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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 controls glucose uptake and glycolysis via HIF1. (A–D) Immunoblot analysis of HIF1α and HIF1β expression in naive P14-LCMV CD8+ T cells ± TCR (gp33-41/anti-CD28) stimulation for 20 h (A and C) and P14-LCMV CTLs (A, B, and D) treated with and without IL-2 (B) or rapamycin (C and D) for 20 h. Phospho-S6K1 and phospho-S6 were used as a measure of mTORC1 activity. (E) Immunoblot analysis of c-myc expression in CTLs treated with or without rapamycin for 20 h. Phospho-S6K1 and phospho-S6 were used as a measure of mTORC1 activity. (F) Flow cytometric analysis of HIF1βWT/WT CD4Cre (WT) and HIF1βflox/flox CD4Cre (HIF1−/−) CD8+ T cells after TCR (2c11) stimulation for 20 h. (G) Immunoblot analysis of WT and HIF1−/− CTLs. (H and I) Analysis of glucose uptake (H) and lactate production (I) in WT and HIF1−/− naive CD8+ T cells after TCR (2c11/anti-CD28) stimulation for 20 h. Glucose uptake in unstimulated WT naive T cells is also shown (uptake in unstimulated HIF1−/− naive T cells is equivalent to WT; not depicted). (J–L) Analysis of glucose uptake (J), Glut1 expression (K), and lactate production (L) in WT versus HIF1−/− CTLs. (M) A comparison of the transcriptional profile of HIF1 WT versus HIF1−/− CTLs was performed by microarray. Shown here are KEGG pathway analysis of genes down-regulated in HIF1−/− CTLs (top) and a heat map of the relative normalized expression of selected genes that are significantly different in expression in WT versus HIF1−/− CTLs, as determined by microarray. 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. Dotted lines indicate that intervening lanes have been spliced out.
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fig3: mTORC1 controls glucose uptake and glycolysis via HIF1. (A–D) Immunoblot analysis of HIF1α and HIF1β expression in naive P14-LCMV CD8+ T cells ± TCR (gp33-41/anti-CD28) stimulation for 20 h (A and C) and P14-LCMV CTLs (A, B, and D) treated with and without IL-2 (B) or rapamycin (C and D) for 20 h. Phospho-S6K1 and phospho-S6 were used as a measure of mTORC1 activity. (E) Immunoblot analysis of c-myc expression in CTLs treated with or without rapamycin for 20 h. Phospho-S6K1 and phospho-S6 were used as a measure of mTORC1 activity. (F) Flow cytometric analysis of HIF1βWT/WT CD4Cre (WT) and HIF1βflox/flox CD4Cre (HIF1−/−) CD8+ T cells after TCR (2c11) stimulation for 20 h. (G) Immunoblot analysis of WT and HIF1−/− CTLs. (H and I) Analysis of glucose uptake (H) and lactate production (I) in WT and HIF1−/− naive CD8+ T cells after TCR (2c11/anti-CD28) stimulation for 20 h. Glucose uptake in unstimulated WT naive T cells is also shown (uptake in unstimulated HIF1−/− naive T cells is equivalent to WT; not depicted). (J–L) Analysis of glucose uptake (J), Glut1 expression (K), and lactate production (L) in WT versus HIF1−/− CTLs. (M) A comparison of the transcriptional profile of HIF1 WT versus HIF1−/− CTLs was performed by microarray. Shown here are KEGG pathway analysis of genes down-regulated in HIF1−/− CTLs (top) and a heat map of the relative normalized expression of selected genes that are significantly different in expression in WT versus HIF1−/− CTLs, as determined by microarray. 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. Dotted lines indicate that intervening lanes have been spliced out.

Mentions: The expression of Glut1 can be controlled by the HIF1α and HIF1β (also known as ARNT or Aryl hydrocarbon receptor [AHR] nuclear translocator) complex (Semenza, 2010). Do CD8+ T cells express HIF1 complexes? Fig. 3 A addresses this issue and shows that TCR-triggered CD8+ T cells expressed both HIF1α and HIF1β. Furthermore, as CD8 T cells differentiated to CTLs, they increased and sustained high levels of HIF1α and HIF1β (Fig. 3 A) in a response that requires sustained IL-2 signaling and mTORC1 activity (Fig. 3 B). TCR-induced HIF1α expression in CD8+ T cells was thus dependent on mTORC1 activity (Fig. 3 C). Similarly, HIF1α protein expression in IL-2–maintained CTLs was dependent on continuous mTORC1 activation (Fig. 3 D). The mTORC1 dependence of HIF1α expression correlates with the requirement for sustained mTORC1 and cytokine signaling to control Glut1 expression and a glycolytic metabolism in CD8+ T cells (Figs. 1 and 2). In this respect, a previous study has implicated c-myc as a major regulator of glucose metabolism in T cells (Wang et al., 2011). However, the expression of c-myc in IL-2–sustained CTLs was not dependent on mTORC1 (Fig. 3 E). The inhibition of mTORC1 with rapamycin thus inhibits Glut1 expression, glucose uptake, and glycolysis in CTLs independently of any effect on c-myc expression.


