Stimulated CD4+ T lymphocytes can differentiate into effector T cell (Teff) or inducible regulatory T cell (Treg) subsets with specific immunological functions. energy generation and biosynthesis (2, 3). To meet these requirements, activated T cells decrease lipid oxidation and undergo a rapid increase in glycolysis (4). At the conclusion of an immune response, decreased glycolysis and increased lipid oxidation can favor the enrichment of long-lived CD8+ memory cells (5, 6). For CD4+ T cells, however, it has been unclear how metabolism may support effector T cells (Teff) Th1, Th2, and Th17 and inducible regulatory T cells (Treg). The mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK) pathways play important and opposing functions in metabolism and immunity. T cell activation stimulates mTOR to increase glycolysis and diminish lipid oxidation (7). This pathway is usually also essential for CD4+ T cell subset differentiation, as mTOR-deficient mice generate Treg, but are severely inhibited in the differentiation of the Teff subsets (8, 9). In contrast, the AMPK pathway acts to prevent mTOR by suppressing mTOR signaling and to promote mitochondrial oxidative metabolism rather than glycolysis (10). Although AMPK activation can be anti-inflammatory (11), its role in T cell metabolism and CD4+ T cell subset differentiation is usually uncertain. In this study, we demonstrate that cell metabolism is usually crucial to regulate CD4+ T cell fate. Direct manipulation of cell metabolism showed that distinct metabolic programs were essential for the survival and specification of Teff and Treg, as Teffs required a glycolytic metabolism with active mTOR, Eletriptan hydrobromide IC50 whereas Treg had high Eletriptan hydrobromide IC50 levels of activated AMPK and required lipid oxidation. Materials and Methods Mice Mice were C57BL/6J and 6C8-wk-old unless indicated. Glut1 transgenic mice were previously described (3). Glut1-myc mice were generated by insertion of a tandem myc tag into exon 3 of Glut1 that encodes an extracellular loop and homologous recombination of this myc-tagged exon into the endogenous Glut1 locus (test, and 0.05 was considered significant. Results and Discussion Distinct metabolic phenotypes of Teff and Treg CD4+ cells The distinct functional requirement of CD4+ Th cells suggests that each subset may require specific metabolic programs to meet their differing dynamic and biosynthetic demands. To examine the metabolic profile of differentiated CD4+ T cells, naive CD4+ T cells were stimulated to replicate Teff (Th1, Th2, or Th17) and inducible Treg culture conditions (Supplemental Fig. 1A) (12). After 3 deb of culture in appropriate conditions, cells were washed and replated for an additional 2 deb in IL-2 alone. The glucose transporter, Eletriptan hydrobromide IC50 Glut1, is usually strongly induced and traffics to the cell surface upon TCF16 T cell activation to promote glycolytic metabolism and T cell growth (3). We examined total manifestation of Glut1 in wild-type mice and surface manifestation of Glut1 using a myc-epitope tagged Glut1 from mice in which a tag had been homologously recombined into the endogenous locus to allow sensitive flow cytometry for endogenous surface Glut1. Both total and cell-surface Glut1 increased between cells stimulated in Treg and Teff conditions, as Treg conditions led to low surface and total Glut1 (Fig. 1by intracellular staining with anti-FoxP3. J. Wild-type CD4+ T cells were stimulated in conditions to enrich for Treg and Th 17 differentiation in the presence or absence of SmM glucose followed by intracellular cytokine and FoxP3 staining. All results are representative of three impartial experiments (n of greater than 5 mice per group) and graphs are displayed as the average and standard deviation with values decided using the Student’s test (* 0.05). Supplemental Physique 2. Lipid oxidation and AMPK activation promote Treg generation in vitro.