit (United States Biochemical, Cleveland, OH) or by dye-labeled primer chemistry (ABI 373 Stretch sequencer, Applied Biosystems, Foster City, CA) performed by the DNA Sequencing Facility (Tufts University, Boston, MA).

VH cDNA libraries

Two phage cDNA libraries containing VH gene sequences derived from µ heavy chain transcripts were used in this study. The construction and detailed characterization of both the BALB/c (c45) and the C57BL/6 (b48) libraries have been described previously (4). Briefly, cells pooled from five nonimmunized adult mice and cultured for 3 days with LPS were used to prepare each library. The poly(A)+ RNAs isolated from these activated spleen cells were primed with a Cµ-specific oligonucleotide, and dscDNA was synthesized. cDNAs were treated with EcoRI methylase, ligated to EcoRI linkers, size fractionated (~800 bp), and cloned into {lambda}gt10. The c45 (BALB/c) and b48 (C57BL/6) libraries contained 75,000 and 24,000 independent VH+/JH+ cDNA clones, respectively. These VH cDNA libraries were amplified once and stored at -80°C in 7% DMSO.

Hybridization probes

The VH10 gene family probe is a 450-bp PstI-DdeI fragment isolated from the MRL-DNA4 cDNA clone described by Kofler (2) and labeled by random priming in the presence of [{alpha}-32P]dCTP (New England Nuclear, Boston, MA). Table IGo describes the oligonucleotide probes for individual VH10 genes used to screen VH cDNA libraries by hybridization. Oligonucleotide probes specific for individual JH segments have been described previously (4).


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  		Table I. Oligonucleotide hybridization probes used to identify VH10 family genes in BALB/c and C57BL/6 VH cDNA libraries

 
Southern blots

Southern blots were prepared as described previously (3). Densitometry of autoradiographs was performed with a model G5–700 densitometer and MultiAnalyst version 1.0.2 software (Bio-Rad, Richmond, CA).

VH cDNA library screening

The representation of VH10 genes within the VH cDNA libraries was determined by plaque lift hybridization (4). Oligonucleotide hybridization probes were end labeled with T4 polynucleotide kinase (Life Technologies, Gaithersburg, MD) and [{gamma}-32P]ATP (New England Nuclear). Oligonucleotide probes and hybridization temperatures used to identify sequences of individual VH10 family genes are described in Table IGo. Following hybridization, the filters were washed three times with 2x SSC-0.2% SDS at room temperature for 30 min and twice with 1x SSC-0.2% SDS at the hybridization temperature for 15 min. The filters were exposed overnight to Kodak XAR film (Eastman Kodak, Rochester, NY) at -80°C with an intensifying screen (Lightning Plus, DuPont, Wilmington, DE). The number of JH+ cDNAs screened was calculated by multiplying the number of plaques by the proportion of plaques determined previously to contain VH+/JH+ cDNAs (33.9% for the BALB/c library and 26.3% for C57BL library) (4). The representation of individual VH10 genes was determined as the number of phage hybridizing to the VH probe divided by the number of JH+ phage screened. Duplicate filters prepared from each plate were hybridized with the same oligonucleotide probe and compared. A plaque was counted as positive when a colocalized radioactive signal was observed on both lifts of a given plate.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Germline structure of the VH10 family

The germline VH10 genes of both Igha (BALB/c) and Ighb (C57BL/10) were cloned and sequenced. The three Igha VH10 clones were designated C6, C8, and C9.5; the numbers correspond to the EcoRI fragment size on which each VH10 gene resides. The two Ighb VH10 clones were designated B4 and B12, corresponding to the 4- and 12-kb germline EcoRI fragments, respectively. The clone designations indicate the strain of origin by including the single letter strain abbreviations, C (BALB) and B (C57BL).

