Kappa Gene Segments Research Articles

Human immunoglobulin selection associated with class switch and possible tolerogenic origins for C delta class-switched B cells.

Current paradigms of peripheral B cell selection suggest that autoreactive B cells are controlled by clonal deletion, anergy, and developmental arrest. We report that changes to the human antibody repertoire likely resulting from these mechanisms both for a well-characterized autoreactivity from antibodies encoded by the V(H)4-34 gene and for other hallmarks of an autoreactive repertoire are apparent mainly for class-switched B cells and not for IgM germinal center, IgM memory, or IgM plasma cells. Other possible indicators of autoreactivity found selected with immunoglobulin class include J(H)6 gene segment usage, increased frequency of B cells with long third hypervariable regions, and distal J(kappa) gene segment bias. Of particular interest is the finding that B cells with these same characteristics are selected into the lineage of B cells that have undergone the unusual class switch from constant region C mu to C delta (C delta-CS). The C delta-CS population also displays an increased frequency of charged amino acids localized to the complementarity-determining regions, further suggesting autoreactivity, and evidence is presented that these B cells had undergone extensive receptor editing. Thus, the C delta-CS lineage may be a "sink" for B cells harboring autoreactive specificities in normal humans. A model for a new tolerizing mechanism that could account for the C delta-CS lineage is presented.

Structure-function relationships for human antibodies to pneumococcal capsular polysaccharide from transgenic mice with human immunoglobulin Loci.

To investigate the influence of antibody structure and specificity on antibody efficacy against Streptococcus pneumoniae, human monospecific antibodies (MAbs) to serotype 3 pneumococcal capsular polysaccharide (PPS-3) were generated from transgenic mice reconstituted with human immunoglobulin loci (XenoMouse mice) vaccinated with a PPS-3-tetanus toxoid conjugate and their molecular genetic structures, epitope specificities, and protective efficacies in normal and complement-deficient mice were determined. Nucleic acid sequence analysis of three MAbs (A7, 1A2, and 7C5) revealed that they use two different V(H)3 genes (A7 and 1A2 both use V3-15) and three different V(kappa) gene segments. The MAbs were found to have similar affinities for PPS-3 but different epitope specificities and CDR3 regions. Both A7 and 7C5 had a lysine at the V(H)-D junction, whereas 1A2 had a threonine. Challenge experiments with serotype 3 S. pneumoniae in BALB/c mice revealed that both 10- and 1- micro g doses of A7 and 7C5 were protective, while only a 10- micro g dose of 1A2 was protective. Both A7 and 7C5 were also protective in mice lacking either an intact alternative (FB(-/-)) or classical (C4(-/-)) complement pathway, but 1A2 was not protective in either strain. Our data suggest that PPS-3 consists of epitopes that can elicit both highly protective and less protective antibodies and that the superior efficacies of certain antibodies may be a function of their structures and/or specificities. Further investigation of relationships between structure, specificity, and efficacy for defined MAbs to PPS may identify antibody features that might be useful surrogates for antibody (and vaccine) efficacy.

Molecular and functional characteristics of a protective human monoclonal antibody to serotype 8 Streptococcus pneumoniae capsular polysaccharide.

The structural characteristics and biological activity of human antibodies that are reactive with the capsular polysaccharides of most serotypes of Streptococcus pneumoniae, including serotype 8, are unknown. This paper describes the generation, molecular structure, and protective efficacy of a human monoclonal antibody (MAb) reactive with the capsular polysaccharide of serotype 8 Streptococcus pneumoniae. We generated the immunoglobulin M(kappa) [IgM(kappa)] MAb D11 by Epstein-Barr virus transformation of peripheral lymphocytes from a Pneumovax recipient. Nucleic acid sequence analysis revealed that MAb D11 uses V3-15/V(H)3 and A20/V(kappa) gene segments with evidence of somatic mutation. In vitro studies revealed MAb D11-dependent complement deposition on the capsule of serotype 8 organisms via either the classical or the alternative complement pathway. In vivo, MAb D11 prolonged the survival of both normal and C4-deficient mice with lethal serotype 8 S. pneumoniae infection. Our findings demonstrate that a serotype-specific human IgM with certain structural and functional characteristics was protective in mice lacking a functional classical complement pathway and show that alternative complement pathway activation is an important determinant of pneumococcal protection.

