The 3D affinities of the OT-I TCR to foreign and self-antigens predict their 2D affinities and reveal imperfect antigen discrimination

T cells use the T cell antigen receptor (TCR) to discriminate between higher-affinity foreign and lower-affinity self peptide-MHC (pMHC) antigens. The OT-I mouse TCR is widely used to study anti-gen discrimination utilising many pMHCs, including foreign and self antigens. Previous studies sug-gested that OT-I T cells achieve near-perfect discrimination between higher and lower affinity antigens. Moreover, these 3D affinities measured in solution did not correlate with the 2D membrane affinities, suggesting a complex relationship between 3D and 2D affinities. In contrast, other TCRs have shown imperfect antigen discrimination and strong correlations between 3D and 2D affinities. To resolve these discrepancies, we extended a protocol for measuring ultra-low TCR/pMHC affinities to accurately de-termine the 3D affinities of the OT-I TCR binding 19 pMHC complexes. These revised 3D affinities now strongly correlate with the 2D affinities, and accurately predict functional responses. Importantly, we now find that the OT-I TCR exhibits enhanced yet imperfect antigen discrimination, similar to other TCRs, allowing it to detect abnormally high levels of low-affinity self-antigens. Finally, we show that discrimination is highest with low-affinity pMHC ligands, a finding predicted by the kinetic-proofreading model of antigen discrimination. This work underscores the ability of T cells to effectively gauge prox-ies for 3D affinity within the 2D cell-cell interface, with significant implications for the mechanisms underlying antigen discrimination. Lay abstractT cells protect the body from infection by distinguishing between foreign and self molecules. They do this using a specialized receptor called the T cell receptor (TCR), which senses differences in how strongly it binds to foreign versus self molecules. Scientists have used a mouse TCR called OT-I to understand this process, concluding that OT-I T cells exhibit near perfect discrimination between foreign and self antigens based on a sharp affinity threshold. However, conflicting results from experiments on other TCRs hinted at a more complicated picture. In this study, we used an improved method to accurately measure OT-I TCR binding affinity to 19 peptides, including foreign and self antigens. In contrast to previous results and like other TCRs, we found that OT-I T cells display enhanced but im-perfect discrimination, enabling them to be activated by high levels of self antigen. The new data also revealed that the ability of T cells to discriminate antigens is particularly high at low affinities. These findings reconcile apparent discrepancies between the OT-I TCR and other TCRs, and have implications for understanding diseases where T cells often respond to self antigen, such as autoimmunity and cancer. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=149 SRC="FIGDIR/small/632665v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@977df5org.highwire.dtl.DTLVardef@1ce7bd3org.highwire.dtl.DTLVardef@12363cdorg.highwire.dtl.DTLVardef@6d5573_HPS_FORMAT_FIGEXP M_FIG C_FIG