A1.25Visualisation and characterisation of citrullinated antigen-specific B cells from peripheral blood of patients with rheumatoid arthritis

Background and objectivesIn Rheumatoid arthritis (RA), anti-citrullinated protein antibodies (ACPA) represent highly disease-specific biomarkers found in the majority of patients. As ACPA have been implicated in disease pathogenesis, it is of crucial relevance to understand the underlying B cell response. In this context, visualisation of citrullinated antigen-specific B cells would allow for a detailed characterisation of the immune response to better understand its development and maintenance. Unfortunately, visualisation of autoreactive B cells in humans has proven extremely difficult.Materials and methodsUsing the CCP2-antigen and its arginine control variant, we developed a multicolour tetramer-based staining method to visualise citrullinated antigen-specific B cells in peripheral blood of RA patients by flow-cytometry. Specificity of the staining was verified by culturing tetramer-positive and -negative B cells isolated by FACS. Citrullinated antigen-reactive B cells were further phenotyped using cell surface markers associated with developmental and functional B cell characteristics. Finally, the frequency of citrullinated antigen-reactive B cells was correlated to ACPA serum levels and in vitro ACPA production.ResultsThe staining procedure successfully separated citrullinated antigen-reactive B cells from non-specific background signals. Already fourteen FACS-sorted tetramer-positive B cells produced detectable amounts of ACPA, whereas no ACPA production was observed in cultures of up to 5000 tetramer-negative B cells. The majority of citrullinated antigen-reactive B cells had a post-germinal centre memory or plasmablast phenotype. Up to 1 in 200 memory B cells were directed against citrullinated antigens, and their frequency correlated with spontaneous ACPA production in culture and ACPA serum titres in vivo.ConclusionsWe show, for the first time, the specific and reliable identification of citrullinated antigen-specific B cells in high frequencies in peripheral blood of RA patients. The majority of this population has a memory phenotype and closely reflects the dynamics of the in vivo ACPA response. These data provide the basis for a detailed characterisation of this disease-specific immune response on a single cell level and could lead to the identification of novel therapeutic targets.