Artikel
Identification and characterization of citrullinated antigen-specific B cells in peripheral blood of patients with rheumatoid arthritis
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Veröffentlicht: | 1. September 2015 |
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Introduction: Immunity to citrullinated antigens is a hallmark of rheumatoid arthritis (RA). We set out to elucidate the biology of this disease-specific immune response by identifying and characterizing citrullinated antigen-specific B cells in peripheral blood of RA patients.
Methods: Differentially labelled streptavidin and extravidin tetramers were conjugated to biotinylated CCP2 or control antigens and used in flow cytometry to identify citrullinated antigen-specific B cells in peripheral blood. Tetramer-positive and -negative B cells were isolated by FACS followed by in vitro culture and analysis of culture supernatants for the presence of ACPA by ELISA. Cells were phenotypically characterized by flow cytometry.
Results: By combining differentially labelled CCP2 tetramers, we successfully separated citrullinated antigen-specific B cells from non-specific background signals. Isolated tetramer-positive B cells, but not tetramer-negative cells, produced large amounts of ACPA upon in vitro stimulation. Phenotypic analyses revealed that citrullinated antigen-specific B cells displayed markers of class-switched memory B cells and plasmablasts, whereas only few cells displayed a naïve phenotype. The frequency of tetramer-positive cells was high (up to 1/500 memory B cells with a median of 1/12,500 total B cells) and correlated with ACPA serum titres and spontaneous ACPA production in culture.
Conclusion: We developed a unique technology to identify and isolate citrullinated antigen-specific B cells from peripheral blood of RA patients. Most cells have a memory phenotype, express IgA or IgG and are present in relatively high frequencies. These data pave the path for a direct and detailed molecular characterization of ACPA-expressing B cells and could lead to the identification of novel therapeutic targets.