Constitutive STAT3 Signaling, in Comparison with STAT5, Enhances CAR T-cell Efficacy and Lowers Systemic Toxicity

Providing cytokine signaling is a key strategy to boost the efficacy of chimeric antigen receptor (CAR) T-cell therapy. However, the individual roles of key downstream mediators, STAT3 and STAT5, remain incompletely understood. In this study, we engineered CAR T cells to express constitutively active mutants of STAT3 (Y640F; caSTAT3) and STAT5 (N642H; caSTAT5) to investigate their individual functions. In vitro, caSTAT3 CAR T cells exhibited enhanced effector function and a robust memory phenotype, with broader transcriptional changes involving both effector- and memory-associated genes compared with caSTAT5 CAR T cells. However, caSTAT3 CAR T cells failed to expand because of activation of apoptosis-related gene programs. In contrast, caSTAT5 CAR T cells demonstrated sustained proliferation over time. Despite the limited in vitro expansion, caSTAT3 CAR T cells exhibited reduced transgene toxicity in vivo and exerted durable antitumor activity in both leukemia and solid tumor models without significant off-tumor toxicity. Titrated expression of caSTAT3 maintained enhanced effector function without inducing apoptosis. On the other hand, caSTAT5 CAR T cells efficiently accumulated in tumors but also infiltrated nontumor tissues, causing lethal systemic toxicity. Co-expression of caSTAT3 and caSTAT5 markedly enhanced the long-term proliferative capacity of CAR T cells, even in the absence of antigen stimulation or cytokine supplementation. These findings elucidate the distinct impacts of STAT3 and STAT5 activation on CAR T-cell behavior and suggest that selective activation of STAT3 at optimal levels may improve CAR T-cell efficacy while minimizing off-tumor toxicities.