Investigating mechanisms of CD8 T cell differentiation in the tumor-draining lymph node

PD-1 pathway targeting antibodies have improved patient outcomes in lung adenocarcinoma (LUAD). Unfortunately, most LUAD patients do not yet benefit from these therapies, and it is not clear why. Robust responses to PD-1/PD-L1 blockade require that the intratumoral CD8 T cells are in a progenitor-exhausted (TPEX) state, as TPEX cells proliferate and give rise to cytotoxic effector CD8 T cells (TEFFs). Yet, we and others have found that tumor-specific TPEX cells are primarily housed in the tumor-draining lymph node (tdLN) associated with the lung. Via migration, these cells continually replenish the tumor migration, underscoring the critical role of the tdLN as a reservoir of stem-like CD8 T cells. However, because the tdLN is the site of long- term maintenance, we hypothesize that the biology of tumor-specific TPEX and their differentiated progeny is shaped by the interactions and signals they receive in this site. Here, we propose in-depth studies on the mechanisms controlling the differentiation and maintenance of TPEX populations in the tdLN. Our proposal integrates genetically engineered LUAD models, CRISPR-based perturbations, and single-cell approaches to dissect this process. Specifically: 1. We will define how KLF2 and T-bet prevent exhaustion by repressing exhaustion-related genes (e.g., TOX) and implementing cytotoxic effector programs as T cells differentiate across the tdLN and tumor. 2. We will determine how IL-21–BATF signaling impacts on TPEX → effector CD8 T cell transitions, and the role of KLF2 in this process. We previously showed IL-21 is provided by T-follicular helper CD4 T cells in the tdLN, and we will leverage models with and without TFH responses to pinpoint how IL-21 signaling promotes CD8 T cell cytotoxicity and limits exhaustion. 3. KLF2 is transiently downregulated by TCR signals. We will determine if KLF2 downregulation is necessary for differentiation in the tdLN and the role that TCR-dependent signals play in maintaining T cell stemness in the tdLN. Our studies will investigate immune signaling pathways and transcriptional networks regulating CD8 T cells in the tdLN, elucidate mechanisms for the provision of IL-21 and its role in driving effector function, and explore the interplay between TCR and KLF2 in shaping CD8 T cell fate. These insights will shed light on immunoregulatory mechanisms that determine whether tumor-specific CD8 T cells maintain anti-tumor functions or become dysfunctional. By illuminating the biology of the tumor-specific TPEX cells in the tdLN reservoir, our goal is to identify entry points for mobilization or reprogramming through targeted interventions, to boost the efficacy of therapies against LUAD. Project Number: 1R01CA308125-01 | Fiscal Year: 2026 | NIH Institute/Center: National Cancer Institute (NCI) | Principal Investigator: Nikhil Joshi | Institution: YALE UNIVERSITY, NEW HAVEN, CT | Award Amount: $696,189 | Activity Code: R01 | Study Section: Basic Cancer Immunobiology Study Section[BCIB] View on NIH RePORTER: https://reporter.nih.gov/project-details/11274810