Mst1 regulates Foxo1 transcription to differentially control memory CD8 T cell differentiation and survival

Abstract Foxo1 is an essential transcription factor required for the differentiation and survival of memory CD8 T cells. Foxo1 activity is heavily regulated by post translational modifications, yet it is unclear whether memory T cell differentiation is regulated in the same manner as survival. Two S/T kinases, Akt1 and Mst1 phosphorylate Foxo1. While Akt1 mediated phosphorylation is known to inhibit Foxo1 transcriptional activity, the effect of Mst1 phosphorylation remains poorly understood. To determine the impact of Mst1 regulation on Foxo1-dependent CD8 T cell differentiation and survival, we generated Foxo1 variants that mimic Mst1 phosphorylation (SD-Foxo1) or disrupt Mst1 phosphorylation (SA-Foxo1). Reconstitution of Foxo1-null T cells with SA- or SD-Foxo1 allowed us to evaluate their effects on early response genes in vitro and memory differentiation and survival in vivo following Listeria monocytogenes infection. We observed significantly higher expression of canonical Foxo1 target genes, including Il7r and Sell when CD8 T cells expressed SA-Foxo1. In contrast, CD8 T cells expressing SD-Foxo1 were defective in inducing canonical Foxo1 targets and in generating memory precursor effector cells post Listeria infection. However, SD-Foxo1 expression promoted survival of memory CD8 T cells as compared to the SA-Foxo1 in vitro. RNAseq analysis revealed that SD-Foxo1 induces an altered Foxo1 transcriptional program including the upregulation of Purinergic Receptor P2X7 (P2RX7), which was recently shown to be required for memory CD8 T cell maintenance and survival. Together, our data suggest that Mst1 regulates a distinct mode of Foxo1 transcription to balance Foxo1-dependent differentiation and survival in CD8 T cells.