Persistent STAT5 activation reprograms the epigenetic landscape in CD4+ T cells to drive polyfunctionality and antitumor immunity [RNA-Seq]
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 with superior antitumor activities. We showed that ectopic expression of a constitutively active form of murine STAT5A (CASTAT5) in tumor-specific CD4+ T cells established a distinct epigenetic and transcriptional landscape, endowing CD4+ T cells polyfunctionality, exhaustion-resistance and tumor-infiltrating capability. Single cell RNA sequencing analysis (scRNAseq) identified a subset of cells with the molecular signature indicative of their role as progenitor polyfunctional T cells. Importantly, T cells engineered to co-express CD19-targeting chimeric antigen receptors (CD19CAR) and CASTAT5 gave rise to polyfunctional CD4+ CAR T cells capable of providing optimal help to CD8+ T cells to achieve durable and curative outcomes in mice with advanced B-cell lymphoma. Evidence of CASTAT5 functional activities in primary human CD4+ T cells underscores its potential clinical relevance. Overall design: The objective of this study was to investigate the functional fate and therapeutic effectiveness of tumor-specific CD4+ T cells expressing a constitutively active form of Stat5a (CASTAT5) via retroviral transduction.  To achieve this goal, we designed a CD4+ T-cell adoptive transfer model system using a murine B-cell lymphoma model and performed ATACseq and bulk RNAseq to interrogate how CASTAT5 reshaped CD4+ T cell phenotypic and functional features using FACS-sorted tumor-specific CD4+ T cells recovered from the spleens and tumors of mice. Our datasets revealed that CASTAT5 induced genome-wide transcriptional and epigenetic remodeling in tumor-specific CD4+ T cells. Subsequent single-cell RNAseq analysis revealed the heterogeneity of CASTAT5-transduced CD4+ T cells and identified a subset of cells bearing a gene signature indicative of progenitor polyfunctional cells. We validated the beneficial effects of CASTAT5-transduced CD4+ T cells in CD19CAR T cell therapy mouse tumor models. We demonstrated that CASTAT5 was also functional in transduced primary human T cells. Mice with established tumors were randomly grouped before receiving the indicated treatment. Sample sizes for the in vivo experiments were determined based on previous studies performed in our lab, and the suitability for statistical analysis and independent repeats. Information regarding the sample size and number of replicates for each experiment is provided in the relevant figure legend.