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

PDK1 regulation of mTOR and hypoxia-inducible factor 1 integrate metabolism and migration of CD8+... A r t i c l e PDK1 regulation of mTOR and hypoxia-inducible factor 1 integrate metabolism and migration of CD8 T cells 1,2 3 3 David K. Finlay, Ella Rosenzweig, Linda V. Sinclair, 3 3 3 Carmen Feijoo-Carnero, Jens L. Hukelmann, Julia Rolf, 4 5 3 Andrey A. Panteleyev, Klaus Okkenhaug, and Doreen A. Cantrell 1 2 School of Biochemistry and Immunology and School of Pharmacy and Pharmaceutical Sciences, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin 2, Ireland 3 4 Division of Cell Signalling and Immunology, College of Life Sciences; and Division of Cancer Research, Medical Research Institute, College of Medicine, Dentistry, and Nursing; University of Dundee, Dundee DD1 4HN, Scotland, UK Laboratory of Lymphocyte Signalling and Development, Babraham Institute, Cambridge CB22 3AT, England, UK 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 glycoly- sis. 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 demon- strates 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. The differentiation of effector CTLs requires expression of the glucose transporter Glut1. CORRESPONDENCE Doreen A. Cantrell: that naive T cells undergo clonal expansion and In this context, it has been reported that rela- [email protected] reprogram their transcriptome to express the key tively high levels of exogenous glucose are OR cytolytic effector molecules that mediate the required to sustain the transcriptional program David K. Finlay: [email protected] CD8 T cell immune response. Moreover, a of CTLs (Cham and Gajewski, 2005; Cham striking feature of CD8 T cells is that they et al., 2008). Abbreviations used: 4OHT, massively increase glucose uptake as they respond During CD8 T cell die ff rentiation, the gly - 4-hydroxytamoxifen; AHR, to an immune challenge and differentiate to colytic switch is initiated by antigen receptors Aryl hydrocarbon receptor; ChIP, chromatin immunopre- cytolytic effectors (Fox et al., 2005; Maciver and co-stimulatory molecules but is then sus- cipitation; PI3K, phosphati- et al., 2008). They also switch from metaboliz- tained by inflammatory cytokines such as IL-2. dylinositol-3 kinase; Pol II, ing glucose primarily through oxidative phos- This cytokine controls the transcriptional pro- RNA polymerase II. phorylation to using the glycolytic pathway. gram that determines CD8 T cell differentia - Glycolysis requires that T cells switch on and tion and promotes ee ff ctor CTL die ff rentiation sustain expression of rate-limiting glycolytic at the expense of memory cell formation (Kalia enzymes such as hexokinase 2, phosphofructo- et al., 2010; Pipkin et al., 2010). In many cells, kinase 1, pyruvate kinases, and lactate dehydrog- growth factors and cytokines control glucose enase and also requires that T cells can sustain © 2012 Finlay et al. This article is distributed under the terms of an Attribution– high levels of glucose uptake by maintaining Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share D.K. Finlay and E. Rosenzweig contributed equally to Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/ this paper. by-nc-sa/3.0/). The Rockefeller University Press $30.00 J. Exp. Med. 2012 Vol. 209 No. 13 2441-2453 www.jem.org/cgi/doi/10.1084/jem.20112607 The Journal of Experimental Medicine metabolism via signaling pathways controlled by phosphati- dylinositol-3 kinase (PI3K) signals and the serine/threonine kinase Akt (also called protein kinase B). However, although PI3K and Akt direct the transcriptional program of CTLs, they are not required for the TCR-mediated initiation of glucose uptake nor are they required for IL-2 to sustain glucose uptake and glycolysis (Macintyre et al., 2011). Rather, this role is controlled by a PI3K-independent mechanism inv olving PDK1 (phosphoinositide-dependent kinase 1; Macintyre et al., 2011). In this context, in CD4 T cells, the serine kinase mTORC1 (mammalian target of rapamycin complex 1) can control glucose metabolism via regulation of HIF1 (hypoxia-inducible factor 1) complexes (Shi et al., 2011). In CD8 T cells, it has been recently reported that the initial glycolytic switch