Fig. 1Go compares 663 nucleotides of five germline VH10 genes to the consensus sequence derived from the five genes. The sequences shown include 56 bp 5' of the octamer sequence through the RSS. The coding sequences of the five VH10 genes are highly conserved, ranging from 97–99% sequence identity. A potential TATA box (TTAAAA) is located 88 bp (87 bp in C6) upstream of the leader exon, and 18 bp 5' of the TATA motif is a consensus octamer sequence. The 56 bp sequenced 5' of the octamer is identical in all five genes except for a single base pair deletion in the C6 gene (position 52, Fig. 1Go). All five VH10 genes have an identical RSS consisting of a consensus heptamer, a 23-bp spacer, and a nonamer sequence, ACACAAACC, differing from the consensus sequence ACAAAAACC (11) only at the fourth position. Based on sequence and organization, all five VH genes appear to be capable of V(D)J recombination, transcription, and expression as part of a functional Ig heavy chain.



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  		FIGURE 1. Germline VH10 family gene sequences of BALB/c and C57BL/10 mice. BALB/c clones are designated C9.5, C8, and C6. C57BL/10 clones are designated B12 and B4. Sequences are compared with the consensus sequence (CON.) derived from the five germline VH10 sequences. Coding sequences are displayed as codons. The leader exon coding sequence spans positions 178–223. The variable region exon spans positions 312–624. The following sequences are in bold type: octamer (nucleotides 57–64), putative TATA box (nucleotides 83–88), RSS heptamer (nucleotides 625–631), and RSS nonamer (655–663). CDR1 (nucleotides 413–427) and CDR2 (nucleotides 470–526) are indicated. Differences from consensus are shown, and those resulting in an amino acid change are capitalized. C6 has single base pair deletions (denoted by a dot) at positions 52 and 147. GenBank accession numbers are: B4, AF064442 (VH10.1b); B12, AF064443 (VH10.2b); C9.5, AF064444 (VH10.1a); C8, AF064445 (VH10.2a); and C6, AF064446 (VH10.3a).

 
VH10 alleles

Comparison of the sequences of the three Igha and two Ighb genes allowed the assignment of alleles. Fig. 2Go summarizes the shared differences from the consensus sequence. B4 and C9.5 share differences from consensus at 14 positions, strongly suggesting that these genes are allelic. Based on these paired differences, we have designated the B4 and C9.5 genes as VH10.1b and VH10.1a, respectively. Similarly, B12 and C8 appear to be allelic based on five shared differences from consensus and will be referred to as VH10.2b and VH10.2a, respectively. The third BALB/c VH10 gene differs from consensus at seven positions. None of these differences is shared with either C57BL/10 gene, and only one, position 529, is shared with the BALB/c gene C8 (VH10.2a). We have, therefore, designated the C6 gene as VH10.3a.



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  		FIGURE 2. Shared nucleotide changes from the consensus sequence. Differences from the consensus sequence that are shared by two germline sequences are summarized. Based on these paired differences, B4 and C9.5 are designated as alleles (VH10.1b and VH10.1a, respectively), and B12 and C8 are designated as alleles (VH10.2b and VH10.2a, respectively). The BALB/c VH10 gene, C6, is designated VH10.3a.

 
Protein sequences

The variable region protein sequences encoded by the five germline VH10 genes are highly conserved, each differing from consensus by only one or two amino acid residues (Fig. 3Go). The largest number of differences between any two of the five sequences is four amino acids (VH10.1a vs VH10.2b). The signal peptide sequences are identical except for a valine encoded by codon -18 of both VH10.1a and VH10.1b. Five partial germline VH10 gene sequences, obtained by PCR amplification of (NZB x NZW)F1 DNA, have been reported (20). These five sequences, one of which is a pseudogene, also reveal a highly conserved set of VH10 genes. The available translated sequences (positions 1–60) differ from the consensus sequence by no more than three amino acids (Fig. 3Go).



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  		FIGURE 3. Amino acid sequences of the germline VH10 family genes of BALB/c and C57BL/10 mice. Sequences are compared with the consensus sequence derived from the five germline VH10 genes. Identity at a position is indicated by a dash. Numbering is according to Kabat et al. (19). CDR1 (residues 31–35) and CDR2 (residues 50–65, with insertions at 52A, 52B, and 52C) are displayed in bold type. Sequences with the 0401 prefix are partial germline sequences obtained by PCR from (NZB x NZW)F1 mice (20). The 0401-4 sequence has a single nucleotide deletion in codon 11 (indicated by X) and is therefore designated a pseudogene ({psi}).

