Comparison of V kappa gene family expression in adult and fetal B cells

The functional B cell repertoires from adult and fetal mice were compared by examining V kappa gene family expression in individual cells. In addition, because little is known about the relative use of the various V kappa gene families in an immune response, adult B cells from several different strains of mice were analyzed. This was accomplished by stimulating B cells with the polyclonal activator, LPS. Activated cells were then analyzed for V kappa gene family expression at the single cell level by in situ hybridization using radiolabeled V kappa gene probes. It was found that all V kappa gene families tested were represented in the LPS-induced adult repertoire with V kappa 1, V kappa 4,5 and V kappa 19 being expressed to the largest degree in all strains tested. The LPS-induced adult V kappa gene family repertoire was then compared to the fetal repertoire and some differences were observed. In particular, a lower proportion of fetal B cells expressed V kappa 1 and a higher proportion of fetal B cells expressed V kappa 4,5 and V kappa 10. Importantly, compared with the adult response there was no evidence in the fetal response for an increased expression of V kappa 21, the family that maps closest to J kappa,C kappa. This is in contrast to what has been shown previously with H chain V region exons in which there was a clear preference for the VH gene families that mapped closest to DH.     

Genetic analysis of human B cell hybridomas expressing a cross-reactive idiotype

We have examined the genetic basis for the expression of a human cross-reactive idiotype (CRI) commonly found on monoclonal IgM rheumatoid factors. The CRI was identified with a monoclonal antibody (17.109) and has been localized previously to the kappa-variable region. By using the human lymphoblastoid cell line WI-L2-729-HF2, and mononuclear cells from several sources, a panel of hybridomas was generated that produced 17.109 CRI-positive Ig. A recently cloned human germ-line V kappa III gene, Humkv305, served as a probe to identify genes which were rearranged and expressed in 17.109 CRI-positive and -negative hybridomas. This probe, when hybridized to human genomic DNA under stringent conditions, identified only two to five germ-line bands. In 10 separate 17.109 CRI-positive hybridoma clones, an additional rearranged V kappa band was identified. The probe did not anneal to rearranged V kappa bands in hybridoma clones that produced kappa-chains lacking the CRI. RNA dot-blot studies provided evidence for expression of genes hybridizing to the Humkv305 probe. The results indicate that the 17.109 CRI is a serologic marker for a single V kappa gene, or a small family of closely related V kappa genes, which is identified by the Humkv305 probe.     

Aberrant recombination events in B cell lines derived from a kappa-deficient human.

We have analyzed the structure of Ig kappa chain genes in B cell lines derived from a human individual who cannot synthesize any kappa chains, and whose Igs all contain lambda chains (1). We have characterized secondary DNA recombination events at two kappa alleles which have undergone misaligned V-J recombinations. One such secondary recombination has joined the flanking sequences of a V kappa and a J kappa 2 gene segment as if it were the reciprocal product of a V-J kappa 2 recombination, and resulted in the displacement of the recombined VJ kappa 1 gene segments from the C kappa locus. The non-rearranged form of the V kappa fragment which had recombined with the J kappa 2 flank was cloned. Nucleotide sequencing of this fragment identified a V kappa gene that differed by at least 38% from all previously sequenced human V kappa genes. The other V-J kappa segment analyzed has undergone a secondary recombination at a different site from that described above, at a site within the intervening sequence between the J kappa and C kappa gene segments, similar to the location of secondary recombinations which have occurred in lambda + B cell lines from mice and humans (2,3). These results prove that multiple recombinations can occur at one J kappa-C kappa locus.     

Evolution of a multigene family of V kappa germ line genes

We have isolated a series of related V kappa germ line genes from a BALB/c sperm DNA library. DNA sequence analysis of four members of this V kappa 24 multigene family implies that three V kappa genes are functional whereas the fourth one (psi V kappa 24) is a pseudogene. The prototype gene (V kappa 24) encodes the variable region gene segment expressed in an immune response against phosphorylcholine. The other two functional genes (V kappa 24A and V kappa 24B) may be expressed against streptococcal group A carbohydrate. The time of divergence of the four genes was estimated by the rate of synonymous nucleotide changes. This implies that an ancestral gene has duplicated approximately 33-35 million years ago and a subsequent gene duplication event has occurred approximately 23 million years ago.     

