VK Genes Research Articles

Recognition of Human Cytomegalovirus by Human Primary Immunoglobulins Identifies an Innate Foundation to an Adaptive Immune Response

Most primates, including humans, are chronically infected with cospecifically evolved, potentially pathogenic CMV. Abs that bind a 10-aa linear epitope (antigenic determinant 2 site 1) within the extracellular domain of human CMV glycoprotein B neutralize viral infectivity. In this study, we show that genes generated by recombinations involving two well-conserved human germline V elements (IGHV3-30 and IGKV3-11), and IGHJ4, encode primary Ig molecules that bind glycoprotein B at this key epitope. These particular V(H), J(H), and V(kappa) genes enable humans to generate through recombination and N nucleotide addition, a useful frequency of primary Igs that efficiently target this critical site on human CMV and thus confer an innate foundation for a specific adaptive response to this pathogen.

Long-Range Interactions between Three Transcriptional Enhancers, Active Vκ Gene Promoters, and a 3′ Boundary Sequence Spanning 46 Kilobases

The mouse immunoglobulin kappa (Igkappa) gene contains an intronic enhancer and two enhancers downstream of its transcription unit. Using chromosome conformation capture technology, we demonstrate that rearranged and actively transcribed Igkappa alleles in MPC-11 plasmacytoma cells exhibit mutual interactions over 22 kb between these three enhancers and Vkappa gene promoters. In addition, the 5' region of the active transcription unit exhibits a continuum of interactions with downstream chromatin segments. We also observe interactions between Ei and E3' with 3' boundary sequences 24 kb downstream of Ed, adjacent to a neighboring housekeeping gene. Very similar interactions between the enhancers are also exhibited by normal B cells isolated from mouse splenic tissue but not by germ line transcriptionally inactive alleles of T cells or P815 mastocytoma cells, which exhibit a seemingly linear chromatin organization. These results fit a looping mechanism for enhancer function like in the beta-globin locus and suggest a dynamic modulation of the spatial organization of the active Igkappa locus. Chromatin immunoprecipitation experiments reveal that the interacting Igkappa gene cis-acting sequences are associated with AP-4, E47, and p65NF-kappaB, potential protein candidates that may be responsible for initiating and/or maintaining the formation of these higher-order complexes. However, S107 plasmacytoma cells that lack NF-kappaB still exhibit mutual interactions between the Igkappa gene enhancers.

Antibody Repertoire Development in Fetal and Neonatal Pigs. VII. Characterization of the Preimmune κ Light Chain Repertoire

Combinatorial diversity is highly restricted in the preimmune porcine H chain repertoire compared with that in humans and mice. This raised the question of whether similar restriction characterized the preimmune L chain repertoire. In this study we present evidence that >90% of all expressed Vkappa genes in the porcine preimmune repertoire belong to three subfamilies of Vkappa genes that share 87% sequence similarity with human IGKV2. This porcine Vkappa family also shares sequence similarity with some, but not all, Vkappa genes from sheep. Hybridization with sperm DNA and sequence analyses of polynucleotides from overlapping bacterial artificial chromosome clones suggest swine possess approximately 60 IGVK2 genes. The latter method also revealed that certain IGKV2 subfamilies are not expressed in the preimmune repertoire. Six members of an IGVK1 family were also expressed as part of the preimmune repertoire, and these shared 87% sequence similarity with human IGVK1. Five Jkappa segments, complete with recombination signal sequences and separated by approximately 300 nt, were identified approximately 3 kb upstream of a single Ckappa. Surprisingly, Jkappa2 accounted for >90% of all framework region 4 sequences in the preimmune repertoire. These findings show that swine use approximately 10 IGVK2 genes from three of six subfamilies and preferentially one Jkappa segment to generate their preimmune kappa repertoire. These studies, like those of porcine Ig constant regions and MHC genes, also indicate unexpected high sequence similarity with their human counterparts despite differences in phylogeny and the mechanism of repertoire diversification.

