Vkappa Gene Research Articles

Conserved cryptic recombination signals in Vkappa gene segments are cleaved in small pre-B cells.

BackgroundThe cleavage of recombination signals (RS) at the boundaries of immunoglobulin V, D, and J gene segments initiates the somatic generation of the antigen receptor genes expressed by B lymphocytes. RS contain a conserved heptamer and nonamer motif separated by non-conserved spacers of 12 or 23 nucleotides. Under physiologic conditions, V(D)J recombination follows the "12/23 rule" to assemble functional antigen-receptor genes, i.e., cleavage and recombination occur only between RS with dissimilar spacer types. Functional, cryptic RS (cRS) have been identified in VH gene segments; these VH cRS were hypothesized to facilitate self-tolerance by mediating VH → VHDJH replacements. At the Igκ locus, however, secondary, de novo rearrangements can delete autoreactive VκJκ joins. Thus, under the hypothesis that V-embedded cRS are conserved to facilitate self-tolerance by mediating V-replacement rearrangements, there would be little selection for Vκ cRS. Recent studies have demonstrated that VH cRS cleavage is only modestly more efficient than V(D)J recombination in violation of the 12/23 rule and first occurs in pro-B cells unable to interact with exogenous antigens. These results are inconsistent with a model of cRS cleavage during autoreactivity-induced VH gene replacement.ResultsTo test the hypothesis that cRS are absent from Vκ gene segments, a corollary of the hypothesis that the need for tolerizing VH replacements is responsible for the selection pressure to maintain VH cRS, we searched for cRS in mouse Vκ gene segments using a statistical model of RS. Scans of 135 mouse Vκ gene segments revealed highly conserved cRS that were shown to be cleaved in the 103/BCL2 cell line and mouse bone marrow B cells. Analogous to results for VH cRS, we find that Vκ cRS are conserved at multiple locations in Vκ gene segments and are cleaved in pre-B cells.ConclusionOur results, together with those for VH cRS, support a model of cRS cleavage in which cleavage is independent of BCR-specificity. Our results are inconsistent with the hypothesis that cRS are conserved solely to support receptor editing. The extent to which these sequences are conserved, and their pattern of conservation, suggest that they may serve an as yet unidentified purpose.

Rap1 Signal Controls B Cell Receptor Repertoire and Generation of Self-Reactive B1a Cells

We previously reported that the mice deficient for SPA-1, a Rap1 GTPase-activating protein, developed hematopoietic stem cell disorders. Here, we demonstrate that SPA-1(-/-) mice show an age-dependent increase in B220(high) B1a cells producing anti-dsDNA antibody and lupus-like nephritis. SPA-1(-/-) peritoneal B1 cells revealed the altered Vkappa gene repertoire, including skewed Vkappa4 usage and the significant Igkappa/Iglambda isotype inclusion indicative of extensive receptor editing. Rap1GTP induced OcaB gene activation via p38MAPK-dependent Creb phosphorylation, and consistently, SPA-1(-/-) immature BM B cells showing high Rap1GTP exhibited the augmented expression of OcaB and Vkappa4 genes. SPA-1(-/-) BM cells could transfer the autoimmunity in association with the generation of peritoneal B220(high) B1a cells in Rag-2(-/-) recipients. Finally, a portion of SPA-1(-/-) mice developed B1 cell leukemia with hemolytic autoantibody. Present results suggest that the regulated Rap1 signal in the immature B cells plays a role in modifying the B cell receptor repertoire and in maintaining the self-tolerance.

Chromatin Structural Analyses of the Mouse Igκ Gene Locus Reveal New Hypersensitive Sites Specifying a Transcriptional Silencer and Enhancer

To identify new regulatory elements within the mouse Igkappa locus, we have mapped DNase I hypersensitive sites (HSs) in the chromatin of B cell lines arrested at different stages of differentiation. We have focused on two regions encompassing 50 kilobases suspected to contain new regulatory elements based on our previous high level expression results with yeast artificial chromosome-based mouse Igkappa transgenes. This approach has revealed a cluster of HSs within the 18-kilobase intervening sequence, which we cloned and sequenced in its entirety, between the Vkappa gene closest to the Jkappa region. These HSs exhibit pro/pre-B cell-specific transcriptional silencing of a Vkappa gene promoter in transient transfection assays. We also identified a plasmacytoma cell-specific HS in the far downstream region of the locus, which in analogous transient transfection assays proved to be a powerful transcriptional enhancer. Deletional analyses reveal that for each element multiple DNA segments cooperate to achieve either silencing or enhancement. The enhancer sequence is conserved in the human Igkappa gene locus, including NF-kappaB and E-box sites that are important for the activity. In summary, our results pinpoint the locations of presumptive regulatory elements for future knockout studies to define their functional roles in the native locus.