Molecular analysis and fine specificity of antibodies against an organophosphorus hapten

We have identified four fine specificity groups reactive with the organophosphorus hapten Soman among 46 hybridomas generated in specific response to immunization with Soman-keyhole limpet hemocyanin (KLH). The different fine specificity groups do not appear to correlate with the use of particular V genes. Molecular analysis of VH genes demonstrates predominant use of VH J558 family members in hybridomas of all fine specificity groups although several different VH genes within this family as well as others are able to contribute. Diversity of VH gene usage was also apparent in primary IgM-producing hybridomas. In contrast, there appears to be restricted L chain usage; a large number (18/46) used the V kappa 1 family. Interestingly, the V kappa 1 family also plays an important role in the memory response to phosphocholine (PC)-KLH, a related organophosphate hapten which shares several structural features with Soman, particularly when coupled to protein carriers. The V kappa 1 C gene appears to predominate in the PC-KLH response. Restriction analysis of DNA from the V kappa 1-positive Soman-KLH-specific hybridomas suggests that a single V kappa 1 gene may be utilized by 17/18 but that this gene is different from V kappa 1 C and may be V kappa 1 A. We propose that members of the V kappa 1 family contribute favorably in generating combining sites that recognize all or part of the structural features shared by the two haptenic structures Soman and PC when they are coupled to protein. This most likely involves recognition of the phenyl linker moiety as the dominant feature. It appears that the L chain rather than the H chain may play a more significant role in forming the phenyl-Soman-specific combining site and perhaps the combining sites for phenyl or ring structures in general.     

Somatic DNA rearrangement generates functional rat immunoglobulin kappa chain genes: the J kappa gene cluster is longer in rat than in mouse

The kappa immunoglobulin (Ig) genes from rat kidney and from rat myeloma cells were cloned and analyzed. In kidney DNA one C kappa species is observed by Southern blotting and cloning in phage vectors; this gene most likely represents the embryonic configuration. In the IR52 myeloma DNA two C kappa species are observed: one in the same configuration seen in kidney and one which has undergone a rearrangement. This somatic rearrangement has brought the expressed V region to within 2.7 kb 5' of the C kappa coding region; the rearrangement site is within the J kappa cluster which we have mapped. The rat somatic Ig rearrangement, therefore, closely resembles that seen in mouse Ig genes. In the rat embryonic fragment two J kappa segments were mapped at 2 and 4.3 kb 5' from the C kappa coding region. Therefore, the rat J kappa cluster extends over about 2.3 kb, a region much longer than the 1.4 kb of the mouse and human J kappa clusters. In the region between C kappa and the expressed J kappa of IR52 myeloma DNA, and XbaI site present in the embryonic kappa gene has been lost. A somatic mutation has therefore occurred in the intervening sequence DNA approx. 0.7 kb 3' from the V/J recombination site. Southern blots of rat kidney DNA hybridized with different rat V kappa probes showed non-overlapping sets of bands which correspond to different subgroups, each composed of 8-10 closely related V kappa genes.     

Structural analysis of a rabbit immunoglobulin kappa 2 J-C locus reveals multiple deletions.

We previously reported that domestic rabbits harbor at least two DNA sequences that hybridize strongly to immunoglobulin kappa C region probes in Southern blots. One of these was cloned from a domestic b4 rabbit and identified as the gene for the nominal b4 allotype kappa chain which is expressed at high levels. We now have cloned (from a b4 rabbit) the other homologous sequence and find that it encodes a kappa chain nearly identical to the kappa 2 chain of "bas" rabbits, which is not normally expressed at detectable levels in domestic rabbits. Sequence analysis of this kappa 2 chain reveals a J kappa -C kappa locus with no obvious coding sequence defects that could explain its low expression. However, several base changes in a putative enhancer region as well as deletions (totalling about 1.5 kb) in the J-C intron might be related to low expression. The comparison between these two kappa genes raises questions about the selective pressures operating during the evolution of this gene system.     

