TCR-ligand dissociation rate is a robust and stable biomarker of CD8+ T cell potency.

Despite influencing many aspects of T cell biology, the kinetics of T cell receptor (TCR) binding to peptide-major histocompatibility molecules (pMHC) remain infrequently determined in patient monitoring or for adoptive T cell therapy. Using specifically designed reversible fluorescent pMHC multimeric complexes, we performed a comprehensive study of TCR-pMHC off-rates combined with various functional assays on large libraries of self/tumor- and virus-specific CD8+ T cell clones from melanoma patients and healthy donors. We demonstrate that monomeric TCR-pMHC dissociation rates accurately predict the extent of cytotoxicity, cytokine production, polyfunctionality, cell proliferation, activating/inhibitory receptor expression, and in vivo antitumor potency of naturally occurring antigen-specific CD8+ T cells. Our data also confirm the superior binding avidities of virus-specific T cells as compared with self/tumor-specific T cell clonotypes (n > 300). Importantly, the TCR-pMHC off-rate is a more stable and robust biomarker of CD8+ T cell potency than the frequently used functional assays/metrics that depend on the T cell's activation state, and therefore show major intra- and interexperimental variability. Taken together, our data show that the monomeric TCR-pMHC off-rate is highly useful for the ex vivo high-throughput functional assessment of antigen-specific CD8+ T cell responses and a strong candidate as a biomarker of T cell therapeutic efficacy.