Structure of rearranged and germ-line rabbit V kappa genes indicated that the CDR3 is encoded by the V kappa gene

The third complementary-determining regions (CDR3) of rabbit kappa chains are unusually long and the length is more heterogeneous when compared to those of the mouse and the human kappa chains. To study how the rabbit kappa light (L) chain genes create diversity and generate CDR3, we analyzed the structure of a rearranged variable kappa gene (Vr) and the variable (Vg) and joining (Jg) regions of the putative precursor genes. Alignment of the Vr gene sequence with that of the Vg and Jg regions allowed precise determination of the recombination event. Five nucleotides between the recombination point and the J2 heptamer were deleted, indicating flexibility in the recombination producing rabbit kappa chains. The entire Vg is contained in the rearranged product demonstrating that neither a D element nor an N sequence addition are required for the CDR3 formation. Comparison of the Vr and the Vg gene sequences show base substitutions suggesting that somatic mutations may contribute to rabbit kappa L chain diversity.     

Generation of heterogeneous rabbit anti-DNP antibodies by gene conversion and hypermutation of rearranged VL and VH genes during clonal expansion of B cells in splenic germinal centers

The mechanisms described here account for development of the heterogeneous high-affinity anti-DNP antibodies that rabbits can produce. Rearranged immunoglobulin light and heavy chain genes from single DNP-specific splenic germinal center B cells were amplified by PCR. We found that in clonal lineages, rearranged V[kappa] and V[H] are further diversified by gene conversion and somatic hypermutation. The positive and negative selection of amino acids in complementarity-determining regions observed allows emergence of a variety of different combining site structures. A by-product of the germinal center reaction may be cells with sequences altered by gene conversion that no longer react with the immunizing antigen but are a source of new repertoire. The splenic germinal center would thus play an additional role in adults similar to that of the appendix and other gut-associated lymphoid tissues of young rabbits.     

Somatic hypermutation of immunoglobulin variable region genes: focus on follicular lymphoma and multiple myeloma

Summary: Analysis of the rearranged immunoglobulin variable region gene hypermutation has provided important information concerning the clonal history and ontogenetic origin of various B-cell lymphoproliferative disorders. Under the selective pressure of antigen, mutational events in immunoglobulin genes will fine tune survival of B-cell clones bearing immunoglobulin with high affinity for antigen. Our studies aimed at analyzing neoplastic disorders originating from germinal and post-germinal center B-cells: follicular lymphoma and multiple myeloma. respectively. Despite the already acknowledged evidence for a selectable distribution of mutations within the clonal immunoglobulin variable heavy chain genes, very little is known about the contribution of light chains in the process of antigen selection. In follicular lymphoma. a more limited pattern of somatic mutation with less evidence of antigen selection was observed in variable K light chain genes (40%) than in their partner heavy chain genes (80%). In myeloma, hypermutation of variable light chain genes, with a distribution suggestive of antigen selection, was frequently observed. Based on these data and recent reports it appears that the light chain expressed by the clonogenic myeloma B-cells plays a pivotal role in the antigen selection process. Additionally, abortive K light chain variable region genes in X-expressing myeloma carried a significant number of somatic mutations indicating that the cell of origin is open to the hypermutation machinery at that particular developmental stage irrespective of antigen selection.     

Diversification of rabbit VH genes by gene-conversion-like and hypermutation mechanisms

Summary: Where, when and how does Vu diversification occur in the rabbit? Early diversification by gene-conversion and somatic hypermutation in rabbit appendix and chicken bursa of Fabricius are similar processes; the chicken bursa and the rabbit appendix have homologous functions. However, diversification in bursa starts during embryonic development whereas it starts in rabbit appendix about 2 weeks after birth in the presence of antigens and superantigens that ma) contribute to positive and negative selection, affect B-cell expansion and mold the repertoire. The biochemical steps leading to diversification by gene conversion are unknown. However elevated levels of RAD51 mRNA in both chicken bursa and young rabbit appendix suggest that repair of double stand breaks may be involved. The base changes found in expressed rabbit V_H sequences derived from rearrangement of known germline V_H genes followed by one or more gene conversions occur with frequencies similar to those found in analyses of somatic hypermutation. The Ser codons in CDR1 and CDR2 of rabbit V_Hl I genes are all AGY rather than TCN, suggesting that they may represent intrinsic hotspots for hypermutation comparable to those described in human and mouse V_Hr. Somatic hypermutation may further refine antibody affinities in rabbit germinal centers.     