Evolutionary relationships and polymorphisms among V κ 10 genes from inbred and wild-derived inbred mice

The V kappa 10 family in BALB/c mice is composed of three members, two of which are utilized in a variety of immune responses. We previously demonstrated that the product of the third gene, V kappa 10C, has never been detected as part of a functional antibody and productive rearrangements are selectively lost during B-cell development. Here we analyzed germline V kappa 10 genes from inbred and wild-derived mice by RFLP and sequencing in order to determine the origin of the V kappa 10C gene, as well as to examine the evolutionary relationships of V kappa 10 genes. Our results demonstrated that the V kappa 10 family is highly conserved across Mus species and subspecies, but that V kappa 10C is rare, being found in only inbred mice of V kappa 10 allelic group b and two of six M. m. domesticus isolates. It was not found in other M. musculus subspecies or M. spretus. V kappa 10A and V kappa 10B were found in all strains, with the exception of one M. m. domesticus isolate, which had only V kappa 10B genes. Overall, V kappa 10A sequences were more highly conserved than V kappa 10B, indicating that different selective pressures may be operating on these genes. The two V kappa 10C sequences from M. m. domesticus were 100% identical to that found in inbred mice. V kappa 10C is more closely related to V kappa 10B than to V kappa 10A and our data suggest that it is a recent duplication of the V kappa 10B gene.     

Characteristics of murine monoclonal anti-CD4. Epitope recognition, idiotype expression, and variable region gene sequence.

We have characterized a series of mouse monoclonal anti-CD4 and describe both their CD4 epitope recognition and Id expression. We also determined the V region gene sequences of these antibodies in an attempt to correlate epitope recognition and Id expression with V region sequence. All of these preparations recognize epitopes that cluster around the HIV gp120 binding site on the human CD4 molecule. However, we observed differences in epitope recognition among the anti-CD4 preparations, based on either competitive inhibition assays or functional assays, such as syncytium inhibition. Analysis of Id specificities using a polyclonal anti-Id generated against anti-Leu 3a indicated that five of the seven monoclonal anti-CD4 expressed a shared Id. Based on V region gene sequences, the V region kappa-chain (V[kappa]) from each of the seven antibodies was encoded by the V[kappa]21 gene family and expressed the J[kappa]4 gene segment. Those preparations that expressed the shared Id with anti-Leu 3a have virtually identical V[kappa] sequences, with a high degree of homology in the CDR. The VH region gene sequences of six of the seven antibodies also shared overall homology and appeared to be encoded by the J558 VH gene family. The seventh anti-CD4 VH region is encoded for by the VHGAM gene family. The majority of these antibodies used JH3 gene segment, although the JH2 and JH4 gene segments were also represented. In addition, several of these antibodies share a common sequence organization within their V-D-J joining regions that appears to involve N and P sequences to generate unique D segments. Together, these data suggest that differences in epitope recognition among the monoclonal anti-CD4 may reflect sequence variability primarily within the CDR3 region of both V[kappa] and VH. The basis for the detection of a shared Id most likely reflects the high degree of homology within the V[kappa] region sequences. In addition, these data, which are based on a limited analysis, suggest the possible restricted use of V region germ-line gene families in the secondary antibody response of BALB/c mice to specific epitopes on the human CD4 molecule.     

Cloning and sequence analysis of the VH and VL regions of an anti-myelin/DNA antibody from a patient with peripheral neuropathy and chronic lymphocytic leukemia

We have cloned and determined the nucleotide sequence of the Ig VH and VL region genes of an IgM kappa mAb that binds to denatured DNA and myelin from a patient (POP) with chronic lymphocytic leukemia and peripheral neuropathy. Sequence analysis indicates that the V region of the kappa L chain gene (PopVK) has 99% homology to a V kappa IIIa germ-line gene and the V region of the mu H chain gene (PopVH) has 96% homology to the VH26 germ-line gene that is a member of the VH3 gene family. It is likely the V kappa and VH genes arose from these respective germ-line genes via somatic mutation or from closely related genes. V kappa III genes have frequently been used by other IgMk mAb especially those with rheumatoid factor activity, and the VH26 gene with no somatic mutation has been used by several anti-DNA antibodies, suggesting the possibility of preferential association of these or related germ-line genes with autoantibodies. The minor differences between the sequences of POP's VH and V kappa genes and sequences used by other autoantibodies, may be responsible for this antibody's crossreactivity with myelin and, as a result, the autoimmune neuropathy.     

