ABSTRACT – Project 2 Glioblastoma (GBM) elicits severe local and systemic immune dysfunction, including T cell lymphopenia, which has remained unexplained and unaddressed for nearly 40 years. Foundational studies characterizing T cell disappearance from the blood and lymphoid organs of both patients and mice with GBM reveal AIDS-level CD4 counts (< 200 cells/μl), substantial reductions in spleen volume (mean of 30%), and up to 5-fold increases in naïve T cell counts in bone marrow. Additional work has connected these observations to the loss of sphingosine 1-phosphate receptor 1 (S1P1) from the T cell surface. Specifically, loss of S1P1 results in sequestration of T cells preferentially in the bone marrow in the context of regressing lymphoid organs such as spleen. In mice, however, S1P1 can be stabilized on T cells by knocking in a receptor with disrupted cytoplasmic serines to prevent β-arrestin recruitment, an early step toward receptor internalization. Importantly, β-arrestin-inhibited, S1P1-stabilized mice implanted with GBM exhibit long-term survival when administered T cell-activating therapies that were previously ineffective. Likewise β-arrestin2 knockout mice, specifically, prove resistant to T cell sequestration in the setting of GBM. Building on these findings, Project 2 proposes to set the groundwork for designing translatable strategies to inhibit β-arrestin2 and stabilize S1P1 as a novel anti-tumor platform. Likewise the project has an early focus on elucidating biomarkers that correctly identify those patients most likely to benefit from intervention and when. The project will test the hypothesis that systemically administered β-arrestin2 small molecule inhibitors developed by the group will effectively hinder S1P1 internalization, stabilize surface T cell S1P1, free T cells from sequestration, and newly license T cell anti-tumor capacities. This hypothesis will be tested by the following Specific Aims: Aim 1: Assess in patients the longitudinal variation in T cell sequestration with tumor resection and treatment and establish predictive biomarkers and contributory mechanisms for T cell S1P1 loss; Aim 2: Establish the relative contribution of β-arrestin2 to S1P1 internalization and screen candidate β-arrestin2 small molecule inhibitors for their ability to stabilize S1P1 and abrogate T cell sequestration; Aim 3. Initiate IND-enabling toxicity and efficacy studies with our leading β-arrestin2 small molecule inhibitor. This study is expected to provide a foundation for therapeutic approaches enabling T cell release, which our data suggest would allow immunotherapies to render a more successful antitumor response.