PROJECT SUMMARY/ABSTRACT Recent studies have revealed an abundance of resident memory T cells (TRM) specific for viral infections in a wide range of tumors, often outnumbering cancer-specific T cells. As these cells lack specificity to tumor antigens, they are spared from the hallmark exhaustion/dysfunction of tumor-specific T cells. To this end, we developed a novel immunotherapy which taps into immunostimulatory virus-specific TRM functions through treatment with viral peptides to break the immunosuppressive tumor environment. Reactivating virus-specific TRM with peptide was sufficient to restrict tumor growth in mice and when combined with checkpoint blockade, promoted durable tumor clearance. Mice that cleared tumors were protected from tumor re-challenge, suggesting anti-tumor immunity was established. Virus-specific TRM represent a major part of the tumor immune environment and can be leveraged therapeutically, yet there is a clinically significant gap in knowledge regarding the mechanisms of tumor clearance and how to optimally harness these cells. The objectives in this proposal are to determine (i) mechanism of tumor cell killing and durable tumor immunity, (ii) the impact of viral specificity on TRM function and therapeutic efficacy, and iii) define the optimal modality to reactivate virus-specific TRM. Using both mouse and human systems, this study addresses an innovative perspective connecting antiviral memory cells to tumor immunotherapy using cutting edge methods. We will complement mouse studies with combinatorial tetramer staining to enable simultaneous profiling of T cells specific for an expanded panel of viruses and vaccines in patient tumors. Completion of these aims will advance our understanding of tumor immunosurveillance and TRM function in mice and humans, and identify new target T cell populations for tumor immunotherapy. This contribution is expected to be significant because it will provide a strong scientific framework to expand the efficacy and utility of virus-specific TRM therapy, and enable the development of novel strategies leveraging these potent immune activating cells.