TGF-β Regulates CD8+ T Cell Memory by Triggering mTORC1Weak-Mediated Activation of the Transcriptional FOXO1-TCF1-Eomes and Metabolic AMPK-ULK1-ATG7 Pathways

CD8⁺ memory T (T_M) cells are essential for vaccine-induced protective immunity. While transforming growth factor beta (TGF-β) triggers CD8⁺ T_M cell differentiation, the underlying molecular mechanism(s) has yet to be uncovered. We therefore used a well-established cell culture protocol to prepare TGF-β-triggered CD8⁺ T_M cells derived from chicken ovalbumin (OVA)-specific T cell receptor (TCR) transgenic OTI mice, and systematically characterized them using Western blotting, confocal microscopy, flow cytometry and Seahorse assay analyses. We found that TGF-β/T cells exhibit a T_M cell phenotype (CD62L⁺KLRG1^-) and display long-term survival upon adoptive transfer into mice. To elucidate the signaling circuitry underpinning the observed transcriptional and metabolic changes required to promote CD8⁺ T_M cell differentiation, we measured the expression of several critical factors and found that TGF-β triggered weak mTORC1 (mTORC1^Weak) signaling. mTORC1^Weak signaling in turn led to an increase in the abundance of key transcriptional (TCF1, FOXO1 and Eomes) and metabolic (AMPK-α1, ATG7, ULK1, SIRT1, OPA1 and LAL) factors and an elevation in mitochondrial mass and reliance on fatty acid oxidation (FAO). Our data thus reveal for the first time that TGF-β regulates CD8⁺ T cell memory by triggering mTORC1^Weak-mediated activation of the transcriptional FOXO1-TCF1-Eomes and metabolic AMPK-ULK1-ATG7 pathways. Given that induction of more qualified CD8⁺ T_M cells is one of the ultimate goals of vaccination, our findings identify additional targets critical to TGF-β-induced T cell memory, which may greatly impact future vaccine development for the treatment of cancer and infectious diseases.