Evolutionary dynamics of the human immunoglobulin κ locus and the germline repertoire of the Vκ genes

We have determined the entire nucleotide sequence of the human immunoglobulin kappa locus, comprising a total of 1,010,706 nucleotides. The 76 Vkappa genes found by a hybridization-based approach and their classification in 7 families were confirmed. A Vkappa orphon located near the locus was also sequenced. In addition, we identified 55 novel Vkappa relics and truncated pseudogenes, which establish 5 new families. Among these 132 Vkappa genes, 46 have open reading frames. According to the databases and the literature, 32 unique Vkappa genes and 5 identical gene pairs form VJ-joints, 27 unique genes and 4 gene pairs are transcribed, and 25 unique genes and 4 gene pairs produce functional proteins. The Vkappa gene locus contains a 360-kb inverted duplication, which harbors 118 Vkappa genes. A comparison of the duplicated Vkappa genes suggests positive selection on the complementarity-determining regions of the duplicated genes by point mutations. The entire duplication unit was divided into 13 blocks, each of which has its distinct nucleotide sequence identity to its duplication counterpart (98.1 - 99.9 %). An inversion-mediated mechanism is suggested to generate the high-homology blocks. Based on the homology blocks and the mutation rates, the inverted duplication is assumed to have taken place approximately 5 million years ago. An orphon Vkappa gene near the kappa locus and a cluster of five Vkappa orphons on chromosome 22 have no counterparts within the kappa locus. This suggests possible mechanisms of the transposition of orphon Vkappa genes.

Overpassing an aberrant V(kappa) gene to sequence an anti-idiotypic abzyme with (beta)-lactamase-like activity that could have a linkage with autoimmune diseases.

A monoclonal antibody 9G4H9 that exhibits a beta-lactamase-like activity was previously obtained in accordance with the idiotypic network theory. This abzyme presents the most catalytic efficiency in amidase activity described in literature (kcat = 0.9 min-1). Some reports have demonstrated that functionality as complex as catalysis may be mimicked in this way. Comparison of the catalytic properties of both enzyme and abzyme previously allowed us to obtain better knowledge about 9G4H9 abzymatic machinery. In attempt to characterize this abzyme, the variable regions of kappa and heavy chain were cloned. We present a 'universal' method to clone the correct Vkappa gene to bypass aberrant Vkappa (abVkappa) produced by MOPC-21-derived hybridomas. Sequences obtained are compared in the GenBank database. The VH and Vkappa genes present some important sequence homology with autoantibodies suggesting a direct relationship between catalytic anti-idiotypic antibody and autoimmunity.

B‐cell repertoire formation: role of the recombination signal sequence in non‐random V segment utilization

V, D, and J gene segments rearrange at different frequencies in vivo. Each rearranging gene segment is flanked by a recombination signal sequence (RSS), which is composed of a conserved heptamer and nonamer, separated by a spacer of conserved length but not conserved sequence. We summarize data from our lab and other labs showing that in many cases, but not all, the RSS can account for differences in recombination frequencies observed in vivo. Our approach is to determine the initial frequency of rearrangement of the V genes in vivo, and then place the RSSs of two V genes into a competition recombination substrate to determine the relative frequency with which the two RSSs support recombination. In one example, we have shown that a polymorphism in the heptamer of a Vkappa gene can result in a significant reduction in recombination frequency. This particular allele is prevalent in Navajos and absent in other populations. We suggest that this single change may play a major role in the high susceptibility of Navajos to Haemophilus influenzae infection, since this Vkappa gene is important in the antibody response to this bacteria. We also describe experiments showing that the sequence of the spacer of the RSS can play an important role in relative recombination frequencies.