Genes encoding Xenopus laevis Ig L chains. Implications for the evolution of kappa and lambda chains.

Xenopus laevis Ig contain two distinct types of L chains, designated rho or L1 and sigma or L2. We have analyzed Xenopus genomic DNA by Southern blotting with cDNA probes specific for L1 V and C regions. Many fragments hybridized to the V probe, but only one or two fragments hybridized to the C probe. Corresponding C, J, and V gene segments were identified on clones isolated from a genomic library prepared from the same DNA. One clone contains a C gene segment separated from a J gene segment by an intron of 3.4 kb. The J and C gene segments are nearly identical in sequence to cDNA clones analyzed previously. The C segment is somewhat more similar and the J segment considerably more similar in sequence to the corresponding segments of mammalian kappa chains than to those of mammalian lambda chains. Upstream of the J segment is a typical recombination signal sequence with a spacer of 23 bp, as in J kappa. A second clone from the library contains four V gene segments, separated by 2.1 to 3.6 kb. Two of these, V1 and V3, have the expected structural and regulatory features of V genes, and are very similar in sequence to each other and to mammalian V kappa. A third gene segment, V2, resembles V1 and V3 in its coding region and nearby 5'-flanking region, but diverges in sequence 5' to position -95 with loss of the octamer promoter element. The fourth V-like segment is similar to the others at the 3'-end, but upstream of codon 64 bears no resemblance in sequence to any Ig V region. All four V segments have typical recombination signal sequences with 12-bp spacers at their 3'-ends, as in V kappa. Taken together, the data suggest that Xenopus L1 L chain genes are members of the kappa gene family.     

An experimental approach to enumerate the genes coding for immunoglobulin variable-regions

Critical to our understanding of the immune system diversity is the determination of the number of germ line V genes. The total number of V genes is given by the product: number of subgroups x number of germ line genes per subgroup. Studies of kappa chains and of embryonic DNA indicate 5-10 V genes per subgroup. Statistical analysis of the limited sequence data of mouse kappa chains suggest about 50 V kappa subgroups. We report here a general approach for direct estimation of the number of VL and VH subgroups expressed in normal spleen, and present data for V kappa. The kappa mRNA of the spleen is a heterogeneous population where different V kappa are linked to the same C kappa, i.e. C kappa equals total V kappa. The ratio C kappa/distinct V kappa approximates the number of subgroups since V kappa of the same subgroup cross hybridize while V kappa of different subgroups do not. This ratio was determined by molecular hybridization of cloned C kappa and V kappa DNA probes with spleen mRNA. The results indicate the expression of 280 V kappa subgroups in mouse. Assuming an average of 7 genes per subgroup, we estimate about 2000 V kappa germ line genes.     

The Z family, a group of transposed human immunoglobulin V kappa genes

A group of highly homologous transposed human V kappa I genes, which we call the Z family, was characterized. To date four members, ZI-ZIV, comprising about 230 kb, have been analyzed on cosmid clones. The largest region (ZI) has a length of 85 kb. The Z regions show extensive homology to each other according to restriction maps and hybridization data. In each Z region a solitary V kappa I gene was found. No V kappa genes of other subgroups were detected by hybridization. The nucleotide sequence of the ZI gene revealed a non-processed V kappa I pseudogene. Hybridization experiments with DNAs from rodent/human cell hybrids and other experimental data indicate that some and possibly all members of the Z family lie outside of the kappa locus which is located on chromosome 2; they have been transposed to other chromosomes. Because of their separation from the J kappa C kappa gene segment, the Z genes can be classified as pseudogenes independent of their sequences. We postulate that the Z family arose by amplification event(s). The Z regions can also be regarded as a small family of very long repetitive sequences.     

Ig V kappa family repertoire of plasma cells derived from autoimmune MRL mice.

