Antibody repertoire diversification through VH gene replacement in mice cloned from an IgA plasma cell.
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PROVIDER: S-EPMC4321288 | BioStudies |
REPOSITORIES: biostudies
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Project description:The genes encoding the variable (V) region of the B-cell antigen receptor (BCR) are assembled from V, D (diversity), and J (joining) elements through a RAG-mediated recombination process that relies on the recognition of recombination signal sequences (RSSs) flanking the individual elements. Secondary V(D)J rearrangement modifies the original Ig rearrangement if a nonproductive original joint is formed, as a response to inappropriate signaling from a self-reactive BCR, or as part of a stochastic mechanism to further diversify the Ig repertoire. VH replacement represents a RAG-mediated secondary rearrangement in which an upstream VH element recombines with a rearranged VHDHJH joint to generate a new BCR specificity. The rearrangement occurs between the cryptic RSS of the original VH element and the conventional RSS of the invading VH gene, leaving behind a footprint of up to five base pairs (bps) of the original VH gene that is often further obscured by exonuclease activity and N-nucleotide addition. We have previously demonstrated that VH replacement can efficiently rescue the development of B cells that have acquired two nonproductive heavy chain (IgH) rearrangements. Here we describe a novel knock-in mouse model in which the prerearranged IgH locus resembles an endogenously rearranged productive VHDHJH allele. Using this mouse model, we characterized the role of VH replacement in the diversification of the primary Ig repertoire through the modification of productive VHDHJH rearrangements. Our results indicate that VH replacement occurs before Ig light chain rearrangement and thus is not involved in the editing of self-reactive antibodies.
| S-EPMC4321307 | BioStudies
Project description:Adult cartilaginous fish express three immunoglobulin (Ig) isotypes, IgM, IgNAR and IgW. Newborn nurse sharks, Ginglymostoma cirratum, produce 19S (multimeric) IgM and monomeric/dimeric IgM1gj, a germline-joined, IgM-related VH, and very low amounts of 7S (monomeric) IgM and IgNAR proteins. Newborn IgNAR VH mRNAs are diverse in the complementarity-determining region 3 (CDR3) with non-templated nucleotide (N-region) addition, which suggests that, unlike in many other vertebrates, terminal deoxynucleotidyl transferase (TdT) expressed at birth is functional. IgW is present in the lungfish, a bony fish sharing a common ancestor with sharks 460 million years ago, implying that the IgW VH family is as old as the IgM VH family. This nurse shark study examined the IgM and IgW VH repertoire from birth through adult life, and analyzed the phylogenetic relationships of these gene families.IgM and IgW VH cDNA clones isolated from newborn nurse shark primary and secondary lymphoid tissues had highly diverse and unique CDR3 with N-region addition and VDJ gene rearrangement, implicating functional TdT and RAG gene activity. Despite the clear presence of N-region additions, newborn CDR3 were significantly shorter than those of adults. The IgM clones are all included in a conventional VH family that can be classified into five discrete groups, none of which is orthologous to IgM VH genes in other elasmobranchs. In addition, a novel divergent VH family was orthologous to a published monotypic VH horn shark family. IgW VH genes have diverged sufficiently to form three families. IgM and IgW VH serine codons using the potential somatic hypermutation hotspot sequence occur mainly in VH framework 1 (FR1) and CDR1. Phylogenetic analysis of cartilaginous fish and lungfish IgM and IgW demonstrated they form two major ancient gene groups; furthermore, these VH genes generally diversify (duplicate and diverge) within a species.As in ratfish, sandbar and horn sharks, most nurse shark IgM VH genes are from one family with multiple, heterogeneous loci. Their IgW VH genes have diversified, forming at least three families. The neonatal shark Ig VH CDR3 repertoire, diversified via N-region addition, is shorter than the adult VDJ junction, suggesting one means of postnatal repertoire diversification is expression of longer CDR3 junctions.
| S-EPMC420240 | BioStudies
Project description:Sharks and skates are representatives of the earliest vertebrates with an immune system based on V(D)J rearrangement. They possess a unique Ig gene organization consisting of 15 to >50 individual IgM loci, each with one VH, two DH, one JH, and one set of constant region exons. The present study attempts to understand how multiple Ig genes are regulated with respect to rearrangement initiation and to targeting during somatic hypermutation. The linkage of three single-copy IgH genes was determined, and single-cell genomic PCR studies in a neonatal animal were used to examine any relationship between relative gene position and likelihood of rearrangement. Our results show that one to three IgH genes are activated independently of linkage or allelic position and the data best fit with a probability model based on the hypothesis that V(D)J rearrangement occurs as a sequence of trials within the B cell. In the neonatal cell set, two closely related IgH, G2A, and G2B, rearranged at similar frequencies, and their membrane forms were expressed at similar levels, like in other young animals. However, older animals displayed a bias in favor of the G2A isotype, which suggests that although rearrangement at G2A and G2B was randomly initiated during primary repertoire generation, the two very similar IgM sequences appear to be differentially expressed with age and exposure to Ag. We performed genomic single-cell PCR on B cells from an immunized individual to study activation-induced cytidine deaminase targeting and found that hypermutation, like V(D)J rearrangement, occurred independently among the many shark IgH.
