PROJECT ABSTRACT Immune checkpoint therapies block inhibitory receptors on T cells to augment anti-tumor immune responses. The Programmed cell Death-1 (PD-1) pathway is a critical inhibitory checkpoint for T cells, and antibodies blocking PD-1 promote immune-mediated identification and clearance of malignant cells. Cancer immunotherapies, including anti-PD-1 antibodies, represent a powerful therapeutic paradigm because they are applicable to a wide variety of tumors and can produce durable clinical responses. Unfortunately, only a fraction of patients demonstrates clinical benefit from current agents. As such, there is an urgent need to develop therapeutics that more effectively target PD-1 signaling as well as other checkpoint inhibitors. We have employed phosphoproteomic protocol to identify proteins that regulate downstream signaling of PD-1. With this approach, we identified the protein PAG as an effector of PD-1 immune checkpoint signaling. We have discovered that PAG is required for PD-1 signaling and inhibition of T cell function and our preliminary genetic and biochemical studies confirm PAG as a target for reversing T cell inhibition. The immediate goal of this proposal is to validate PAG as checkpoint inhibitor candidate. Our long-term goal is to translate our findings and to generate novel immunotherapies for patients with malignancies. We will accomplish these goals with the following aims: Aim 1. Define the mechanism by which PD-1 induces PAG phosphorylation to promote immune checkpoint activation. Using high resolution microscopy, we will determine how PD-1 and PAG cluster at the immunological synapse. Using mutagenesis, we will define the requirement for specific tyrosine within the cytoplasmic region of PAG for PD-1 function and immune synapse clustering. Aim 2. To assess PAG-mediated immune inhibitory potential, in vivo. We will assess tumor growth in syngeneic models using PAG knockout mice. Aim 3. To develop anti-PAG functional antibodies. We will develop strategies to target PAG in vivo. Together, these studies will uncover the mechanism by which PAG mediates inhibitory signals in T cells and its value as a novel target for immune checkpoint blockade.