ABSTRACT
Acute systemic lupus erythematosus (SLE) courses with surges of antibody-secreting cells (ASCs) whose origin, diversity and contribution to serum autoantibodies remain unknown. Here, deep sequencing, proteomic profiling of autoantibodies and single-cell analysis demonstrated highly diversified ASCs punctuated by clones expressing the variable heavy-chain region VH4-34 that produced dominant serum autoantibodies. A fraction of ASC clones contained autoantibodies without mutation, a finding consistent with differentiation outside the germinal centers. A substantial ASC segment was derived from a distinct subset of newly activated naive cells of considerable clonality that persisted in the circulation for several months. Thus, selection of SLE autoreactivities occurred during polyclonal activation, with prolonged recruitment of recently activated naive B cells. Our findings shed light on the pathogenesis of SLE, help explain the benefit of agents that target B cells and should facilitate the design of future therapies.
Subject(s)
Antibody Diversity/immunology , Antibody-Producing Cells/immunology , Autoantibodies/immunology , Cell Proliferation , Lupus Erythematosus, Systemic/immunology , Acute Disease , Amino Acid Sequence , Antibody Diversity/genetics , Antibody-Producing Cells/metabolism , Autoantibodies/genetics , Autoantibodies/metabolism , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Base Sequence , Clone Cells/immunology , Clone Cells/metabolism , Flow Cytometry , Humans , Immunoglobulin G/blood , Immunoglobulin G/genetics , Immunoglobulin G/immunology , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Heavy Chains/immunology , Immunoglobulin Heavy Chains/metabolism , Immunoglobulin Variable Region/genetics , Immunoglobulin Variable Region/immunology , Immunoglobulin Variable Region/metabolism , Influenza Vaccines/immunology , Lupus Erythematosus, Systemic/genetics , Lupus Erythematosus, Systemic/metabolism , Molecular Sequence Data , Proteome/analysis , Proteome/immunology , Proteomics/methods , Sequence Homology, Amino Acid , Single-Cell Analysis/methods , Tandem Mass Spectrometry , Tetanus Toxoid/immunologyABSTRACT
We describe a proteomics approach that identifies antigen-specific antibody sequences directly from circulating polyclonal antibodies in the serum of an immunized animal. The approach involves affinity purification of antibodies with high specific activity and then analyzing digested antibody fractions by nano-flow liquid chromatography coupled to tandem mass spectrometry. High-confidence peptide spectral matches of antibody variable regions are obtained by searching a reference database created by next-generation DNA sequencing of the B-cell immunoglobulin repertoire of the immunized animal. Finally, heavy and light chain sequences are paired and expressed as recombinant monoclonal antibodies. Using this technology, we isolated monoclonal antibodies for five antigens from the sera of immunized rabbits and mice. The antigen-specific activities of the monoclonal antibodies recapitulate or surpass those of the original affinity-purified polyclonal antibodies. This technology may aid the discovery and development of vaccines and antibody therapeutics, and help us gain a deeper understanding of the humoral response.
