SAT0031 Detection and isolation of antigen specific b cells in patients with rheumatoid arthritis

Background The majority of autoimmune diseases, e.g. rheumatoid arthritis (RA), are characterized by autoantibodies that are produced by B cells. We have identified several novel post-translationally modified epitopes in collagen type II (CII), a major cartilage constituent that are autoreactive. In mouse models, autoantibodies that bind to these epitopes can either induce or protect against inflammatory arthritis. However, very little is known about the frequencies or phenotype the B cells that produce these autoantibodies in humans. Objectives The aim of this project is to study the B cells that produce autoantibodies reactive to CII-epitopes in patients with RA. Methods Clinical data and peripheral blood were collected from patients with RA (n=100). Titres of CII-reactive serum-autoantibodies were determined by Luminex. The different subsets of B cells that expressed a CII-reactive B cell receptor (BCR) were analysed and isolated in patients with positive (n=10) or negative (n=5) titres for autoantibodies recognizing the CII-epitopes cyc48 (CII-F4-R-Cit) and cyc49 (CII-F4-Cit-R) as well as in healthy controls (n=3) using flow cytometry. Results In patients with detectable autoantibodies to cyc48 and cyc49, their titres were 13761±3815 and 5585±1992 (mean ± SEM), respectively. The corresponding titres in the patients negative for these autoantibodies were 122±55 and 234±75. In autoantibody-positive patients, the frequencies of cells expressing a BCR reactive for cyc48 and cyc49 in different subsets were: transitional cells 0.88±0.18% and 1.33±0.33%; naive cells 0.09±0.0008% and 0.13±0.06 and memory cells 0.31±0.25 and 0.30±0.12%. All frequencies were well above those detected in patients without cyc48/49 titres and in healthy controls. A higher proportion (p=0.03) of the cyc48/49 positive patients were treated with methotrexate compared to cyc48/49 negative patients, no other clinical differences were recorded. CII-reactive single B cells were isolated in order to clone the BCR Conclusions In patients with manifest RA there is a relatively high and detectable frequency of transitional B cells that express a joint-specific BCR, which suggests that the deletion of autoreactive B cells in the bone marrow in RA patients is defective. As there is a decrease in the proportion of naive CII-specific B cells, a substantial amount of the autoreactive cells are deleted in the periphery. However, peripheral B-cell tolerance is incomplete, as CII-specific B cells are enriched in the pool of memory B cells. Disclosure of Interest None declared