V kappa gene family usage was determined in the resident in vivo-activated plasma cells of individual diseased MRL mice by using in situ hybridization. In this way, the entire autoimmune repertoire could be analyzed. Autoantibody levels and extent of glomerulonephritis were also measured, so that the severity of disease could be assessed. It was found that V kappa expression was highly variable from mouse to mouse. Some animals displayed a V kappa family repertoire similar to mitogen-stimulated cells and consistent with the size of the families. These animals tended to have lower disease indices. Other animals, which had higher disease indices, displayed considerable over- or underutilization of individual V kappa families. However, no particular V kappa families were repeatedly biased in their expression, as was found at the VH level with J558. Importantly, in the 10% of animals that expressed VH J558 exclusively, four or more V kappa families were expressed and multiple antiself specificities were produced. The data are most consistent with a number of J558 genes being expanded in a variety of self-specificities. However, because only VH J558 is expressed in these sicker animals, nonspecific polyclonal activation is highly unlikely. These results underscore the continuing evolution of the autoimmune repertoire, with considerable diversity at early stages followed by a highly selected repertoire in which a potential role for nonspecific polyclonal activation is virtually excluded.     

Genetics and primary structure of V kappa gene segments encoding antibody to streptococcal group A carbohydrate. Comparison of V kappa gene structure with idiotope expression

Two gene segments, V kappa-25-39 and V kappa-25-47, that encode antibody to streptococcal group A carbohydrate in A/J mice were found to be more than 95% homologous in nucleotide sequence in both coding and noncoding regions. It was previously shown that V kappa-25-39 encodes immunoglobulins that express the IdX and IdI-1 idiotopes, whereas V kappa-25-47 encodes IdX+, and IdI-1- immunoglobulins. V kappa gene segments that were clearly allelic to V kappa-25-47 are used to encode IdX+, IdI-1- anti-group A carbohydrate antibodies by C.B20 mice and likely by C57BL/6 mice. Murine strains that are deficient in IdI-1 idiotope expression were investigated by Southern blotting with a 5' probe from V kappa-25-39. Two IdI-1-deficient strains, CE/J and C58/J, had a grossly altered V kappa gene segment structure compared with the A/J prototype. In contrast, the IdI-1-deficient strain, C57BL/6, was indistinguishable from A/J with the 5'V kappa-25-39 probe, indicating that more subtle genetic changes account for the loss of IdI-1 expression in C57BL/6 mice. The evolution of V kappa-25-39 and V kappa-25-47 gene segments was deduced by comparison with the homologous V kappa 24B gene segment of Mus pahari. V kappa-25-39 and V kappa-25-47 likely have recently duplicated once in A/J and related strains of laboratory mice and may have duplicated again in CE/J mice. Thus, individual members of the V kappa 24 gene family, to which V kappa-25-39 and V kappa-25-47 belong, are preserved while the number of gene copies expands or contracts. This fact is strong evidence that evolutionary forces have maintained the V kappa 24 gene family, all of which encode antibody specific for carbohydrate found in bacterial pathogens.     

Variant (6;15) translocations in murine plasmacytomas involve a chromosome 15 locus at least 72 kb from the c-myc oncogene.

The variant (6;15) translocations in murine plasmacytomas join the myc oncogene-bearing band of chromosome 15 and the immunoglobulin kappa band of chromosome 6. We recently cloned a region from chromosome 15 linked to C kappa and have now used probes from that region to define the major locus of plasmacytoma variant translocations, which we denote pvt-1. In five of nine plasmacytomas we analysed, the 6;15 translocation resulted from reciprocal recombination between the C kappa locus and a 4.5-kb region of pvt-1. Moreover, nearby we located the region shown by others to have undergone a complex (15;12;6) translocation in plasmacytoma PC7183. All the chromosome 6 breakpoints fell between 1 and 3 kb 5' to C kappa but only two were near J kappa genes. Thus the J kappa -C kappa region appears to be a recombination 'hot spot' in lymphocytes, but the breaks are unlikely to be mediated via V/J recombination enzymes. Comparison of a cloned 108-kb region across pvt-1 and another of 52 kb across c-myc established that the pvt-1 breakpoints lie at least 72 kb from the c-myc promoters. Since c-myc is expressed at a substantial level, the 6;15 translocation apparently activates c-myc. Activation may occur directly, at a remarkable distance along the chromosome, or indirectly, via a putative pvt-1 gene product.