Factors that influence formation of B cell repertoire.

V, D, and J gene segments rearrange at different frequencies in vivo. In my laboratory, we are interested in determining the reasons for this unequal rearrangement of V genes during B cell development, and also in gaining insights into the mechanisms that control recombination. Every V, D, and J gene segment is flanked on its recombining side(s) by a recombination signal sequence (RSS), which is composed of a conserved heptamer and nonamer, separated by a spacer of conserved length. In this article, we summarize data showing that in many cases the RSS can account for differences in recombination frequencies observed in vivo. The approach that we use is to determine the frequency of initial rearrangement of the V genes in vivo. The RSSs of two V genes are then placed into a competition recombination substrate to determine the relative frequency with which the two RSSs recombine. In one example, we have shown that a single base pair polymorphism in the RSS of a Vkappa gene may play a major role in susceptibility to Haemophilus influenzae type b infection.

Development of a functional monoclonal single-chain variable fragment antibody against Venezuelan equine encephalitis virus.

We have generated a single-chain variable fragment (ScFv) antibody, from a previously well-characterized monoclonal antibody (MAb) to Venezuelan equine encephalitis (VEE) virus, 5B4D-6. The variable regions of the heavy (V(H)) and light (V(L)) chain antibody genes, were connected by a DNA linker and cloned in the phagemid vector pCANTAB5E. The ScFv clone in Escherichia coli strain TG-1, 5B4D-6-6, was expressed as a approximately 30 kDa ScFv protein and higher molecular weight fusion products which were functional in recognizing VEE virus by enzyme-linked immunosorbent assay (ELISA). Results were reproduced in Escherichia coli strain HB2151, where clone D66 was expressed mainly as soluble periplasmic protein. The D66 ScFv antibody bound VEE virus strongly as determined by ELISA. Nucleotide sequence analysis of 5B4D-6-6 ScFv indicated that the Vkappa gene belonged to family XVI, subgroup V, while the V(H) gene was unique in its sequence, though its amino acid sequence could be subgrouped as IA. The deduced protein sequence of D66 was highly homologous to published murine ScFv protein sequences. This work demonstrates, for the first time, cloning of a functional ScFv antibody against VEE virus.

Enhanced mutational activity of Vkappa gene rearrangements in systemic lupus erythematosus.

To determine the differential impact of somatic hypermutation and selective influences on the light chain repertoire in systemic lupus erythematosus (SLE), the frequency and pattern of somatic hypermutations were compared between the productive and nonproductive Vkappa gene repertoire manifested by individual CD19(+) B cells in a patient with SLE. The mutational frequency of nonproductive rearrangements in the SLE patient was significantly (P < 0.001) increased (3.7 x 10(-2)) compared to normals (4.8 x 10(-3)). Similarly, the mutational frequency of the productive Vkappa rearrangements was also significantly increased in the SLE patient (2.8 x 10(-2) vs 1.1 x 10(-2)) (P < 0.001). There were no differences in the R/S ratios of mutations in productive and nonproductive Vkappa rearrangements. Moreover, a variety of mutational "hot spots" were noted, but, unexpectedly, in the FRs. As in normals, mutations were found most frequently in RGYW/WRCY sequences accounting for 39.3% (nonproductive) and 40.1% (productive) of all mutations. Of note, nonproductive Vkappa rearrangements harbored significantly more mutations than productive rearrangements (P < 0.05) indicating that there was overall selection against mutations in the expressed repertoire. This was most apparent in the CDR3. These data are most consistent with the conclusion that, in this SLE patient, the mutational machinery was markedly enhanced compared to normals, but with no subsequent positive selection of mutations. The enhanced mutational activity may play a role in the emergence of autoreactivity in this SLE patient.

The variable genes and gene families of the mouse immunoglobulin kappa locus.

