An AMBRA1, ULK1 and PP2A regulatory network regulates cytotoxic T cell differentiation via TFEB activation

The scaffold protein AMBRA1, which participates in the autophagy pathway, also promotes CD4⁺ T cell differentiation to Tregs independent of autophagy through its interactor PP2A. Here we have investigated the role of AMBRA1 in CD8⁺ T cell differentiation to cytotoxic T cells (CTL). AMBRA1 depletion in CD8⁺ T cells was associated with impaired expression of the transcription factors RUNX3 and T-BET that drive CTL differentiation and resulted in impaired acquisition of cytotoxic potential. These effects were recapitulated by pharmacological inhibition of the AMBRA1 activator ULK1 or its interactor PP2A. Based on the ability of PP2A to activate TFEB, we hypothesized a role for TFEB in the CTL differentiation program regulated by AMBRA1. We show that TFEB modulates RUNX3 and T-BET expression and the generation of killing-competent CTLs, and that AMBRA1 depletion, or ULK1 or PP2A inhibition, suppresses TFEB activity. These data highlight a role for AMBRA1, ULK1 and PP2A in CTL generation, mediated by TFEB, which we identify as a new pioneering transcription factor in the CTL differentiation program.