induced in response to antigen receptor triggering is mediated by c-myc and is independent of HIF1 (Wang et al., 2011). It thus remains to be determined whether the mTORC1–HIF1 pathway plays any role in controlling CD8 T cell metabolism. Never- theless, mTORC1 does play an essential role in CD8 T cells to integrate inputs from nutrients, antigen, and cytokine receptors to control T cell die ff rentiation (Powell and Delgoffe, 2010). For example, inhibition of mTORC1 activity in effec - tor CD8 T cells can divert these cells to a memory fate (Araki et al., 2009). Moreover, mTORC1 signaling controls expression of cytolytic effector molecules in CTLs (Rao et al., 2010) and dictates the tissue-homing properties of these cells by regulating the expression of chemokine and adhesion receptors (Sinclair et al., 2008). However, the molecular Figure 1. mTORC1 regulates glucose uptake and glycolysis in TCR- mechanisms used by mTORC1 to control CD8 T cell diff - stimulated CD8 T cells. (A) Immunoblot analysis of Glut1 expression in erentiation are not fully understood; neither are the signaling 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 processes that activate mTORC1. Here it is pertinent that assayed for glucose uptake (B) and lactate production (C). (D) Immunoblot mTORC1 activity in CD8 T cells is proposed to be con- analysis of naive OTI CD8 T cells ± TCR (SIINFEKL) stimulation for 20 h. trolled by PI3K and Akt (Rao et al., 2010). If this model were mTORC1 activity was determined by analyzing the phosphorylation of correct, then the PI3K–Akt independence of glucose metab- target sequences on S6K1 (T389 and S241/242) and phosphorylation of olism in CD8 T cells would argue against a role for mTORC1 the S6K1 substrate S6 ribosomal protein. PTEN was used as a loading in CD8 T cell metabolism. The caveat is that models propos- control. (E–G) Immunoblot analysis (E) and analysis of glucose uptake ing PI3K control of mTORC1 activity in T cells are based (F) and lactate production (G) for naive P14-LCMV CD8 T cells ± TCR on experiments with the PI3K inhibitors wortmannin and (gp33-41/anti-CD28) stimulation with or without rapamycin for 20 h. LY294002, drugs which have very well-documented o- ff target For all panels, data are mean ± SEM or representative of at least three experiments. All metabolic assays were preformed in triplicate (**, P < 0.01; effects; of most concern is that they can directly inhibit ***, P < 0.001). Molecular mass is indicated in kilodaltons. mTOR catalytic function (Brunn et al., 1996). The possibility thus remains that mTORC1 is a key regulator of glucose metabolism in CD8 T cells but is activated via PI3K–Akt- RESULTS mTORC1 regulates glucose uptake and glycolysis independent pathways. in TCR- and IL-2–stimulated CD8 T cells Accordingly, the focus of the present study is the regulation + + and role of mTORC1 in CD8 T cells. The data establish that TCR triggering of naive CD8 T cells with peptide–MHC PI3K- and Akt-independent mechanisms mediated by PDK1 complexes induced expression of the glucose transporter Glut1 and a concomitant increase in glucose uptake and lactate control mTORC1 activity in CD8 T cells. They also expose that a PI3K- and Akt-independent pathway mediated by PDK1 output (Fig. 1, A–C). TCR triggering also activated mTORC1 and mTORC1 controls expression of HIF1 transcriptional com- activity (Fig. 1 D), as judged by assessing the impact of TCR ligation on phosphorylation of mTORC1 substrate sequences plexes in CD8 T cells. This mTORC1–HIF1 pathway is re- quired to sustain glucose metabolism and glycolysis in ee ff ctor on S6K1 (T389 and S421/424) and the phosphorylation of CTLs and strikingly functions to couple mTORC1 to a diverse the S6K1 substrate S6 ribosomal protein (S235/236). The in- hibition of mTORC1 activity with rapamycin blocked TCR- transcriptional program that controls expression of cytolytic induced increases in Glut1 expression and glucose uptake and ee ff ctor molecules and essential chemokine and adhesion recep - tors that regulate T cell trac ffi king. reduced lactate production (Fig. 1, E–G). TCR-primed CD8 2442 mTOR/HIF1 controls CTL metabolism and migration | Finlay et al. A r t i c l e 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 