| S-EPMC3190574 | BioStudies
Project description:VH replacement occurs through RAG-mediated secondary recombination between a rearranged VH gene and an upstream unrearranged VH gene. Due to the location of the cryptic recombination signal sequence (cRSS, TACTGTG) at the 3' end of VH gene coding region, a short stretch of nucleotides from the previous rearranged VH gene can be retained in the newly formed VH-DH junction as a "footprint" of VH replacement. Such footprints can be used as markers to identify Ig heavy chain (IgH) genes potentially generated through VH replacement. To explore the contribution of VH replacement products to the antibody repertoire, we developed a Java-based computer program, VH replacement footprint analyzer-I (VHRFA-I), to analyze published or newly obtained IgH genes from human or mouse. The VHRFA-1 program has multiple functional modules: it first uses service provided by the IMGT/V-QUEST program to assign potential VH, DH, and JH germline genes; then, it searches for VH replacement footprint motifs within the VH-DH junction (N1) regions of IgH gene sequences to identify potential VH replacement products; it can also analyze the frequencies of VH replacement products in correlation with publications, keywords, or VH, DH, and JH gene usages, and mutation status; it can further analyze the amino acid usages encoded by the identified VH replacement footprints. In summary, this program provides a useful computation tool for exploring the biological significance of VH replacement products in human and mouse.
| S-EPMC3918983 | BioStudies
Project description:Coordination of V rearrangements between loci on homologous chromosomes is critical for Ig and TCR allelic exclusion. The Ataxia Telangietasia mutated (ATM) protein kinase promotes DNA repair and activates checkpoints to suppress aberrant Ig and TCR rearrangements. In response to RAG cleavage of Ig? loci, ATM inhibits RAG expression and suppresses further V?-to-J? rearrangements to enforce Ig? allelic exclusion. Because V recombination between alleles is more strictly regulated for TCR? and IgH loci, we evaluated the ability of ATM to restrict biallelic expression and V-to-DJ recombination of TCR? and IgH genes. We detected greater frequencies of lymphocytes with biallelic expression or aberrant V-to-DJ rearrangement of TCR? or IgH loci in mice lacking ATM. A preassembled DJ? complex that decreases the number of TCR? rearrangements needed for a productive TCR? gene further increased frequencies of ATM-deficient cells with biallelic TCR? expression. IgH and TCR? proteins drive proliferation of prolymphocytes through cyclin D3 (Ccnd3), which also inhibits VH transcription. We show that inactivation of Ccnd3 leads to increased frequencies of lymphocytes with biallelic expression of IgH or TCR? genes. We also show that Ccnd3 inactivation cooperates with ATM deficiency to increase the frequencies of cells with biallelic TCR? or IgH expression while decreasing the frequency of ATM-deficient lymphocytes with aberrant V-to-DJ recombination. Our data demonstrate that core components of the DNA damage response and cell cycle machinery cooperate to help enforce IgH and TCR? allelic exclusion and indicate that control of V-to-DJ rearrangements between alleles is important to maintain genomic stability.
| S-EPMC4157102 | BioStudies
Project description:In developing B cells, the immunoglobulin heavy chain (IgH) locus is thought to move from repressive to permissive chromatin compartments to facilitate its scheduled rearrangement. In mature B cells, maintenance of allelic exclusion has been proposed to involve recruitment of the non-productive IgH allele to pericentromeric heterochromatin. Here, we used an allele-specific chromosome conformation capture combined with sequencing (4C-seq) approach to unambigously follow the individual IgH alleles in mature B lymphocytes. Despite their physical and functional difference, productive and non-productive IgH alleles in B cells and unrearranged IgH alleles in T cells share many chromosomal contacts and largely reside in active chromatin. In brain, however, the locus resides in a different repressive environment. We conclude that IgH adopts a lymphoid-specific nuclear location that is, however, unrelated to maintenance of allelic exclusion. We additionally find that in mature B cells-but not in T cells-the distal VH regions of both IgH alleles position themselves away from active chromatin. This, we speculate, may help to restrict enhancer activity to the productively rearranged VH promoter element.
