Heavy Chain Variable Gene Usage Research Articles

Sequencing the B cell receptor repertoires of antibody-deficient individuals with and without infection susceptibility

PurposeMost individuals with antibody deficiency (hypogammaglobulinemia) need immunoglobulin replacement therapy (IgG-RT) from healthy plasma donors to stay clear of infections. However, a small subset of hypogammaglobulinemic patients do not require this substitution therapy. We set out to investigate this clinical conundrum by asking whether the peripheral B cell receptor repertoires differ between antibody-deficient patients who do and do not need IgG-RT. MethodsWe sequenced and analyzed IgG and IgM heavy chain B cell receptor repertoires from peripheral blood mononuclear cells (PBMCs) isolated from patients with low serum IgG concentrations who did or did not require IgG-RT. ResultsCompared to the patients who did not need IgG-RT, those who needed IgG-RT had higher numbers of IgG antibody clones, higher IgM diversity, and less oligoclonal IgG and IgM repertoires. The patient cohorts had different heavy chain variable gene usage, and the patients who needed IgG-RT had elevated frequencies of IgG clones with higher germline identity (i.e., fewer somatic hypermutations). ConclusionAntibody-deficient patients with infection susceptibility who needed IgG-RT had more diverse peripheral antibody repertoires that were less diverged from germline and thus may not be as optimal for targeting pathogens, possibly contributing to infection susceptibility.

Sequencing the B Cell Receptor Repertoires of Antibody-Deficient Individuals With and Without Infection Susceptibility

PurposeMost individuals with antibody deficiency (hypogammaglobulinemia) need immunoglobulin replacement therapy (IgG-RT) from healthy plasma donors to stay clear of infections. However, a small subset of hypogammaglobulinemic patients do not require this substitution therapy. We set out to investigate this clinical conundrum by asking whether the peripheral B cell receptor repertoires differ between antibody-deficient patients who do and do not need IgG-RT.MethodsWe sequenced and analyzed IgG and IgM heavy chain B cell receptor repertoires from peripheral blood mononuclear cells (PBMCs) isolated from patients with low serum IgG concentrations who did or did not require IgG-RT.ResultsCompared to the patients who did not need IgG-RT, those who needed IgG-RT had higher numbers of IgG antibody clones, higher IgM diversity, and less oligoclonal IgG and IgM repertoires. The patient cohorts had different heavy chain variable gene usage, and the patients who needed IgG-RT had elevated frequencies of IgG clones with higher germline identity (i.e., fewer somatic hypermutations).ConclusionAntibody-deficient patients with infection susceptibility who needed IgG-RT had more diverse peripheral antibody repertoires that were less diverged from germline and thus may not be as optimal for targeting pathogens, possibly contributing to infection susceptibility.

Sequence-Based Discovery Demonstrates That Fixed Light Chain Human Transgenic Rats Produce a Diverse Repertoire of Antigen-Specific Antibodies.

We created a novel transgenic rat that expresses human antibodies comprising a diverse repertoire of heavy chains with a single common rearranged kappa light chain (IgKV3-15-JK1). This fixed light chain animal, called OmniFlic, presents a unique system for human therapeutic antibody discovery and a model to study heavy chain repertoire diversity in the context of a constant light chain. The purpose of this study was to analyze heavy chain variable gene usage, clonotype diversity, and to describe the sequence characteristics of antigen-specific monoclonal antibodies (mAbs) isolated from immunized OmniFlic animals. Using next-generation sequencing antibody repertoire analysis, we measured heavy chain variable gene usage and the diversity of clonotypes present in the lymph node germinal centers of 75 OmniFlic rats immunized with 9 different protein antigens. Furthermore, we expressed 2,560 unique heavy chain sequences sampled from a diverse set of clonotypes as fixed light chain antibody proteins and measured their binding to antigen by ELISA. Finally, we measured patterns and overall levels of somatic hypermutation in the full B-cell repertoire and in the 2,560 mAbs tested for binding. The results demonstrate that OmniFlic animals produce an abundance of antigen-specific antibodies with heavy chain clonotype diversity that is similar to what has been described with unrestricted light chain use in mammals. In addition, we show that sequence-based discovery is a highly effective and efficient way to identify a large number of diverse monoclonal antibodies to a protein target of interest.

Proteomic Analysis of Pemphigus Autoantibodies Indicates a Larger, More Diverse, and More Dynamic Repertoire than Determined by B Cell Genetics

In autoantibody-mediated diseases such as pemphigus, serum antibodies lead to disease. Genetic analysis of B cells has allowed characterization of antibody repertoires in such diseases but would be complemented by proteomic analysis of serum autoantibodies. Here, we show using proteomic analysis that the serum autoantibody repertoire in pemphigus is much more polyclonal than that found by genetic studies of B cells. In addition, many B cells encode pemphigus autoantibodies that are not secreted into the serum. Heavy chain variable gene usage of serum autoantibodies is not shared among patients, implying targeting of the coded proteins will not be a useful therapeutic strategy. Analysis of autoantibodies in individual patients over several years indicates that many antibody clones persist but the proportion of each changes. These studies indicate a dynamic and diverse autoantibody response not revealed by genetic studies and explain why similar overall autoantibody titers may give variable disease activity.