In this report 118 mouse Vkappa genes are described which, together with the 22 Vkappa genes reported previously (T. Kirschbaum et al., Eur. J. Immunol. 1998. 28: 1458-1466) amount to 140 genes that had been cloned and sequenced in our laboratory. For 73 of them cDNAs are known, i. e. they have to be considered functional genes, although 10 genes of this group have 1-bp deviations from the canonical promoter, splice site or heptanucleotide recombination signal sequences. Twenty Vkappa genes have been defined as only potentially functional since they do not contain any defect, but no cDNAs have been found (yet) for them. Of the 140 Vkappa genes 47 are pseudogenes. There are indications that two to five Vkappa genes or pseudogenes exist in the kappa locus which we have not yet been able to clone. The 140 Vkappa genes and pseudogenes were assigned to 18 gene families, 4 of them being one-member families. This differs from previous enumerations of the families only by the combination of the Vkappa9 and Vkappa10 families and by the addition of the Vkappa dv gene as a new separate family. Sequence identity usually was 80% or above within the gene families and 55-80% between genes of different families. Many of the mouse Vkappa gene families show significant homologies to the human ones, indicating that in evolution Vkappa gene diversification predated the divergence of the primate and rodent clades.

Repertoire of human antibodies against the polysaccharide capsule of Streptococcus pneumoniae serotype 6B.

We examined the repertoire of antibodies to Streptococcus pneumoniae 6B capsular polysaccharide induced with the conventional polysaccharide vaccine in adults at the molecular level two ways. In the first, we purified from the sera of seven vaccinees antipneumococcal antibodies and determined their amino acid sequences. Their VH regions are mainly the products of VH3 family genes (candidate genes, 3-23, 3-07, 3-66, and 3-74), but the product of a VH1 family gene (candidate gene, 1-03) is occasionally used. All seven individuals have small amounts of polyclonal kappa+ antibodies (Vkappa1 to Vkappa4 families), although kappa+ antibodies are occasionally dominated by antibodies formed with the product of the A27 Vkappa gene. In contrast, lambda+ anti-6B antibodies are dominated by the antibodies derived from one of 3 very similar Vlambda2 family genes (candidate genes, 2c, 2e, and 2a2) and Clambda1 gene product. The Vlambda2(+) antibodies express the 8.12 idiotype, which is expressed on anti-double-stranded-DNA antibodies. In one case, Vlambda is derived from a rarely expressed Vlambda gene, 10a. In the second approach, we studied a human hybridoma (Dob1) producing anti-6B antibody. Its VH region sequence is closely related to those of the 3-15 VH gene (88% nucleotide homology) and JH4 (92% homology). Its VL region is homologous to the 2a2 Vlambda2 gene (91%) and Jlambda1/Clambda1. Taken together, the V region of human anti-6B antibodies is commonly formed by a VH3 and a Vlambda2 family gene product.

Diversity of Ig light chain variable region gene expression in fetal lambs.

The usage of lambda and kappa light chain variable region genes in peripheral fetal lamb B cells was assessed at intervals from 61 to 146 days of gestation (term = 150 days). Transcripts of 12 distinct Vlambda genes were identified, eight of which belonged to the previously described families I or II, while four segments defined a new Vlambda family, provisionally called family VI. A total of six different Vkappa gene segments were identified and grouped into four families, while two Jkappa gene segments were expressed. Southern blot analysis indicated that the sheep Igkappa locus probably contains approximately 10 Vkappa genes and suggests that the V gene cluster has been duplicated. Throughout fetal ontogeny, Vkappa transcripts contained more diverse CDR3 sequences than did Vlambda transcripts, where the V-J junctions were nearly always invariant and canonical. There was also a shift in usage of Vkappa genes and in the pattern of Vkappa-Jkappa gene recombination during fetal ontogeny.

Immunoglobulin and T cell receptor gene rearrangement patterns in acute lymphoblastic leukemia are less mature in adults than in children: implications for selection of PCR targets for detection of minimal residual disease

