Murine T-cell receptor OT-I exhibits imperfect discrimination between foreign and self-antigens.

T cells use their T-cell receptors (TCRs) to discriminate between higher-affinity foreign and lower-affinity self-peptide-MHC (pMHC) antigen complexes. The OT-I mouse TCR is widely used to study antigen discrimination between foreign and self-pMHC antigens, and previous work suggested it achieved near-perfect discrimination between higher- and lower-affinity antigens. However, other TCRs show imperfect discrimination. To resolve these discrepancies, we developed in this study a protocol for measuring ultra-low TCR-pMHC binding affinities to determine the 3D solution affinities of OT-I TCR for 19 pMHCs. These revised 3D affinities now strongly correlate with 2D membrane affinities and predict T-cell functional responses. Our results indicate that OT-I exhibits enhanced yet imperfect discrimination, similar to other TCRs, explaining how T cells can detect abnormally high levels of low-affinity self-antigens. We also show that OT-I discrimination is consistent with the kinetic proofreading model, which highlights that discrimination is most effective for low-affinity pMHC ligands. Our work underscores the ability of T cells to gauge proxies for 3D affinity within the 2D interface, with implications for the mechanisms underlying antigen discrimination.