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 controls glucose uptake and glycolysis via HIF1. (A–D) Immunoblot analysis of HIF1α and HIF1β expression in naive P14-LCMV CD8+ T cells ± TCR (gp33-41/anti-CD28) stimulation for 20 h (A and C) and P14-LCMV CTLs (A, B, and D) treated with and without IL-2 (B) or rapamycin (C and D) for 20 h. Phospho-S6K1 and phospho-S6 were used as a measure of mTORC1 activity. (E) Immunoblot analysis of c-myc expression in CTLs treated with or without rapamycin for 20 h. Phospho-S6K1 and phospho-S6 were used as a measure of mTORC1 activity. (F) Flow cytometric analysis of HIF1βWT/WT CD4Cre (WT) and HIF1βflox/flox CD4Cre (HIF1−/−) CD8+ T cells after TCR (2c11) stimulation for 20 h. (G) Immunoblot analysis of WT and HIF1−/− CTLs. (H and I) Analysis of glucose uptake (H) and lactate production (I) in WT and HIF1−/− naive CD8+ T cells after TCR (2c11/anti-CD28) stimulation for 20 h. Glucose uptake in unstimulated WT naive T cells is also shown (uptake in unstimulated HIF1−/− naive T cells is equivalent to WT; not depicted). (J–L) Analysis of glucose uptake (J), Glut1 expression (K), and lactate production (L) in WT versus HIF1−/− CTLs. (M) A comparison of the transcriptional profile of HIF1 WT versus HIF1−/− CTLs was performed by microarray. Shown here are KEGG pathway analysis of genes down-regulated in HIF1−/− CTLs (top) and a heat map of the relative normalized expression of selected genes that are significantly different in expression in WT versus HIF1−/− CTLs, as determined by microarray. 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. Dotted lines indicate that intervening lanes have been spliced out.
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fig3: mTORC1 controls glucose uptake and glycolysis via HIF1. (A–D) Immunoblot analysis of HIF1α and HIF1β expression in naive P14-LCMV CD8+ T cells ± TCR (gp33-41/anti-CD28) stimulation for 20 h (A and C) and P14-LCMV CTLs (A, B, and D) treated with and without IL-2 (B) or rapamycin (C and D) for 20 h. Phospho-S6K1 and phospho-S6 were used as a measure of mTORC1 activity. (E) Immunoblot analysis of c-myc expression in CTLs treated with or without rapamycin for 20 h. Phospho-S6K1 and phospho-S6 were used as a measure of mTORC1 activity. (F) Flow cytometric analysis of HIF1βWT/WT CD4Cre (WT) and HIF1βflox/flox CD4Cre (HIF1−/−) CD8+ T cells after TCR (2c11) stimulation for 20 h. (G) Immunoblot analysis of WT and HIF1−/− CTLs. (H and I) Analysis of glucose uptake (H) and lactate production (I) in WT and HIF1−/− naive CD8+ T cells after TCR (2c11/anti-CD28) stimulation for 20 h. Glucose uptake in unstimulated WT naive T cells is also shown (uptake in unstimulated HIF1−/− naive T cells is equivalent to WT; not depicted). (J–L) Analysis of glucose uptake (J), Glut1 expression (K), and lactate production (L) in WT versus HIF1−/− CTLs. (M) A comparison of the transcriptional profile of HIF1 WT versus HIF1−/− CTLs was performed by microarray. Shown here are KEGG pathway analysis of genes down-regulated in HIF1−/− CTLs (top) and a heat map of the relative normalized expression of selected genes that are significantly different in expression in WT versus HIF1−/− CTLs, as determined by microarray. 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. Dotted lines indicate that intervening lanes have been spliced out.
Mentions: The expression of Glut1 can be controlled by the HIF1α and HIF1β (also known as ARNT or Aryl hydrocarbon receptor [AHR] nuclear translocator) complex (Semenza, 2010). Do CD8+ T cells express HIF1 complexes? Fig. 3 A addresses this issue and shows that TCR-triggered CD8+ T cells expressed both HIF1α and HIF1β. Furthermore, as CD8 T cells differentiated to CTLs, they increased and sustained high levels of HIF1α and HIF1β (Fig. 3 A) in a response that requires sustained IL-2 signaling and mTORC1 activity (Fig. 3 B). TCR-induced HIF1α expression in CD8+ T cells was thus dependent on mTORC1 activity (Fig. 3 C). Similarly, HIF1α protein expression in IL-2–maintained CTLs was dependent on continuous mTORC1 activation (Fig. 3 D). The mTORC1 dependence of HIF1α expression correlates with the requirement for sustained mTORC1 and cytokine signaling to control Glut1 expression and a glycolytic metabolism in CD8+ T cells (Figs. 1 and 2). In this respect, a previous study has implicated c-myc as a major regulator of glucose metabolism in T cells (Wang et al., 2011). However, the expression of c-myc in IL-2–sustained CTLs was not dependent on mTORC1 (Fig. 3 E). The inhibition of mTORC1 with rapamycin thus inhibits Glut1 expression, glucose uptake, and glycolysis in CTLs independently of any effect on c-myc expression.

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