Objective To investigate the occurrence and frequency of anti-citrullinated protein antibodies (ACPA) to cyclic citrullinated type II collagen (COL2) epitope with a capacity to bind joint cartilage. Methods Luminex immunoassay was used to analyze serum antibody reactivity to 10 COL2-citrullinated peptides (ACC10) and corresponding arginine peptide controls in rheumatoid arthritis (RA), osteoarthritis (OA), and healthy individuals’ cohorts. Top ten “promiscuous” sera (cross-reactive with all ACC10) and top ten “private” sera (restrictedly reactive with one ACC10 peptide) from RA and OA cohorts were selected. Enzyme-linked immunosorbent assay (ELISA) was used to detect response to native COL2. Sera were analyzed with naive and arthritic joints from DBA/1J mice by immunohistochemistry, using monoclonal ACPAs and COL2 reactive antibodies with human Fc as comparison. Staining specificity was confirmed with C1 (a major antibody epitope on COL2) mutated mice and competitive blocking with epitope-specific antibodies. Results All patient sera bound ACC10 compared with control peptides but very few (3/40) bound native triple-helical COL2. Most sera (27/40) specifically bound to arthritic cartilage, whereas only one private RA serum bound to healthy cartilage. Despite very low titers, private sera from both RA and OA showed an epitope-specific response, documented by lack of binding to cartilage from C1-mutated mice and blocking binding to wild-type cartilage with a competitive monoclonal antibody. As a comparison, monoclonal ACPAs visualized typical promiscuous, or private reactivity to joint cartilage and other tissues. Conclusion ACPA from RA and OA sera, reactive with citrullinated non-triple-helical COL2 peptides, can bind specifically to arthritic cartilage.

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ObjectivesTo identify the arthritogenic B cell epitopes of glucose-6-phosphate isomerase (GPI) and their association with rheumatoid arthritis (RA).MethodsIgG response towards a library of GPI peptides in patients with early RA, pre-symptomatic individuals and population controls, as well as in mice, were tested by bead-based multiplex immunoassays and ELISA. Monoclonal IgG were generated, and the binding specificity and affinity were determined by ELISA, gel size exclusion chromatography, surface plasma resonance and X-ray crystallography. Arthritogenicity was investigated by passive transfer experiments. Antigen-specific B cells were identified by peptide tetramer staining.ResultsPeptide GPI293-307was the dominant B cell epitope in K/BxN and GPI-immunised mice. We could detect B cells and low levels of IgM antibodies binding the GPI293-307epitopes, and high affinity anti-GPI293-307IgG antibodies already 7 days after GPI immunisation, immediately before arthritis onset. Transfer of anti-GPI293-307IgG antibodies induced arthritis in mice. Moreover, anti-GPI293-307IgG antibodies were more frequent in individuals prior to RA onset (19%) than in controls (7.5%). GPI293-307-specific antibodies were associated with radiographic joint damage. Crystal structures of the Fab–peptide complex revealed that this epitope is not exposed in native GPI but requires conformational change of the protein in inflamed joint for effective recognition by anti-GPI293-307antibodies.ConclusionsWe have identified the major pathogenic B cell epitope of the RA-associated autoantigen GPI, at position 293–307, exposed only on structurally modified GPI on the cartilage surface. B cells to this neo-epitope escape tolerance and could potentially play a role in the pathogenesis of RA.