Human single chain Fv antibodies and a complementarity determining region-derived peptide binding to amyloid-beta 1–42

A library of phage-displayed human single-chain Fv (scFv) antibodies was selected against the human amyloid-beta peptide (Aβ42). Two new anti-Aβ42 phage-displayed scFvs antibodies were obtained, and the sequences of their V H and Vκ genes were analyzed. A synthetic peptide based on the sequence of Ig heavy chain (V H) complementarity-determining region (HCDR3) of the clone with the highest recognition signal was generated and determined to bind to Aβ42 in ELISA. Furthermore, we showed for the first time that an HCDR3-based peptide had neuroprotective potential against Aβ42 neurotoxicity in rat cultured hippocampal neurons. Our results suggest that not only scFvs recognizing Aβ42 but also synthetic peptides based on the V H CDR3 sequences of these antibodies may be novel potential candidates for small molecule-based Alzheimer's disease (AD) therapy.

Editing anti-DNA B cells by Vlambdax.

Receptor editing is performed by replacement of Vκ genes that contribute to autoreactivity. In addition, the Cκ locus can be deleted by Vκ rearrangement to intronic or 3′ of Cκ RS sequences (also referred to as κ deletion elements). B cells that delete the Cκ can then express λ light chains. However, the λ locus, either of man or mouse, does not allow V gene replacement. Nor does it appear to be deleted. Therefore, editing of autoreactive λ B cells may require alternative pathways. We have found that in anti-DNA heavy chain transgenic mice (tgs) VH3H9/56R, B cells that express anti-DNA receptors comprised of λ1 in association with an anti-DNA heavy chain often coexpress a κ chain that prevents DNA binding. We speculate that such isotypically included cells may have low anti-DNA receptor densities, a feature that may lead to self-tolerance. Here we describe a mechanism of preventing DNA binding by expression of a rarely used member of the Vλ family, Vλx. The λx B cells of the tgs also express CD25 and may represent B cells that have exhausted light chain editing possibilities.

Production of human antibodies from transgenic mice.

AbstractMice transgenic for human immunoglobulin genes offer the opportunity to investigators to derive fully human antibodies using well-established hybridoma technology. Several different strains of such mice are available, and can be obtained under certain conditions from various commercial entities (1–3). The brand names of these mice include XenoMouse®, HuMAb® Mouse, TC™ mouse, and KM™ mouse. The leading transgenic mice all share certain properties. These include the following: 1. the endogenous murine heavy-chain locus has been inactivated by knocking out either all of the JH genes or the Cμ gene (4); 2. the endogenous murine kappa light-chain locus has been inactivated by knocking out the Jκ and/or the Cκ genes; 3. the endogenous murine lambda light-chain locus remains intact and functional; 4. the human heavy-chain transgene contains, at minimum, the majority of the human VH genes, all the D genes, all the JH genes, Cμ Cδ, and at least one Cγ gene, together with regulatory elements; and 5. the human kappa light-chain transgene contains the majority of the human Vκ genes, all the Jκ genes, and the Cκ gene, together with regulatory elements. KeywordsTissue GrinderTransgenic Mouse StrainFusion PlateHuman Heavy ChainHuman Immunoglobulin GeneThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Analysis of V kappa genes in rheumatoid arthritis (RA) synovial B lymphocytes provides evidence for both polyclonal activation and antigen-driven selection.

To define mechanisms of sustained activation of synovial B lymphocytes in RA, we studied hybridomas established from the local synovial B cell repertoire of two RA patients for V kappa gene expression and for antigen-binding specificity. The analyses revealed that members of the main V kappa families (I, II and III) were utilized at frequencies consistent with random V kappa gene family use. Furthermore, although the hybridomas expressed genes frequently seen in response to other self- and exogenous antigens, only one V kappa I- and two of three V kappa III-expressing hybridomas exhibited reactivity with self-antigens. Nucleotide sequence analysis revealed that all hybridomas, with the exception of rheumatoid factor (RF)-producing hybridomas, expressed V kappa genes highly related to known germ-line genes (99.3-100% homology) and that diversity was generated by deletions and random nucleotide insertions at the V kappa-J kappa junction. Examination of the few nucleotide changes seen with the V kappa genes revealed a predominance of silent to replacement changes. Moreover, most of these changes can be attributable either to allotypic variations or to limited random nucleotide replacements independent of antigen selection. In contrast, one IgG-RF (B4D8) exhibited predominantly replacement nucleotide changes in the complementarity-determining regions, suggestive of antigen-driven selection. The random expression of immunoglobulin variable region genes with no, or little, evidence of mutation in the synovial B lymphocyte repertoire, including natural polyreactive antibodies, alongside mutated IgG-RF, suggest that both polyclonal activation and antigen-driven responses occur in RA synovia.

