Gene expression profiles of CAR-T cells expressing constitutively active mutants of STAT3 and STAT5 [STAT3_3+5 _CART_rested]

Augmenting cytokine signaling represents a pivotal strategy to enhance the therapeutic efficacy of chimeric antigen receptor (CAR) T cell therapy. However, the distinct roles of the downstream transcription factors STAT3 and STAT5 remain insufficiently defined. To elucidate their individual contributions, we engineered CAR-T cells to express constitutively active mutants of STAT3 (Y640F; caSTAT3) or STAT5 (N642H; caSTAT5). In vitro, caSTAT3 CAR-T cells demonstrated superior effector functions and a pronounced memory-associated phenotype, accompanied by broader transcriptomic alterations compared to their caSTAT5 counterparts. Despite impaired proliferative capacity in vitro, caSTAT3 CAR-T cells exhibited reduced systemic toxicity and sustained antitumor activity across multiple in vivo tumor models. In contrast, caSTAT5 CAR-T cells showed efficient intratumoral accumulation but also disseminated into non-malignant tissues, resulting in fatal toxicity. Notably, co-expression of caSTAT3 and caSTAT5 synergistically promoted both effector function and long-term persistence of CAR-T cells. Collectively, these findings provide mechanistic insights into the differential roles of STAT3 and STAT5 signaling in shaping the functional and safety profiles of CAR-T cell therapies.