Foxp3 expression diversifies the metabolic capacity and enhances the efficacy of CD8 T cells in adoptive T cell therapy

CD4 regulatory T cells overwhelm conventional T cells in the tumor microenvironment (TME) thanks to a Foxp3-driven metabolic program that allows them to use alternate substrate and engage different metabolic pathways. In this study, we proposed to overexpress Foxp3 in CD8 T cells (CTL) and examine how it affected their antitumor properties. We show that Foxp3 overexpressing (Foxp3UP) CTLs proliferated and markedly accumulated in the TME while showing enhanced cytotoxicity and antitumor efficacy but not suppressor activity. Interestingly, tumor-infiltrating Foxp3UP CTLs exhibited a T-cell effector and leukocyte migration genetic signature, and positive enrichment in glycolysis, fatty acid (FA) metabolism and oxidative phosphorylation (OXPHOS) pathways. Intratumoral Foxp3UP CTLs showed an increased expression of Glut1, enhanced glucose and FA uptake and intracellular lipid accumulation. Interestingly, Foxp3UP CTLs compensated for the loss of mitochondrial respiration-driven ATP production by activating aerobic glycolysis. Moreover, in limiting nutrient conditions these cells engaged FA oxidation to drive OXPHOS for their energetic demands. Further, their ability to couple glycolysis and OXPHOS allowed then to sustain proliferation under glucose restriction. Our findings demonstrate a hitherto unknown role of Foxp3 in the adaptation of CTLs to TME that can be used to enhance their efficacy in adoptive T cell therapy Overall design: Tumor bearing mice were treated with a mix of Foxp3UP and Mock CD8 T cells. After 5 days of treatment tumor cell suspensions containing transferred CTLs were surface stained with mAbs against distinctive population biomarkers (CD8, CD45.1 and/or CD90.1) in the presence of purified anti-CD16/32 mAb (Fc Block) and then with SYTOX Blue dead dye. Sorting of CTL subsets was performed in MoFloAstrio EQ (Beckman-coulter). RNA from transferred CD8 T cells was isolated using Maxwell 16 LEV simplyRNA tissue kit (Promega) and RNA libraries were prepared for sequencing using standard Illumina protocols.