Amyloid-beta peptide-specific single chain Fv antibodies isolated from an immune phage display library

A single-chain fragment variable (scFv) antibody library displayed on phage was constructed using spleen cells from mice immunized with human amyloid-beta peptide (Aβ42). This first anti-Aβ42 scFv immune antibody library was selected against human Aβ42. A number of positive clones were obtained, and sequences of V H and Vκ genes were analyzed using ExPASy and BLAST computer tools. This analysis revealed that only two unique clones with identical V H and Vκ complementarity determining region (CDR) (except HCDR2) and identical germline genes were selected, indicating that oligoclonal immune response was occurring in Aβ42-immunized mice. Aβ42-specific scFv antibodies selected from this first immune anti-Aβ42 phage antibody library may be an important tool for the development of therapeutic molecules for Alzheimer's disease (AD).

Germline transcripts of immunoglobulin light chain variable regions are structurally diverse and differentially expressed

The murine pre-B cell line R2-bfl, which can be induced to differentiate in vitro, was used to study germline transcription of variable regions of the light chain loci. RNA from these cells was subjected to a 3′-RACE and germline transcripts from 17 individual Vκ gene segments belonging to 12 Vκ families were characterized. Germline transcripts of all three Vλ regions were similarly analyzed. The synchronous differentiation of R2-bfl cells was then used to investigate the order of appearance of germline transcripts of the V and JC clusters of both light chain loci. This was taken as indicator for accessibility of a particular locus to rearrangement. Germline transcripts of the JCκ cluster and the Vκ family most proximal to JCκ was detectable already at day 0, while transcripts of the most distal Vκ family became apparent after initiation of differentiation at day 1. Transcripts of the JCλ cluster could be found at day 2, whereas transcripts of the Vλ region were already present at day 1. Thus, the λ locus becomes accessible to rearrangement later during development than κ, confirming and extending our previous findings. The V and JC clusters open at the same stage of development although slight asynchronicities were found for the Vλ and the distal Vκ gene segments.

The influence of maternal immunization on light chain response to Haemophilus influenzae type b vaccine

The repertoire of human antibodies to Haemophilus influenzae type b polysaccharide (Hib PS) has been extensively characterized. In response to Hib PS vaccines, both IgG1 and IgG2 antibodies are produced. Based on the Vκ gene used for the antibodies, heamophilus antibodies can be divided into A2 and non-A2 antibodies. A2 antibodies account for the majority of heamophilus antibodies, are generally more functional, and are specific for heamophilus polysaccharide. In contrast, non-A2 antibodies cross-react with Escherichia coli K100. Studies of placental transfer showed that IgG1 antibodies cross the placenta better than IgG2 antibodies and the A2 antibodies better than non-A2 antibodies. Babies born to mothers who were immunized with a heamophilus vaccine had a high level of antibodies to Hib PS but the antibody level decreased during the first 6 months. The children also responded to a heamophilus conjugate vaccine.

The kinetics of V–J joining throughout 3.5 megabases of the mouse Igκ locus fit a constrained diffusion model of nuclear organization

To gain insight into the nuclear organization of the mouse Igκ locus and how it may relate to the formation of synapses during recombination, we have studied the kinetics of rearrangement of different Vκ gene families to Jκ gene segments in the pre-B cell line, 103bcl2. Remarkably, Vκ gene families separated by more than 3.5 Mb from Jκ gene segments rearranged with nearly identical kinetics to those as close as 18 kb to Jκ gene segments. These results fit a model of nuclear organization in which the entire VκJκ region resides within a single nuclear subcompartment and is capable of exhibiting multiple reversible contacts through diffusion and Brownian motion.