expres- sion 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 dehy- drogenase (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. mTORC1 controls glucose uptake and glycolysis via HIF1 The expression of Glut1 can be controlled by the HIF1 and HIF1 (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 HIF1 and HIF1. Furthermore, as CD8 T cells differ - entiated to CTLs, they increased and sustained high levels of HIF1 and HIF1 (Fig. 3 A) in a response that requires sustained IL-2 signaling and mTORC1 activity (Fig. 3 B). TCR-induced HIF1 expression in CD8 T cells was thus dependent on mTORC1 activity (Fig. 3 C). Similarly, HIF1 protein expression in IL-2–maintained CTLs was dependent on continuous mTORC1 activation (Fig. 3 D). The mTORC1 Figure 2. mTORC1 regulates glucose uptake and glycolysis in IL-2– dependence of HIF1 expression correlates with the require- maintained CTLs. (A) Immunoblot analysis for Glut1 expression in naive ment for sustained mTORC1 and cytokine signaling to control + OTI CD8 T cells ± TCR (SIINFEKL) stimulation for 20 h and also mature OTI Glut1 expression and a glycolytic metabolism in CD8 T cells CTLs. The black line indicates that intervening lanes have been spliced out. (Figs. 1 and 2). In this respect, a previous study has implicated (B) Immunoblot analysis for Glut1 expression in CTLs treated with or with- out IL-2 for 20 h. 4EBP1 was used as a loading control. (C and D) Analysis of c-myc as a major regulator of glucose metabolism in T cells glucose uptake (C) and lactate production (D) in P14-LCMV CTLs treated (Wang et al., 2011). However, the expression of c-myc in IL- with or without IL-2 for 20 h. (E and F) Analysis of lactate production 2–sustained CTLs was not dependent on mTORC1 (Fig. 3 E). (E) and glucose uptake (F) in P14-LCMV CTLs treated with or without rapa- The inhibition of mTORC1 with rapamycin thus inhibits mycin for 20 h. (G) Immunoblot analysis for Glut1 expression in P14-LCMV Glut1 expression, glucose uptake, and glycolysis in CTLs CTLs treated with or without rapamycin for 20 h. 4EBP1 was used as a load- independently of any effect on c-myc expression. ing control. (A–G) Data are mean ± SEM or representative of at least three experiments. All metabolic assays were preformed in triplicate (**, P < 0.01; The HIF1 complex does not initiate but sustains ***, P < 0.001). Molecular mass is indicated in kilodaltons. (H) SILAC-based proteomic analysis of P14-LCMV CTLs treated with and without rapamycin glycolytic metabolism in CD8 T cells for 48 h. Shown is the relative expression of rate-limiting glycolytic en- To explore a causal link between mTORC1, HIF1, and gly- zymes (rapamycin/untreated). Data are mean ± SEM for three experiments colysis, we deleted functional HIF1 transcriptional complexes and were analyzed by ANOVA (**, P < 0.01; ***, P < 0.001). CD8 was used in CD8 T cells. In these experiments, mice with floxed as a control protein with unchanged expression. HIF1 alleles were backcrossed to transgenic mice expressing Cre recombinase under the control of the CD4 promoter to TCR and IL-2 triggering, as judged by their ability to nor- mally up-regulate expression of CD25, CD71, and CD44 and (CD4Cre). These mice produced a normal complement of undergo blastogenesis (Fig. 3 F and not depicted). The deletion peripheral / T cells in the thymus, lymph nodes, and spleen (not depicted). HIF1-null CD8 T cells activated in response of HIF1 was confirmed by immunoblot analysis (Fig. 3 G). JEM Vol. 209, No. 13 2443 Figure 3. mTORC1 controls glucose uptake and glycolysis via HIF1. (A–D) Immunoblot analysis of HIF1 and HIF1 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 WT/WT flox/flox / + HIF1 CD4Cre (WT) and HIF1 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. 