Construction and diversity analysis of a murine IgE phage surface display library.

To make further investigation of the IgE antibody repertoire in Trichosanthin (TCS) allergic responses, a murine IgE phage surface display library was constructed (3.0 x 10(5) independent clones). We first constructed the V epsilon cDNA library (4.6 x 10(5) independent clones) and V kappa cDNA library (3.0 x 10(5) independent clones). Then, the V epsilon and V kappa gene segments were amplified from both libraries by PCR respectively, and assembled into Fab fragment by SOE PCR. The phage library containing Fabs was thus constructed. The diversity of V epsilon from this library was analyzed and proved. Fab clones with high specificity to TCS have been screened out.

Analysis of the kappa light chain variable region in multiple myeloma.

The study of immunoglobulin heavy chain gene rearrangements in multiple myeloma has revealed extensive divergence from the germline sequences, but no intraclonal diversity with disease evolution. Our study investigated the state of the rearranged kappa light chain variable region (V kappa) gene segments as well as abortive V kappa family gene usage in cases of multiple myeloma expressing lambda light chain. We studied 11 cases of kappa and five cases of lambda light chain-expressing multiple myeloma. Total cellular RNA was extracted from the bone marrow of patients with overt disease and subjected to reverse transcription-polymerase chain reaction (RT-PCR) analysis to amplify clonally rearranged variable region sequences. Direct nucleotide sequencing by the dideoxy-chain termination method was performed on the RT-PCR products. We did not observe preferential usage of certain V kappa gene families. Mutation frequencies of the V kappa segments varied in number. In the majority of cases, extensive somatic mutations occurred within the complementarity determining regions (CDRs) of V kappa, whereas only a limited degree of divergence from the germline was observed in others. In all cases studied. replacement mutations tended to cluster in the CDRs, a finding compatible with an antigen-driven somatic hypermutation process. In 3/5 cases of lambda light-chain expressing multiple myeloma, abortively rearranged V kappa gene segments were amplified from genomic DNA; in two cases a non-templated nucleotide insertion rendering the V kappa sequences out-of-frame was observed, and in the third a stop codon was identified in the open reading frame of the V kappa sequence. Somatic mutations were observed in all cases of abortive V kappa genes studied; however, their distribution does not suggest selection by antigen. We conclude that somatic mutations observed in the V kappa regions of myeloma cells are of variable extent and suggest operation of the antigen selection process. Lack of or minimal somatic hypermutation in a few cases may be in some way implicated in the biological heterogeneity of the disease.

Yeast artificial chromosome contigs reveal that distal variable-region genes reside at least 3 megabases from the joining regions in the murine immunoglobulin kappa locus.

The immunoglobulin kappa gene locus encodes 95% of the light chains of murine antibody molecules and is thought to contain up to 300 variable (V kappa)-region genes generally considered to comprise 20 families. To delineate the locus we have isolated 29 yeast artificial chromosome genomic clones that form two contigs, span > 3.5 megabases, and contain two known non-immunoglobulin kappa markers. Using PCR primers specific for 19 V kappa gene families and Southern analysis, we have refined the genetically defined order of these V kappa gene families. Of these, V kappa 2 maps at least 3.0 Mb from the joining (J kappa) region and appears to be the most distal V kappa gene segment.

Antibody response against poly (Glu60Ala30Tyr10) terpolymer and bacterial levan in kappa-deficient mice.