Rearrangement of only one human IGHV gene is sufficient to generate a wide repertoire of antigen specific antibody responses in transgenic mice

In recent years, mice carrying human IG transgenes are being generated for the production of human monoclonal antibodies as an alternative approach to the conventional use of mouse or chimeric-humanized antibodies. Theoretically, the size of the repertoire of human antibodies that these mice could produce would be critically dependent on the number of human V genes introduced in the transgene. This could be the case for BABkappa and BABkappa,lambda transgenic mice, which carry several genes from the human IGK (BABkappa), and IGK and IGL (BABkappa,lambda) loci, but only five human IGHV genes and the entire IGHD-IGHJ cluster linked to two human IGHC (IGHM-IGHD) genes. We analyzed the expressed human IG genes in 30 IgM-secreting hybridomas generated from transgenic mice immunized either with soluble proteins (human IgM coupled to KLH) or with cells (human PBMC, tumour cell lines or rat cells transfected with human CD69). The results show that all hybridoma cells analyzed rearranged exclusively the IGHV1-2 gene, in contrast with naive spleen B cells that used three out of the five IGHV genes present in the transgene. The configuration of the rearranged CDR3 region revealed a much higher heterogeneity in the heavy chains. A variety of IGHJ and IGHD genes were used in hybridomas, and somatic mutations were also seen in some hybrids. Regarding the rearranged light chains genes, it was a much higher variety in the use of V and J genes in both, kappa and lambda chains, than in the heavy chain, and also in the level of mutation. The results indicate that only one IGHV gene is sufficient to generate a wide repertoire of antigen specific antibody responses. Thus, efforts aimed at the generation of new transgenic mice should focus more on the integrity of the D/J region and on the DNA regions regulating somatic hypermutation, rather than on the number of V genes present in the transgene.     

Analysis of Ig kappa light chain gene variable regions expressed in the rheumatoid synovial B cells

Sequence analysis of antibody variable (V) regions can provide an insight regarding whether B cells have gone through an antigen-driven process of affinity maturation. In this study, we analyzed 16 V-regions of immunoglobulin (Ig) kappa light chain genes obtained from a cDNA library of a rheumatoid arthritis (RA) synovial tissue. A salient feature of our results is the high frequency utilization of germline V kappa I family genes, especially the O2/O12 gene (38%). All kappa V-regions showed extensive somatic hypermutation with 5.4% of an average mutation rate. Replacement to silent mutation (R/S) ratio in the complementarity determining region (CDR) was > 2.9 in 12 out of 16 clones, indicating that the majority of the RA synovial B cells had undergone affinity maturation. However, the four other clones showed R/S ratios of < 2.9 in the CDR despite a high mutation rate. In contrast to the previous reports, long CDR3 was not a characteristic feature of these clones. In summary, these data show the high frequency utilization of the germline O2/O12 gene and a high rate of mutation with an evidence of antigen selection in most of the Ig kappa genes expressed in the RA synovium.     

The repertoire of human antibody to the Haemophilus influenzae type b capsular polysaccharide.

