Additional Relevant Details AbstractThe inhibitory receptor CD94-NKG2A is expressed on subsets of NK cells and cytotoxic CD8+ T cells, but not CD4+ T helper cells. It’s ligand is the non-classical MHC class I molecule HLA-E, commonly over-expressed in human cancers. We previously demonstrated that high HLA-E expression in ovarian carcinoma and NSCLC may neutralize the survival benefit of T cell infiltration, suggesting that NKG2A could represent an immune checkpoint that tumors may exploit for immune evasion. We thus examined NKG2A expression on human and mouse Tumor Infiltrating Lymphocyte (TIL) populations and tested blockade of this receptor in mouse tumor models. Multicolor flow cytometry analysis showed higher frequency of NKG2A+CD8+ T cells in TIL samples obtained from biopsies of human head- and neck- carcinomas (n=17) compared to peripheral blood CD8+ T cells. Frequency of NKG2A+TIL was higher in patients with measurable immune reactivity to HPV16-viral antigens than in patients without tumor immunity (14.8% vs 4.7%, respectively). Interestingly, only subsets of the NKG2A+ T cells co-expressed other inhibitory receptors, including PD-1 and TIM3, suggesting the existence of a ‘dedicated’ T cell subset for NKG2A. In our mouse tumor models we observed similar findings and, moreover, demonstrated that therapeutic vaccines comprising tumor antigens increased the frequency of NKG2A+-positive CD8+ TIL up to 80%. Similarly, expression of Qa-1, the mouse homologue of HLA-E, on tumor cells also increased after therapeutic vaccination. As a proof of concept that the NKG2A - Qa-1 axis limits the efficacy of immunotherapy, NKG2A blocking antibodies were applied in the TC-1 model together with therapeutic peptide vaccination. Combination treatment with vaccine and NKG2A blockade significantly improved the therapeutic efficacy compared to the vaccine alone, both in terms of survival (P<0.05, median survival 44 vs 31 days) as well as overall response rate, defined as decreased tumor size of more than 30% (93.8% vs 43.8%, respectively). Similar results were obtained in Qa-1-deficient mice, suggesting that Qa-1 on tumor cells is of importance. In conclusion, inhibitory receptor NKG2A is enriched on CD8+ TIL and functions as an immune checkpoint that restrains therapeutic efficacy of a cancer vaccine.