Dissecting the Mechanism for Transient ER Stress-Induced Anti-Tumor T Cell Response - ABSTRACT The development of T cell immunotherapies over the past decade revealed that the reinvigoration of anti-tumor immunity could be achieved by manipulating mitochondrial dynamics in T cells. The endoplasmic reticulum (ER) and the mitochondria coordinate to maintain cellular homeostasis, metabolism, and death. While persistent ER stress responses cause mitochondrial collapse, moderate ER stress conditions promote mitochondrial function. Strategies to boost anti-tumor T cell function by targeting ER-mitochondrial crosstalk have not been exploited yet. We used carbon monoxide (CO), a short-lived gaseous molecule, to test if engaging in moderate ER stress conditions can improve T cells' mitochondrial and anti-tumor functions. Using melanoma antigen-specific T cells, we identified that CO-induced transient activation of the ER stress sensor ‘protein kinase R-like endoplasmic reticulum kinase (PERK)' dramatically increases anti-tumor T cell function by engaging autophagy. Using a dual reporter, the LC3-GFP-RFP mouse, we found that, in response to transient ER stress (imposed by CO), T cells substantially increase the LC3-GFPpos population (that prepare themselves to undergo active autophagy) compared to those that fail to enter the process (LC3-GFPneg). We noted that the LC3-GFPpos T cells inherited healthier mitochondria and showed improved tumor control. Moreover, LC3-GFPpos and LC3-GFPneg T cells showed distinct metabolic epigenetic profiles. Overall, our preliminary data highlights that transient ER stress- activated autophagy pathways modify mitochondrial function and epigenetically reprogram T cells towards a superior antitumor phenotype. Based on the existing literature and our preliminary data, CO-induced transient PERK activation followed by autophagy influences a combination of events, including mitochondrial quality, metabolic, and epigenetic programming, leading to the reinvigoration of exhausted anti-tumor T cells and increased T cell effector function in the TME. Experiments are proposed to test this hypothesis with the following aims: 1) To determine how CO-induced mitochondrial quality control mechanisms regulate T cell effector function. 2) To determine how autophagy-induced altered metabolite ratios epigenetically reprogram anti-tumor T cells. 3) To determine if CO induced autophagy improves the anti-tumor efficacy of human TILs and CAR-T cells. We believe the successful completion of the proposal will: a) help us identify mitochondrial signaling molecules that regulate T cell effector functions to improve tumor immunotherapy. b) will identify the dysfunctional mitochondria-derived metabolites and the epigenetic programs that regulate the gene expression profile associated with effector functions and longevity of the T cells in the TME. c) Finally, successful completion of the proposed studies will provide a comprehensive mechanistic understanding and testing strategies (such as using CO) with TILs and TCR/CAR-transduced T cells, which will lead to improved translational outcomes required for treating solid tumors and drastically reduce the cost of ACT.
