It is believed that the scope of promising cancer immunotherapy applications is limited because anti-tumor T cells are inhibited in the microenvironment of solid tumors. The overall goal of this proposal is to understand the mechanism of tumor protection and to accomplish the targeted elimination of tumor-protecting immunosuppressive molecules. This is crucial in order to render novel tumor immunotherapies more effective. The central hypothesis of this proposal is that tumor cells in hypoxic cancerous tissues, protected from immune damage because anti-tumor T cells are inactivated by the combined action of at least two hypoxia-dependent mechanisms: (1) extracellular adenosine produced by the hypoxic tumor inhibits T cells via signaling through their surface A2A and A2B adenosine receptors; and (2) the hypoxia-inducible transcription factor 1-alpha (HIF-1alpha) which governs the expression of genes that contribute to the immunosuppressive effects of tumor hypoxia. Our preliminary results - findings of much improved rejection of large tumors in mice with inactivated A2A adenosine receptor - strongly support this hypothesis. Here we plan to further clarify this mechanism of tumor protection by using several recently developed unique mice with T cell-specific deletion of A2A receptor, A2B receptor, and HIF-1alpha genes. Both immunogenic and weakly immunogenic tumors will be tested in different models of CD8+ T cell-mediated rejection by endogenous and adoptively transferred anti-tumor T cells. We expect that targeted deletion of these molecules will render anti-tumor T cells resistant to inhibition in the tumor microenvironment and, thus, facilitate the destruction of tumors. The proposed studies may allow dissection of the complex tumor microenvironment by reducing it to a manageable genetic, immunological, and biochemical analysis of only three molecules - A2A receptor, A2B receptor, and HIF-1alpha- and may also offer a novel and feasible strategy to improve cancer immunotherapy.