Antigen-specificity and DTIC before peptide-vaccination differently shape immune-checkpoint expression pattern, anti-tumor functionality and TCR repertoire in melanoma patients

We have recently described that DNA-damage inducing drug DTIC, administered before peptide (Melan-A and gp100)-vaccination, improves anti-tumor CD8(+) Melan-A-specific T-cell functionality, enlarges the Melan-A(+) TCR repertoire and impacts the overall survival of melanoma patients. To identify whether the two Ags employed in the vaccination differently shape the anti-tumor response, herein we have carried out a detailed analysis of phenotype, anti-tumor functionality and TCR repertoire in treatment-driven gp100-specific CD8(+) T cells, in the same patients previously analyzed for Melan-A. We found that T-cell clones isolated from patients treated with vaccination alone possessed an Early/intermediate differentiated phenotype, whereas T cells isolated after DTIC plus vaccination were late-differentiated. Sequencing analysis of the TCRBV chains of 29 treatment-driven gp100-specific CD8(+) T-cell clones revealed an oligoclonal TCR repertoire irrespective of the treatment schedule. The high anti-tumor activity observed in T cells isolated after chemo-immunotherapy was associated with low PD-1 expression. Differently, T-cell clones isolated after peptide-vaccination alone expressed a high level of PD-1, along with LAG-3 and TIM-3, and were neither tumor-reactive nor polyfunctional. Blockade of PD-1 reversed gp100-specific CD8(+) T-cell dysfunctionality, confirming the direct role of this co-inhibitory molecule in suppressing anti-tumor activity, differently from what we have previously observed for Melan-A(+)CD8(+) T cells, expressing PD-1 but highly functional. These findings indicate that the functional advantage induced by combined chemo-immunotherapy is determined by the tumor antigen nature, T-cell immune-checkpoints phenotype, TCR repertoire diversity and anti-tumor T-cell quality and highlights the importance of integrating these parameters to develop effective immunotherapeutic strategies.