Human antibody to the Haemophilus influenzae capsular polysaccharide (Hib CP) is restricted in diversity in the individual and the population with a limited number of variable region genes encoding antibody. Antibody to the Hib CP shows restricted isoelectric focusing gel patterns and light chain usage with frequent restriction to use of only kappa light chains. Shared cross-reactive idiotypes are expressed on antibody. The heavy chain of antibody to the Hib CP is predominantly encoded by two members of the VH3 family--LSG 6.1/M85-like and VH26/30P1-like. In VH the CDR1, based on complete identity in LSG 6.1/M85-like antibodies, CDR2, based on the suggestion of mutation in this region, and CDR3, based on conserved CDR3 usage in unrelated individuals, may be important for antigen binding. Six or more different VL gene families encode antibody. The predominant antibody of the majority of individuals uses the A2-V kappa II gene in germline or near germline configuration, which encodes an idiotype designated HibId-1. Antibody can also be encoded by V kappa I, non-A2 V kappa II, V kappa III, V kappa IV, V lambda II, and V lambda VII genes. Although different VL genes can be used, unrelated individuals appear to use the same V kappa III (A27), V lambda II (V lambda 2.1 and V lambda VII (4A) genes. The VL diversity accounts for differences in fine binding specificity, with A2-V kappa II genes not encoding E. coli K100 CP cross-reactive antibodies and V lambda VII genes and some of the non-A2 V kappa genes encoding cross-reactive antibodies. The arginine in CDR3 of both antibody kappa and lambda light chains and the asparagine in CDR2 of VL sequences and in CDR1 of LSG6.1-M85 VH sequences of antibody appear to be important residues for antigen binding. A relatively limited degree of somatic mutation has occurred in the non-A2 VL genes, V lambda VII, and the VH genes. Further studies comparing the polymorphism of germline V genes to antibody-encoding V genes are needed to clarify this issue. Research comparing this repertoire to repertoires directed to other bacterial CP and to self antigens and defining structure-antigen binding relationships is in progress.     

A simple and rapid protocol for the sequence determination of functional kappa light chain cDNAs from aberrant-chain-positive murine hybridomas

This protocol describes the application of a polymerase chain reaction to allow the cloning and sequencing of new functional kappa light chain cDNAs from murine hybridomas co-expressing aberrant endogenous kappa chain mRNAs. The presence of kappa light chain aberrant mRNAs can hinder or even prevent determination of the sequence of functional murine kappa light chain cDNAs amplified by PCR from hybridomas. The method described here employs a panel of kappa primers in the presence of molar excess of a primer complementary to the complementary determining region (CDR) 3 of the known aberrant chain sequence. Analysis of the PCR products reveals two bands for some reactions: one the functional, full-length kappa chain cDNA (approximately 400 bp) and another shorter (approximately 100 bp) band corresponding to short aberrant chain kappa CDR3-constant region. The full-length product is gel purified and cloned prior to sequencing and aligned with V-region germline sequences available in NCBI and GenBank databases. This method is used routinely in our laboratory and demonstrates consistency and reliability for sequence determination of kappa light chain V-gene cDNA of mAbs to diverse antigens. This protocol is a rapid and convenient method for determining the sequence of murine V kappa region genes from hybridomas expressing aberrant kappa chain mRNAs.     

Multiple germline functional VL genes contribute to the IgL repertoire in ducks

In the immunoglobulin light chain gene loci of nearly all bird species examined to date, there is only a single functional variable gene segment that can recombine with joining gene segments. Thus, Ig light chain diversity relies on gene conversion using pseudogenes as sequence donors to modify the single rearranged variable gene. In the present study, we have sequenced a bacterial artificial chromosome (BAC) clone containing the entire duck Igλ light chain gene locus. Although only a single pair of J_λ and C_λ was found, 88 V_λ gene segments were identified upstream of the J_λ and C_λ segments. Among the identified V_λ gene segments, 79 appear to be pseudogenes, the remaining 9 are structurally intact and all are able to functionally rearrange with the J_λ. Phylogenetic analyses suggest that the 9 functional variable genes may have been derived from a single gene through duplication events. Although these multiple functional variable gene segments can be subject to VJ recombination, both gene conversion and somatic hypermutation are also actively involved in the generation of diversity in duck Igλ light chains. These data provide significant insight into understanding the duck Ig system.     