Restoration of Overexpressed Variable Heavy Chain 2 Transcripts with Tumor Necrosis Factor Inhibitors in Ankylosing Spondylitis

To the Editor: Ankylosing spondylitis (AS) is a chronic inflammatory disorder characterized by progressive and destructive arthritis of the spine and pelvis1. B cells are involved in the pathogenesis of autoimmune diseases through antibody production, cytokine release, and antibody presentation to auto-reactive T cells. In AS, the role of B cells in the pathogenesis is still incompletely understood2. It has been hypothesized that the production of high affinity monoreactive autoantibodies in autoimmune disease could arise from intrinsic abnormalities in the generation of immunoglobulin genes3. Immunoglobulin gene usage can be regarded as an important factor of pathogenesis of autoimmune diseases. Investigation of variable heavy chain (VH) gene usage is important for determining whether usage of particular gene families is distorted. Several studies have investigated the VH gene usage in various autoimmune diseases, including systemic lupus erythematosus4, myasthenia gravis5, rheumatoid arthritis (RA)6, Sjogren syndrome7, and AS8 … Address correspondence to Dr. S-C. Shim, Division of Rheumatology, Department of Medicine, Daejeon Rheumatoid and Degenerative Arthritis Center, Chungnam National University Hospital, 6 munwha-ro Jung-gu, Daejeon, South Korea. E-mail: shimsc{at}cnuh.co.kr

Correction: Antibody Repertoire Development in Fetal and Neonatal Piglets. XI. The Relationship of Variable Heavy Chain Gene Usage and the Genomic Organization of the Variable Heavy Chain Locus

Correction: Antibody Repertoire Development in Fetal and Neonatal Piglets. XI. The Relationship of Variable Heavy Chain Gene Usage and the Genomic Organization of the Variable Heavy Chain Locus

Homologous regions of autoantibody heavy chain complementarity-determining region 3 (H-CDR3) in patients with pemphigus cause pathogenicity

Pemphigus is a life-threatening autoimmune disease in which antibodies specific for desmogleins (Dsgs) cause loss of keratinocyte cell adhesion and blisters. In order to understand how antibodies cause pathogenicity and whether there are commonalities among antibodies in different patients that could ultimately be used to target specific therapy against these antibodies, we characterized Dsg-specific mAbs cloned by phage display from 3 patients with pemphigus vulgaris and 2 with pemphigus foliaceus. Variable heavy chain gene usage was restricted, but similar genes were used for both pathogenic and nonpathogenic mAbs. However, the heavy chain complementarity-determining region 3 (H-CDR3) of most pathogenic, but not nonpathogenic, mAbs shared an amino acid consensus sequence. Randomization of the H-CDR3 and site-directed mutagenesis indicated that changes in this sequence could block pathogenicity but not necessarily binding. In addition, for 2 antibodies with longer H-CDR3s, a tryptophan was critical for pathogenicity but not binding, a result that is consistent with blocking the tryptophan acceptor site that is thought to be necessary for Dsg-mediated adhesion. These studies indicate that H-CDR3 is critical for pathogenicity of a human autoantibody, that a small region (even 1 amino acid) can mediate pathogenicity, and that pathogenicity can be uncoupled from binding in these antibodies.

Antibody Repertoire Development in Fetal and Neonatal Piglets. XI. The Relationship of Variable Heavy Chain Gene Usage and the Genomic Organization of the Variable Heavy Chain Locus

In this study, we have mapped the 3' H chain V region (V(H)) genes and those in the H chain diversity, H chain joining, and 5' portion of the H chain constant locus. We show that swine possess only two functional H chain diversity segments and only one functional H chain joining segment. These data help to explain more than a decade of observations on the preimmune repertoire of this species and reveal the vulnerability of swine to natural or designed mutational events. The results are consistent with earlier studies on the region containing Enh, Cmu, and Cdelta while revealing that the ancestral IgG3 is the most 5' Cgamma gene. We also observed a recent duplication ( approximately 1.6 million years ago) in the V(H) locus that contains six of the seven V(H) genes that comprise 75% of the preimmune repertoire. Because there are no known transfers of immune regulators or Ags that cross the placenta as in mice and humans, fetal V(H) usage must be intrinsically regulated. Therefore, we quantified V(H) usage in fetal piglets and demonstrated that usage is independent of the position of V(H) genes in the genome; the most 3' functional V(H) gene (IGHV2) is rarely used, whereas certain upstream genes (IGHV14 and IGHV15) are predominately used early in fetal liver but seldom thereafter. Similar to previous studies, three V(H) genes account for 40% of the repertoire and six for approximately 70%. This limited combinatorial diversity of the porcine V(H) repertoire further emphasizes the dependence on CDR3 diversity for generating the preimmune Ab repertoire of this species.