2444 mTOR/HIF1 controls CTL metabolism and migration | Finlay et al. A r t i c l e A previous study has suggested that the TCR-induced glyco - of mTORC1 with rapamycin, which down-regulates HIF1 lytic switch is regulated by c-myc and is not HIF1 dependent complexes and severely impairs glucose metabolism and gly- (Wang et al., 2011). The present data confirm this and show colysis in CD8 T cells, does not block the proliferation of that TCR-induced glucose uptake and lactate output were CTLs (Fig. 4 B). normal in HIF1-null cells (Fig. 3, H and I). However, strik- A key role for mTORC1 in CD8 T cells is to promote ingly, activated HIF1-null CD8 T cells could not sustain effector differentiation of CTLs by driving expression of high levels of glucose uptake as they differentiated to become genes encoding cytolytic effector molecules such as perforin, cytolytic effectors in response to IL-2. HIF1 -null immune- granzymes, and IFN-. We therefore interrogated the data to activated CD8 T cells maintained in the presence of IL-2 determine whether HIF1 transcriptional complexes mediate thus had greatly decreased Glut1 expression and glucose uptake mTORC1 control of any of the key molecules that control and produced significantly less lactate compared with control CTL die ff rentiation. It was thus striking that immune-activated IL-2–maintained CTLs (Fig. 3, J–L). The ability of IL-2 to HIF1-null CD8 T cells had lost expression of perforin and sustain glucose uptake and glycolysis is thus strictly depen- multiple granzymes (Fig. 4 C). This was not a global block in dent on HIF1. In this context, HIF1 can complex with CD8 T cell differentiation as HIF1 complexes were not AHR. However, CTLs do not express high levels of the AHR, required for expression of other CD8 effector molecules such and there is little evidence of functional AHR signaling. More- as IFN-, Fas ligand, or lymphotoxin (Fig. 4, C and D). More- over, AHR-null CTLs have normal glucose uptake and lactate over, activated HIF1-null CD8 T cells retained expression output (not depicted). of the transcription factors T-bet and Blimp-1, which drive mTORC1 can regulate glycolysis by controlling expres- T cell die ff rentiation (Fig. 4 E and Tables S1 and S2). The failure sion of Glut1 and the expression of key glycolytic enzymes of immune-activated HIF1-null CD8 T cells to express (Fig. 2, G and H). Is the role of HIF1 in CD8 T cells perforin was confirmed independently by quantitative PCR restricted to control of Glut1 expression? To explore this issue, analysis of perforin mRNA levels and Western blot analysis we used Affymetrix microarray analysis to transcriptionally of perforin protein expression (Fig. 4, F and G). Consistent profile HIF1 -null CTLs. Approximately 11,517 annotated with a role for mTORC1–HIF1 in controlling perforin ex- genes were expressed in CTLs, and the impact of HIF1 loss pression, protein levels for perforin were also decreased after was a decrease in the expression of <5% of these genes and an rapamycin treatment in WT CTLs (Fig. 4 G). We also assessed increase in the expression of another 6%. The full list of genes whether the expression of HIF1 complexes was rate limiting changing in expression is detailed in Tables S1 and S2. We for perforin gene expression by examining the impact of then used the functional annotation tools within DAVID hypoxia on the expression of perforin in WT CTLs. In these Bioinformatics Resources 6.7 (Huang et al., 2009a,b) to per- experiments, CTLs were switched from normoxic (21%) to form gene annotation enrichment analysis and KEGG path- hypoxic (1%) oxygen for 24 h. The switch to hypoxia strik- way mapping of the significantly changed genes in the ingly increased expression of HIF1 and also expression of HIF1-null cells. This analysis indicated that one significant Glut1, a direct HIF1 transcriptional target (Fig. 4 H). Impor- impact caused by the loss of HIF1 was down-regulation of tantly hypoxia also induced expression of perforin (Fig. 4 H). multiple genes encoding proteins that control glycolysis and These data may explain earlier observations that CTLs cul- pyruvate metabolism (Fig. 3 M). HIF1-null cells thus cannot tured under hypoxic conditions display increased cytotoxic sustain expression of key rate-limiting glycolytic enzymes: function (Caldwell et al., 2001). hexokinase 2, pyruvate kinase 2, phosphofructokinase, and What is the mechanism for HIF1 control of perforin gene lactate dehydrogenase. The ability of HIF1 complexes to sus- expression? HIF1 regulates the expression of Glut1 by direct tain the T cell glycolytic switch thus extends beyond a simple binding to the Glut1 promoter. Similarly, HIF1-regulated gly- model of HIF1 