In murine species, the kappa (+)-bearing immunoglobulins dominate the antibody (Ab) repertoire with a kappa/lambda ratio of 95:5. The aim of the present study is to investigate the characteristics of the antibody response in kappa-deficient (K-/-) mice immunized with a T-dependent synthetic antigen, poly(Glu60Ala30Tyr10) (GAT) and a T-independent antigen, bacterial levan (BL). K-/- mice were obtained by targeted deletion of the J kappa C kappa gene segments. In response to GAT, K-/- mice respond by producing increasing amounts of anti-GAT Ig lambda 1 and Ig lambda 2 in the primary as well as secondary response, although anti-GAT specific monoclonal antibodies (mAb) raised in K-/- mice are mostly of IgM isotype. The GAT public idiotype, GATIdX, present on all GAT-specific Ab bearing kappa light chain, is not detected in the sera of K-/- mice or on any of the anti-GAT lambda 1 mAb. In response to BL, the amount of Ig lambda 1+ Ab in K-/- mice is comparable to the amount of Ig kappa + Ab in normal mice. However, lambda 2+ Ab are detected neither in wild-type nor in K-/- mice. Like kappa + Ab, the majority of lambda 1+ mAb are specific for beta 2-6 fructosan present in BL and rye levan and, to some extent, express the BL-specific idiotype, A48ld. Our results show that important compensatory mechanisms occur in kappa-deficient mice, restoring their ability to mount immune responses against a variety of T-dependent and T-independent antigens by the alternative usage of the clonally restricted lambda repertoire.

Analysis of the structural correlates for antibody polyreactivity by multiple reassortments of chimeric human immunoglobulin heavy and light chain V segments.

Polyreactive antibodies (Abs) constitute a major proportion of the early Ab repertoire and are an important component of the natural defense mechanisms against infections. They are primarily immunoglobulin M (IgM) and bind a variety of structurally dissimilar self and exogenous antigens (Ags) with moderate affinity. We analyzed the contribution of Ig polyvalency and of heavy (H) and light (L) chain variable (V) regions to polyreactivity in recombinatorial experiments involving the VH-diversity(D)-JH and V kappa-J kappa gene segments of a human polyreactive IgM, monoclonal antibody 55 (mAb55), and those of a human monoreactive anti-insulin IgG, mAb13, in an in vitro C gamma l and C kappa human expression system. These mAbs are virtually identical in their VH and V kappa gene segment sequences. First, we expressed the VH-D-JH and V kappa-J kappa genes of the IgM mAb55 as V segments of an IgG molecule. The bivalent recombinant IgG Ab bound multiple Ags with an efficiency only slightly lower than that of the original decavalent IgM mAb55, suggesting that class switch to IgG does not affect the Ig polyreactivity. Second, we coexpressed the mAb55-derived H or kappa chain with the mAb13-derived kappa or H chain, respectively. The hybrid IgG Ab bearing the mAb55-derived H chain V segment paired with the mAb13-derived kappa V segment, but not that bearing the mAb13-derived H chain V segment paired with the mAb55-derived kappa V segment, bound multiple Ags, suggesting that the Ig H chain plays a major role in the Ig polyreactivity. Third, we shuffled the framework 1 (FR1)-FR3 and complementarity determining region 3 (CDR3) regions of the H and kappa chain V segments of the mAB55-derived IgG molecule with the corresponding regions of the monoreactive IgG mAb13. The mAb55-derived IgG molecule lost polyreactivity when the H chain CDR3, but not the FR1-FR3 region, was replaced by the corresponding region of mAb13, suggesting that within the H chain, the CDR3 provides the major structural correlate for multiple Ag-binding. This was formally proved by the multiple Ag-binding of the originally monoreactive mAb13-derived IgG molecule grafted with the mAb55-derived H chain CDR3. The polyreactivity of this chimeric IgG was maximized by grafting of the mAb55-derived kappa chain FR1-FR3, but not that of the kappa chain CDR3.(ABSTRACT TRUNCATED AT 400 WORDS)

Light chain isotype regulation in the horse. Characterization of Ig kappa genes.

