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Ag tetramers prepared from linear epitopes represent a promising strategy for the identification of small subsets of Ag-reactive immune cells. This is challenging given the requirement for identification and validation of linear epitopes and the complexity of autoantibody responses, including the broad spectrum of autoantibody specificities and the contribution of isotype to pathogenicity. Therefore, we tested a two-tiered peptide microarray approach, coupled with epitope mapping of known autoantigens, to identify and characterize autoepitopes using the BXD2 autoimmune mouse model. Microarray results were verified through comparison with established age-associated profiles of autoantigen specificities and autoantibody class switching in BXD2 and control (C57BL\/6) mice and high-throughput ELISA and ELISPOT analyses of synthetic peptides. Tetramers were prepared from two linear peptides derived from two RNA-binding proteins (RBPs): lupus La and 70-kDa U1 small nuclear ribonucleoprotein. Flow cytometric analysis of tetramer-reactive B cell subsets revealed a significantly higher frequency and greater numbers of RBP-reactive marginal zone precursor, transitional T3, and PDL-2+CD80+ memory B cells, with significantly elevated CD69 and CD86 observed in RBP+ marginal zone precursor B cells in the spleens of BXD2 mice compared with C57BL\/6 mice, suggesting a regulatory defect. This study establishes a feasible strategy for the characterization of autoantigen-specific B cell subsets in different models of autoimmunity and, potentially, in humans.<\/jats:p>","DOI":"10.4049\/jimmunol.1402335","type":"journal-article","created":{"date-parts":[[2015,4,18]],"date-time":"2015-04-18T02:21:39Z","timestamp":1429323699000},"page":"5022-5034","source":"Crossref","is-referenced-by-count":26,"title":["General Approach for Tetramer-Based Identification of Autoantigen-Reactive B Cells: Characterization of La- and snRNP-Reactive B Cells in Autoimmune BXD2 Mice"],"prefix":"10.1093","volume":"194","author":[{"given":"Jennie A","family":"Hamilton","sequence":"first","affiliation":[{"name":"Division of Clinical Immunology and Rheumatology, Department of Medicine, University of Alabama at Birmingham , Birmingham, AL 35294"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jun","family":"Li","sequence":"additional","affiliation":[{"name":"Division of Clinical Immunology and Rheumatology, Department of Medicine, University of Alabama at Birmingham , Birmingham, AL 35294"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qi","family":"Wu","sequence":"additional","affiliation":[{"name":"Division of Clinical Immunology and Rheumatology, Department of Medicine, University of Alabama at Birmingham , Birmingham, AL 35294"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"PingAr","family":"Yang","sequence":"additional","affiliation":[{"name":"Division of Clinical Immunology and Rheumatology, Department of Medicine, University of Alabama at Birmingham , Birmingham, AL 35294"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bao","family":"Luo","sequence":"additional","affiliation":[{"name":"Division of Clinical Immunology and Rheumatology, Department of Medicine, University of Alabama at Birmingham , Birmingham, AL 35294"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hao","family":"Li","sequence":"additional","affiliation":[{"name":"Division of Clinical Immunology and Rheumatology, Department of Medicine, University of Alabama at Birmingham , Birmingham, AL 35294"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"John E","family":"Bradley","sequence":"additional","affiliation":[{"name":"Division of Clinical Immunology and Rheumatology, Department of Medicine, University of Alabama at Birmingham , Birmingham, AL 35294"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Justin J","family":"Taylor","sequence":"additional","affiliation":[{"name":"Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center , Seattle, WA 98109"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Troy D","family":"Randall","sequence":"additional","affiliation":[{"name":"Division of Clinical Immunology and Rheumatology, Department of Medicine, University of Alabama at Birmingham , Birmingham, AL 35294"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"John D","family":"Mountz","sequence":"additional","affiliation":[{"name":"Division of Clinical Immunology and Rheumatology, Department of Medicine, University of Alabama at Birmingham , Birmingham, AL 35294"},{"name":"Department of Medicine, Birmingham, Alabama VA Medical Center , Birmingham, AL 35233"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hui-Chen","family":"Hsu","sequence":"additional","affiliation":[{"name":"Division of Clinical Immunology and Rheumatology, Department of Medicine, University of Alabama at Birmingham , Birmingham, AL 35294"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2015,5,15]]},"reference":[{"key":"2025030706220089100_r1","doi-asserted-by":"crossref","first-page":"3428","DOI":"10.1172\/JCI23587","article-title":"Identification of autoantibody clusters that best predict lupus disease activity using glomerular proteome arrays","volume":"115","author":"Li","year":"2005","journal-title":"J. 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