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Asset Tag: Immune repertoire

July 7, 2019  |  

In-depth determination and analysis of the human paired heavy- and light-chain antibody repertoire.

High-throughput immune repertoire sequencing has emerged as a critical step in the understanding of adaptive responses following infection or vaccination or in autoimmunity. However, determination of native antibody variable heavy-light pairs (VH-VL pairs) remains a major challenge, and no technologies exist to adequately interrogate the >1 × 10(6) B cells in typical specimens. We developed a low-cost, single-cell, emulsion-based technology for sequencing antibody VH-VL repertoires from >2 × 10(6) B cells per experiment with demonstrated pairing precision >97%. A simple flow-focusing apparatus was used to sequester single B cells into emulsion droplets containing lysis buffer and magnetic beads for mRNA capture; subsequent emulsion RT-PCR generated VH-VL amplicons for next-generation sequencing. Massive VH-VL repertoire analyses of three human donors provided new immunological insights including (i) the identity, frequency and pairing propensity of shared, or ‘public’, VL genes, (ii) the detection of allelic inclusion (an implicated autoimmune mechanism) in healthy individuals and (iii) the occurrence of antibodies with features, in terms of gene usage and CDR3 length, associated with broadly neutralizing antibodies to rapidly evolving viruses such as HIV-1 and influenza.

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July 7, 2019  |  

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. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

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July 7, 2019  |  

Genomic organization of the zebrafish (Danio rerio) T cell receptor alpha/delta locus and analysis of expressed products.

In testing the hypothesis that all jawed vertebrate classes employ immunoglobulin heavy chain V (IgHV) gene segments in their T cell receptor (TCR)d encoding loci, we found that some basic characterization was required of zebrafish TCRd. We began by annotating and characterizing the TCRa/d locus of Danio rerio based on the most recent genome assembly, GRCz10. We identified a total of 141 theoretically functional V segments which we grouped into 41 families based upon 70 % nucleotide identity. This number represents the second greatest count of apparently functional V genes thus far described in an antigen receptor locus with the exception of cattle TCRa/d. Cloning, relative quantitative PCR, and deep sequencing results corroborate that zebrafish do express TCRd, but these data suggest only at extremely low levels and in limited diversity in the spleens of the adult fish. While we found no evidence for IgH-TCRd rearrangements in this fish, by determining the locus organization we were able to suggest how the evolution of the teleost a/d locus could have lost IgHVs that exist in sharks and frogs. We also found evidence of surprisingly low TCRd expression and repertoire diversity in this species.

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July 7, 2019  |  

Immunoglobulin gene analysis as a tool for investigating human immune responses.

The human immunoglobulin repertoire is a hugely diverse set of sequences that are formed by processes of gene rearrangement, heavy and light chain gene assortment, class switching and somatic hypermutation. Early B cell development produces diverse IgM and IgD B cell receptors on the B cell surface, resulting in a repertoire that can bind many foreign antigens but which has had self-reactive B cells removed. Later antigen-dependent development processes adjust the antigen affinity of the receptor by somatic hypermutation. The effector mechanism of the antibody is also adjusted, by switching the class of the antibody from IgM to one of seven other classes depending on the required function. There are many instances in human biology where positive and negative selection forces can act to shape the immunoglobulin repertoire and therefore repertoire analysis can provide useful information on infection control, vaccination efficacy, autoimmune diseases, and cancer. It can also be used to identify antigen-specific sequences that may be of use in therapeutics. The juxtaposition of lymphocyte development and numerical evaluation of immune repertoires has resulted in the growth of a new sub-speciality in immunology where immunologists and computer scientists/physicists collaborate to assess immune repertoires and develop models of immune action.© 2018 The Authors. Immunological Reviews Published by John Wiley & Sons Ltd.

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