Horse Ig kappa genes have been characterized to determine whether there may be a structural basis for the low level of kappa expression in this species. The overall organization of the J kappa-C kappa locus is remarkably similar to that of the mouse and human loci. A single C kappa exon is separated by 2.9 kb from five J kappa segments, four of which seem functional and three of which are associated with canonical recombination signal sequences. A highly conserved intron enhancer was identified upstream of the C kappa exon and a single restriction fragment in horse genomic DNA hybridized strongly with the mouse downstream kappa enhancer. Germ-line and splenic cDNA V kappa sequences were characterized and found to encode N-terminal amino acid sequences previously found by protein sequencing. Hybridization of horse genomic DNA with the horse V kappa-1 germ-line gene and a variety of mouse V kappa-region probes provided evidence for at least 20 V kappa gene segments. The results indicate that the horse possesses a functional kappa locus with a complement of variable region gene segments potentially as large as that previously found at the lambda locus although the exact numbers of functional variable region genes remains to be established for both loci. This finding suggests that the predominance of lambda-chains in horse Ig may not be simply a result of a lack of functional kappa genes or of a disproportionate number of lambda vs kappa variable region genes. The results are discussed in terms of various models of isotype regulation.

Limited junctional diversity in kappa light chains. Junctional sequences from CD43+B220+ early B cell progenitors resemble those from peripheral B cells.

Among all adult T and B cell Ag receptor chains, only Ig light chains lack N regions. It is thought that this is due to the fact that light chain genes rearrange after heavy chain genes, and that terminal deoxynucleotidyl transferase, the enzyme that adds N regions, is not longer expressed at that stage. However, this concept has been challenged recently by the demonstration that 3 to 10% of B cell precursors (CD43+B220+) appear to rearrange their light chains at approximately the same time as they undergo VH-->DJ rearrangements. To examine N region addition in B cell precursors undergoing early kappa-chain rearrangement, we PCR amplified rearranged V kappa 21 genes from the CD43+B220+ bone marrow cells and compared them to sequences obtained from whole bone marrow and spleen. Unexpectedly, all three populations showed approximately 10% N region containing junctions, most consisting of only one N nucleotide. Thus, even the B cell precursors that rearrange light chains at this early stage of development lack much N region diversity. Twelve percent of the sequences unambiguously contained P regions, which were from 1 to 5 nucleotides in length. All but 2 of the 41 productive rearrangements had the commonly observed CDR3 length of nine amino acids. Many (71%) of the sequences were out of frame. CDR3 length was very restricted in nonproductive rearrangements too, and deletion of nucleotides from V kappa and J kappa gene segments was limited. Thus, even at the level of nonproductive rearrangements, junctional diversity is minimal for kappa-chains.

Antibody response elicited by T-dependent and T-independent antigens in gene targeted kappa-deficient mice.

Animal models substantially contribute to the understanding of the pathogenesis of various human diseases, including those associated with genetic defects. Our study investigated the characteristics of antibody responses elicited by T-dependent and T-independent antigens in mice rendered kappa-deficient by targeted deletion of the J kappa C kappa gene segments. It is known that in normal murine species the kappa repertoire dominates the antibody repertoire (kappa/lambda ratio = 95:5). Our results indicate that the kappa gene deletion causes the alternative usage of lambda 1 (93%) and lambda 2 (7%) light chains, confirming previous studies demonstrating that in kappa-deficient mice all B cells express Ig lambda receptors. The anti-trinitrophenylbenzene (TNP) response in K-/- mice was compensated for by lambda 1 and lambda 2 bearing Igs. However, isoelectric focusing analysis of anti-TNP antibodies showed a considerably more restricted pattern of lambda anti-TNP antibodies in K-/- as compared with kappa antibodies in normal mice. No major differences were observed in the affinity for the hapten of kappa or lambda 1 or lambda 2 mAbs obtained from 129/Sv and K-/- mice. Furthermore, lambda 1 and lambda 2 chains can reconstitute the expression of an idiotype (460Id) borne on kappa anti-TNP antibodies. The 460Id was detected both in polyclonal and monoclonal anti-TNP antibodies obtained from K-/- mice. Our results clearly showed that the kappa anti-TNP repertoire is compensated by the lambda repertoire even though the latter is clonally restricted in K-/- mice.