regulation of Glut1 and glucose uptake. colytic enzymes are direct HIF1 target genes. This raises the question of whether or not HIF1 regulation of perforin gene The HIF1 complex regulates the CD8 T cell transcriptional expression is direct or indirect. There was no evidence for program but is not essential for T cell proliferation HIF1-binding sites in the perforin promoter. Moreover, there The massive up-regulation of glucose metabolism and the was no change in the recruitment of RNA polymerase II switch to glycolysis under normoxia that accompanies the (Pol II) to the transcription start site and distal exons of the immune activation of CD8 T cells are thought to be essential perforin gene in HIF1-null CD8 T cells (Fig. 4 I). Hence the to meet the metabolic demands caused by the rapid prolifera - loss of perforin mRNA in HIF1-null CD8 T cells (Fig. 4 F) + + tion of clonally expanding CD8 T cells. However, CD8 was not a consequence of the failure to recruit the appro- T cells deleted of HIF1 transcriptional complexes proliferated priate transcription factors to the perforin locus. These data and clonally expanded normally (Fig. 4 A). Moreover, tran- argue that the HIF1 effect on perforin expression is indirect. scriptional profiling of HIF1 -null CTLs using Affymetrix In this regard, one possibility we considered was that the loss microarray analysis did not reveal any negative impact of HIF1 of perforin expression might be an indirect consequence of deletion on cell cycle progression, cell survival, or mitosis failed glucose uptake. Here it is relevant that a previous study (Tables S1 and S2). The failure to see proliferative defects in has shown that glucose deprivation prevents perforin expres- HIF1-null T cells is consistent with observations that inhibition sion in immune-activated CD8 T cells (Cham et al., 2008). JEM Vol. 209, No. 13 2445 + Figure 4. The HIF1 complex regulates the CD8 T cell transcriptional program but is not essential for T cell proliferation. (A and B) Proliferation analysis / of WT and HIF1 CTLs (A) and P14-LCMV CTLs treated with and without rapamycin (B). Cells were seeded at 0.3 × 10 /ml, and CTL numbers were counted after 24 h. Data are mean ± SEM of five experiments. (C) Heat map showing the relative normalized expression of selected genes that are significantly different in expression in / WT versus HIF1 CTLs, as determined by microarray. (D) Real-time PCR analysis of IFN- expression in WT / and HIF1 CTLs. Data are mean ± SEM of three experi- ments in triplicate. (E) Immunoblot analysis of T-bet and / Blimp1 expression in WT and HIF1 CTLs. Data are representative of two experiments. (F) Real-time PCR / analysis of Perforin mRNA expression in WT and HIF1 CTLs. Data are mean ± SEM of three experiments in trip- licate (**, P < 0.01). (G) Immunoblot analysis of Perforin / protein expression in WT and HIF1 CTLs (top) and P14-LCMV CTLs treated ± rapamycin for 20 h (bottom). Data are representative of at least three experiments. (H) IL-2–maintained CTLs were placed in either hypoxic (1%) or normoxic (20%) oxygen for 24 h before being subjected to immunoblot analysis for HIF1, Glut1, and perforin expression. Data are representative of three experiments. (I) ChIP was performed with anti–Pol II, and the changes in Pol II binding to the Perforin transcription start site and the second exon were quantified by real- time PCR. Data were normalized to input DNA amounts and plotted as fold over the values for Pol II binding to the HPRT proximal promoter. Data are mean ± SEM of three experiments performed in duplicate. (J) P14-LCMV T cells were activated for 2 d with gp33-41 and then cultured for a further 4 d with IL-2 in different glucose concentrations. Cells were then subjected to immunoblot analysis for perforin expression. Data are representative of two experiments. Molecular mass is indicated in kilo- daltons. Black lines (solid or dotted) indicate that inter- vening lanes have been spliced out. The present data (Fig. 4 J) show that CTLs maintained in immune-activated CTLs (Fig. 5 A). Moreover, the expression 2 mM glucose express substantially lower levels of perforin of the gene encoding the cell adhesion molecule CD62L compared with the control cells maintained in 10 