Glioblastoma multiforme (GBM) is a deadly disease with a grim prognosis, with median survival at diagnosis of less than a year and a half.1 Standard treatment with the DNA alkylating drug temozolomide yields incremental improvements in survival but better therapies remain needed.1 While anecdotal reports of individual patient benefit after receiving cancer vaccines for malignant glioma are promising,2-5 the overall record of vaccines for the treatment of this disease has been marked by failure.6 Tumors express large numbers of mutated genes with novel amino acid sequences that function as readily targeted neo-antigens in cancer vaccines.7-10 However, in glioblastoma such cancer vaccine trials have been conducted subsequent to standard of care treatment with temozolomide.11 Cancer vaccines for other malignancies are often given after standard alkylating chemotherapies.12 While some recent reports have indicated synergy between drugs like temozolomide and immune therapy,5,13-15 our results indicate that the direct anti-proliferative effect of drugs like temozolomide is the dominant effect on vaccine driven immune responses, diminishing their quantity and quality. These observations led to our central hypothesis that the anti-proliferative effect of temozolomide causes cells with high affinity antigen receptors to die preferentially in response to vaccination. Furthermore, immune therapy strategies that spare lymphocytes from exposure to alkylating drugs could yield improved results and true synergy between chemotherapy and experimental immune therapy. This project aims to: (1) use pre-clinical modeling to develop translational clinical strategies that overcome the obstacle of temozolomide anti-proliferative effect by generating large of tumor specific lymphocytes either in vivo before exposure to alkylating chemotherapy or ex vivo; (2) use knowledge of mechanisms of temozolomide resistance to protect lymphocytes from alkylating chemotherapeutic drugs before vaccination. To achieve (1) we will use a combination of standard immunological techniques combined with modern bioinformatics to generate large numbers of neo-antigen specific T cells. These cells will be generated either in non-temozolomide treated mice (simulating temozolomide naive glioma patients) or in vitro (simulating ex vivo expansion of glioma patient PBMC) and transferred into temozolomide treated mice bearing gliomas. Point (2) will use gene delivery of drug resistance genes to protect lymphocytes from the anti-proliferative effects of temozolomide. This knowledge could be combined with strategies such as those in (1) or more traditional vaccine approaches to yield greater synergy between temozolomide chemotherapy and immune therapy. The successful completion of this project will lead to solid pre-clinical leads in overcoming a major obstacle to the use of cancer vaccines in glioblastoma and other malignancies.