PROJECT SUMMARY Immune checkpoint receptors (e.g. CTLA-4, PD-1, Tim-3) are expressed on dysfunctional or “exhausted” CD8+ tumor-infiltrating lymphocytes (TILs) that exhibit defective effector functions (cytotoxicity and pro-inflammatory cytokine production) and are thus poor mediators of tumor clearance. In the last decade, immune checkpoint blockade (ICB) has achieved durable responses in many cancers, including melanoma, lung, and renal cancer. Despite this success, current estimates indicate that only 12% of all cancer patients respond to ICB. These observations underscore the remaining unmet clinical need in cancer treatment and the need to understand what constitutes effective response to ICB in order to improve response rates. Through examination of the population and single-cell RNA profiles of CD8+ TILs upon ICB, we have identified stem-like CD8+ TILs that are integral for the response to ICB. These cells are tumor antigen-specific, exhibit polyfunctional effector capacity, and increase in proportion upon various ICBs across different cancer types. Although the transcription factor TCF-1 plays an important role in the maintenance and effector function of these cells, we have found that TCF-1 expression in CD8+ T cells is not requisite for positive response to ICB in all tumor contexts. These observations underscore the relevance of stem-like CD8+ TILs for effective response to ICB and the need to better understand how TCF-1 and additional factors regulate their biology. Stem cells reside in niches where crosstalk between stem cells and other cells in the niche regulates not only their maintenance and function but also the function of niche cells. We have found that tumor-associated dendritic cells (DCs) are altered when TCF-1 is absent in mature CD8+ T cells. We thus hypothesize that 1) stem-like CD8+ TILs reside within niches in the tumor micro-environment (TME) where they interact with and modulate antigen-presenting cells; 2) this intercellular communication circuit within the niche may be required for effective priming of anti-tumor T cell responses; and 3) modulation of stem-like CD8+ TILs may positively or negatively influence this communication circuit, thus affecting the efficacy of ICB. Our overarching goal is to understand the cell-autonomous regulation of stem-like CD8+ TILs, their crosstalk with other cells in the TME, and how together they govern the activation of proficient anti- tumor CD8+ T cell responses and ICB efficacy. Accordingly, we propose the following aims: 1) Determine the role of TCF-1 in the generation of proficient anti-tumor T cell responses; 2) Test the role of novel candidate regulators of stem- and effector-like CD8+ TILs, and 3) Characterize the crosstalk between stem-like CD8+ TILs and the TME.