mM glucose. (L-selectin) was increased in HIF1-null cells (Fig. 5, A and B). This could explain why an early study noted that glucose Immune-activated CD8 T cells down-regulate CD62L gene deprivation can limit the cytolytic function of effector CTLs transcription, and thus CTLs normally express low levels of (MacDonald and Koch, 1977). this adhesion molecule (Fig. 5, B and C; Sinclair et al., 2008). However, CD62L mRNA and protein levels were high in HIF1 regulation of chemokines and chemokine receptors activated HIF1-null CD8 T cells (Fig. 5, B and C). CCR7 One unexpected outcome from the microarray experiment is a chemokine receptor that coordinates the migration of came from the bioinformatic pathway analysis of the genes T cells into lymph nodes and is normally down-regulated in whose expression was increased in HIF1-null T cells. This CTLs. PCR analysis confirmed the microarray data: immune- analysis indicated that immune-activated HIF1-null CD8 activated HIF1-null T cells retained high levels of CCR7 T cells up-regulated the expression of genes involved in cyto- mRNA compared with normal CTLs (Fig. 5 D). kine/cytokine receptor interactions (Fig. 5 A). Closer data CD62L and CCR7 are expressed at high levels in naive interrogation revealed that these HIF1-regulated genes enco- and memory T cells and are essential for lymphocyte trans- ded chemokines, chemokine receptors, and adhesion mole - migration from the blood into secondary lymphoid tissue. cules. For example, loss of HIF1 transcriptional complexes CD62L and CCR7 loss is thus part of the program that redi- increased expression of mRNA encoding the chemokine rects effector T cell trafficking away from lymphoid tissue receptors S1P , CXCR4, CXCR3, CCR5, and CCR7 in toward sites of inflammation. In this respect, inhibition of 2446 mTOR/HIF1 controls CTL metabolism and migration | Finlay et al. A r t i c l e T cells not only retained expression of the secondary lym- phoid organ-homing receptors but also increased expression of inflammatory chemokine receptors such as CXCR3 and CCR5 (Fig. 5 A). These pleiotropic effects make it more dif - ficult to predict the impact of HIF1 loss on T cell trafficking in vivo. Accordingly, the in vivo lymph node–homing ability of activated control or HIF1-null CD8 T cells was com- pared. Strikingly, activated CD8 T cells that lacked HIF1- mediated transcription retained the capacity to home to secondary lymphoid organs and accumulated in the lymph nodes (Fig. 5 E). There is therefore a dominant requirement for HIF1 transcriptional complexes for the normal program- ming of effector CD8 T cell trafficking. Is HIF1 regulation of CD62L gene expression direct, or is the impact of HIF1 on CD62L expression an indirect consequence of the inability of HIF1-null cells to maintain glucose uptake? The experi- ment in Fig. 5 F addresses this issue and shows that CD8 T cells stimulated by antigen and IL-2 in culture media with low glucose levels fail to down-regulate CD62L expression. PI3K- and Akt-independent control of mTORC1 activity and HIF1 expression in CD8 T cells The insight that HIF1 transcription factor complexes mediate mTORC1 control of the expression of CD62L, CCR7, and S1P raises a question. How does the mTORC1–HIF1 pathway that controls T cell trafficking connect to a well- documented PI3K–Akt–Foxo pathway that also controls the expression of these key trafficking molecules? The retention Figure 5. HIF1 regulation of chemokines and chemokine receptors. / (A) A comparison of the transcriptional profile of WT versus HIF1 CTLs of high levels of CCR7 and CD62L expression by immune- was performed by microarray. Shown here are KEGG pathway analysis of activated HIF1-null CD8 T cells thus phenocopies the / genes up-regulated in HIF1 CTLs (top) and a heat map showing the impact of inhibiting PI3K–Akt signaling in activated CD8 relative normalized expression of selected genes that are significantly T cells (Sinclair et al., 2008; Waugh et al., 2009; Macintyre / different in expression in WT versus HIF1 CTLs, as determined by et al., 2011). PI3K–Akt control of CD62L, CCR7, and S1P microarray. (B) Real-time PCR analysis of CD62L expression in WT and expression reflects that the expression of these molecules