PROJECT SUMMARY/ABSTRACT Cancer remains a leading cause of mortality within the United States with overall cancer incidence predicted to rise. Immunotherapies have ushered in a new age of cancer treatment, harnessing the potency of the immune system to restrict cancer without many of the side effects of conventional therapies. These immunotherapies have largely targeted T cell responses that are specific for tumor antigens. While immunotherapies leveraging tumor-specific T cells have proven powerful tools in combatting cancer, their success if context-dependent, displaying limited efficacy in many solid tumors. Part of this phenomenon results from tumor-specific T cells receiving chronic T cell receptor (TCR) signaling (caused by recognition of cognate tumor antigens) within the tumor micro-environment (TME). Chronic TCR signaling in the TME permanently renders tumor-specific T cells functionally inert, suggesting a probable limit to which we can exploit these tumor-specific T cells before they are therapeutically unviable. However, T cells are diverse in human tumors, with many incapable of recognizing tumor or tumor-associated antigens. Though these “bystander” T cells have been discounted given their lack of TCR specificity for tumors, they are spared from TCR-mediated dysfunction within the TME. Thus, bystander T cells are a promising target for novel tumor therapies. Outside of tumors, bystander T cells can become activated into innate-like killers when exposed to combinations of pro-inflammatory cytokines (a phenomenon called bystander activation); but it remains unclear if this program can be harnessed intratumorally to elicit tumor killing. Using approaches incorporating TCR-defined bystander T cells, multiple solid tumor models, and in vitro and in vivo manipulations, I have found that intratumoral bystander activation is indeed possible but differs across tumor types. My objectives are 1) to identify the mechanisms of bystander activation (and subsequent cytotoxicity) that can be harnessed for tumor killing, 2) to pinpoint and overcome tumor-intrinsic regulatory mechanisms that blunt bystander activation, and 3) to translate these findings to other tumor types. To achieve these objectives, I will employ my expertise in high-parameter single-cell analyses and animal models to uncover the mechanisms that shape bystander T cell fate and function in the TME. Hypothesis: My central hypothesis is that bystander T cells can be therapeutically leveraged in solid tumors. AIM 1: In models that permit TME bystander activation, test the hypothesis that indirect- and direct-killing mechanisms of activated bystander T cells contribute to tumor restriction. AIM 2: In models that blunt TME bystander activation, test the hypothesis that immunosuppressive pathways curb bystander activation and can be blocked to promote anti-tumor responses. My proposed experiments will elucidate the role of bystander T cells in tumors and their therapeutic viability, with the goal of developing interventions to improve cancer outcomes, which is in alignment with the mission of the NCI and NIH. .

PROJECT NARRATIVE T cells with specificity for tumor antigens are the primary target of most immunotherapies; however, T cells specific for tumor-unrelated antigens, termed “bystanders,” are often found in human solid tumors. Despite being a major component of the tumor infiltrate, it is unknown if bystander T cells can be broadly activated to elicit antitumor functions. This project seeks to define the mechanisms that permit or restrict the functions of tumor-infiltrating bystander T cells so that this population can be harnessed as a therapeutic agent for cancers.