Glioblastoma multiforme (GBM) is among the most lethal of cancers, with average survival being only 12-15 months after diagnosis. Standard front line therapy currently includes surgical removal of the tumor, followed by radiation and chemotherapy. While these approaches can help to extend survival, GBM frequently invades surrounding healthy tissues and local recurrence is rapid. For this reason, it is critical to establish experimental model systems and develop immunotherapeutic strategies that enhance anti-tumor immunity to remove glioma cells left behind following surgery. Previous work has shown that enhancement of anti-tumor killer T cell responses via dendritic cell vaccination correlates with a positive immune response in select GBM patients. However, the mechanisms by which killer T cell responses are enhanced or inhibited remain poorly defined. To reconcile this, we have made use of the GL261 syngeneic glioma model system, which enables the study of brain tumor growth in the C57BL/6 mouse strain. Along with possessing an intact immune system, this mouse strain allows for the use of powerful genetic and transgenic mouse resources. Using an engineered GL261 cell line (GL261-Quad Cassette) expressing model T cell epitopes, preliminary studies have demonstrated that a tumor-specific killer T cell response can be generated toward the GL261 glioma in vivo. While increased infiltration of tumor antigen specific CD8 T cells is correlated with a significant reduction in tumor volume, as determined by 3D volumetric MRI, this natural response is insufficient to eradicate the tumor in nearly all cases. This proposal aims to address the hypothesis that enhancing effective antigen presentation and killer T cell recruitment into the CNS will result in the clearance of established gliomas. To accomplish this, we propose the use of a novel vaccination approach using a TMEV picornavirus that has been engineered to express tumor-specific antigens. In preliminary studies, intracranial vaccination of GL261-Quad Cassette tumors with engineered TMEV-Ova was effective at generating anti-tumor CD8 T cells and at inhibiting tumor progression, clearing the tumor completely in four of five cases. In Specific Aim 1, we will evaluate the effectiveness of this vaccination strategy when administered by less invasive routes to determine if tolerance can be broken despite immune privilege of the central nervous system (CNS). In Aim 2, we will define the immune effector molecules involved in the generation of protective immunity, as well as the respective contributions of cells of the innate and adaptive immune systems as sources of these molecules. Finally, in Specific Aim 3 we will utilize novel transgenic mice generated by our laboratory to uncover the cellular source of antigen processing and presentation in the CNS, a crucial step in the development of effective vaccines against CNS tumors. Through the completion of this work, we will gain insight into the mechanisms by which killer T cells contribute to tumor immunity, as well as potential therapeutic strategies that could be employed to enhance this protective immunity for human glioblastoma patients.