Targeting NOX1 to Reprogram T Cell Activation and Metabolism for Enhanced CAR T Efficacy 2251163

Abstract Introduction T-cell exhaustion is a critical roadblock to achieving durable remission with adoptive cell therapies. The immunosuppressive tumor microenvironment limits T-cell persistence and function, demanding new reprogramming techniques. Our genome-wide CRISPR screen in CD8+ T cells identified NOX1 as a novel, negative effector regulator. We hypothesize that NOX1 suppresses signaling downstream of the TCR, and its therapeutic inhibition will improve T-cell-based immunotherapies, particularly the next generation of CAR T therapies Methods We generated NOX1 knockout (KO) T cells from primary human T cells via CRISPR-Cas9, with KO efficiency validated by Western Blot. Mechanistic studies compared NOX1 KO to controls using TCR-induced calcium flux and ROS production for existing and induced ROS. Functional outcomes were assessed via cytotoxicity against melanoma lines and cytokine production. The enhanced anti-tumor efficacy was validated in a syngeneic in vivo mouse model Results Genetic knockout of NOX1 in T cells resulted in functional enhancement. NOX1 KO significantly reduced basal intracellular ROS levels and exhibited stronger, sustained TCR-induced calcium flux, confirming a key role in TCR signaling, relatively proximal to the TCR signaling event. NOX1 deficient T cells displayed increased cytotoxicity against B16 melanoma cells in vitro, with enhanced production of effector cytokines. This translated into improved anti-tumor efficacy and survival in a corresponding in vivo mouse model Conclusion NOX1 functions as a critical negative regulator that imposes a redox-dependent threshold, limiting TCR-initiated calcium flux and dampening T-cell activation. By demonstrating that NOX1 deletion enhances signaling, overcomes metabolic deficiencies, and achieves potent anti-tumor efficacy in vivo, we establish NOX1 as a novel therapeutic target. This work provides the strong rationale for genetically inhibiting NOX1 as a way to enhance durability and function of T-cell therapies to overcome T-cell exhaustion Funding Source N/A Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)