Failure of receptor revision in the autoreactive B cells in patients with systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a prototype of autoimmune diseases characterized by B-cell hyper-reactivity and production of various autoantibodies. It has recently been found that the recombination activating gene (RAG) is re-expressed in activated mature B cells, and RAG-expressing B cells revise their surface immunoglobulin or die by failure of productive secondary rearrangement, leading to elimination of the autoreactive B cells. We have investigated the expression of RAG proteins in mature B cells in patients with SLE. Although normal mature B cells did not express RAG proteins spontaneously, activated B cells expressed RAGs. In SLE, whole mature B cells spontaneously expressed RAG proteins excessively. Because we had found that failure of receptor editing of the Vk gene with autoreactive potential (A30-Vk2) is associated with the development of anti-DNA antibody in SLE, we next focused on anti-DNA autoantibody secreting B cells. We found that anti-DNA autoantibody secreting B cells failed to re-express RAGs spontaneously, even after activation with mitogen. Thus, anti-DNA autoantibody secreting B cells did not revise their immunoglobulin gene nor die via apoptosis, leading them to develop and persist with autoantibody secretion. These findings indicate the importance of appropriate RAG re-expression for the elimination of autoreactive B cells even at the mature stage, and the absence of such events in anti-DNA secreting B cells may be important for autoantibody production in SLE.

The kappa gene repertoire of human neonatal B cells

The kappa chain repertoire of individual IgD + human neonatal B cells was analyzed using a single cell PCR technique. A total of 104 productive and 90 non-productive VκJκ rearrangements from three cord blood B cell samples were sequenced and compared to the adult IgM + peripheral B cell VκJκ repertoire. All six Vκ families were present in neonatal B cells, but the distribution was not random. In the non-productive repertoire Vκ2 and Vκ6 families were less frequent, Vκ1 and Vκ3 families were as frequent, and Vκ4 and Vκ5 families were more frequent than expected from random chance. Notably, the Vκ2 family was negatively selected into the productive repertoire. In contrast, the Vκ1 family was positively selected because of positive selection of three specific genes, O12/O2, L12a and L9. B3 (Vκ4) and B2 (Vκ5) were over-represented in the non-productive repertoire and then were expressed less frequently in the productive repertoire. In contrast, the Vκ3 family gene, A27, was also over-represented in the non-productive repertoire but not further selected into the productive repertoire. Compared to the adult repertoire, junctional diversity was less marked because of a diminished influence of TdT activity, whereas the mean CDR3 length was comparable to that of normal adult B cells. Comparison of the distribution of Vκ and Jκ genes with those found in normal adult subjects suggested that there was less receptor editing in neonatal B cells. When neonatal CD5 + B cells were compared with CD5 − IgD + B cells, it was noted that the Vκ gene A30 was used only in CD5 + B cells in both the productive and non-productive repertoires. The results indicate that the usage of Vκ genes by neonatal B cells is biased by both intrinsic molecular processes and selection. The evidence of selection indicates that the Vκ repertoire is shaped by self antigens, since exposure to exogenous antigens is limited at the time of birth.

Rearrangement of the human heavy chain variable region gene V3-23 in transgenic mice generates antibodies reactive with a range of antigens on the basis of VHCDR3 and residues intrinsic to the heavy chain variable region.

To formulate a 'logic' for how a single immunoglobulin variable region gene generates antibodies with different antigen specificity and polyreactivity, we analysed chimeric antibodies produced in transgenic mice carrying the germ-line human V3-23 gene, multiple diversity (D) and joining (J) gene segments. Hybridomas producing antibodies encoded by the V3-23 gene in combination with different mouse Vkappa genes were obtained by fusion of splenocytes from transgenic mice. All antibodies had human mu-chains and mouse light chains, were multimeric in structure and expressed the human V3-23 gene. Nucleotide sequence analyses of genes encoding the heavy and light chains of 12 antibodies in relation to antigen specificity highlighted the importance of heavy chain variable region CDR3 in determining reactivity with different antigens. However, the results also suggest that non-CDR3 sequences intrinsic to the V3-23 gene itself may be involved in, or determine, the binding of the chimeric antibodies to some of the antigens tested in the current study.

