Project Summary/Abstract Programmed cell death 1 (PD-1) and its ligands, PD-L1 and PD-L2, are highly expressed in human and murine thymus. Despite this knowledge, the function of the PD-1 signaling pathway during T cell development has been severely understudied. In the past decade, immunotherapies inhibiting the PD-1:PD-L1 axis have produced remarkable improvements in the clinical management of several malignancies, at least in part by restoring T cell receptor (TCR) signaling within the immunosuppressive tumor microenvironment. However, because TCR signaling is essential for thymocyte development, we postulate PD-1/PD-L1 inhibitors may have profound effects on the function and specificity of newly generated T cells. These thymus-specific actions may highlight a key unexplored mechanism by which PD-1 blockade elicits anti-tumor immune responses. Leveraging this pathway for pediatric cancer patients may be particularly beneficial considering their high rate of thymic T cell production. Our preliminary data illustrate that hematopoietic stem cells (HSCs) administered in conjunction with anti-PD-1 therapy expands the T cell pool and helps overcome treatment-resistance in murine glioblastoma. This may suggest anti-PD-1 therapy acts within the thymus to promote the proliferation and maturation of HSC-derived thymocytes. Importantly, pediatric high-grade glioma (HGG) is the prevailing cause of cancer-related death in children which emphasizes the need for new therapies. Thus it is our priority to investigate this novel pathway with the goal of improving the standard of care for pediatric HGG and other childhood tumors. Our objective is to characterize how inhibiting PD-1 signaling modifies thymocyte development in health and HGG. The central hypothesis of this proposal is that PD-1 inhibition increases thymic T cell production and promotes the positive selection, or survival, of tumor-specific TCRs. Aim 1 will determine the impact of PD-1 blockade on the proliferation, selection, and output of developing thymocytes in healthy and glioma-bearing mice. Aim 2 will assess the thymic contribution towards the therapeutic response in the context of PD-1 inhibiton and HSC transfer. This work is significant because no prior study has investigated how thymus-specific PD-1 blockade impacts anti-tumor immune responses. The results from this study have the potential to substantially influence clinical decision making and treatment regimens for pediatric HGG patients. This project is innovative because it will utilize nontransgenic mouse models to more accurately define how PD-1 inhibition affects thymus physiology in health and disease. In summary this proposal will comprehensively characterize how the PD-1 pathway modulates T cell development and will investigate a novel thymic mechanism that may revolutionize our understanding of anti-PD-1 therapy in cancer.