 
Number of germline VH10 genes

Kofler (2) originally described the VH10 gene family as consisting of strongly hybridizing EcoRI fragments of three sizes (9.5, 8, and 6 kb) in Igha strains and of two sizes (12 and 4 kb) in Ighb strains (Fig. 4Go). By densitometric analysis of Southern blots of EcoRI-digested liver DNA, the germline intensities of the two C57BL and three BALB/c VH10 bands were found comparable (data not shown). Since the five cloned VH10 genes, each corresponding to a germline band, have >97% nucleotide sequence identity to the hybridization probe used, we conclude that each VH10 Southern blot band represents the same number of VH10 genes.



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  		FIGURE 4. Southern blot of germline and rearranged VH10 genes. Southern blot of BALB/c and C57BL/10 liver tissue and the pre-B cell lines F100 and M4. Each lane contains 10 µg of DNA digested with EcoRI. The blot was hybridized with the VH10 family probe.

 
To determine whether each of the BALB and C57BL VH10-hybridizing Southern blot bands represents only a single germline VH10 gene segment, we analyzed pre-B cell lines with a rearranged VH10 gene segment. These two cell lines, CXXB F100 (F100) and CXXB M4 (M4), are Ab-Abelson murine leukemia virus-transformed bone marrow cells derived from (BALB x C57BL)F1 mice (Igha/Ighbheterozygotes). The status (germline, rearranged, or deleted) of VH family genes in both F100 and M4 have been reported previously (3, 21). Both cell lines have rearranged a VH10 gene of the Ighb allele. We reasoned that since mouse VH genes are typically (15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25) kb apart (22, 23), a fragment smaller than 15 kb containing a rearranged VH10 gene is likely to contain a single VH gene. The cell line M4 has retained its haploid set of BALB/c-derived germline VH10a fragments at 9.5, 8, and 6 kb. M4 has rearranged VH10.2b and retained VH10.1b in its germline context (Fig. 4Go). The retention of VH10.1b suggests that the germline organization of Ighb VH10 genes is 5'-VH10.1b-VH10.2b-3' (3). The cell line F100 has also retained a haploid Igha VH10 family. The Ighb allele of F100, however, has rearranged the 5' member of the two VH10 fragments (VH10.1b) and, in the process, deleted the more 3' VH10 gene (VH10.2b) (3). Using densitometry, the rearranged VH10.1b in F100 and the rearranged VH10.2b in M4 were compared with the unrearranged VH10 genes within the same cell line (Fig. 5Go). Based on the comparable intensities of the unrearranged and rearranged VH genes, we conclude that each of the five germline VH10-hybridizing EcoRI fragments represents a single VH10 coding region.



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  		FIGURE 5. Densitometric analysis of VH10 gene copy number. Band intensities of Southern blot lanes (Fig. 4Go) containing DNA from cell lines M4 and F100 were quantified. For each lane, the value of each band was normalized to the value obtained for the 9.5-kb band (VH10.1a gene), which was arbitrarily set at 1.0. Data were obtained as volumes (OD x mm x mm), and values for the 9.5-kb bands are 4.40 (F100) and 2.92 (M4).

 
Hybridization probes for individual VH10 genes

To determine which of the germline VH10 genes contributes to the high representation of VH10 sequences in the preimmune repertoire (4, 5, 6), we designed oligonucleotide hybridization probes capable of distinguishing the individual VH10 genes within each haplotype (Table IGo). The hybridization temperatures required for single gene specificity were determined by plaque hybridization of sets of VH10 family probe hybridizing phage isolated from the VH cDNA libraries. Nineteen VH10+ phage isolated from the C57BL/6 VH cDNA library were hybridized with either the VH10.1b or the VH10.2b oligonucleotide probe. As shown in Fig. 6Go, three phage hybridized with only the VH10.1b probe, while the remainder hybridized specifically with the VH10.2b probe. Eighteen VH10+ phage were isolated similarly from the BALB/c VH cDNA library and hybridized with probes specific for each of the three BALB/c germline genes. All 18 BALB/c VH10+ phage hybridized with either the VH10.3a- or VH10.2a-specific probe, but none hybridized with the VH10.1a probe. To obtain a VH10.1a cDNA to serve as a specificity control, several thousand cDNAs were screened with the VH10.1a probe, and a hybridizing phage was isolated. This phage is included in the panel of BALB/c-derived cDNAs (position D5; Fig. 6Go, upper panel).



