Project Summary Anti-PD-1 therapy reinvigorates exhausted CD8 T cells, which can lead to complete tumor eradication as early as 3 weeks. Yet, despite robust T cell reinvigoration in >78% of patients, less than 40% are cured. The goal of this proposal is to understand why robust reinvigoration of exhausted CD8 T cells by αPD-1 therapy does not necessarily translate to clinical efficacy. Exhausted CD8 T cells (TEX) are a major cell type responding to PD-1 blockade. We and others have shown that αPD-1 therapy (αPD-1) reinvigorates exhausted CD8 T cells, as defined by enhanced proliferation and cytokine production. TEX are heterogeneous with progenitor and terminally differentiated TEX that have different roles in anti-tumor immune responses. Progenitor TEX (ProgEX) replenish terminally differentiated TEX (TermEX), which in turn provide anti-tumor activity. Thus, similar to stem cells, ProgEX represent a reservoir for CD8 T cell responses against the tumor. Importantly, it is the ProgEX, rather than TermEX, that respond to αPD-1 and almost exclusively contribute to the early burst of CD8 T cell reinvigoration. Reinvigoration of TEX results in accelerated differentiation of ProgEX to TermEX, resulting in a numerically greater pool of TermEX and improved tumor control. However, at the same time, the accelerated differentiation induced by αPD-1 places increased stress on ProgEX homeostasis. This raises the possibility that a ProgEX niche is key for providing the cells and signals necessary to prevent the depletion of ProgEX, and may prove crucial for the efficacy of PD-1 blockade. Our central hypothesis is that depletion of ProgEX after αPD-1 impairs clinical efficacy and that a tumor- associated niche is important in maintaining the pool of ProgEX. In Aim 1, we will test the hypothesis that αPD-1 results in enhanced differentiation of ProgEX and that that depletion of ProgEX is associated with clinical progression. We will use combinatorial tetramers and single cell RNA+TCR sequencing to understand how αPD-1 alters the pool of melanoma-specific ProgEX, and how the size of ProgEX pool in turn, impacts the clinical efficacy of αPD-1. In Aim 2, we test the hypothesis that tertiary lymphoid structures serve as a tumor- associated niche for ProgEX, and that increased number or size of ProgEX niches after αPD-1 is associated with the preservation of ProgEX and clinical efficacy. We will use multiparameter immunofluorescence and spatial transcriptomics to define the cellular composition of the ProgEX niche, the transcriptional circuits utilized by ProgEX in the niche, and the importance of the ProgEX niche in preserving the pool of ProgEX.