Adenosine Uptake through the Nucleoside Transporter ENT1 Suppresses Antitumor Immunity and T-cell Pyrimidine Synthesis

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. Although 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. Blocking or deleting host ENT1 significantly enhanced CD8+ T-cell-dependent antitumor 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, IFNγ, IL2, TNFα, 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 cocultured with cognate antigen/HLA-matched melanoma cells. Mechanistically, ENT1-mediated adenosine uptake inhibited the activity of phosphoribosyl pyrophosphate synthetase in activated T cells, thereby suppressing production of uridine 5'-monophosphate and its derivatives required for DNA and RNA synthesis. In summary, this study identified ENT1-mediated adenosine uptake as an important mechanism of adenosine-mediated immunosuppression and pyrimidine starvation that can be targeted to enhance antitumor T-cell responses. Significance: ENT1 is a potential therapeutic target to overcome immunosuppression induced by extracellular adenosine and to increase the activity of PD-1 blockade.