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  		FIGURE 6. Analysis of VH10 cDNAs for individual VH and JH segment use. Phage were isolated from BALB/c and C57BL/6 VH cDNA libraries based on hybridization with a VH10 gene family probe. Phage isolates where plated, and the resulting plaques (~5 mm in diameter) were screened by plaque lift hybridization using. One phage in the BALB/c panel (at position 5-D) was initially isolated using the gene-specific VH10.1a probe (Table IGo). As a negative control, position 5-C (BALB/c) is a plaque that failed to hybridize with any of the four JH probes.

 
The hybridization results shown in Fig. 6Go demonstrate that the probes are highly specific for the individual germline genes. In addition, all of the VH10 hybridizing phage are recognized by one of the specific probes, supporting the conclusion that the expressed VH10 genes in BALB and C57BL mice are all derived from the five germline genes described in this report. Each VH10+ phage hybridized to only one of four JH segment probes, confirming the clonality of the phage isolates (Fig. 6Go).

VH10 gene use

The representation of each germline VH10 gene segment in the adult splenic repertoire was determined by using the VH10-specific hybridization probes to screen the BALB/c and C57BL/6 VH cDNA libraries by plaque lift hybridization (Table IIGo). We have shown that this approach provides a reproducible assessment of the functionally rearranged, unmutated IgM VH regions available in the preimmune repertoire (4, 5). Screening 1200–1800 JH+ phage from the BALB/c library with probes specific for each of the three germline VH10 genes revealed a large disparity of usage among certain of these highly homologous gene segments. The VH10.2a and VH10.3a genes were represented at 3.6 and 1.9% of the JH+ cDNAs, respectively. By contrast, the VH10.1a gene sequence was found in only 0.2% (4 of 1797) of the JH+ cDNAs screened. The C57BL/6 VH cDNA library was screened with probes specific for each of the two germline Ighb VH10 genes. The VH10.1b-specific probe hybridized to 2.3% (224 of 9784) JH+ cDNAs, and the VH10.2b-specific probe hybridized to 7.2% (659 of 9152) JH+ cDNAs. The values obtained for the use of individual VH10 genes within the total VH cDNA libraries (Table IIGo) are entirely consistent with the detailed analysis of the panels of phage selected by hybridization with the VH10 family probe (Fig. 6Go).


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  		Table II. Representation of VH10 gene family members in splenic VH cDNA libraries1

 
cDNA sequences

To verify that cDNAs identified by hybridization with the gene-specific VH probes are derived from the expected germline VH10 gene, we chose randomly one cDNA representing each of the five germline VH10 genes for sequence analysis. Each cDNA sequence corresponded to an in-frame VDJ rearrangement of the germline VH10 gene matching the specific probe used. All VH and JH sequences were identical with the germline counterpart (data not shown).

Junctional sequences of VH10.2b rearrangements

Usage of the VH10.2b gene is extraordinarily high (7.2%). We therefore examined the junctional sequences of a set of VH10.2b rearrangements (cDNAs) for evidence of Ag selection (CDR3 length or recurrent use of certain amino acids) or for biases in the rearrangement process (D segment use, presence of N regions). The junctional sequences of 16 VH10.2b rearrangements, all in-frame and using a variety of JH and DH germline segments, are shown in Fig. 7Go. With one exception, each junction is unique and is therefore the product of a clonally distinct B cell. The exception is a pair of VH10.2b-JH1 cDNA sequences (B-4 and B-5 in Fig. 7Go) that have identical junctional sequences. B-4 and B-5 could be the result of the independent cDNA synthesis of µ transcripts from the same B cell clone, two copies of the same phage from the amplified phage library, or the products of two independent rearrangements resulting in identical junctional sequences. For our analyses, we consider B-4 and B-5 to represent the same rearrangement event.



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  		FIGURE 7. Junctional cDNA sequences of VH10.2b-JH rearrangements. The cDNA sequences (except for clone v7) correspond to the C57BL phage isolates shown in Fig. 6Go. Germline JH sequences (5') are in bold type. The assignment, nomenclature, and reading frame (RF) designations of germline DH segments are from the reports by Ichihara et al. (24) and Feeney and Riblet (25). Underlined nucleotides can be from either of the adjacent germline segments and have been arbitrarily assigned to the more 5' segment.

