Project Summary Pancreatic cancer is the third leading cause of cancer-related deaths in the United States with a 5-year survival rate of 12%. Despite the success of immune-modulating therapies such as immune checkpoint blockade (ICB) in other cancers such as melanoma, single agent ICB is decidedly ineffective in pancreatic ductal adenocarcinoma (PDAC), leaving surgery and palliative chemotherapy the only options for over 80% of patients. CD8 T cells are the key effectors of anti-tumor immune responses that recognize and eliminate early malignant cells and can enforce durable responses that prevent recurrence and metastatic spread through adaptive immune memory. In PDAC, chronic exposure to cellular constituents in the tumor microenvironment (TME) leads to the terminal dysfunction of CD8 T cells, termed “exhaustion”. Exhausted CD8 T cells (TEX) have reduced proliferative capacity, an inability to persist and proliferate, and limited protective capacity compared to functional CD8 T cells which is a major barrier to the success of immunotherapy in PDAC. Stelekati et. al. has recently shown that antagonizing surface checkpoint molecules (such as PD-1) in chronic infection can synergize with overexpression of one micro-RNA (miR), miR-29a, in CD8 T cells to abrogate TEX differentiation and restore antigen-specific cytotoxicity and T cell memory In parallel, the Datta lab at UMMSM has recently uncovered TNF signaling to drive T cell dysfunction in PDAC through a myeloid TNF-TNFR2 dependent manner. The goal of this project is to determine the contribution of TNFR2-miR-29a to CD8 T cell dysfunction in PDAC to generate durable T-cell mediated antitumor activity. I hypothesize that miR-29a regulates TEX differentiation by downregulating immunosuppressive signaling and provides a promising intervention to overcome resistance to immune checkpoint blockade in PDAC. Using the canonical KPC model of PDAC engineered to express the neoantigen Ova, I retrovirally overexpress (OE) miR-29a in antigen-specific OT-1 CD8 T cells and observed improved tumor clearance, along with altered phenotypic states. Consequently, in this project I aim to: (1) alter the differentiation of terminally exhausted cells to a progenitor and memory-like phenotype that express Ly108, TCF-1, and CD127, which has shown to be the subset responsive to a-PD-1 checkpoint blockade therapy. I will then validate the translational importance of these findings by developing an effective combination strategy using miR-29a in mice; (2) determine the precise contribution of myeloid-derived TNF-TNFR2 signaling upstream of terminal TEX differentiation in the PDAC tumor microenvironment. By validating this novel mechanistic link between myeloid immunosuppression and terminal T cell exhaustion, this work may uncover tolerogenic circuitries characteristic of immunosuppressive TME’s such as PDAC. Taken together, this work will enhance our understanding of T cell dysfunction in the PDAC TME and elucidate a nov...