High-throughput sequencing of the paired human immunoglobulin heavy and light chain repertoire

Each B-cell receptor consists of a pair of heavy and light chains. High-throughput sequencing can identify large numbers of heavy- and light-chain variable regions (VH and VL) in a given B-cell repertoire, but information about endogenous pairing of heavy and light chains is lost after bulk lysis of B-cell populations. Here we describe a way to retain this pairing information. In our approach, single B cells (>5 × 104 capacity per experiment) are deposited in a high-density microwell plate (125 pl/well) and lysed in situ. mRNA is then captured on magnetic beads, reverse transcribed and amplified by emulsion VH:VL linkage PCR. The linked transcripts are analyzed by Illumina high-throughput sequencing. We validated the fidelity of VH:VL pairs identified by this approach and used the method to sequence the repertoire of three human cell subsets—peripheral blood IgG+ B cells, peripheral plasmablasts isolated after tetanus toxoid immunization and memory B cells isolated after seasonal influenza vaccination.

[1]  Ron Diskin,et al.  Sequence and Structural Convergence of Broad and Potent HIV Antibodies That Mimic CD4 Binding , 2011, Science.

[2]  Zoltán Konthur,et al.  V-gene amplification revisited - An optimised procedure for amplification of rearranged human antibody genes of different isotypes. , 2010, New biotechnology.

[3]  R. White,et al.  High-Throughput Sequencing of the Zebrafish Antibody Repertoire , 2009, Science.

[4]  Mario Roederer,et al.  Focused Evolution of HIV-1 Neutralizing Antibodies Revealed by Structures and Deep Sequencing , 2011, Science.

[5]  Andreas Radbruch,et al.  Blood-borne human plasma cells in steady state are derived from mucosal immune responses. , 2009, Blood.

[6]  P. S. Andersen,et al.  Isolation of human antibody repertoires with preservation of the natural heavy and light chain pairing. , 2006, Journal of molecular biology.

[7]  P. Wilson,et al.  Tools to therapeutically harness the human antibody response , 2012, Nature Reviews Immunology.

[8]  M. Nussenzweig,et al.  Predominant Autoantibody Production by Early Human B Cell Precursors , 2003, Science.

[9]  Mark Mulligan,et al.  Pandemic H1N1 influenza vaccine induces a recall response in humans that favors broadly cross-reactive memory B cells , 2012, Proceedings of the National Academy of Sciences.

[10]  Y. Louzoun,et al.  Rep‐Seq: uncovering the immunological repertoire through next‐generation sequencing , 2012, Immunology.

[11]  S. Reddy,et al.  Systems analysis of adaptive immunity by utilization of high-throughput technologies. , 2011, Current opinion in biotechnology.

[12]  Patrick C. Wilson,et al.  Rapid cloning of high-affinity human monoclonal antibodies against influenza virus , 2008, Nature.

[13]  Seung Hyun Kang,et al.  Monoclonal antibodies isolated without screening by analyzing the variable-gene repertoire of plasma cells , 2010, Nature Biotechnology.

[14]  N. Fischer,et al.  Sequencing antibody repertoires: the next generation. , 2011, mAbs.

[15]  Sean A Beausoleil,et al.  Proteomics-directed cloning of circulating antiviral human monoclonal antibodies , 2012, Nature Biotechnology.

[16]  J. D. Capra,et al.  Rapid generation of fully human monoclonal antibodies specific to a vaccinating antigen , 2009, Nature Protocols.

[17]  Andrew D. Ellington,et al.  Antibody Repertoires in Humanized NOD-scid-IL2Rγnull Mice and Human B Cells Reveals Human-Like Diversification and Tolerance Checkpoints in the Mouse , 2012, PloS one.

[18]  Sean A Beausoleil,et al.  A proteomics approach for the identification and cloning of monoclonal antibodies from serum , 2012, Nature Biotechnology.

[19]  Hongmei Yang,et al.  Frequencies of human influenza-specific antibody secreting cells or plasmablasts post vaccination from fresh and frozen peripheral blood mononuclear cells. , 2009, Journal of immunological methods.

[20]  Hiroyuki Kishi,et al.  Single-cell microarray for analyzing cellular response. , 2005, Analytical chemistry.

[21]  Veronica Sanchez-Freire,et al.  Microfluidic single-cell real-time PCR for comparative analysis of gene expression patterns , 2012, Nature Protocols.

[22]  A. Chaffotte,et al.  Measurements of the true affinity constant in solution of antigen-antibody complexes by enzyme-linked immunosorbent assay. , 1985, Journal of immunological methods.

[23]  Marie-Paule Lefranc,et al.  IMGT/V-QUEST: the highly customized and integrated system for IG and TR standardized V-J and V-D-J sequence analysis , 2008, Nucleic Acids Res..

[24]  I. Barnea,et al.  Antibody internalization studied using a novel IgG binding toxin fusion. , 2007, Journal of immunological methods.

[25]  P. Lipsky,et al.  Secondary Immunization Generates Clonally Related Antigen-Specific Plasma Cells and Memory B Cells , 2010, The Journal of Immunology.

[26]  T. Sugiyama,et al.  Cell‐microarray analysis of antigen‐specific B‐cells: Single cell analysis of antigen receptor expression and specificity , 2007, Cytometry. Part A : the journal of the International Society for Analytical Cytology.

[27]  Stephen L. Hauser,et al.  Naive antibody gene-segment frequencies are heritable and unaltered by chronic lymphocyte ablation , 2011, Proceedings of the National Academy of Sciences.

[28]  J. C. Love,et al.  Screening individual hybridomas by microengraving to discover monoclonal antibodies , 2009, Nature Protocols.

[29]  Helene Andersson-Svahn,et al.  PCR amplification and genetic analysis in a microwell cell culturing chip. , 2009, Lab on a chip.

[30]  Samuel Aparicio,et al.  High-throughput microfluidic single-cell RT-qPCR , 2011, Proceedings of the National Academy of Sciences.

[31]  Hiroyuki Kishi,et al.  Single lymphocyte analysis with a microwell array chip , 2007, Cytometry. Part A : the journal of the International Society for Analytical Cytology.

[32]  Marie-Paule Lefranc,et al.  IMGT/3Dstructure-DB and IMGT/DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF , 2009, Nucleic Acids Res..

[33]  H. Nakasone,et al.  Single-cell analysis of T-cell receptor repertoire of HTLV-1 Tax-specific cytotoxic T cells in allogeneic transplant recipients with adult T-cell leukemia/lymphoma. , 2010, Cancer research.

[34]  L A Herzenberg,et al.  Frequent occurrence of identical heavy and light chain Ig rearrangements. , 1997, International immunology.

[35]  M. Nussenzweig,et al.  Differential regulation of self-reactivity discriminates between IgG+ human circulating memory B cells and bone marrow plasma cells , 2011, Proceedings of the National Academy of Sciences.