 
All junctions examined have nontemplated (N) nucleotides at the V-D and/or D-J boundaries. The amino acid sequences of the 15 independent CDR3 sequences (residues 95–102, Fig. 8Go) are heterogeneous and range in length from 6–11 amino acids. The mean length of 8.9 amino acids is similar to the mean CDR3 length (8.5) observed for mouse heavy chains (26). Only one of the CDR3 sequences encodes an arginine (clone C-4, position 98). Thus, the most striking structural features associated with CDR3 of DNA binding Abs, longer length and frequent presence of arginine (27, 28, 29), are not common in the VH10.2b rearrangements found in the C57BL primary repertoire. However, other biases in CDR amino acid composition have been observed, including the presence of asparagine (17, 18). Of the 15 independent VH10.2b CDR sequences examined, seven (47%) contain at least one asparagine (Fig. 8Go). In contrast, only one of 15 comparable µ chain cDNAs representing three other VH gene families (J558, Q52, and 3609) encodes a CDR3 asparagine (5). In addition, among the VDJ junctions of conventional B cells reported by Kantor et al. (7), only 10 of 59 (17%) encode an asparagine. Thus, the VH10.2b CDR3s encode asparagine more frequently than expected.



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  		FIGURE 8. Translated amino acid sequence of VH10.2b CDR3s. Sequences derived from the junctional sequences in Fig. 7Go. Numbering is according to Kabat et al. (19). Asparagine residues are in bold type.

 
Six of the 15 VH10.2b junctions encode the basic amino acid histidine. Although not generally associated with DNA binding Abs, histidine residues of a zinc finger DNA binding motif have been shown to contact both bases and phosphate backbone (30). Interestingly, in five of the six histidine-encoding CDR3 sequences the histidine is at position 95 and results from the retention of the CA dinucleotide present between the 3' end of the coding region and the RSS (Fig. 7Go). In two other sequences the retention of the CA results in glutamine that, like arginine and asparagine, has the potential to interact with nucleic acid bases (31).

Specificities of VH10-encoded Abs

Twenty-three VH10-encoded Abs of known specificity have been reported (Fig. 9Go). Thirteen VH10 Abs bind some form of DNA, nine of which bind ssDNA. Other specificities include a variety of foreign and self Ags. Although the sample size is limited, the relative use of individual VH10 genes in the preimmune repertoire appears to be faithfully reflected by the VH10-encoded Abs reported in the literature (Fig. 9Go). With respect to the seven BALB/c-derived Abs in Fig. 9Go, three are encoded by VH10.2, three are encoded by VH10.3, and one is encoded by either VH10.2 or VH10.3. This distribution mirrors the preimmune repertoire that has comparable (within twofold) VH10.2 and VH10.3 use. Based on our results (Table IIGo), we calculate that the BALB/c VH10.1 allele represents only about 4% of all VH10 genes expressed in the preimmune splenic repertoire. Consistent with this limited contribution to the preimmune repertoire, none of the reported BALB/c Abs is encoded by VH10.1a. A similar correlation of preimmune and expressed repertoires can be made for C57BL Abs. Consistent with the threefold greater use of VH10.2b in the preimmune repertoire, four of the six VH10-encoded Abs of C57BL origin are encoded by VH10.2b.



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  		FIGURE 9. Specificities of mAbs encoded by VH10 genes. Spontaneous autoantibodies derived from autoimmune mouse strains are designated auto. Abs induced by LPS are indicated. All other Abs were induced by immunization. GL gene refers to the germline gene segment used but does not indicate the absence of mutations. The 0401-13 germline gene is a partial germline sequence from (NZB x NZW)F1 mice (20). The partial sequence of mAb 185b is insufficient to distinguish between VH10.2 and VH10.3. The partial sequence of mAb 136/9 is insufficient to assign a germline gene. Germline VH10 gene sequences are not available for NOD mice (Ab 193020). The sequence of mAb dC1 was submitted to GenBank by O’Connor et al. in 1998. Abs that are members of clonally related sets of hybridomas (i.e., derived from a common B cell) are indicated by an asterisk. Sequences from these related sets that are not listed have the following accession numbers: Z22080 and Z22093 (related to 17p.80), L30140 (related to 1255), U55465 (related to 373p.114), and U55546 and U55544 (related to 452p.108).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The three BALB/c and two C57BL VH10 germline genes appear to be functional based on sequence analysis and their ability to be expressed as properly rearranged µ transcripts by LPS-activated spleen cells. For all five genes, the elements known to be involved in VH gene promoter function have identical sequences and organization. In addition, the VH10 genes of both strains have previously been shown to be located within the same subregion of the Igh locus (3, 46). Thus, the structure and cis-regulatory elements of these genes suggest that all five are capable of comparable levels of rearrangement and transcription.