is / / HIF1 CTLs. (C) Analysis of CD62L surface expression on WT and HIF1 regulated by the Foxo1 transcription factor (Fabre et al., 2008; CTLs by flow cytometry. (D) Real-time PCR analysis of CCR7 expression in / / Kerdiles et al., 2009). Foxo1 is inactivated by Akt, which WT and HIF1 CTLs. (E) WT and HIF1 CTLs were labeled with CFSE or phosphorylates Foxo1, resulting in its nuclear exclusion and CellTracker orange (CMTMR) and mixed at a ratio of 1:1 before being in- / jected into C57BL/6 host mice. Values indicate recovery of WT or HIF1 retention in the cytosol. Akt inhibition results in dephosphor- cells as a percentage of the total recovered transferred cells from the ylation of Foxo1 and restores its nuclear location and tran- blood and lymph nodes 4 h after transfer. Each dot indicates a mouse; scriptional activity and restores expression of CD62L and horizontal bars indicate mean. (F) P14 T cells were activated for 2 d with CCR7 in CTLs (Waugh et al., 2009; Macintyre et al., 2011). cognate peptide and then cultured for a further 4 d with IL-2 in different Accordingly, a key question is whether mTORC1 and/or glucose concentrations. Cells were then analyzed for the surface expres- expression of HIF1 complexes regulate Akt activity and Foxo sion of CD62L by flow cytometry. In B and D, mean ± SEM of three phosphorylation in T cells. It is also pertinent to question experiments performed in triplicate is shown; in C and F, data are whether PI3K and Akt control mTORC1 activity in CD8 representative of at least three experiments (*, P < 0.05; **, P < 0.01). T cells. Lymphocyte signaling models frequently position PI3K–Akt signaling as an upstream obligatory regulator of mTORC1 with rapamycin can restore expression of CD62L mTORC1 activity. However, the present results showing that and CCR7 in effector T cells and reprogram their trafficking mTORC1 controls glucose metabolism in T cells are incon- such that they regain the ability to home to secondary lym- sistent with these models because they are discrepant with phoid organs (Sinclair et al., 2008). The retention of CD62L observations that PI3K and Akt do not control glucose uptake + + and CCR7 on immune-activated HIF1-null CD8 T cells in CD8 T cells (Macintyre et al., 2011). Moreover, the present thus raises the possibility that these cells may retain the migra- data herein show that the mTORC1–HIF1 pathway controls tory properties of naive T cells and preferentially home to expression of the rate-limiting glycolytic enzymes (Figs. 2 H secondary lymphoid tissues. However, HIF1-null CD8 and 3 M). In contrast, microarray analysis of Akt-regulated JEM Vol. 209, No. 13 2447 genes in T cells failed to find any evidence that Akt controlled glucose metabolism or glycolysis (Macintyre et al., 2011). To explore the links between PI3K, Akt, mTORC1, and HIF1, we addressed two questions. Do mTORC1 and HIF1 regulate Akt activity and Foxo phosphorylation? Is PI3K and Akt activity required for mTORC1 activation and HIF1 expression? In the context of the first question, long-term inhibition of mTORC1 can destabilize mTORC2 complexes in some cell systems and suppress Akt function (Sarbassov et al., 2006). Indeed, rapamycin treatment of CD4 T cells activated with CD3 and CD28 antibodies does diminish Akt S473 phosphorylation, although the impact of this on Akt activity has not been fully assessed (Lee et al., 2010; Delgoffe et al., 2011). If this were true in CTLs, then long-term treat- ment of T cells with rapamycin would result in the loss of Foxo phosphorylation and cause cells to regain Foxo tran- scriptional activity and thus CD62L and CCR7 expression. We therefore examined the impact of long-term inhibition of mTORC1 with rapamycin on Foxo phosphorylation and localization in CTLs. We also assessed Akt–Foxo and mTORC1 signaling in IL-2–maintained HIF1-null T cells to assess whether loss of HIF1 complexes compromised Foxo phosphorylation/inactivation. Fig. 6 (A and B) shows that inhibition of Akt decreased Foxo phosphorylation (Fig. 6 A) and restored nuclear localization of Foxos in CTLs (Fig. 6 B). Figure 6. mTORC1 and HIF1 do not regulate Akt activity or Foxo In contrast, Akt remained active, i.e., phosphorylated on T308 phosphorylation. (A) Immunoblot