| S-EPMC3737562 | BioStudies
Project description:In developing B cells the immunoglobulin heavy chain (IgH) locus is thought to move from repressive to permissive chromatin compartments to facilitate its scheduled rearrangement. In mature B cells, maintenance of allelic exclusion has been proposed to involve recruitment of the non-productive IgH allele to pericentromeric heterochromatin. Here we used an allele-specific chromosome conformation capture combined with sequencing (4C-seq) approach to unambigously follow the individual IgH alleles in mature B lymphocytes. Despite their physical and functional difference, productive and non-productive IgH alleles in B cells and unrearranged IgH alleles in T cells share many chromosomal contacts and largely reside in active chromatin. In brain, however, the locus resides in a different, repressive environment. We conclude that IgH adopts a lymphoid-specific nuclear location that is however unrelated to maintenance of allelic exclusion. We additionally find that in mature B cells - but not in T cells – the distal VH regions of both IgH alleles position themselves away from active chromatin. This, we speculate, may help to restrict enhancer activity to the productively rearranged VH promoter element. Allele specific analysis of the nuclear organization of the IgH locus in resting and activated B cells and T cells and fetal brain cells
| E-GEOD-47128 | BioStudies
Project description:VH replacement refers to RAG-mediated secondary recombination of the IgH genes, which renews almost the entire VH gene coding region but retains a short stretch of nucleotides as a VH replacement footprint at the newly generated VH-DH junction. To explore the biological significance of VH replacement to the antibody repertoire, we developed a Java-based VH replacement footprint analyzer program and analyzed the distribution of VH replacement products in 61,851 human IgH gene sequences downloaded from the NCBI database. The initial assignment of the VH, DH, and JH gene segments provided a comprehensive view of the human IgH repertoire. To our interest, the overall frequency of VH replacement products is 12.1%; the frequencies of VH replacement products in IgH genes using different VH germline genes vary significantly. Importantly, the frequencies of VH replacement products are significantly elevated in IgH genes derived from different autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, and allergic rhinitis, and in IgH genes encoding various autoantibodies or anti-viral antibodies. The identified VH replacement footprints preferentially encoded charged amino acids to elongate IgH CDR3 regions, which may contribute to their autoreactivities or anti-viral functions. Analyses of the mutation status of the identified VH replacement products suggested that they had been actively involved in immune responses. These results provide a global view of the distribution of VH replacement products in human IgH genes, especially in IgH genes derived from autoimmune diseases and anti-viral immune responses.
| S-EPMC4105631 | BioStudies
Project description:Characterization of the diversified immunoglobulin (Ig) repertoire may provide insight into pathways that shape an efficient antibody (Ab) repertoire for immune response against human immunodeficiency virus (HIV) infection. This study aimed to profile characteristics of the plasmablast repertoire during chronic HIV infection. Ig variable regions of plasmablasts from both chronically HIV-infected donors (HIVDs) previously treated with antiretroviral therapy (ART) and healthy donors (HDs) were amplified by single-cell PCR to establish the basis for further repertoire analysis. We compared the plasmablast repertoires expressed in multiple chronically HIVDs after ART treatment cessation and HDs. We also examined the non-productive repertoire to identify the indication of the immediate products of the rearrangement machinery without an impact of selection during HIV infection. We found multiple differences between the productive repertoires of HIVD and HD subjects, including biased usages of VH3-49, VH1-2, VH3-33, VH3-74, and VH5-51 in VH and D1-7, D1-14, D1-20, and D5-5/18 in D segments in the HIVD group, as well as shorter and preferential glycine usages in CDRH3 regions. Gene selections were also detected in light chains. Notably, differences between productive rearrangements of HIVDs and HDs outnumbered those between productive and non-productive rearrangements within HIVDs. HIV infection may exert a dominant impact on the development of the plasmablast repertoire. The impact of selection is of limited significance in shaping the plasmablast repertoire. Overall, the data indicate that the environment in which the plasmablasts live can affect the distribution of the VH and VL genes in the repertoire and the amino acid compositions of the expressed Abs.
| S-EPMC7026028 | BioStudies
Project description:VH replacement occurs through RAG-mediated recombination between the cryptic recombination signal sequence (cRSS) near the 3' end of a rearranged VH gene and the 23-bp RSS from an upstream unrearranged VH gene. Due to the location of the cRSS, VH replacement leaves a short stretch of nucleotides from the previously rearranged VH gene at the newly formed V-D junction, which can be used as a marker to identify VH replacement products. To determine the contribution of VH replacement products to mouse antibody repertoire, we developed a Java-based VH Replacement Footprint Analyzer (VHRFA) program and analyzed 17,179 mouse IgH gene sequences from the NCBI database to identify VH replacement products. The overall frequency of VH replacement products in these IgH genes is 5.29% based on the identification of pentameric VH replacement footprints at their V-D junctions. The identified VH replacement products are distributed similarly in IgH genes using most families of VH genes, although different families of VH genes are used differentially. The frequencies of VH replacement products are significantly elevated in IgH genes derived from several strains of autoimmune prone mice and in IgH genes encoding autoantibodies. Moreover, the identified VH replacement footprints in IgH genes from autoimmune prone mice or IgH genes encoding autoantibodies preferentially encode positively charged amino acids. These results revealed a significant contribution of VH replacement products to the diversification of antibody repertoire and potentially, to the generation of autoantibodies in mice.
| S-EPMC3585286 | BioStudies