PROJECT SUMMARY Glioblastoma (GBM) is the most common primary brain tumor in adults and is characterized by aggressive growth, significant neurologic morbidity and eventually death. Temozolomide (TMZ) is the most widely accepted chemotherapy drug used to treat GBM, although its efficacy is limited in almost 50% of GBM patients. Therefore, novel treatment approaches, such as immune checkpoint inhibitors are being investigated for GBM. Immune checkpoints are immune inhibitory responses that are co-opted by tumor cells to down regulate T cell targeting of tumor antigens. Antibodies to programmed cell death 1 (PD-1) allow for T cell activation against tumor neoantigens. This approach has had tremendous success in the treatment of solid tissue tumors and are now being tested for efficacy in GBM. This research proposal will investigate the mechanisms by which dose-modified TMZ may prime host T cells and tumor neoantigen expression to improve response to PD-1 immune checkpoint blockade. The rationale for this proposal include several factors. First, TMZ has been found to be advantageous when used in combination with immunotherapy for GBM due to conditioning of host immunity during the recovery from TMZ-induced lymphopenia. Second, immune checkpoint inhibitors enhance anti-tumor T cell responses against tumor neoantigens. This phenomenon can be leveraged since TMZ is known to be mutagenic and result in an increase burden of tumor neoantigens in malignant glioma. Third, combination TMZ and PD-1 blockade has a unique role for resistant phenotypes of GBM since these tumors have an increase in PD-L1 expression. This proposal will test the hypothesis that TMZ induces host immunologic and tumor genetic changes that will result in increased response to PD-1 blockade. This hypothesis will be tested by completing three aims. The first aim will determine the effects of TMZ dose and timing on host immunologic changes and the impact on PD-1 blockade efficacy for GBM. This aim will be tested by evaluating PD-1 and PD-L1 expression on host immune cells after various TMZ dosing strategies, and evaluating the effects of combining TMZ with PD-1 blockade on host immunologic function and tumor infiltrating lymphocytes in a syngeneic murine GBM model. The second aim will evaluate the utility of combining dose-modified TMZ and PD-1 blockade for resistant GBM tumors. This aim will utilize an MGMT-mediated TMZ resistant tumor model to determine the efficacy of combinatorial treatment for resistant phenotypes of GBM. The final aim will explore the mechanism by which dose-modified TMZ alters the tumor mico-environment and changes response to PD-1 blockade. The experiments will test the differences tumor genetic mutations and T cell clonality in tumors that respond to PD- 1 blockade compared to those that are resistant. Tumors that have been pre-treated with TMZ will also be tested to determine how post-treatment tumors may differ in response to immune checkpoint blockade indepen...