Staff Scientist Benaroya Research Institute Seattle, Washington, United States
Abstract Text: The shared epitope amino acid sequence motif in the beta chain of HLA-DR is the primary genetic risk factor for anti-citrullinated protein antibody (ACPA)-positive rheumatoid arthritis (RA). Among common HLA-DRB1 alleles, the shared epitope is found in DRB1*04:01, DRB1*04:04, and DRB1*01:01 proteins. DRB1*04:01-restricted T cell epitopes have been previously found in RA autoantigens such as vimentin, α-enolase, and aggrecan. However, less is known about DRB1*01:01-restricted T cell epitopes in RA. To this end, 244 citrullinated peptides covering all possible citrullination sites in vimentin, α-enolase, aggrecan, fibrinogen-α, and fibrinogen-β were synthesized and tested for binding to DRA-DRB1*01:01 protein in a competition binding assay. Thirty-nine peptides were identified that bound DRA-DRB1*01:01 with an IC50 < 50 µM. The immunogenicity of these peptides was evaluated with 12 blood samples from ACPA-positive RA participants. PBMCs or CD8-depleted PBMCs were stimulated with peptides for 15-18 days and stained with the corresponding DRA-DRB1*01:01 tetramers. CD8-depletion enhanced expansion of tetramer-positive T cells. Two T-cell epitopes from aggrecan and one from fibrinogen-β were detected in 50% or more of the samples. The citrullinated-Agg 553 epitope was most often observed and typically had the most robust expansion. All three predominant T cell epitopes were further verified by isolating tetramer-positive T cell clones. Citrullinated-Agg 553 had previously been shown to be a predominant DRB1*04:01-restricted T cell epitope, thus finding an overlap in predominant T cell epitopes between shared epitope alleles. These results provide a panel of tetramers spanning multiple citrullinated antigens for studying DRB1*01:01-restricted T cell responses in ACPA-positive RA subjects.