Compartmentalization of lambda subtype expression in the B cell repertoire of mice with a disrupted or normal C kappa gene segment.

The establishment of the B cell repertoire depends on two major parameters. The first is determined by mechanistic processes that give rise to a great diversity of B cell receptors from a combination of multiple gene segments. The second is dominated by selective processes that recruit B cell clones via their immunoglobulin receptors. To assess the impact of these parameters on the composition of B cell repertoire, we constructed a mouse model displaying a B cell repertoire limited in its diversity. To this end, we disrupted the C kappa segment by gene targeting. B cells from such mutant mice do not express the kappa light chain. Their light chain repertoire is therefore limited by the expression of only four main lambda light chains: lambda 1, lambda 2(V2), lambda 2(Vx) and lambda 3. In this study we described the proportions of each lambda subtype in various lymphoid compartments. Our results show that the lambda 1 subtype is dominant in the spleen and the bone marrow. Moreover, lambda 1 prevalence is independent of the wild or mutant C kappa genotype. These results suggest that the mechanistic processes are mainly responsible for the bias in lambda subtype expression. On the other hand, the lambda 2(V2) and/or lambda 3 subtypes are expressed at higher levels in the peritoneal cavity. Their prevalence is again observed regardless of the C kappa genotype and seems to be due to B1 cells. These results suggest that different mechanistic processes could control lambda subtype expression in B1 and B2 cell lineages.

VH4.21-encoded natural autoantibodies with anti-i specificity mirror those associated with cold hemagglutinin disease.

The Ig VH regions of virtually all human pathogenic cold agglutinin (CA) anti-i/l autoantibodies are encoded by a single Ig VH gene segment, VH4.21, in conjunction with a highly variable CDR3 structure. The anti-I specificity is often associated with V kappa III-encoded L chains, whereas anti-i autoantibodies appear to use a broader array of kappa and lambda VL gene segments. B cells expressing VH4.21 are abundantly present in adult lymphoid tissues from healthy individuals but their relationship with B cells secreting pathogenic CA is unknown. Herein we have analyzed the distribution of VH4.21-expressing B cells in fetal, neonatal, and adult B cell populations using the monoclonal anti-VH4.21 antibody 9G4. In addition, we have analyzed the anti-i and anti-I binding capacity and V regions of 19 VH4.21-encoded mAb secreted by cord and adult blood-derived cell lines from healthy individuals. The results show that VH4.21 expressing B cells are overrepresented in all repertoires studied and are evenly distributed over cord blood CD5+ and CD5- B cell populations. VH4.21-encoded H chains strongly predispose for anti-i binding capacity, regardless of the VL regions or H chain CDR3 structure. The avidity of some of these antibodies was similar to those of pathogenic CA. In addition, we found evidence for monospecific anti-I binding in antibodies encoded by other members of the VH4 gene family. We conclude that VH4.21-encoded antibodies with anti-i specificity from the normal B cell repertoire mirror their pathogenic counterparts and that naturally occurring anti-I antibodies may be encoded by a more diverse array of VH4 genes.

B lymphocytes may escape tolerance by revising their antigen receptors.

