Project description:CD4 T cells were activated using CD3/CD28 antibodies in the presence of IL2 and IL12, to generate Th1 cells. Th1 cells were maintained and expanded in IL2 and IL12, on D5 Th1 cells were sorted for CD4+ expression and DAP1 exclusion. After flow sorting, live TH1 cells were resuspended (1e6 per ml) in methionine free RPMI, supplemented with 10% dialysed FBS, IL2 and IL12, and with L-methionine (100μM, or 1μM). Cells were cultured for 5 hrs before collection for proteomics processing.
Project description:This study provides evidence on the molecular mechanisms by which P2RX7 signaling promotes Th1 cell differentiation. P2RX7 induces T-bet expression and aerobic glycolysis in splenic CD4+ T cells that respond to malaria, at a time prior to Th1/Tfh polarization. Cell-intrinsic P2RX7 signaling sustains the glycolytic pathway and causes bioenergetic mitochondrial stress in activated CD4+ T cells. We also show in vitro the phenotypic similarities of Th1-polarized CD4+ T cells that do not express P2RX7 and those in which the glycolytic pathway is pharmacologically inhibited. In addition, ATP synthase blockade in vitro and the consequent inhibition of oxidative phosphorylation, which forces cells to use aerobic glycolysis, is sufficient to promote rapid CD4+ T cell proliferation and polarization to the Th1 profile in the absence of P2RX7. These data demonstrate that P2RX7-mediated metabolic reprograming for aerobic glycolysis is a key event for Th1 cell differentiation and suggest that ATP synthase inhibition is a fundamental mechanism by which P2X7 signaling induces the Th1 response.
