Cutting edge: distinct glycolytic and lipid oxidative metabolic programs are essential for effector and regulatory CD4+ T cell subsets

Abstract

Stimulated CD4(+) T lymphocytes can differentiate into effector T cell (Teff) or inducible regulatory T cell (Treg) subsets with specific immunological roles. We show that Teff and Treg require distinct metabolic programs to support these functions. Th1, Th2, and Th17 cells expressed high surface levels of the glucose transporter Glut1 and were highly glycolytic. Treg, in contrast, expressed low levels of Glut1 and had high lipid oxidation rates. Consistent with glycolysis and lipid oxidation promoting Teff and Treg, respectively, Teff were selectively increased in Glut1 transgenic mice and reliant on glucose metabolism, whereas Treg had activated AMP-activated protein kinase and were dependent on lipid oxidation. Importantly, AMP-activated protein kinase stimulation was sufficient to decrease Glut1 and increase Treg generation in an asthma model. These data demonstrate that CD4(+) T cell subsets require distinct metabolic programs that can be manipulated in vivo to control Treg and Teff development in inflammatory diseases.

FIGURE 1

Teff and Treg CD4⁺ T cell subsets exhibit distinct metabolic profiles. CD4⁺ T cells from wild-type (A, C–E) or Glut1-myc (B) knockin mice were cultured to enrich Teff (Th1, Th2, Th17) and Treg subsets. After 3 d, cells were washed and cultured 2 d in IL-2 alone and analyzed by immunoblot (A), surface staining of endogenous Glut1-myc (B) with anti-Myc Ab followed by gating on each T cell subset as determined by intracellular cytokine staining, glycolysis (C), mitochondrial potential by tetramethylrhodamine ethyl ester staining (D), and palmitate oxidation (E). Naive CD4⁺ T cells or cells activated in the presence of the CPTI inhibitor Etx are shown as controls. F, Percentage of splenic cytokine⁺ CD4⁺ T cells from >1 y aged Glut1 transgenic (n = 6) and nontransgenic (n = 5) mice following stimulation intracellular cytokine staining for IFN-γ, IL-4, and IL-17. The percentage of Foxp3⁺ cells was assessed ex vivo by intracellular staining. Results are from three independent experiments with average and SD determined using the Student t test. *p ≤ 0.05.

FIGURE 2

Lipid oxidation promotes Treg generation and suppresses Teff function and survival. A, CD4⁺ T cells were stimulated in conditions to enrich for Th1 cells and Treg in the presence or absence of Etx and assessed by intracellular cytokine and Foxp3 staining. CD4⁺ T cells were cultured in Teff and Treg skewing cultures in the presence or absence of exogenous FA, washed, and examined for function by intracellular cytokine staining or in a T cell suppressor assay (B) and expression of CD4⁺ lineage commitment transcription factors by flow cytometry (C). D, Th cell subsets were generated, washed, and replated an additional 2 d in IL-2 and a further 2 d in the presence or absence of FA in parallel wells to measure cell viability and cytokine production in each condition. The number of viable cytokine-producing cells was then calculated for each condition. Results are representative of three independent experiments with average and SD determined using the Student t test. *p ≤ 0.05.

FIGURE 3

AMPK and mTOR regulate lipid oxidation and enrich Treg differentiation in vivo. A, CD4⁺ T cells were stimulated with anti-CD3 and anti-CD28 for 18 h, washed, and replated in the presence of anti-CD3 alone, 25 nM rapamycin (Rapa), 2 ng/ml TGFb, 200 μM Etx, or 10 mM FA for an additional 48 h followed by intracellular Foxp3 staining. B, Immunoblot of Th cultures, naive CD4⁺ T cells, and natural Tregs. C–E, Mice were sensitized to OVA in the presence of Met or a PBS followed by aerosol challenge 21 d later and analyzed by intracellular Glut1 staining in CD4⁺ CD44^lowT cells (solid gray) and CD4⁺ CD44^high T cells in the draining lymph node of the lung from mice treated with (black line) or without (gray line) Met (C). In addition, the percentage (D) and number (E) of CD4⁺ Foxp3⁺ cells and total CD4⁺ T cells in the draining lymph node following aerosol challenge were determined. Results are representative of three independent experiments with average and SD determined using the Student t test. *p ≤ 0.05.