Head and neck squamous cell carcinoma (HNSC) related to tobacco and alcohol consumption is aggressive and responds poorly to available therapy. Even the new PD-1/PD-L1 immune checkpoint blockade is insufficient to control progression due to other highly immunosuppressive conditions in the tumor microenvironment (TME). Compelling evidence implicates the ecto-5'-nucleotidase (NT5E) CD73 as another important immune checkpoint, as high CD73 expression in the TME of many tumors, including HNSC, is linked to poor patient survival. Several consistent TME conditions, such as hypoxia and treatment, cause an increase in extracellular (e)ATP, and CD73 serves at the last, rate-limiting step in concert with CD39 and other ectoenzymes, to convert eATP to adenosine (eADO). eADO activates adenosine receptor (AR) A2AR to suppress T cell activation, while A2BR promotes immunosuppression via cancer-associated fibroblasts (CAF) and myeloid cells present in the TME. Studies, including our own, show that CD73 and A2BR, and to a lesser extent A2AR, are expressed in the highly immunosuppressive TME of progressive HNSC of both clinical and preclinical samples, suggesting their role in the immunosuppression. However, the contributions of molecules downstream of CD73 are poorly understood. Recent rapid advances in spatial analysis of the TME have revealed a critical role of immune effector to target cell distance in generating productive antitumor immunity with impact on clinical outcomes. Because eADO is short-lived, the spatial relationship between CD73 on one hand and A2AR or A2BR on the other hand, is likely to affect the specific AR activated by eADO. We discovered in HNSC clinical specimens that A2AR is rather limited and located remotely from CD73, which supports the hypothesis that the major immunosuppressive impact of CD73-AR immune checkpoint is mediated primarily through the CD73-A2BR axis. Our plan is to: (1) dissect how the CD73-A2BR pathway regulates the immune landscape in the TME of ectopic preclinical HNSC models during progression and treatment; (2) develop effective CD73-ADO-A2BR ICB regimens in preclinical models of orthotopic and in primary carcinogen-induced tongue HNSC; (3) assess the relationships between HNSC patient peripheral blood myeloid precursors, levels and distribution of CD73 and A2BR on myeloid and CAF subsets in the TME, patients’ three-year post-operative outcomes. Impact: The proposed study will identify CD73-A2BR-dependent mechanisms of immunosuppression in HNSC preclinical models and develop translatable therapeutic intervention. The results will provide highly valuable insights into the effects of CD73-A2BR axis on disease progression and the therapeutic potential of blocking it.