The VH10 genes of BALB and C57BL are highly similar, ranging from 97% (9-bp differences) to 99% (2-bp differences) sequence identity in the VH-coding regions. The coding regions of the two-gene VHX24 family of BALB/c are similarly conserved, having only seven nucleotide differences (47). Six germline VGAM3.8 family genes have been cloned and have 93–99% sequence identity (48). Greater diversity is found within the VHS107 family of BALB/c (23). VHS107 includes four genes, one of which (V3) has structural defects and is clearly a pseudogene. The other three VHS107 genes (V1, V11, and V13) differ by 21–42 nucleotides (86–93% identity) in their VH-coding sequences.

The analysis of shared (paired) differences from the VH10 consensus sequence allowed the unambiguous assignment of VH10 alleles. Allelic relationships among VH gene families of different haplotypes are not always decipherable, since expansion (or loss) of a family may occur after subspecies (strain) separation (49). For example, allelic relationships between BALB and C57BL VH genes of the VHS107 family could not be established with certainty (50).

The reason for the threefold difference in usage of the two C57BL VH10 genes (7.2 vs 2.3%) is not obvious. Since both genes have identical promoters and RSS, there is no basis to suggest differential rearrangement efficiency or transcription rate as an explanation. It is possible that one or more of the four amino acid differences encoded by these two genes might influence the association of heavy and light chains or BCR-mediated cell selection at one or more stages of B cell differentiation. One VH10 gene examined, the VH10.1 allele of BALB/c, encoded only 0.2% of splenic VH cDNAs. Consistent with this finding, none of the seven reported VH10-encoded BALB/c Abs use the VH10.1 isotype (Fig. 9Go). This relative rarity of VH10.1a cDNAs is interesting given that VH10.1a encodes only two amino acid differences (at positions 38 and 53) compared with the frequently expressed C57BL/6 allele (VH10.1b). At position 53 (CDR2), VH10.1a encodes asparagine, the consensus amino acid encoded at this position by four of the five germline VH10 genes. At position 38 (framework region 2), however, VH10.1a is unique and encodes a cysteine instead of the arginine encoded by the four other germline VH10 genes (Fig. 3Go). If the low representation of VH10.1a is, in fact, due to the encoded protein, the cysteine in framework region 2 is probably involved, since position 38 is quite conserved and is nearly always an arginine or lysine. Furthermore, of the expressed mouse Ab H chains in the sequence collection of Kabat et al. (19), only the anti-Smith Ag mAb Z26 (51) has a cysteine at this framework position. The germline VHS107 V3 alleles of C57BL/10 and CBA/J mice also encode a cysteine at position 38; however, both genes are considered nonfunctional due to defective RSS (52). Therefore, we suggest that the limited contribution of VH10.1a to the BALB/c repertoire may be due to the ability of the cysteine at position 38 to interfere with correct heavy chain folding and/or heavy-light chain interactions.

The first VH10 family member was identified as a gene encoding a ssDNA-specific autoantibody produced by a hybridoma derived from a MRL-lpr/lpr mouse (1). Subsequent studies have identified eight additional mAbs that bind ssDNA and have a VH10-encoded heavy chain (Fig. 9Go). Two Z-DNA-specific mAbs produced in C57BL/6 mice immunized with Z-DNA both express heavy chains encoded by VH10 genes (35). One of these anti-Z-DNA Abs, Z22, is encoded by VH10.2b, and the other, Z44, is encoded by VH10.1b. Thus, although VH10-encoded heavy chains are found in Abs with a variety of specificities for foreign and non-DNA autoantibodies, VH10 genes are considered to be within a subset of V genes with a proclivity for encoding DNA-binding Abs (53).

