PROJECT SUMMARY Pancreatic ductal adenocarcinoma (PDAC) is a deadly malignancy characterized by poor response to all existing therapies. Although immunotherapy has shown great promise against multiple deadly cancers, single-agent checkpoint immunotherapy has been largely ineffective in PDAC. This lack of response is in part attributed to its extensive inflammatory, desmoplastic stromal reaction and hypoxic microenvironment. Although tumor hypoxia induces adaptive changes in both cancer cells and the surrounding stroma, most studies on the role of hypoxia in tumorigenesis have focused on cancer cell-intrinsic properties. The impact of hypoxia on stromal cells themselves and their interactions with cancer cells has remained largely unknown. The major cell populations found within the PDAC stroma—macrophages and fibroblasts—are known to be large producers of immunosuppressive factors, some of which can negatively affect cytotoxic CD8+ T cell activity in tumors. CD8+ T cells play an essential role in the anti-tumor response and the efficacy of immunnotherapies relies on the amount of CD8+ T cell infiltration within the tumor bed. Based on our preliminary observation that macrophages and inflammatory fibroblasts are enriched in hypoxic tumor regions while T cells are excluded from hypoxic areas in PDAC, I hypothesize that hypoxia suppresses infiltration and activation of CD8+ T cells by modulating their interactions with macrophages and fibroblasts. In Specific Aim 1, I will determine whether macrophages are critical for hypoxia-mediated CD8+ T cell exclusion and suppression by treating CD8+ T cells with conditioned media derived from macrophage cultures under hypoxia and depleting macrophages in syngeneic orthotopic PDAC tumors. In Specific Aim 2, I will determine how fibroblasts regulate CD8+ T cell recruitment and function in hypoxic tumor regions by blocking a factor secreted from fibroblasts in syngeneic orthotopic PDAC tumors, injecting a hypoxia probe into mice, and assessing CD8+ T cell infiltration and activation in hypoxic and normoxic tumor regions. The results of my studies will further the field’s understanding of how hypoxia drives an immunosuppressive microenvironment via the crosstalk between CD8+ T cells, macrophages, and fibroblasts, and thus aid in the development of effective immunotherapeutic strategies.