Antigen-specific T-cell frequency and phenotype mirrors disease activity in DRB1*04:04+ rheumatoid arthritis patients

Rheumatoid arthritis (RA) is associated with high-risk HLA class II alleles known as the "RA shared epitope." Among prevalent shared epitope alleles, study of DRB1*04:04 has been limited. To define relevant epitopes, we identified citrullinated peptide sequences from synovial antigens that were predicted to bind to HLA-DRB1*04:04 and utilized a systematic approach to confirm their binding and assess their recognition by CD4 T cells. After confirming the immunogenicity of 13 peptides derived from aggrecan, cartilage intermediate layer protein (CILP), α-enolase, vimentin, and fibrinogen, we assessed their recognition by T cells from a synovial tissue sample, observing measurable responses to 8 of the 13 peptides. We then implemented a multicolor tetramer panel to evaluate the frequency and phenotype of antigen-specific CD4 T cells in individuals with anti-citrullinated protein antibody-positive RA and controls. In subjects with RA, CILP-specific T-cell frequencies were significantly higher than those of other antigens. The surface phenotypes exhibited by antigen-specific T cells were heterogeneous, but Th1-like and Th2-like cells predominated. Stratifying based on disease status and activity, antigen-specific T cells were more frequent and most strongly polarized in RA subjects with high disease activity. In total, these findings identify novel citrullinated epitopes that can be used to interrogate antigen-specific CD4 T cells and show that antigen-specific T-cell frequency is elevated in subjects with high disease activity.