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PDK1 regulation of mTOR and hypoxia-inducible factor 1 integrate metabolism and migration of CD8+ T cells.

Finlay DK, Rosenzweig E, Sinclair LV, Feijoo-Carnero C, Hukelmann JL, Rolf J, Panteleyev AA, Okkenhaug K, Cantrell DA - J. Exp. Med. (2012)

Bottom Line: We also show that PI3K- and Akt-independent pathways mediated by mTORC1 regulate the expression of HIF1 (hypoxia-inducible factor 1) transcription factor complex.This mTORC1-HIF1 pathway is required to sustain glucose metabolism and glycolysis in effector CTLs and strikingly functions to couple mTORC1 to a diverse transcriptional program that controls expression of glucose transporters, multiple rate-limiting glycolytic enzymes, cytolytic effector molecules, and essential chemokine and adhesion receptors that regulate T cell trafficking.These data reveal a fundamental mechanism linking nutrient and oxygen sensing to transcriptional control of CD8+ T cell differentiation.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin 2, Ireland. finlayd@tcd.ie

ABSTRACT
mTORC1 (mammalian target of rapamycin complex 1) controls transcriptional programs that determine CD8+ cytolytic T cell (CTL) fate. In some cell systems, mTORC1 couples phosphatidylinositol-3 kinase (PI3K) and Akt to the control of glucose uptake and glycolysis. However, PI3K-Akt-independent mechanisms control glucose metabolism in CD8+ T cells, and the role of mTORC1 has not been explored. The present study now demonstrates that mTORC1 activity in CD8+ T cells is not dependent on PI3K or Akt but is critical to sustain glucose uptake and glycolysis in CD8+ T cells. We also show that PI3K- and Akt-independent pathways mediated by mTORC1 regulate the expression of HIF1 (hypoxia-inducible factor 1) transcription factor complex. This mTORC1-HIF1 pathway is required to sustain glucose metabolism and glycolysis in effector CTLs and strikingly functions to couple mTORC1 to a diverse transcriptional program that controls expression of glucose transporters, multiple rate-limiting glycolytic enzymes, cytolytic effector molecules, and essential chemokine and adhesion receptors that regulate T cell trafficking. These data reveal a fundamental mechanism linking nutrient and oxygen sensing to transcriptional control of CD8+ T cell differentiation.

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mTORC1 regulates glucose uptake and glycolysis in TCR-stimulated CD8+ T cells. (A) Immunoblot analysis of Glut1 expression in naive OTI CD8+ T cells ± TCR (SIINFEKL) stimulation for 20 h. (B and C) Naive P14-LCMV CD8+ T cells ± TCR (gp33-41/anti-CD28) stimulation were assayed for glucose uptake (B) and lactate production (C). (D) Immunoblot analysis of naive OTI CD8+ T cells ± TCR (SIINFEKL) stimulation for 20 h. mTORC1 activity was determined by analyzing the phosphorylation of target sequences on S6K1 (T389 and S241/242) and phosphorylation of the S6K1 substrate S6 ribosomal protein. PTEN was used as a loading control. (E–G) Immunoblot analysis (E) and analysis of glucose uptake (F) and lactate production (G) for naive P14-LCMV CD8+ T cells ± TCR (gp33-41/anti-CD28) stimulation with or without rapamycin for 20 h. For all panels, data are mean ± SEM or representative of at least three experiments. All metabolic assays were preformed in triplicate (**, P < 0.01; ***, P < 0.001). Molecular mass is indicated in kilodaltons.
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fig1: mTORC1 regulates glucose uptake and glycolysis in TCR-stimulated CD8+ T cells. (A) Immunoblot analysis of Glut1 expression in naive OTI CD8+ T cells ± TCR (SIINFEKL) stimulation for 20 h. (B and C) Naive P14-LCMV CD8+ T cells ± TCR (gp33-41/anti-CD28) stimulation were assayed for glucose uptake (B) and lactate production (C). (D) Immunoblot analysis of naive OTI CD8+ T cells ± TCR (SIINFEKL) stimulation for 20 h. mTORC1 activity was determined by analyzing the phosphorylation of target sequences on S6K1 (T389 and S241/242) and phosphorylation of the S6K1 substrate S6 ribosomal protein. PTEN was used as a loading control. (E–G) Immunoblot analysis (E) and analysis of glucose uptake (F) and lactate production (G) for naive P14-LCMV CD8+ T cells ± TCR (gp33-41/anti-CD28) stimulation with or without rapamycin for 20 h. For all panels, data are mean ± SEM or representative of at least three experiments. All metabolic assays were preformed in triplicate (**, P < 0.01; ***, P < 0.001). Molecular mass is indicated in kilodaltons.

