Adenosine uptake through equilibrative nucleoside transporter-1 suppresses antitumor immunity by pyrimidine starvation

Immunosuppression by adenosine is an important cancer immune checkpoint. Extracellular adenosine signals through specific receptors and can be transported across the cell membrane through nucleoside transporters. While adenosine receptors are well-known to regulate tumor immunity, the impact of adenosine transporters remains unexplored. In this study, we investigated the effect on tumor immunity of equilibrative nucleoside transporter-1 (ENT1), the major regulator of extracellular adenosine concentrations. Using selective inhibitors and gene targeting approaches, we demonstrated that blocking or deleting host ENT1 significantly enhanced CD8+ T cell-dependent anti-tumor responses. Tumors inoculated into ENT1-deficient mice showed increased infiltration of effector CD8+ T cells with an enhanced cytotoxic transcriptomic profile and significant upregulation of granzyme B, IFN-g, IL-2, TNF-a and CXCL10. ENT1-deficiency was further associated with decreased tumor-infiltrating T regulatory cells and CD206high macrophages, and suppressed CCL17 production. ENT1-deficiency notably potentiated the therapeutic activity of PD-1 blockade. T cells upregulated ENT1 upon activation, and blocking ENT1 enhanced their function when co-cultured with cognate antigen/HLA-matched melanoma cells. Mechanistically, ENT1-mediated adenosine uptake inhibited the activity of phosphoribosylpyrophosphate synthetase (PRPS) in activated T cells, thereby suppressing production of uridine 5′-monophosphate (UMP) and its derivatives required for DNA and RNA synthesis. In summary, our study identified ENT1-mediated adenosine uptake as an important and previously unappreciated mechanism of adenosine-mediated immunosuppression via pyrimidine starvation that can be targeted to enhance antitumor T cell responses. MCA205 tumors were implanted in C57Bl/6 WT and ENT1-deficient mice and CD45+ CD8+ T cells were isolated after 14 days by FACS-based cell sorting. At least 30 000 purified CD8+ T cells per condition (4 ENT1KO samples and 3 WT samples) were stored at -80°C in trizol stabilizing solution and submitted for Illumina next-generation sequencing to the IRIC Genomics Platform.