Gene expression profiles of CAR-T cells expressing constitutively active mutants of STAT3 and STAT5 [STAT3_5_CART_in_vivo]

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. Overall design: Human CD8+ T cells were retrovirally transduced with an anti-mesothelin CAR construct (ss1-28z) alone, or in combination with either constitutively active STAT3 (caSTAT3) or STAT5 (caSTAT5). NSG mice inoculated with AsPC-1 cells were individually infused with the three types of CAR-T cells, and subcutaneous tumors were harvested 12 to 15 days post-infusion. In each experiment, CD8+ CAR-T cells were isolated from the tumors, total RNA was extracted, and gene expression profiles were analyzed by RNA sequencing.