Mentions: TCR triggering of naive CD8+ T cells with peptide–MHC complexes induced expression of the glucose transporter Glut1 and a concomitant increase in glucose uptake and lactate output (Fig. 1, A–C). TCR triggering also activated mTORC1 activity (Fig. 1 D), as judged by assessing the impact of TCR ligation on phosphorylation of mTORC1 substrate sequences on S6K1 (T389 and S421/424) and the phosphorylation of the S6K1 substrate S6 ribosomal protein (S235/236). The inhibition of mTORC1 activity with rapamycin blocked TCR-induced increases in Glut1 expression and glucose uptake and reduced lactate production (Fig. 1, E–G). TCR-primed CD8+ T cells cultured in IL-2 clonally expanded and differentiated to CTLs. One role for IL-2 is to sustain glucose uptake and glycolysis in TCR-primed T cells. CTLs cultured in IL-2 thus had high levels of Glut1 expression (Fig. 2 A), high levels of glucose uptake, and high levels of lactate output. The expression of Glut1 was lost when CTLs were deprived of IL-2 (Fig. 2 B). Moreover, the removal of IL-2 caused CTLs to decrease glucose uptake and lactate output, i.e., glycolysis (Fig. 2, C and D). Strikingly, when CTLs were treated with rapamycin, they also decreased lactate output, which would be consistent with a model whereby rapamycin treatment inhibits glycolysis (Fig. 2 E). Consistent with such a model, rapamycin-treated T cells showed severely decreased glucose uptake, loss of Glut1 expression, and decreased expression of several essential glycolytic enzymes such as hexokinase 2, phosphofructokinase 1, pyruvate kinases, and lactate dehydrogenase (Fig. 2, F–H). mTORC1 thus integrates both TCR and IL-2 signaling to induce and sustain Glut1 expression and glucose uptake. mTORC1 also controls expression of key glycolytic enzymes in activated CD8+ T cells.


PDK1 regulation of mTOR and hypoxia-inducible factor 1 integrate metabolism and migration of CD8+ T cells.

Finlay DK, Rosenzweig E, Sinclair LV, Feijoo-Carnero C, Hukelmann JL, Rolf J, Panteleyev AA, Okkenhaug K, Cantrell DA - J. Exp. Med. (2012)

mTORC1 regulates glucose uptake and glycolysis in TCR-stimulated CD8+ T cells. (A) Immunoblot analysis of Glut1 expression in naive OTI CD8+ T cells ± TCR (SIINFEKL) stimulation for 20 h. (B and C) Naive P14-LCMV CD8+ T cells ± TCR (gp33-41/anti-CD28) stimulation were assayed for glucose uptake (B) and lactate production (C). (D) Immunoblot analysis of naive OTI CD8+ T cells ± TCR (SIINFEKL) stimulation for 20 h. mTORC1 activity was determined by analyzing the phosphorylation of target sequences on S6K1 (T389 and S241/242) and phosphorylation of the S6K1 substrate S6 ribosomal protein. PTEN was used as a loading control. (E–G) Immunoblot analysis (E) and analysis of glucose uptake (F) and lactate production (G) for naive P14-LCMV CD8+ T cells ± TCR (gp33-41/anti-CD28) stimulation with or without rapamycin for 20 h. For all panels, data are mean ± SEM or representative of at least three experiments. All metabolic assays were preformed in triplicate (**, P < 0.01; ***, P < 0.001). Molecular mass is indicated in kilodaltons.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3526360&req=5

