Immunotherapy via checkpoint blockade could be an attractive option for Glioblastoma (GBM), a disease with limited treatment options. A clinical response to checkpoint immunotherapy in other cancer settings is often dependent upon a pre-existing immune infiltrate. However, glioblastoma is commonly poorly infiltrated by effector immune cells. Thus, the translation of adjunct approaches that enhance T-cell infiltration and/or lift the immunosuppressive tumor microenvironment could vastly expand the population of GBM patients exhibiting durable responses to immunotherapy. Toward this goal, we hypothesize that perturbation of the GBM microenvironment with focused ultrasound (FUS), applied in energy regimes designed to elicit partial thermal ablation or microbubble cavitation, can stimulate immunologic responses that are both intrinsically therapeutic and synergistic with translatable immunotherapies. Indeed, our pilot studies in melanoma indicate FUS application can elicit tumor growth control and improved survival via trafficking of activated lymphocytes from lymph nodes to the tumor. This proposal is comprised of 2 specific aims that will serve to define differences in the innate and adaptive immune responses that are elicited by applying different FUS energy regimes to tumors, identify barriers to tumor immunity, and ascertain treatment protocols that more effectively combine FUS energy regimes with adjunct immunotherapies for treating GBM. Specific Aim 1 will be to determine the impact of the selected FUS energy regimes on discrete factors that influence the sequential steps involved in the activation, expansion, and recruitment of dendritic cells (DC) to the tumor microenvironment. Specific Aim 2 will be to assess the ability of selected FUS regimens to promote the trafficking and extravasation of T cells into the GBM tumor microenvironment, and ask whether hypothesis-driven selection of agents that promote trafficking can augment T cell presence and persistence within tumors. This will allow us to understand barriers to access for T cells expanded by vaccination or after adoptive transfer. Going forward, this will be a critical aspect in optimizing the combination of FUS with anti-tumor immunotherapy. We believe the systematic and directed approach proposed here is more likely to lead to successful clinical therapies for GBM patients with limited T cell infiltration.