The presence of polyfunctional CD4⁺ T cells is often associated with favorable antitumor immunity. We report here that persistent activation of signal transducer and activator of transcription 5 (STAT5) in tumor-specific CD4⁺ T cells drives the development of polyfunctional T cells. We showed that ectopic expression of a constitutively active form of murine STAT5A (CASTAT5) enabled tumor-specific CD4⁺ T cells to undergo robust expansion, infiltrate tumors vigorously, and elicit antitumor CD8⁺ T cell responses in a CD4⁺ T cell adoptive transfer model system. Integrated epigenomic and transcriptomic analysis revealed that CASTAT5 induced genome-wide chromatin remodeling in CD4⁺ T cells and established a distinct epigenetic and transcriptional landscape. Single-cell RNA sequencing analysis further identified a subset of CASTAT5-transduced CD4⁺ T cells with a molecular signature indicative of progenitor polyfunctional T cells. The therapeutic significance of CASTAT5 came from our finding that adoptive transfer of T cells engineered to coexpress CD19-targeting chimeric antigen receptor (CAR) and CASTAT5 gave rise to polyfunctional CD4⁺ CAR T cells in a mouse B cell lymphoma model. The optimal therapeutic outcome was obtained when both CD4⁺ and CD8⁺ CAR T cells were transduced with CASTAT5, indicating that CASTAT5 facilitates productive CD4 help to CD8⁺ T cells. Furthermore, we provide evidence that CASTAT5 is functional in primary human CD4⁺ T cells, underscoring its potential clinical relevance. Our results implicate STAT5 as a valid candidate for T cell engineering to generate polyfunctional, exhaustion-resistant, and tumor-tropic antitumor CD4⁺ T cells to potentiate adoptive T cell therapy for cancer.

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