PROJECT SUMMARY The goal of this project is to evaluate novel therapeutics that block PSGL-1 (P-selectin glycoprotein-1), which we identified as a key inhibitory receptor (IR) that is expressed on T cells, and address the impact(s) on immune cell functions in the tumor microenvironment (TME). PSGL-1 is a conserved ligand for the selectin family of adhesion molecules, P, E, and L, that is expressed on most hematopoietic cells and regulates leukocyte migration when fully glycosylated. T cells are the only cells that express the non-selectin binding form of PSGL- 1 that serves as a receptor for the lymph node chemokines, CCL19 and CCL21, and the PD-L1 homologue, VISTA (B7-H5). We showed that PSGL-1-deficient (Selplg-/-) CD8 T cells fail to acquire the hallmarks of exhaustion compared to their wild-type (WT) counterparts after infection with chronic lymphocytic choriomeningitis virus and significantly limit tumor growth in two murine models of melanoma, one that is nonresponsive to anti-PD-1 treatment. We identified that TILs from Selplg-/- mice have greater anti-tumor function, including cytotoxicity and cytokine production. Moreover, expression of multiple inhibitory receptors (IRs) that distinguish exhausted T cells (TEX) were downregulated on Selplg-/- T cells, including PD-1, BTLA, CD160, LAG3, and TIM3. Notably, PSGL-1 ligation on naïve T cells (TN) and TEX by an agonist mAb in the context of T cell receptor stimulation induced/augmented expression of multiple IRs, underscoring an integral connection of PSGL-1 to immune inhibitory pathways and suggesting a link to the recently identified inhibitory gene module that regulates the coordinated transcription of IRs. We identified that a monovalent Fab of the same agonist mAb dramatically improved T cell responses as did treatment with a rPSGL-1 fusion protein, indicating a capacity to block PSGL-1 function. Notably, blocking PSGL-1 did not lead to widespread inflammation or toxicity. We hypothesize that PSGL-1 blockade has the potential to augment anti-tumor T cell responses by limiting expression levels of multiple IRs, and alter immunosuppression in the TME by impacting other immune cells. We propose to evaluate the effects of PSGL-1 blockade on mouse and human T cell anti-tumor responses using rPSGL-1 and Fab anti-PSGL-1 as comparators to novel mouse and human anti-PSGL-1 blocking mAbs. With these tools, we will analyze the impacts of blocking PSGL-1 on IR expression and functions of T cells and immune cells in the TME, as well as potential synergy with anti-PD-1. In the context of these studies, we will assess the role of VISTA as the relevant PSGL-1 ligand in the TME. These studies will address the potential of targeting PSGL-1 for reversal of T cell exhaustion, induction of anti-tumor immunity, and translational potential.