Organization and structure of wild rabbit kappa2 genes: implications for regulation of kappa expression

In domestic populations, the rabbit kappa light chains are known to be encoded by two loci which are unequally expressed. The kappa1 chains account for the majority of total serum kappa chains, and display an unusual complex polymorphism. In order to study the evolution and the putative correlations between the expression, the organization and the structure of the kappa genes, we analysed the kappa loci in wild rabbit populations. The kappa genes of b95, b97 and b98 allotypes are organized in two loci similar to that of domestic rabbits. The structure of the constant region of the kappa2 locus was determined from a wild rabbit which expresses b95 allotype kappa1 chains. The Ckappa2bas2 of b95 displays a single silent mutation when compared to Ckappa2bas2 associated with b4 and one amino acid change relative to Ckappa2bas1 chain. Therefore, in contrast to the kappa1 locus, the constant regions of the kappa2 locus display strong conservation during evolution. A model based on conformation of the kappa chains is discussed to explain the evolution and expression of the two kappa loci.     

Activation-induced cytidine deaminase induces DNA break repair events more frequently in the Ig switch region than other sites in the mammalian genome

Activation-induced cytidine deaminase (AID) produces DNA breaks in immunoglobulin genes during antibody diversification. Double-stranded breaks (DSB) in the switch region mediate class switch recombination, and contribute to gene conversion and somatic hypermutation in the variable regions. However, the relative extent to which AID induces DSB in these regions or between these and other actively expressed sequences is unknown. Here, we exploited an enhancer-trap plasmid that identifies DSB in actively expressed loci to investigate the frequency and position of AID-induced vector integration events in mouse hybridoma cells. Compared to control cells, wild-type AID stimulates plasmid integration into the genome by as much as 29-fold. Southern and digestion-circularization PCR analysis revealed non-uniformity in the integration sites, with biases of 30- and 116-fold for the immunoglobulin kappa light chain and mu heavy chain genes, respectively. Further, within the immunoglobulin mu gene, 73% of vector integrations map to the mu switch region, an enhancement of five- and 12-fold compared to the adjacent heavy chain variable and mu gene constant regions, respectively. Thus, among potential highly transcribed genes in mouse hybridoma cells, the immunoglobulin heavy and light chain genes are important AID targets, with the immunoglobulin mu switch region being preferred compared to other genomic sites.     

Molecular analysis of the V kappa III-J kappa junctional diversity of polyclonal rheumatoid factors during rheumatoid arthritis frequently reveals N addition.

Much interest was stirred in recent years by the evidence that rheumatoid factors (RF) variable regions are encoded by a restricted set of V genes, with little or no somatic mutations, that are often overexpressed in the fetal repertoire. This is reminiscent of what has been observed for natural autoantibodies. However, these data come from studies of monoclonal RF (mRF) isolated from patients with lymphoproliferative disorders who usually do not present autoimmune symptoms. The molecular characterization of RF during autoimmune diseases such as rheumatoid arthritis (RA) has been hampered for some time because of their polyclonality; recently using the polymerase chain reaction method, we have demonstrated that RF kappa variable regions from a patient with RA were encoded by V kappa III genes known to code for mRF but that these genes had undergone somatic mutations with a pattern suggesting an antigen-driven maturation. Because an important role of the light chain third complementarity-determining region (CDR3) in anti-IgG reactivity and idiotype expression has already been suspected for RF, we now report the molecular characterization of the junction regions of these rearranged V kappa gens. Surprisingly, our data show that in 55% of the cases there is addition of a proline and/or glycine amino acid residue at the recombination site between V kappa and J kappa. The sequence analysis of our patients' germ-line Vg and J kappa 4 genes segments and their flanking regions demonstrates that the additional codons are not readily explicable by recombination between germ-line sequences and probably result from an N addition process. Since we could not find such an additional codon in 15 previously published mRF kappa chains we suggest that "pathogenic" RF during RA and mRF derive from different, although overlapping, B cell subsets. Moreover, since additional codons at the recombination site of V kappa and J kappa seem exceptional in expressed human kappa chains and because the resulting amino acid residue is a proline in most cases, we think that RF kappa chain CDR3 is under a very strong selective pressure during RA.     