There is significant support for the idea that B cells that survive in the periphery and constitute the preimmune repertoire require BCR-mediated signaling during their development (8, 54, 55, 56). The strongest evidence that BCR-mediated selection influences individual V gene use is based on comparisons of productive and nonproductive rearrangement ratios (10, 15, 57). Although the ligands involved are not yet defined, it has been reported recently that selection for cells expressing functional heavy chains is inefficient when conducted in vitro (58). Because the promoter and RSS elements are identical in the five germline VH10 genes examined, we suggest that differences in the expression of individual VH10 genes may reflect differential interactions with selecting ligands.

We examined the CDR3 sequences of VH10.2b-encoded cDNAs for evidence that cells expressing this VH segment are selectively expanded by a restricted set of Ags and, in particular, for structural features associated with DNA binding. Our analysis revealed that VH10.2b rearrangements display the high level of diversity normally associated with rearrangements in preimmune B cells (59). Only one of the 15 independent VH10.2b rearrangements we examined encodes a CDR3 arginine, a feature found in 42% of the monoclonal IgM and 70% of the IgG DNA-binding Abs reported by Krishnan and co-workers (29). However, these investigators noted that in heavy chains, the association of CDR3 arginines with DNA binding appears to be restricted to certain germline VH genes. Consistent with this, of the 13 reported VH10-encoded DNA-binding Abs (Fig. 9Go), only two have heavy chains with CDR3 arginines (Z44 and Jel 318). However, the VH10.2b rearrangements examined here do appear to be biased for asparagine, an amino acid capable of forming stable hydrogen bonds with nucleic acid bases (30) and associated with DNA-binding Abs (17, 18). We also found that VH10.2b rearrangements frequently (7 of 15) retain the CA dinucleotide encoded between the VH-coding region and the RSS, resulting in either histidine (5 of 15) or glutamine (2 of 15) at position 95 (Figs. 7Go and 8Go). Interestingly, among the five germline VH10 genes studied, this CA is unique to the VH10.2b gene (Fig. 1Go). Given the apparent bias for polar (asparagine and glutamine) and basic (histidine) amino acids in CDR3, we speculate that the ligand(s) involved in the expansion of VH10.2b-expressing B cells may include DNA and/or have structural similarities to DNA Ags.

Studies of the preimmune repertoire in our laboratory have identified the V{kappa}1-A gene as the most frequently used mouse V{kappa} segment and demonstrated that BCR-mediated selection contributes to the high frequency of V{kappa}1-A expression (10). Thus, the small VH10 and V{kappa}1 gene families include the most overused mouse V segments. Like VH10, V61-A chains are frequently found in DNA-binding Abs (17, 28). For example, 24% of 94 DNA-binding Abs recently reported by Krishnan et al. use V{kappa}1 (23). Of the seven VH10-encoded ssDNA binding autoantibodies with known light chains, five use V{kappa}1 (Fig. 9Go). In view of the frequent expression of VH10 and V{kappa}1 genes in the preimmune repertoire and their association with DNA binding, it is noteworthy that a high proportion (1.2%) of LPS-activated spleen cells from normal mice express ssDNA-binding Abs (60). Although the role of Abs encoded by VH10 and/or V{kappa}1 genes in normal physiology and autoimmunity remains unclear, our results point to BCR-mediated selection as having a prominent role in ensuring a large representation of these Abs in the preimmune B cell repertoire.


   Acknowledgments
 
We thank Drs. B. David Stollar and Roy Riblet for critically reviewing this manuscript and providing helpful insights and suggestions. Dr. R. Kofler generously provided the MRL-DNA4 cDNA.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant GM36064 (to P.H.B.) and National Institutes of Health Training Grant 5T32AI07077 (to E.A.W. and B.B.H.). Back

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

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. E-mail address: Back

4 Abbreviations used in this paper: RSS, recombination signal sequence; BCR, B cell receptor for antigen; CDR, complementarity-determining region of immunoglobulin. Back

Received for publication July 28, 1998. Accepted for publication October 22, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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