Project Summary/Abstract Successful anti-tumor immune response requires coordination between the arms of immunity against multiple tumor antigens, as shown through studies of animal models and human immune responses. Key to cancer cell elimination are T lymphocytes, and in particular cytotoxic CD8 T cells, which serve to recognize antigens presented on tumor cells on an HLA class I molecule, through their T cell receptors (TCRs). Correspondingly, recent advances in immunotherapy geared at boosting T cell cytotoxicity through immune checkpoint blockade (ICB) have transformed the treatment landscape for many patients suffering from solid tumors. However, in melanoma, ICB induces an objective response only in around 40% of patients. The limited efficacy of ICB has been previously linked to the presence of dysfunctional T cells within the tumor microenvironment (TME), persisting in a state of terminal exhaustion. There is thus a pressing need to better understand the determinants of T cell responses against tumors. To investigate these mechanisms, our lab has extensively characterized the landscape of anti-tumor T cells infiltrating melanoma and RCC biopsies, establishing that tumor-specific T cells are found overwhelmingly in the exhausted T cell compartment. Still, the required antigenic diversity and spatial interactions of such tumor-specific T cells determining anti-tumor response in vivo is poorly understood. Additionally, the relative importance of antigen class, particularly neoantigens and tumor-associated antigens (TAAs), to effective anti-tumor T cell responses is incompletely understood. In Specific Aim 1, I thus propose to generate patient-derived xenograft (PDX) murine models of 3 melanoma patient tumors, to test the required diversity of TCRs chosen from tumor-infiltrating T cells using markers associated with tumor specificity to exert immune pressure on a heterogeneous tumor like melanoma. By infusing T cells transduced with varying numbers of putative patient tumor-specific TCRs, or control non-tumor-specific TCRs, I will be able to assess the diversity of TCRs required to control tumor growth. Further, in Specific Aim 2, I will investigate the role of antigen class in tumor control using one of these melanoma PDX models, in combination with a cell-barcoding system, to track the relative immune-induced selection on the tumor of TCRs specific against either neoantigens or TAAs in vivo. Finally, in Specific Aim 3, I will utilize spatial sequencing techniques to study the localization of CD8 T cells first within my T cell-challenged PDX models, to characterize tumor-intrinsic immunosuppressive interactions and infiltration of T cells in my PDX models. I will then investigate the interactions and associated phenotypes of tumor-specific T cells in the native TME of patient melanoma biopsies from recurrent tumors. Achievement of these aims will lead to a pipeline that could isolate putative tumor-specific T cells through cellular markers ...