In order to gain insight into immunoglobulin (Ig) and T cell receptor (TCR) gene rearrangements in adult acute lymphoblastic leukemia (ALL), we studied 48 adult patients: 26 with precursor-B-ALL and 22 with T-ALL. Southern blotting (SB) with multiple DNA probes for the IGH, IGK, TCRB, TCRG, TCRD and TAL1 loci revealed rearrangement patterns largely comparable to pediatric ALL, but several differences were found for precursor-B-ALL patients. Firstly, adult patients showed a lower level of oligoclonality in the IGH gene locus (five out of 26 patients; 19%) despite a comparable incidence of IGH gene rearrangements (24 out of 26 patients; 92%). Secondly, all detected IGK gene deletions (n = 12) concerned rearrangements of the kappa deleting element (Kde) to Vkappa gene segments, which represent two-thirds of the Kde rearrangements in pediatric precursor-B-ALL and only half of the Kde rearrangements in mature B cell leukemias. Thirdly, a striking predominance of immature Ddelta2-Ddelta3 cross-lineage recombinations was observed (seven out of 16 TCRD rearrangements; 44%), whereas more mature Vdelta2-Ddelta3 gene rearrangements occurred less frequently (six out of 16 TCRD rearrangements; 38% vs >70% in pediatric precursor-B-ALL). Together these data suggest that the Ig/TCR genotype of precursor-B-ALL is more immature and more stable in adults than in children. We also evaluated whether heteroduplex analysis of polymerase chain reaction (PCR) products of rearranged Ig and TCR genes can be used for identification of molecular targets for minimal residual disease (MRD) detection. Using five of the major gene targets (IGH, IGK, TCRG, TCRD and TAL1 deletion), we compared the SB data and heteroduplex PCR results. High concordance between the two methods ranging from 96 to 100% was found for IGK, TCRG and TAL1 genes. The concordance was lower for IGH (70%) and TCRD genes (90%), which may be explained by incomplete or 'atypical' rearrangements or by translocations detectable only by SB. Finally, the heteroduplex PCR data indicate, that MRD monitoring is possible in almost 90% of adult precursor-B-ALL and >95% of adult T-ALL patients.

The 3' part of the immunoglobulin kappa locus of the mouse.

A detailed restriction map of a 430-kb contig comprising the single Ckappa, the 5 Jkappa and the adjoining 22 Vkappa gene segments is presented. The first 12 Vkappa genes following the JkappaCkappa region belong to the Vkappa21 family, the subsequent ones to the closely related families Vkappa8 and Vkappal 9/ 28. Previous difficulties in cloning all Vkappa21 genes can now be explained by the presence of a duplicated region in this part of the locus. The structure was established by analysis of yeast artificial chromosome, bacterial artificial chromosome and cosmid clones and by the so-called long template PCR technique. The distance between Ckappa and the proximal Vkappa21 gene is 22 kb and the average distances between the Vkappa genes are about 20 kb. Of the 12 Vkappa21 genes 5 were sequenced for the first time and 8 of the 12 genes were found to be expressed. Of the 10 Vkappa8 and Vkappa19/28 germline genes 9 are new; expression products of 8 of the 10 genes were known. The known 5', 3' polarities allow to specify for the 22 Vkappa genes whether they are rearranged to the JkappaCkappa element by a deletion or an inversion mechanism. Also the formation of interesting rearrangement products in classical cell lines as MPC11, MOPC41 and PC 7043 can be explained now. The non-Vkappa sequence L10 whose rearrangement by inversion has been described earlier (Hoechtl and Zachau, Nature 1983. 302: 260-263) was now localized downstream of JkappaCkappa.

Heterogeneity in junctional regions of immunoglobulin kappa deleting element rearrangements in B cell leukemias: a new molecular target for detection of minimal residual disease.

Virtually all immunoglobulin kappa (IGK) gene deletions are mediated via rearrangements of the so-called kappa deleting element (Kde). Kde rearrangements occur either to Vkappa gene segments (Vkappa-Kde rearrangements) or to the heptamer recombination signal sequence in the Jkappa-Ckappa intron. Kde rearrangements were analyzed by the polymerase chain reaction (PCR) and heteroduplex analysis in 130 B-lineage leukemias: 63 precursor-B-acute lymphoblastic leukemias (ALL) and 67 chronic B cell leukemias. To obtain detailed information about Kde rearrangements, we sequenced 109 of the 189 detected junctional regions. Vkappa gene family usage in the Vkappa-Kde rearrangements in our series of B-lineage leukemias was comparable to Vkappa gene family usage in functional Vkappa-Jkappa rearrangements in normal and malignant mature B cells, except for a higher frequency of VkappaII family usage in precursor-B-ALL. Junctional region sequencing of the Kde rearrangements in precursor-B-ALL revealed a mean insertion of 4.7 nucleotides and a mean deletion of 9.5 nucleotides, resulting in an extensive junctional diversity, whereas in chronic B cell leukemias the insertion (1.9) and deletion (6.0) were significantly lower. The relatively extensive junctional diversity of the Kde rearrangements in precursor-B-ALL allowed us to design leukemia/patient-specific oligonucleotide probes, which were proven to be useful for detection of minimal residual disease (MRD) with sensitivities of 10(-4) to 10(-5). Kde rearrangements occur in approximately 50% of precursor-B-ALL cases and are likely to remain stable during the disease course, because Kde rearrangements are assumed to be 'end-stage' rearrangements, which cannot easily be replaced by continuing rearrangement processes. These findings indicate that junctional regions of Kde rearrangements in precursor-B-ALL represent new valuable patient-specific PCR targets for detection of MRD.

