Kappa Families Research Articles

Characterization of immunoglobulin light chain utilization and variable family diversity in rainbow trout

This study characterizes immunoglobulin light chain (IgL) expression and variable family usage in rainbow trout. IgL transcripts were generated by 5’ RACE from both immune and TNP-KLH immunized fish. Phylogenetic analysis revealed that the IgL variable regions clustered into seven different families: three kappa families (two newly described in this study), three sigma families, and a single lambda family. IgL1 and IgL3 transcripts expressing identical variable regions were identified and genomic analysis revealed that the two isotypes are co-localized on chromosomes 7, 15, 18, and 21 allowing for potential rearrangement between clusters. Fish were immunized with TNP-KLH (n = 5) and percent expression of IgL1, IgL2, IgL3, and IgL4 measured by qRT-PCR from immune tissues and magnetically sorted TNP-specific lymphocyte populations. In all samples IgL1 constituted 80–95% of the transcripts. The percentage of anti-TNP specific IgL1 transcripts was measured in naïve, unsorted, and TNP-specific cell populations of TNP-KLH fish (n = 3) and found to be significantly higher in the TNP positive cell population (21%) compared to the naïve population (1%; p = 0.02) suggesting that there is a selection of TNP specific IgL sequences.

Organization of the human immunoglobulin lambda light-chain locus on chromosome 22q11.2.

The maps of the human immunoglobulin heavy-chain and kappa light-chain loci have recently been completed. We have now completed a map of the human lambda locus (IGL) located on chromosome 22q11.2. We mapped 52 V lambda genes from 10 V lambda families and 7 J lambda and C lambda genes on a 1140 kb contig constructed from eight YACs and 129 cosmid clones. The V lambda genes are arranged within 800 kb. Genes of the different V lambda families are organized in three clusters, V lambda II and III families (cluster A); V lambda I, V, VII and IX families (cluster B); V lambda IV, VI, VIII and X families (cluster C), in contrast to the dispersed organization of the different VH and V kappa families within the human VH and V kappa loci. We note that the most frequently used V lambda families (V lambda II and III) are proximal to the J lambda and C lambda genes. The VpreB gene, encoding part of the surrogate light chain, the GGT2 gene and the BCRL4 pseudogene were also mapped within the lambda locus.

Restricted kappa chain expression in early ontogeny: biased utilization of V kappa exons and preferential V kappa-J kappa recombinations.

To determine the extent of kappa chain diversity in the preimmune repertoire early in development, kappa cDNA libraries were analyzed from 15-d old fetal omentum, 18-d-old fetal liver, and 3-wk old bone marrow. An anchored polymerase chain reaction approach was used to avoid bias for particular V kappa families. From the sequence analysis of 27 bone marrow clones, 10 different families and 20 unique V kappa genes were identified. In contrast, the V kappa expression in the fetus is highly restricted and clearly differs from the broader distribution see in 3-wk-old bone marrow. Although several V kappa families were represented in the fetal library including V kappa 9, V kappa 10, V kappa 4,5, V kappa 8, and V kappa 1, one or two members of individual families were observed repeatedly. The fetal liver and omentum libraries were found to be largely overlapping. Given the V kappa families/exons identified in the fetal sequences, the mechanism of kappa rearrangements in the early repertoire appears to occur predominantly by inversion. Importantly, the fetal repertoire was further restricted by dominant V kappa-J kappa combinations such as V kappa 4,5-J kappa 5, V kappa 9-J kappa 4, and V kappa 10-J kappa 1. Since in some cases independent rearrangements could be established, the results indicate a bias for particular V kappa-J kappa joins. The results also suggest that clonal expansion/selection in the fetal repertoire takes place after light chain rearrangement as opposed to at the pre-B cell level in the bone marrow. The restriction observed in kappa light chain expression together with known restrictions in gene usage and junctional diversity at the heavy chain level indicate a remarkably conserved fetal repertoire.