To explore mechanisms that prevent autoreactivity in nonautoimmune mice, endogenous immunoglobulin (Ig) light (L) chains that associate with a transgenic anti-DNA heavy chain were analyzed. The antibodies from splenic B cell hybridomas of such mice did not bind double-stranded DNA (dsDNA) and their L chain sequences showed a biased use of V kappa and J kappa gene segments. The 44 L chains in this survey were coded for by just 18 germline genes. Six of the genes, each belonging to a different V kappa group, were used more than once and accounted for three fourths of all sequences. Based on the distribution of V kappa genes, the L chain repertoire in this line of transgenic mice was estimated at 37 V kappa genes. The most frequently observed gene, a member of the V kappa 12/13 group, was identified in 16 hybrids. In addition, the majority of V kappa genes used J kappa 5. We interpret the skewed representation of V kappa and J kappa gene segments to result from negative selection. Based on the data, we suggest that V kappa rearrangements giving rise to anti-dsDNA reactivity are removed from the repertoire by a corrective mechanism capable of editing self-reactive Ig.

B cell development in mice that lack one or both immunoglobulin kappa light chain genes.

We have generated mice that lack the ability to produce immunoglobulin (Ig) kappa light chains by targeted deletion of J kappa and C kappa gene segments and the intervening sequences in mouse embryonic stem cells. In wild type mice, approximately 95% of B cells express kappa light chains and only approximately 5% express lambda light chains. Mice heterozygous for the J kappa C kappa deletion have approximately 2-fold more lambda+ B cells than wild-type littermates. Compared with normal mice, homozygous mutants for the J kappa C kappa deletion have about half the number of B cells in both the newly generated and the peripheral B cell compartments, and all of these B cells express lambda light chains in their Ig. Therefore, homozygous mutant mice appear to produce lambda-expressing cells at nearly 10 times the rate observed in normal mice. These findings demonstrate that kappa gene assembly and/or expression is not a prerequisite for lambda gene assembly and expression. Furthermore, there is no detectable rearrangement of 3' kappa RS sequences in lambda+ B cells of the homozygous mutant mice, thus rearrangements of these sequences, per se, is not required for lambda light chain gene assembly. We discuss these findings in the context of their implications for the control of Ig light chain gene rearrangement and potential applications of the mutant animals.

Vχ gene segments rearranged in chronic lymphocytic leukemia are distributed over a large portion of the Vχ locus and do not show somatic mutation

The structure of the human V kappa locus has been thoroughly investigated, but how the germ-line V kappa gene segment repertoire is actually sampled in kappa chain gene rearrangements is not known. In order to begin to answer this question we have polymerase chain reaction (PCR) amplified the rearranged V kappa genes from 26 kappa-expressing cases of chronic lymphocytic leukemia (CLL), followed by cloning and sequencing of the PCR product. All four V kappa gene families were represented amongst rearranged genes. In 25 out of 32 cases, the sequence of the rearranged gene matches perfectly that of 1 of 11 different known germ-line V kappa genes, indicating that no somatic mutation has occurred. Of the remaining 7 rearranged V kappa genes, 4 differ from known germ-line genes by only one or two amino acid residues; and 3 differ from each other and from all known sequences by 5 or more residues, suggesting that somatic mutation has occurred in these 3 cases. We conclude that: (a) in at least three-quarters of cases the rearranged genes are unmutated; (b) there is preferential usage of individual V kappa genes but not of V kappa gene families; and (c) the V kappa genes used are widely dispersed in the V kappa locus.

A rheumatoid factor from a normal individual encoded by VH2 and V kappa II gene segments.