Preferrential rearrangement in normal rabbits of the 3' VHa allotype gene that is deleted in Alicia mutants; somatic hypermutation/conversion may play a major role in generating the heterogeneity of rabbit heavy chain variable region sequences

The rabbit is unique in having well-defined allotypes in the variable region of the heavy chain. Products of the VHa locus, (with alleles a1, a2, and a3), account for the majority of the serum immunoglobulins. A small percentage of the serum immunoglobulins are a-negative. In 1986, Kelus and Weiss described a mutation that depressed the expression of the Ig VH a2 genes in an a1/a2 rabbit. From this animal the Alicia rabbit strain was developed and the mutation was termed ali. We previously showed, using Southern analysis and the transverse alternating field electrophoresis technique, that the difference between the ali rabbit and normal is a relatively small deletion including some of the most 3' VH genes. The most JH proximal 3' VH1 genes in DNA from normal rabbits of a1, a2 and a3 haplotypes encode a1, a2 and a3 molecules respectively, and it has been suggested that these genes are responsible for allelic inheritance of VHa allotypes. The present study suggests that the 3' end of the VH locus probably plays a key role in regulation of VH gene expression in rabbits because VH gene(s) in this region are the target(s) of preferential VDJ rearrangements. This raises the possibility that mechanisms such as somatic gene conversion and hypermutation are at work to generate the antibody repertoire in this species. Our data support the view that the 3' VH1 gene may be the preferred target for rearrangement in normal rabbits, and for the normal chromosome in heterozygous ali animals. However, homozygous ali rabbits with a deletion that removed the a2-encoding VH1 on both chromosomes do survive, rearrange other VH genes and produce normal levels of immunoglobulins as well as a significant percentage of B cells which bear the a2 allotype. This challenges the view that one VH gene, VH1, is solely responsible for the inheritance pattern of VHa allotypes.     

Mapping of the duplicated rabbit immunoglobulin kappa light chain locus.

The rabbit has two isotypic forms of the immunoglobulin kappa light chain, K1 and K2, which probably arose by duplication. In the normal rabbit, only traces of K2 light chains are produced. However, K2 levels are elevated in allotype-suppressed rabbits and in the Basilea strain which does not produce K1 because of a K1 mRNA splice site mutation. Previous cloning and sequencing showed that each isotype has its own set of J kappa genes but it was not known whether the two isotypes utilize shared or separate sets of V kappa genes. In addition, although genetic linkage of allotypes associated with the K1 and K2 genes has been demonstrated, physical linkage had not been previously demonstrated by overlapping cosmid or phage clones. We used pulsed field and transverse alternating field electrophoresis to obtain megabase maps and to estimate the size of the duplication of the rabbit kappa light chain locus. We found that the two C kappa genes are about 1 megabase apart. One explanation for the poor expression of K2, could be great physical distance from V kappa genes. However, we found that there are V kappa, J kappa and C kappa 2 genes within a approximately 105-kb fragment. Thus, physical distance of V kappa from C kappa 2 may not be the basis for poor K2 expression.     

Receptor revision plays no major role in shaping the receptor repertoire of human memory B cells after the onset of somatic hypermutation.

In order to determine whether V gene replacement accompanies somatic hypermutation in the germinal center (GC) reaction in the human, we analyzed V(kappa)J(kappa) and V(lambda)J(lambda) joints and the kappa-deleting element in single lambda(+) naive and post GC B cells for rearrangements at the kappa and lambda loci. Among 265 lambda(+) post GC B cells, not a single unequivocal and only two potential examples of a cell that switched to lambda light chain expression after accumulation of (unfavorable) mutations in its productive V(kappa) rearrangement were observed. Taking the PCR efficiency into account, the frequency of such cells is likely below 3 %. In addition, heavy and light chain gene rearrangements were amplified and sequenced from the oligoclonal population of IgD-only peripheral blood post GC B cells which display extensive intraclonal sequence diversity. Among 61 IgD-only B cells belonging to 15 clones with intraclonal diversity, no combination of V gene rearrangements indicating receptor revision during clonal expansion was observed. Moreover, among 124 and 49 V(H) genes amplified from IgD-only and class-switched B cells, respectively, not a single example of V(H) revision through V(H) hybrid generation was detected. These results suggest that in the human GC reaction V gene replacement either does not usually accompany somatic hypermutation or is mostly counterselected.