TCR-Ligand Dissociation-Rate, a Reliable and Determining Parameter, Identifying Tumor-Reactive CD8 T Cells of High Avidity and Functional Potency

Background & Aims: Kinetics of T-cell receptor (TCR) binding to peptide-MHC (pMHC) contribute to T-cell activation and various cellular responses. However, due to technical reasons, the characterization of T-cells is usually only done with functional assays, without assessment of TCR-pMHC binding kinetics, thus limiting the information on the overall quality and clinical relevance of TCR-ligand binding properties. With the aim to broadly evaluate possible correlations between T-cell function and TCR binding kinetics in humans, we undertook a large-scale analysis of combined multiple functional readouts (i.e. CD107a degranulation, cytokine production and proliferation) and off-rate measurements by NTAmers (ref. 1) to characterize tumor- and virus-specific CD8 T-cell clones (n=400) isolated from melanoma patients and healthy donors.

Results: Our results show that, within an antigen-specific repertoire, TCR-ligand off-rates accurately predicted single-functional and poly-functional avidities (i.e. ligand potency) as well as co-receptors expression/modulation of CD8 T-cells. Importantly, the TCR-pMHC dissociation-rate represented a constant and highly reliable bio-physical parameter, contrasting to functional avidity which depended on the activation status of the T-cells following re-stimulation. Remarkably, we found that the TCR-ligand off-rate repertoire depended on the antigenic-specificity, as CD8 T-cells specific for the cancer-testis antigen NY-ESO-1_157-165 displayed longer TCR-ligand interactions than T-cells specific for the tumor/differentiation antigen Melan-A_26-35, and both these tumor-specific TCRs exhibited significantly lower avidities than those specific for persistent herpes virus antigens (CMV/pp65_495-503, EBV/BMFL1_259-267).

Conclusions: Our data from a large library of human T-cell clones demonstrate that the TCR-ligand dissociation-rate is a stable/reliable and determining parameter of T-cell responsiveness, arguing that it should be systematically assessed for meaningful monitoring of human T-cell responses. Furthermore, our TCR-ligand off-rate comparisons highlighted strong binding differences between non-self/virus- and self/tumor-specific repertoires. Our data from individual clones suggest that the tumor-specific repertoires contains rare but nevertheless promising TCRs with sufficiently slow off-rate and high functional avidity to potentially confer protective immune responses against cancer.

(ref. 1) Hebeisen et al., Cancer Res, 75(10):1983, 2015