The molecular basis of T cell receptor recognition of citrullinated tenascin-C presented by HLA-DR4

CD4⁺ T cell autoreactivity against citrullinated (cit) self-epitopes presented by HLA-DRB1 is associated with rheumatoid arthritis (RA) pathogenesis. We understand the molecular bases of T cell receptor (TCR) recognition of cit-fibrinogen, cit-vimentin, and cit-α-enolase epitopes, and the role of citrulline in shaping TCR repertoire usage. Nevertheless, how TCRs recognize other cit-epitopes, including tenascin-C (TNC) and how alternative citrullination positions may modulate the T cell recognition remains unclear. Here, we examined TNC^1014,1016cit peptide, which contains citrullination at position P-1 and P2, to study the underlying TCR-HLA-DRB1^∗04:01-TNC^1014,1016cit molecular interactions. Crystal structure of HLA-DRB1^∗04:01^{TNC1014,1016cit} at 2.4 Å resolution revealed a conserved peptide binding register to the established HLA-DRB1^∗04:01-peptide structures, where both citrullines protruded upward. Next, we determined the crystal structure of a RA patient-derived TRAV35⁺/TRBV10-2⁺ (PB) TCR in complex with HLA-DRB1^∗04:01^{TNC1014,1016cit} at 3.2 Å resolution. The CDR3α loop (¹⁰⁹VGNTN¹¹³) of PB TCR formed a secondary helical conformation at the N-terminus of the peptide binding cleft, allowing extensive interactions between the P-1 and P2 citrullines of TNC^1014,1016cit peptide. Surface plasmon resonance, tetramer staining, and CD69 activation assays revealed that the PB TCR did not cross-react to other RA autoantigens, and the P-1-Cit, P2-Cit, and P5-Tyr of TNC^1014,1016cit are the key determinants underlying the strict specificity of the PB TCR. Collectively, we provide molecular insight into citrullination in modulating TCR recognition.