Errata

Microbiology and ImmunologyVolume 53, Issue 1 p. 58-58 Free Access Errata This article corrects the following: Variable gene family usage of protective and non-protective anti-Vibrio cholerae O1 LPS antibody heavy chains Terri K. Wade, William F. Wade, Volume 52Issue 12Microbiology and Immunology pages: 611-620 First Published online: December 3, 2008 First published: 15 January 2009 https://doi.org/10.1111/j.1348-0421.2008.00110.xCitations: 2AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat In Microbiology and Immunology, the footnotes for Tables 2, 3 and 4 were incorrect.1 The correct table legends and their footnotes should be as follows: Table 2 Hybridomas investigated in the present study No footnote. Table 3 O1 LPS binding and vibriocidal capacity of the anti-O1 LPS mAbs ELISA (OD) and vibriocidal data were generated using a standard volume from supernatants of individual hybridomas that contained different concentrations of mAb. The effective concentration of mAb was calculated by dividing the concentration of mAb (μg/mL) by the reciprocal of the end-point dilution. The lack of vibriocidal activity was because IgA does not fix complement. Other isotypes (e.g. IgM, IgG1) of mAbs that fix complement that were not vibriocidal were also denoted as negative (neg). Table 4 Variable heavy chain gene usage (family member) of the anti-O1 LPS mAbs The V family usage was determined from the DNA sequences of the mAbs using the Ig BLAST program from NCBI (http://www.ncbi.nlm.nih.gov/igblast). The cDNA sequences for all the mAbs are available upon request. The specific family member for individual mAb that was assigned and the percent identity to that member are: mAb 22 (Vh9.1, 99.0%), mAb 23 (Vh5, 7183.a15.24, 99.3%), mAb 24 (Vh1, J558.8, 99.7%), mAb 26 (Vh5, 7183.a47.7, 96.2%), mAb 35 (Vh1, J558.47, 100%), mAb 36 (Vh1, J558.45, 99.7%), mAb 40 (Vh6, J606.4.82, 99.7%), mAb 47 (Vh5, 7183.a47.76, 99.0%), mAb 55 (Vh3, 36–60, 96.5%), mAb 58 (Vh1, J558.8, 99.7%), mAb 1 (Vh1, J558.29, 95.2%), mAb 6 (Vh11, 100%), mAb 9 (Vh5, 7183.a33.55, 95.6%), mAb 11 (Vh5, 7183.a37.59, 96.6%), mAb 16 (Vh9.1, 100%), mAb 18 (Vh3, 36–60, 92.5%) and mAb 19 (Vh5, 7183.a47.24, 96%). Reference 1 Wade T.K., Wade W.F. (2008) Variable gene family usage of protective and non-protective anti-Vibrio cholerae O1 LPS antibody heavy chains. Microbiol Immunol 52: 611– 20. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar Citing Literature Volume53, Issue1January 2009Pages 58-58 ReferencesRelatedInformation

Subsets with restricted immunoglobulin gene rearrangement features indicate a role for antigen selection in the development of chronic lymphocytic leukemia

AbstractWe recently identified a chronic lymphocytic leukemia (CLL) subgroup using the immunoglobulin variable heavy-chain (VH) gene VH3-21 with almost identical heavy-chain complementarity determining region 3s (HCDR3s) and preferential variable light-chain (VL) gene usage, suggesting recognition of a common antigen epitope in this subset. To further explore the B-cell receptors (BCRs) in CLL, we characterized 407 VH rearrangements amplified from 346 CLLs regarding VH, diversity (D), and joining (JH) gene usage and performed multiple alignment of the HCDR3 sequences. These analyses revealed 3 small subsets (2 VH1-69 groups, 7 cases; and 1 VH1-2 group, 5 cases) with highly restricted HCDR3 features including identical VH/D/JH usage, HCDR3 lengths, and shared N-sequences, in addition to the VH3-21 group (22 cases). Furthermore, another 3 groups (9 VH1-3+ cases, 3 VH1-18+ cases, and 5 VH4-39+ cases) had essentially identical VH/D/JH use and similar HCDR3 lengths but less conserved N-regions. Analysis in all 6 of these subgroups showed restriction in VL gene use, whereas no association between VH and VL usage was found in cases without HCDR3 similarities. Altogether, structurally similar HCDR3s associated with preferential VL gene usage implies selection of BCRs, especially in subsets showing high HCDR3 similarities, thus pointing to restricted antigen recognition sites and possibly involvement of specific antigens in CLL development.