fig1: mTORC1 regulates glucose uptake and glycolysis in TCR-stimulated CD8+ T cells. (A) Immunoblot analysis of Glut1 expression in naive OTI CD8+ T cells ± TCR (SIINFEKL) stimulation for 20 h. (B and C) Naive P14-LCMV CD8+ T cells ± TCR (gp33-41/anti-CD28) stimulation were assayed for glucose uptake (B) and lactate production (C). (D) Immunoblot analysis of naive OTI CD8+ T cells ± TCR (SIINFEKL) stimulation for 20 h. mTORC1 activity was determined by analyzing the phosphorylation of target sequences on S6K1 (T389 and S241/242) and phosphorylation of the S6K1 substrate S6 ribosomal protein. PTEN was used as a loading control. (E–G) Immunoblot analysis (E) and analysis of glucose uptake (F) and lactate production (G) for naive P14-LCMV CD8+ T cells ± TCR (gp33-41/anti-CD28) stimulation with or without rapamycin for 20 h. For all panels, data are mean ± SEM or representative of at least three experiments. All metabolic assays were preformed in triplicate (**, P < 0.01; ***, P < 0.001). Molecular mass is indicated in kilodaltons.
Mentions: TCR triggering of naive CD8+ T cells with peptide–MHC complexes induced expression of the glucose transporter Glut1 and a concomitant increase in glucose uptake and lactate output (Fig. 1, A–C). TCR triggering also activated mTORC1 activity (Fig. 1 D), as judged by assessing the impact of TCR ligation on phosphorylation of mTORC1 substrate sequences on S6K1 (T389 and S421/424) and the phosphorylation of the S6K1 substrate S6 ribosomal protein (S235/236). The inhibition of mTORC1 activity with rapamycin blocked TCR-induced increases in Glut1 expression and glucose uptake and reduced lactate production (Fig. 1, E–G). TCR-primed CD8+ T cells cultured in IL-2 clonally expanded and differentiated to CTLs. One role for IL-2 is to sustain glucose uptake and glycolysis in TCR-primed T cells. CTLs cultured in IL-2 thus had high levels of Glut1 expression (Fig. 2 A), high levels of glucose uptake, and high levels of lactate output. The expression of Glut1 was lost when CTLs were deprived of IL-2 (Fig. 2 B). Moreover, the removal of IL-2 caused CTLs to decrease glucose uptake and lactate output, i.e., glycolysis (Fig. 2, C and D). Strikingly, when CTLs were treated with rapamycin, they also decreased lactate output, which would be consistent with a model whereby rapamycin treatment inhibits glycolysis (Fig. 2 E). Consistent with such a model, rapamycin-treated T cells showed severely decreased glucose uptake, loss of Glut1 expression, and decreased expression of several essential glycolytic enzymes such as hexokinase 2, phosphofructokinase 1, pyruvate kinases, and lactate dehydrogenase (Fig. 2, F–H). mTORC1 thus integrates both TCR and IL-2 signaling to induce and sustain Glut1 expression and glucose uptake. mTORC1 also controls expression of key glycolytic enzymes in activated CD8+ T cells.

Bottom Line: We also show that PI3K- and Akt-independent pathways mediated by mTORC1 regulate the expression of HIF1 (hypoxia-inducible factor 1) transcription factor complex.This mTORC1-HIF1 pathway is required to sustain glucose metabolism and glycolysis in effector CTLs and strikingly functions to couple mTORC1 to a diverse transcriptional program that controls expression of glucose transporters, multiple rate-limiting glycolytic enzymes, cytolytic effector molecules, and essential chemokine and adhesion receptors that regulate T cell trafficking.These data reveal a fundamental mechanism linking nutrient and oxygen sensing to transcriptional control of CD8+ T cell differentiation.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin 2, Ireland. finlayd@tcd.ie

ABSTRACT
mTORC1 (mammalian target of rapamycin complex 1) controls transcriptional programs that determine CD8+ cytolytic T cell (CTL) fate. In some cell systems, mTORC1 couples phosphatidylinositol-3 kinase (PI3K) and Akt to the control of glucose uptake and glycolysis. However, PI3K-Akt-independent mechanisms control glucose metabolism in CD8+ T cells, and the role of mTORC1 has not been explored. The present study now demonstrates that mTORC1 activity in CD8+ T cells is not dependent on PI3K or Akt but is critical to sustain glucose uptake and glycolysis in CD8+ T cells. We also show that PI3K- and Akt-independent pathways mediated by mTORC1 regulate the expression of HIF1 (hypoxia-inducible factor 1) transcription factor complex. This mTORC1-HIF1 pathway is required to sustain glucose metabolism and glycolysis in effector CTLs and strikingly functions to couple mTORC1 to a diverse transcriptional program that controls expression of glucose transporters, multiple rate-limiting glycolytic enzymes, cytolytic effector molecules, and essential chemokine and adhesion receptors that regulate T cell trafficking. These data reveal a fundamental mechanism linking nutrient and oxygen sensing to transcriptional control of CD8+ T cell differentiation.

Show MeSH
Related in: MedlinePlus