A Minor Group of Rheumatoid Factors Isolated from a Patient with Rheumatoid Arthritis is Derived from Somatically Mutated VK1 Genes Further Evidence That Rheumatoid Factors During Autoimmune Diseases Undergo an Antigen Driven Maturation

To better understand the structural basis for rheumatoid factor [RF] activity and the origin of autoantibodies in human autoimmune diseases, we isolated the RF producing B cells from the peripheral blood and from the synovial fluid of a patient suffering from rheumatoid arthritis [RA]. We previously demonstrated that a significant fraction of these RF were derived from three V kappa III genes known to encode most of the monoclonal RF light chain variable regions. To get more insight into the actual repertoire of RF-V kappa genes during RA, we analyzed the nucleotide sequences of RF light chain variable regions of other V kappa families. Using two sets of polymerase chain reactions in order to amplify the cDNA derived from RF producing cells from the same patient KRA, we isolated only three different rearranged V kappa-J kappa complexes: slkv5, slkv7 and bkv42, all derived from V kappa I germ-line genes not previously known to be associated with RF activity; this suggests that the repertoire of VL genes coding for RF during RA is more diverse than the one involved in the generation of paraprotein RF during monoclonal lymphoid proliferations, although there remains a possible bias in favor of the V kappa III family. Moreover, each of these genes is somatically mutated with a pattern suggesting a selective pressure of the antigen. Particularly interesting is the additional proline residue at the V kappa-J kappa junction of bkv42, an unorthodox feature that we found previously in more than 50% of RF V kappa III-J kappa gene complexes. Finally, the homogeneity of some non conservative mutations suggests the existence of a restricted set of pathogenic epitopes driving the production of RF during RA.

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.

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.

Molecular heterogeneity of auto‐anti‐idiotypic antibodies in MLR‐lpr/lpr mice

The VH and V kappa gene families expressed by 20 monoclonal auto-anti-idiotypes (Ab2) derived from unmanipulated MLR-lpr/lpr mice were determined by Northern blotting. Complete variable region sequences of six Ab2, along with three additional V kappa-JH Ab2 sequences, were obtained. These auto-anti-idiotypes arose spontaneously in the animals, and they bound specifically to an idiotypic determinant (Id/r) on mAb 28/12, a monoclonal IgG2b MLR-lpr/lpr anti-small nuclear ribonucleoprotein antibody. The 16 Ab2 heavy chains belonged to 7 different VH gene families, and the 10 Ab2 light chains were derived from 8 V kappa families. The light chains of two Ab2 were approximately 99% identical; the remaining variable region sequences were highly heterogeneous. There was no correlation between primary amino acid sequence of either heavy or light chain and idiotypic properties of the auto-anti-idiotypes. Six Ab2 used VH or V kappa genes that are identical to known germ-line genes. A high proportion of the spontaneous auto-anti-idiotypes was shown to have autoantibody activity (anti-DNA, anti-ribonucleoprotein), or specific binding reactions with lipopolysaccharide of Salmonella RE, or both properties. The structural diversity of spontaneous MLR-lpr/lpr auto-anti-idiotypes differs sharply from the structural homogeneity reported for Ab2 induced in normal animals against syngeneic Ab1. Our results suggest that auto-anti-idiotypes might arise independently of an immunogenic stimulus from an Ab1.

Induced rearrangement of kappa genes in the BLIN-1 human pre-B cell line correlates with germline J-C kappa and V kappa transcription.

The human pre-B acute lymphoblastic leukemia cell line, BLIN-1, has been previously shown to undergo kappa light chain rearrangement in vitro, making it a valuable resource for analyzing pre-B to B cell differentiation. We have examined the recombination potential of BLIN-1 by characterizing several independently derived kappa-expressing subclones for DNA rearrangement and V kappa gene usage. Analysis of five kappa-expressing subclones (all having the same heavy chain rearrangement) demonstrated independent kappa light chain rearrangement events by DNA hybridization analysis. Northern blot analysis using probes recognizing the four different V kappa families revealed that two subclones used the most proximal V kappa (V kappa IV), one subclone used a V kappa I, and one subclone used a V kappa II. By polymerase chain reaction analyses, we detected transcripts from rearranged V-J-C kappa genes as well as transcripts from germline J-C kappa and V kappa in BLIN-1 cells induced to rearrange the kappa locus. kappa germline transcripts were also detected in normal developing B cell populations in fetal liver and bone marrow. Our collective results indicate that: (a) BLIN-1 can be induced to rearrange the kappa locus, and this correlates with the expression of germline kappa locus transcripts that may play a role in activating or targeting gene rearrangement; and (b) active rearrangement and usage of V genes representing different kappa families suggest that, like in the mouse, repertoire diversification in humans occurs in the presence of a fixed heavy chain rearrangement.