A novel approach to study variable heavy chain gene usage in response to the capsular polysaccharide of Neisseria meningitidis serogroup C

The molecular mechanisms involved in the relatively poor immune response in the elderly are not clearly understood. Qualitative aspects of the immune response could be a possible explanation for the differential response to T-independent antigens in young adults and elderly. This study is directed towards elucidating the differential usage of variable heavy chain by young adult and elderly derived sequences in response to the capsular polysaccharide of Neisseria meningitidis serogroup C. We currently report findings of a preliminary study designed to test the feasibility of a novel approach to isolate antigen-specific B cells. Paramagnetic beads coated with an anti-idiotypic antibody, which mimics the capsular polysaccharide of N. meningitidis serogroup C, were used to select B cells. Analysis of the gene usage data indicates some unexpected differences in the use of variable chain heavy chain in the case of young adult versus elderly sequences. The elderly derived sequences use a more diverse array of V H gene families in contrast to the young adult sequences, where the V H gene family usage is restricted. Nearly half the young adult sequences utilize V H3-15 germline sequence while only 25% of the elderly sequences use this germline sequence. There were interesting differences in the types of JH chain and the composition and length of CDR3 utilized by the two groups. Together, these significant differences may contribute towards the poor immune response to T-independent antigens in the elderly. These data validate the techniques used for these studies and suggest that it is pertinent to use this approach towards future investigations to elucidate gene usage in response to an antigen.

VH Gene Analysis of Primary Cutaneous B-Cell Lymphomas: Evidence for Ongoing Somatic Hypermutation and Isotype Switching

Primary cutaneous B-cell lymphomas are B-cell non-Hodgkin’s lymphomas that arise in the skin. The major subtypes discerned are follicle center cell lymphomas, immunocytomas (marginal zone B-cell lymphomas), and large B-cell lymphomas of the leg. In this study, we analyzed the variable heavy chain (VH) genes of 7 of these lymphomas, ie, 4 follicle center cell lymphomas (diffuse large-cell lymphomas) and 3 immunocytomas. We show that all these lymphomas carry heavily mutated VH genes, with no obvious bias in VH gene usage. The low ratios of replacement versus silent mutations observed in the framework regions of 5 of the 7 lymphomas suggest that the structure of the B-cell antigen receptor was preserved, as in normal B cells that are selected for antibody expression. Moreover, evidence for ongoing mutation was obtained in 3 immunocytomas and in one lymphoma of large-cell type. In addition, in 1 immunocytoma, both IgG- and IgA-expressing clones were found, indicative of isotype switching. Our data provide insight into the biology of primary cutaneous B-cell lymphomas and may be of significance for their classification.

VH Gene Analysis of Primary Cutaneous B-Cell Lymphomas: Evidence for Ongoing Somatic Hypermutation and Isotype Switching

Primary cutaneous B-cell lymphomas are B-cell non-Hodgkin's lymphomas that arise in the skin. The major subtypes discerned are follicle center cell lymphomas, immunocytomas (marginal zone B-cell lymphomas), and large B-cell lymphomas of the leg. In this study, we analyzed the variable heavy chain (VH) genes of 7 of these lymphomas, ie, 4 follicle center cell lymphomas (diffuse large-cell lymphomas) and 3 immunocytomas. We show that all these lymphomas carry heavily mutated VH genes, with no obvious bias in VH gene usage. The low ratios of replacement versus silent mutations observed in the framework regions of 5 of the 7 lymphomas suggest that the structure of the B-cell antigen receptor was preserved, as in normal B cells that are selected for antibody expression. Moreover, evidence for ongoing mutation was obtained in 3 immunocytomas and in one lymphoma of large-cell type. In addition, in 1 immunocytoma, both IgG- and IgA-expressing clones were found, indicative of isotype switching. Our data provide insight into the biology of primary cutaneous B-cell lymphomas and may be of significance for their classification.

Heavy chain variable gene usage by human B-1 lymphocytes and polyreactive autoantibodies

To evaluate the role of B-1 cells and polyreactive autoantibodies in the development of adult immune repertoire, it is necessary to assess their immunoglobulin heavy chain variable-gene usage. We thus screened 28 independently derived human polyreactive MAbs from fetal and adult splenic B lymphocytes for their VH-region usage. We demonstrate that the polyreactivity of the IgM antibodies secreted by B-1 cells is not the result of the expression of particular variable region gene families. All six VH families are represented roughly in proportion to their estimated family size. Furthermore, the representation of the six families appears similar in polyreactive MAbs derived from fetal or adult lymphocytes.