Development of pathogenic anti-DNA antibodies in patients with systemic lupus erythematosus.

The anti-DNA response is a hallmark of systemic lupus erythematosus (SLE). The precise mechanisms leading to anti-DNA antibody (Ab) production remain to be studied. Nonetheless, it is becoming clear that anti-DNA Abs cause inflammatory lesions not only via deposition of circulating immune complexes (IC) consisting of anti-DNA Ab and antigens (Ags), but also via in situ IC formation by cationic anti-DNA Abs. It is intriguing that cationic anti-DNA Abs are encoded by a unique germline Vkappa gene, A30, which encodes an extraordinary cationic light chain, whereas somatic mutations did not induce a cationic shift of electrical charge in human lupus nephritis, suggesting that the usage of a specific germline gene may confer the cationic charge (or pathogenicity) on anti-DNA Abs and that somatic mutations induce the affinity maturation of Abs. Whether cationic anti-DNA Abs will develop depends at least partly on the presence or absence of the germline A30 gene, since patients who lack this gene in the germline Vkappa repertoire did not develop severe lupus nephritis. Receptor editing, a mechanism for changing the affinity of the B cell Ag receptor [surface immunoglobulin (Ig) receptor] to avoid self-reactivity actually seems defective in patients with SLE because normal B cells edited the A30 gene, whereas SLE B cells express A30 mRNA. Thus, along with the importance of somatic mutations, polymorphisms of Ig Vkappa locus, and genetic predisposition, the failure of receptor editing may contribute to the development of pathogenic anti-DNA responses in humans.

Restricted V gene repertoire in the secondary response to insulin in young BALB/c mice.

Previous data on the genetic origin of insulin Abs in BALB/c mice are derived from immune responses following a single or primary immunization. This primary response, however, may not represent the repertoire established by chronic insulin administration, which is used in the treatment of diabetes mellitus and in protocols to alter beta cell destruction in pre-insulin-dependent (type 1) diabetes mellitus. In this study, the genetic composition and structure of 16 anti-insulin mAbs from the secondary response of a single BALB/c mouse were determined. In contrast to studies on other protein Ags, the V genes in the secondary response to insulin show only a low level of somatic mutation and the majority of the response is represented by restricted sets of V(H) and Vkappa genes. The restriction of V genes is not chiefly due to oligoclonal expansion but reflects recurrent usage of structurally similar V regions of independent origin. Secondary anti-insulin V(H) genes in 12 of 16 mAbs are derived from related J558 subsets, and 8 of 16 mAbs used the Vkappa19.34 gene without mutations in CDRLs. The CDHR3s of half of the mAbs are generated from an uncommon second reading frame of DSP2, suggesting that the amino acid motif STMIT may contact insulin and contribute to selection of these B cells. A total of 14 of 15 mAbs bind autologous rodent insulin with 40 to 100% of the activity on human insulin, and all mAbs bind proinsulin. These data show that a preferred set of V(H), D, and Vkappa gene segments dominate secondary responses to insulin and suggest that specific structural interactions with epitopes that are shared with autologous insulin shape this secondary response.

Human cord blood kappa repertoire.

