No abstract

CD8 T-cell response efficiency critically depends on the TCR binding strength to peptide-MHC, i.e., the TCR binding avidity. A current challenge in onco-immunology lies in the evaluation of vaccine protocols selecting for tumor-specific T-cells of highest avidity, offering maximal immune protection against tumor cells and clinical benefit. Here, we investigated the impact of peptide and CpG/adjuvant doses on the quality of vaccine-induced CD8 T-cells in relation to binding avidity and functional responses in treated melanoma patients. Using TCR-pMHC binding avidity measurements combined to phenotype and functional assays, we performed a comprehensive study on representative tumor antigen-specific CD8 T-cell clones (n = 454) from seven patients vaccinated with different doses of Melan-A/ELA peptide (0.1 mg vs. 0.5 mg) and CpG-B adjuvant (1-1.3 mg vs. 2.6 mg). Vaccination with high peptide dose favored the early and strong in vivo expansion and differentiation of Melan-A-specific CD8 T-cells. Consistently, T-cell clones generated from those patients showed increased TCR binding avidity (i.e., slow off-rates and CD8 binding independency) readily after 4 monthly vaccine injections (4v). In contrast, the use of low peptide or high CpG-B doses required 8 monthly vaccine injections (8v) for the enrichment of anti-tumor T-cells with high TCR binding avidity and low CD8 binding dependency. Importantly, the CD8 binding-independent vaccineinduced CD8 T-cells displayed enhanced functional avidity, reaching a plateau of maximal function. Thus, T-cell functional potency following peptide/CpG/IFA vaccination may not be further improved beyond a certain TCR binding avidity limit. Our results also indicate that while high peptide dose vaccination induced the early selection of Melan-A-specific CD8 T-cells of increased functional competence, continued serial vaccinations also promoted such high-avidity T-cells. Overall, the systematic assessment of T-cell binding avidity may contribute to optimize vaccine design for improving clinical efficacy.

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-1157-165 displayed longer TCR-ligand interactions than T-cells specific for the tumor/differentiation antigen Melan-A26-35, and both these tumor-specific TCRs exhibited significantly lower avidities than those specific for persistent herpes virus antigens (CMV/pp65495-503, EBV/BMFL1259-267). 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 Disclosures No relevant conflicts of interest to declare.