Project Summary/Abstract The evasion of immune-mediated cytotoxicity is a hallmark feature of tumorigenesis. To this end, therapeutic inhibition of the immune checkpoint Programmed Cell Death Protein 1 (PD-1) has shown considerable promise in the management of several malignancies. In pancreatic cancer, however, despite a strong association between expression of the PD-1 ligand (PD-L1) and poor survival, checkpoint inhibitors have shown limited efficacy as a monotherapy. Recent efforts have therefore focused on identifying additional factors in the tumor microenvironment that modulate PD-L1/PD1 expression in hopes of improving drug response. Our group has previously identified stromal-derived Transforming Growth Factor Beta (TGFβ) as a crucial repressor of anti- tumor immune function, particularly with respect to cytotoxic T lymphocytes. Despite these observations, pharmacologic inhibition of TGFβ signaling has also been ineffective in clinical trials. When examining a small cohort of pancreatic cancer patients, we found an inverse association between PD-L1 expression and that of SMAD4, a downstream target of TGFβ signals. In accordance with these observations, exogenous TGFβ1 repressed the acquisition of PD-L1 in vitro. Similarly, neoplastic mouse models with genetic ablation of TGFβ signaling developed increased PD-L1 expression in the pancreas, and failed to mount a full anti-tumor immune response. Using an established model of metastatic pancreatic cancer, we therefore administered pharmacologic inhibitors of PD-1 (Invivomab) and/or TGFBR1 (Galunisertib). While neither monotherapy had an effect, after two months, mice receiving both Invivomab and Galunisertib had no overt evidence of disease. Histologically, the pancreata of these mice had near complete regression of neoplasms, with abnormal tissues displaying increased lymphocyte infiltration, Granzyme deposition, and apoptosis. Combined, these data suggest that TGFβ pathway blockade augments immune checkpoint inhibition, and may be a reasonable approach to overcome drug resistance in the clinic. Given the substantial preclinical efficacy of this approach, it is essential to further understand the means through which TGFβ and PD-L1/PD-1 signals are regulated, as well as the merits to their inhibition in select patient populations. Through the experiments detailed in this proposal, we will gain valuable insight into these events in hopes of extending survival and reducing cancer- associated morbidity in PDAC patients.