To determine the Vkappa gene utilization in cord blood, we made libraries of Igkappa sequences from two cord blood cDNA samples. The rearranged sequences were amplified using random amplification of cDNA ends PCR, ensuring unbiased amplification of all Vkappa genes. Although the human kappa locus contains approximately 38 potentially functional V genes, we observed that approximately 75% of the 146 sequences from our two samples used only nine Vkappa genes. Using leader-specific primers, we also amplified VkappaI and VkappaIII rearrangements from genomic DNA from one of these individuals. Nonproductive rearrangements give an approximation of the relative frequency of gene rearrangement. Some of the genes that were overused in the cDNA libraries were also observed to rearrange frequently, but others did not show high rearrangement frequencies, suggesting that cellular selection caused their increase in the periphery. Surprisingly, we observed a high frequency of rearrangements using L9, which has been reported to be a defective Vkappa gene. Sequence analysis of the unrearranged gene revealed two new functional alleles of this gene. We observed that N nucleotides were present in 29% of the productive sequences from cord blood DNA and RNA. To determine the actual rate of N region addition, we analyzed V-J junctions of rearrangements of two nonfunctional V genes. Forty-six percent of those cord blood sequences contained N regions. In comparison, 57% of junctions of the rearranged nonfunctional gene from adult PBMC contained N regions. Finally, we observed that CDR3 length heterogeneity was more pronounced for VkappaIII genes than for all of the other Vkappa families.

Molecular mechanisms and selective influences that shape the kappa gene repertoire of IgM+ B cells.

To analyze the human kappa chain repertoire and the influences that shape it, a single cell PCR technique was used that amplified Vkappa Jkappa rearrangements from genomic DNA of individual human B cells. More than 350 productive and 250 nonproductive Vkappa Jkappa rearrangements were sequenced. Nearly every functional Vkappa gene segment was used in rearrangements, although six Vkappa gene segments, A27, L2, L6, L12a, A17, and O12/O2 were used preferentially. Of these, A27, L2, L6, and L12a showed evidence of positive selection based on the variable region and not CDR3, whereas A17 was overrepresented because of a rearrangement bias based on molecular mechanisms. Utilization of Jkappa segments was also nonrandom, with Jkappa1 and Jkappa2 being overrepresented and Jkappa3 and Jkappa5 underrepresented in the nonproductive repertoire, implying a molecular basis for the bias. In B cells with two Vkappa Jkappa rearrangements, marked differences were noted in the Vkappa segments used for the initial and subsequent rearrangements, whereas Jkappa segments were used comparably. Junctional diversity was generated by n-nucleotide addition in 60% and by exonuclease trimming in 75% of the Vkappa Jkappa rearrangements analyzed. Despite this large degree of diversity, a strict CDR3 length was maintained in both productive and nonproductive rearrangements. More than 23% of the productive rearrangements, but only 7% of the nonproductive rearrangements contained somatic hypermutations. Mutations were significantly more frequent in Vkappa sequences derived from CD5- as compared with CD5+ B cells. These results document that the gene segment utilization within the Vkappa repertoire is biased by both intrinsic molecular processes as well as selection after light chain expression. Moreover, IgM+ memory cells with highly mutated kappa genes reside within the CD5- but not the CD5+ B cell compartment.

Structural and affinity studies of IgM polyreactive natural autoantibodies.

Natural polyreactive autoantibodies (NAA) are an important component of the normal B cell repertoire. One intriguing characteristic of these Abs is their binding to various dissimilar Ags. It has been generally assumed that these Abs bind the Ags with low affinity, and are encoded by germline genes. We have used surface plasmon resonance to determine binding of avidities, and conducted a structural analysis of five murine monoclonal natural autoantibodies displaying a typical polyreactive binding pattern against cytoskeleton Ags and DNA. We show that 1) all the five Abs bind the different Ags with kinetic constants similar to those observed for immune Abs; 2) they express a restricted set of V(H) and V(L) genes, since the same V(H) gene is expressed by three out of the five, and one particular Vkappa gene was expressed twice. In addition, a single D gene segment was used by three of the five Abs; and 3) they express, in most cases, genes in a close germline configuration. Our amino acid sequence and modeling studies show that the distribution of exposed side chains in the NAA paratopes is close to the general pattern observed in the complementarity-determining regions (CDRs) of variable domains from immune Abs. Although CDR3 regions of the heavy chain have been postulated to play a major role in determining polyreactivity on the basis of recombinatorial experiments, our results failed to show any distinctive particularity of this region in terms of length or charge when compared with classical immune Abs.