PROJECT SUMMARY/ABSTRACT Targeting the immune system to destroy cancer cells using immunotherapy has rapidly emerged as a promising avenue for treating cancer, and has resulted in robust clinical responses in a subset of patients. While the initial success stories have provided proof of concept that the immune system can be harnessed to treat cancer, the majority of patients do not achieve long lasting or even initial benefit, highlighting the need to better understand the barriers to successfully using the immune system to eliminate cancer cells. The focus of this proposal is non- small cell lung cancer (NSCLC), which has only shown 20-40% of patients having an objective response to immune based therapies to date. A major obstacle to improving current immunotherapies in lung cancer is a lack of understanding of the overarching T cell response during tumorigenesis. Indeed, few studies have been able to longitudinally dissect the CD8 T cell response in a physiological time frame in the native lung microenvironment. Previous work has identified a population of less dysfunctional T cells in the tumor that express the transcription factor T cell factor 1 (TCF-1), and it is this population that is thought to mediate productive anti-tumor responses. However, how to generate these cells when they are lacking, the developmental trajectories of these cells during tumorigenesis, and how to best target these cells for therapeutic purposes remain unclear. The goal of this project is to address these critical questions surrounding the TCF-1+ subset of anti-tumor CD8 T cells using an autochthonous mouse model of lung adenocarcinoma that recapitulates both the time scale and anatomical progression of human NSCLC. Utilizing cutting edge methodologies and techniques, including single cell RNA sequencing, parabiosis and proximity labeling, we will define the ontogeny, differentiation, function, and determinants that dictate the fate of responding anti-tumor TCF-1+ CD8 T cells. In Aim 1, we will build on our preliminary data demonstrating heterogeneity within the TCF- 1+ CD8 T cell compartment by elucidating functionality and transcriptional status of anti-tumor CD8 T cell populations. We will compare the transcriptomic data we generate in Aim 1 to existing human single cell RNA sequencing to determine the relevance of the CD8 T cell states we identify in our model. In Aim 2 we will determine the role of tumor-T cell interactions in T cell phenotype and fate using a proximity labeling system to identify CD8 T cells that have recently interacted with tumor cells. In Aim 3, we will define the relationship of lymph node and lung TCF-1+ CD8 T cells, and will attempt to therapeutically harness anti-tumor CD8 T cells within the dLN to seed more TCF-1+ CD8 T cells into the tumor. Taken together, our aims are positioned to redefine our understanding of the anti-tumor CD8 T cell response and potentially identify new therapeutic avenues to treat cancer.