PROJECT SUMMARY/ABSTRACT Cancer remains the second leading cause of death in the US. Immunotherapy seeks to bolster immune cells’ ability to target malignant cells and has brought immense improvements in the field. One important inhibitory protein in T cells, Programmed Cell Death Protein 1 (PD-1), has become an invaluable target for cancer immunotherapy. While anti-PD-1 antibody therapy is extremely successful in some patients, in many others, it fails to help or causes complications, including cancer hyper-progression and immune-related adverse events. Study of the inhibitory transmembrane protein Phosphoprotein Associated with Glycosphingolipid Rich Microdomains 1 (PAG), a downstream target of PD-1 signaling, will help us better understand the PD-1 pathway, and offer another, perhaps more nuanced, target to potentially improve response rates and/or avoid immune- related adverse events. As a link between lipid-rich/signaling-protein-rich membrane regions and the actin cytoskeleton, PAG is an exciting and novel target for manipulating immune function. Prior therapeutic methods of immune manipulation all disrupt ligand binding or enzyme function. In contrast, innovative use of an anti-PAG antibody to simply disrupt appropriate PAG localization within the synapse could disturb immune synapse architecture. Synapse organization is tightly regulated to prevent inappropriate immune responses, but the precise interaction between cytoskeletal dynamics and synaptic organization is not fully understood. Investigating the role of PAG in this process could provide added clarity. To test the hypothesis that PAG works with actin to control T cell synapse organization and facilitate T cell receptor (TCR) and PD-1 signaling, PAG will be studied in a human T cell line and a murine PAG-knockout model. Methods will include confocal and TIRF microscopy, FRET and proximity ligation assay, flow cytometry, ELISA, adhesion and cytotoxicity assays, and murine tumor models. To understand the contribution of the actin binding domain of PAG on its localization, protein-protein interactions, and T cell activation and effector functions, the primary hypothesis will be examined through the following specific aims: Aim 1. Define the role of the PAG-actin link in driving T cell synapse architecture and stability. Aim 2. Determine the impact of PAG-actin interactions on T cell functions downstream of TCR and PD-1 signaling. Aim 1 will demonstrate the role of PAG in T cells synapse anatomy, and the physical impacts of a PAG-targeting therapy. Aim 2 will illuminate which PD-1 downstream targets are dependent on the PAG-actin link, and provide evidence for whether PAG and PD-1 could serve as good co-targets in cancer therapy regimens. Ultimately, this study will illuminate crucial control mechanisms associated with T cell synapse organization, opening more avenues of targeting the immune synapse. Furthermore, the mentorship and resources in Dr. Adam Mor’s lab and the Columbia MSTP,...