To gain insight into the immunoglobulin variable-region repertoire of anti-IgG antibodies (rheumatoid factors [RF]), we characterized the VH and V kappa gene segments utilized in an IgM-RF-secreting lymphoblastoid cell line (SSH23) isolated from a normal individual. The cell line SSH23 was established by Epstein-Barr virus transformation of peripheral blood non-T mononuclear cells. First-strand complementary DNA (cDNA) was generated and used for polymerase chain reaction amplification of the heavy and light chain variable domains. The amplified variable domains were sequenced and compared with an extensive database of germline and cDNA V gene segments. The VH sequence was found to be identical to a previously described fetal VH2 incomplete cDNA and to differ by only 3 nucleotides from a JH proximal germline VH2 gene segment. To our knowledge, this is the first example of a VH2 rheumatoid factor. The V kappa 2-J kappa 4 light chain contains an uncommon 10-amino acid third complementarity-determining region (CDR 3). Utilization of preimmune fetal VH gene segments and unusual light chain junctional diversity appear to be features shared by many physiologic and pathologic rheumatoid factors.

Ongoing V kappa-J kappa recombination after formation of a productive V kappa-J kappa coding joint.

V kappa genes of man can recombine with the J kappa gene segments either by an inversion or by a deletion mechanism. Back-to-back fusion products of the respective recombination signal sequences (signal joints) are retained on the chromosome after the formation of a V kappa-J kappa coding joint by an inversion. Our knowledge of the structure of the human kappa locus and the application of the polymerase chain reaction allowed us now to establish a direct relationship between different kappa recombination products in the lymphoid cell line JI. Two consecutive inversions fully explain the existence of two coding joints and two signal joints on the same chromosome of this cell line. Although the initially formed coding joint is productively rearranged and expressed, a second V kappa-J kappa rearrangement took place which leads to an aberrant joint. In this process a J kappa gene segment of the signal joint that had been created in the first V kappa-J kappa joining was used as the recombination target. The sequence of the two rearrangements is unequivocal since a product of the first (productive) reaction is a partner in the second (aberrant) one.

Correlation of antibody multireactivity with variable region primary structure among murine anti-erythrocyte autoantibodies.

A high proportion of the antibodies in the preimmune repertoire bind to several unrelated antigens and are considered to be multireactive. This property is reportedly associated with the antibodies produced by CD5+ B lymphocytes. Because many antibodies specific for bromelain-treated mouse red blood cells (BrMRBC) derive from CD5+ B cells, we tested monoclonal antibodies of this specificity for multireactivity. Two variable region combinations, VH11/V kappa 9 and VH12/V kappa 4, account for greater than 80% of this repertoire, but none of these antibodies exhibited a multireactive phenotype. In contrast, three anti-BrMRBC binding antibodies belonging to the J558 family (BrM1, BrM8, and CH12) showed varying degrees of multireactivity, and bound both highly negatively and positively charged antigens. The amino acid sequences of the VH regions of these antibodies are highly homologous (greater than 85% identical) and they possess large VH-D-J junctions with extensive N-region insertions. The kappa chains of two of these antibodies utilize an identical V kappa gene segment, while the third uses a very different V kappa with only 50% homology. The entire H chain V regions of these antibodies are unusually basic, with isoelectric points of 9.5-10, a feature which might be important in promoting interactions with acidic epitopes. The multireactive antibodies also contain regions with a high concentration of hydroxylside chain amino acids, especially in their VH-D-J junctions. This region also contains acidic amino acid residues, which may be important in binding of positively charged epitopes. We propose that an open, accessible binding site and a charge polarity may be features which facilitate the binding of charged epitopes, providing a structural basis for multireactivity of at least some antibodies.

Utilization of V kappa families and V kappa exons. Implications for the available B cell repertoire.

A molecular cloning approach was used to determine the relative utilization of 2 individual V kappa 21 genes, 13 V kappa gene families, and the 4 functional J kappa gene segments among splenic B cells of nonimmunized BALB/c mice. Based on the observed frequency of individual V kappa gene expression, we estimate that the mouse genome encodes 150 to 180 functional V kappa genes, and we suggest that most functional V kappa exons are expressed at comparable frequencies in the preimmune antibody repertoire. In contrast, clear differences in J kappa segment utilization were observed, J kappa 4 being consistently underrepresented with respect to the other J kappa segments.