PROJECT SUMMARY Head and neck cancer, including oral cavity squamous cell carcinoma (OCSCC), is the sixth leading cause of cancer and a major cause of morbidity and mortality. Unfortunately, the five-year survival rate of OCSCC has only slightly improved in the past 30 years. Recently, immune checkpoint inhibition (ICI) therapies have emerged as a promising treatment to improve OCSCC outcomes, yet only 14-22% of OCSCC patients respond to ICI. Thus, delineating the mechanisms of ICI resistance represents an opportunity to identify novel targets that may increase ICI efficacy and improve OCSCC patient survival. CD8 T cells are the primary immune cell type that kills malignant cells during ICI, and their dysfunction may contribute to ICI resistance. During the chronic response to a tumor, CD8 T cells enter an exhausted cell state – a dysfunctional phenotype characterized by decreased ability to lyse target cells. Expression of the transcription factor thymocyte selection-associated HMG box (TOX) is critical for inducing CD8 T cell exhaustion and is enriched in the exhausted CD8 T cell subset within single cell RNA-seq datasets of OCSCC patient tumor samples. The genes that are directly transcriptionally regulated by TOX that promote CD8 T cell dysfunction and may be therapeutically targeted, however, remain poorly defined. In addition to TOX, malignant cell signals may contribute to CD8 T cell dysfunction and ICI resistance. We discovered a hybrid epithelial/mesenchymal (HEM) malignant cell state in OCSCC that localizes to the tumor edge adjacent to CD8 T cells and is associated with exhaustion of these adjacent CD8 T cells. The mechanisms of HEM cells underlying this association are not well understood. The primary hypothesis of this proposal is that CD8 T cell dysfunction in OCSCC is driven by the direct gene targets of TOX in CD8 T cells and by immunosuppressive contact and paracrine signaling from HEM cells. The goal of this proposal is to define mechanisms of CD8 T cell dysfunction that may be targeted to improve ICI through the following Aims: In Aim 1, the direct gene targets of TOX and their role in CD8 T cell dysfunction will be defined in vitro using CD8 T cells isolated from OCSCC samples. In Aim 2, patient-derived CD8 T cells will be co-cultured with patient-matched OCSCC tumor-derived organoids or cancer cell lines expressing a model antigen. In these co-cultures, HEM- specific membrane and secreted proteins will be knocked-down in the malignant cells to determine their role in suppressing CD8 T cells. The proposed research will provide mechanistic insight into CD8 T cell dysfunction in OCSCC, thereby supporting development of new strategies to therapeutically activate CD8 T cells and improve ICI and OCSCC outcomes. Importantly, during this fellowship, I will pursue scientific and clinical activities under the direct mentorship of a comprehensive fellowship advisory committee composed of physician-scientists and experts in cancer and im...