Editors and Editing of Anti-DNA Receptors

Receptor editing is a means by which immature bone marrow B cells can become self-tolerant. Rearrangements of heavy (H) and/or light (L) chain genes are induced by encounter with autoantigens to change the specificity from self to nonself. We have developed site-directed transgenic mice (sd-tg) whose transgenes code for the H chain of antibodies that bind DNA. B cells that express the transgenic H chain associate mainly with four of the 93 functional Vκ genes of the mouse. Numerous aspartate residues that might inhibit DNA binding by the V H domain distinguish these L chain Vκ sequences, but engaging these Vκ editors often requires multiple rearrangements. Among the edited B cells is a subset of multispecific cells that express multiple receptors. One consequence of multispecificity is partial autoreactivity; these multispecific B cells may contribute to autoimmunity.

The PU.1 and NF-EM5 binding motifs in the Igkappa 3' enhancer are responsible for directing somatic hypermutations to the intrinsic hotspots in the transgenic Vkappa gene.

Somatic hypermutation is a key mechanism in generating Ig with higher affinities to antigen, a process known as affinity maturation. Using Igkappa transgenes, the 3' enhancer (kappaE3') has been shown to play an important role in introducing hypermutations. In order to identify the cis-acting elements that regulate hypermutagenesis, we have generated transgenic substrates containing mutations/deletions in the kappaE3' region. Here, we report that base substitutions in the kappaE3', either in the PU.1 or in the NF-EM5 binding motif, not only reduce the mutation rate but also disrupt the directed mutagenesis in the intrinsic hotspots of the Igkappa transgene.

Characteristics of immunoglobulin gene usage of the xenoantibody binding to gal-alpha(1,3)gal target antigens in the gal knockout mouse.

Natural antibodies that react with galactose-alpha(1,3)galactose [galalpha(1,3)gal] carbohydrate epitopes exist in humans and Old World primates because of the inactivation of the alpha1,3-galactosyltransferase (alpha1,3GT) gene in these species and the subsequent production of antibodies to environmental microbes that express the galalpha(1,3)gal antigen. The Gal knockout (Gal o/o) mouse, produced by homologous disruption of the alpha1,3GT gene, spontaneously makes anti-galalpha(1,3)gal antibodies and can be used to study the genetic control of humoral immune responses to this carbohydrate epitope. Six hybridomas that produce monoclonal antibodies (mAbs) to galalpha(1,3)gal were generated in Gal o/o mice. The mAbs were tested to characterize the binding activity with flow cytometry using pig aortic endothelial cells and ELISA with galalpha(1,3)gal carbohydrates. The VH and VK genes of these hybridomas were cloned, sequenced, and analyzed. The mAbs showed distinct patterns of antibody binding to galalpha(1,3)gal antigens. The VH genes that encode the mAb binding activity were restricted to a small number of genes expressed in their germline configuration. Four of six clones used closely related progeny of the same VH germline gene (VH441). Comparison of the mouse gene VH441 to the human gene IGHV3-11, a gene that encodes antibody activity to galalpha(1,3)gal in humans, demonstrates that these two genes share a nonrandom distribution of amino acids used at canonical binding sites within the variable regions (complimentary determining regions 1 and 2) of their immunoglobulin VH genes. These results demonstrate the similarity of the Gal o/o mice and humans in their immune response to galalpha(1,3)gal epitopes. Gal o/o mouse can serve as a useful model for examining the genetic control of antibody/antigen interactions associated with the humoral response to pig xenografts in humans.

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 κlocus, comprising a total of 1,010,706 nucleotides. The 76 Vκgenes found by a hybridization-based approach and their classification in 7 families were confirmed. A Vκorphon located near the locus was also sequenced. In addition, we identified 55 novel Vκrelics and truncated pseudogenes, which establish 5 new families. Among these 132 Vκgenes, 46 have open reading frames. According to the databases and the literature, 32 unique Vκgenes 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 Vκgene locus contains a 360-kb inverted duplication, which harbors 118 Vκgenes. A comparison of the duplicated Vκgenes 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 ∼ 5 million years ago. An orphon Vκgene near the κlocus and a cluster of five Vκorphons on chromosome 22 have no counterparts within the κlocus. This suggests possible mechanisms of the transposition of orphon Vκgenes.

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.