Contribution of the VH11 gene family to mitogen‐responsive B cell repertoire in C57BL/6 mice

The contribution of VH11 gene family to the development of the primary B cell repertoire has been studied by analyzing 1.8 x 10(4) mitogen induced B lymphocyte colonies. The data demonstrate that VH11 family is predominantly expressed among neonatal splenic as well as adult peritoneal B cell colonies, both rich in Ly-1+ B cells. VH11 gene family expression among B splenocytes decreases during ontogeny and VH11 family pairs stochastically with different V kappa families among mitogen-activated neonatal B cell colonies, which are representative of an antigen unselected B cell repertoire. Thus, an increased VH11 expression among peritoneal and neonatal B cells points towards its biased expression among Ly-1+ B lymphocytes. The restricted V gene rearrangements and VH11-V kappa 9 pairing observed among anti-bromelain-treated mouse red blood cells autoantibodies are likely to be an outcome of both intrinsic gene recombination processes per se as well as selection by an autoantigen and/or local selective environmental factors.

The Mouse Immunoglobulin Kappa Light-Chain Genes Are Located in Early- and Late-Replicating Regions of Chromosome 6

The murine immunoglobulin kappa (kappa) light-chain multigene family includes the constant region (C kappa), joining-region genes, and approximately 30 kappa-variable (V kappa) region families. The entire region occupies an estimated 1,000 to 3,000 kilobases, and some V kappa families have been linked by recombinant inbred mapping. The C kappa gene and 14 V kappa families replicated differently among cell lines of lymphoid and nonlymphoid origin. In nonlymphoid cells, the C kappa gene replicated earlier than the V kappa families. A transition from replication during the second third of S phase for the C kappa gene to later replication during S for V kappa families was observed. The V kappa family (V kappa 21) that maps closest to the C kappa gene, replicated during the first half of the S phase; most of the other V kappa families replicated during the second half of S, and some replicated during the last quarter of the S phase. In lymphoid cells, the kappa locus replicated earlier in the pre-B than in the B-cell lines. In one pre-B-cell line, 22D6, the kappa genes examined replicated at the beginning of the S phase. In the B-cell lines, the EcoRI segment containing the transcribed gene replicated near the beginning of the S phase. Other V kappa families replicated within the first two-thirds of S phase. Some linked V kappa families replicated at similar times. In the B-cell lines, a transition from replication at the beginning of S for the transcribed C kappa and V kappa genes and surrounding DNA sequences to later replication for the other V kappa families was observed. However, in contrast to the non-lymphoid cell lines, the replication of this locus occurred predominantly during the first half of S. The kappa locus contains both early- and late-replicating genes, and early replication is usually associated with transcriptional activity. The results are discussed with respect to the organization of transcriptionally active chromatin domains.

The mouse immunoglobulin kappa light-chain genes are located in early- and late-replicating regions of chromosome 6.

The murine immunoglobulin kappa (kappa) light-chain multigene family includes the constant region (C kappa), joining-region genes, and approximately 30 kappa-variable (V kappa) region families. The entire region occupies an estimated 1,000 to 3,000 kilobases, and some V kappa families have been linked by recombinant inbred mapping. The C kappa gene and 14 V kappa families replicated differently among cell lines of lymphoid and nonlymphoid origin. In nonlymphoid cells, the C kappa gene replicated earlier than the V kappa families. A transition from replication during the second third of S phase for the C kappa gene to later replication during S for V kappa families was observed. The V kappa family (V kappa 21) that maps closest to the C kappa gene, replicated during the first half of the S phase; most of the other V kappa families replicated during the second half of S, and some replicated during the last quarter of the S phase. In lymphoid cells, the kappa locus replicated earlier in the pre-B than in the B-cell lines. In one pre-B-cell line, 22D6, the kappa genes examined replicated at the beginning of the S phase. In the B-cell lines, the EcoRI segment containing the transcribed gene replicated near the beginning of the S phase. Other V kappa families replicated within the first two-thirds of S phase. Some linked V kappa families replicated at similar times. In the B-cell lines, a transition from replication at the beginning of S for the transcribed C kappa and V kappa genes and surrounding DNA sequences to later replication for the other V kappa families was observed. However, in contrast to the non-lymphoid cell lines, the replication of this locus occurred predominantly during the first half of S. The kappa locus contains both early- and late-replicating genes, and early replication is usually associated with transcriptional activity. The results are discussed with respect to the organization of transcriptionally active chromatin domains.