Enhanced <i>FOS</i> expression improves tumor clearance and resists exhaustion in NR4A3-deficient CAR T cells under chronic antigen exposure

The dysfunction of chimeric antigen receptor (CAR) T cells in the tumor microenvironment is a major obstacle to their therapeutic efficacy against solid tumors. Through single-cell RNA sequencing analysis of tumor-infiltrating T cells from patients with glioma, NR4A family genes were identified as closely associated with T cell exhaustion and were coexpressed with dysfunctional genes <i>HAVCR2</i> and <i>TIGIT</i>. Notably, CAR T cells with <i>NR4A3</i> knockdown exhibited enhanced cytotoxic activity against tumors, leading to improved tumor clearance and prolonged survival in vivo. However, the promoted antiexhausted phenotype diminished with prolonged tumor burden. This decline in T cell function correlates with the compensatory down-regulation of <i>FOS</i> induced by chronic antigen exposure following <i>NR4A3</i> knockdown. Overexpressing <i>FOS</i> alongside <i>NR4A3</i> knockdown robustly boosted the antitumor responses of CAR T cells by skewing their phenotypes and transcriptional profiles away from exhaustion and toward increased effector function. These findings offer a promising strategy for the clinical modification of CAR T cell therapy.