analysis of phosphorylated AKT and and S473, and Foxos remained highly phosphorylated and Foxos in P14-LCMV CTLs treated with and without Akti1/2 or rapamycin excluded from the nucleus of rapamycin-treated CTLs (Fig. 6, for 24 h. (B) P14-LCMV CTLs were treated with and without rapamycin or A and B). Additionally, Akt phosphorylation (T308) and the Akti1/2 for 24 h and subjected to nuclear/cytoplasmic fractionation before immunoblot analysis for Foxo1 and Foxo3a expression. Purity of phosphorylation of Foxo transcription factors on Akt sub- cytoplasmic and nuclear fractions was confirmed by I B and Smc1 strate sites (T24/32) were unaffected in HIF1 -null CTLs, / expression. (C) Immunoblot analysis of WT or HIF1 CTLs for Akt–Foxo demonstrating that Akt signaling is not altered by HIF1 dele- / and mTORC1 signaling. HIF1 CTLs were treated with rapamycin as a tion (Fig. 6 C). It was also evident that the phosphorylation negative control for mTORC1 activity. For all panels, data are representa- of mTORC1 substrates S6K1 (p70 S6-kinase 1) and 4EBP1 tive of at least three experiments. Molecular mass is indicated in kilodal- (eIF4E-binding protein 1), on T389 and S65, respectively, tons. Dotted lines indicate that intervening lanes have been spliced out. and downstream signaling to S6 ribosomal protein (an S6K1 substrate) were normal in HIF1-null CD8 T cells (Fig. 6 C). Therefore, mTORC1 and HIF1 regulate the expression of inhibitor, Akti1/2, or a p110 inhibitor, IC87114, potently CD62L and CCR7 and T cell trafficking but do not control inhibited Akt activity in CTLs as judged by the loss of phos- Akt–Foxo phosphorylation and localization. phorylated Akt on T308 and S473 and inhibition of the phos- What about the second question? Are PI3K and Akt phorylation of the Akt substrates, Foxo transcription factors activity required for mTORC1 activation and HIF1 expres- (Fig. 7, A and B). However, neither Akti1/2 nor IC87114 + + sion in CD8 T cells? To address this issue, we used comple- prevented mTORC1 activity in CD8 T cells as neither com- mentary genetic and pharmacological strategies to block PI3K pound blocked the phosphorylation of mTORC1 substrates S6K1 and 4EBP1 or S6 phosphorylation in CTLs (Fig. 7 A). and Akt and then monitored the impact of these perturba- tions on mTORC1 activity by assessing the phosphoryla- Moreover, CTLs that had WT PI3K p110 catalytic subunits D910A tion of mTORC1 substrate sequences on S6K1 (T389 and substituted with a catalytically inactive mutant (p110 ) S421/424) and 4EBP1 (S35/47) and the phosphorylation did not activate Akt in response to IL-2 but showed normal of the S6K1 substrate S6 ribosomal protein (S235/236). The rapamycin-sensitive phosphorylation of S6K1 and S6 (Fig. 7 C). data show that IL-2–maintained CTLs contain high levels Further evidence that mTORC1 activity is independent of of active Akt phosphorylated on threonine 308 and high PI3K and Akt is that the expression of HIF1 was not regu- levels of mTORC1 signaling (Fig. 7 A). mTORC1 inhibition lated by Akt and PI3K inhibitors (Fig. 7 D). These data reveal with rapamycin abolished the phosphorylation of S6K1 on that PI3K–Akt activity is dispensable for mTORC1 activity T389 and S421/424 and blocked S6 phosphorylation. In and HIF1 expression in CTLs. If mTORC1 activity is not mediated by Akt, then what is CD8 T cells, Akt is activated via a PI3K complex containing the p110 catalytic subunit (Macintyre et al., 2011). The Akt the alternative pathway? One candidate is the serine/threonine 2448 mTOR/HIF1 controls CTL metabolism and migration | Finlay et al. A r t i c l e Figure 7. PI3K and Akt do not regulate mTORC1 activity. (A and B) CTLs were cultured in the presence or absence of Akti1/2, IC87114, rapamycin, or LY294002 for 60 min (A and B) or 24 h (A) and subjected to immunoblot analysis with the indicated antibodies. (C) CTLs generated from WT or D910A p110 mice were subjected to immunoblot analysis with or without rapamycin treatment (30 min). Data are representative of two experiments. (D) CTLs were cultured in the presence or absence of Akti1/2, IC87114, or rapamycin for 24 h and subjected to immunoblot analysis with the indicated flox/flox Flox WT/WT antibodies. (E–G) CTLs generated from PDK1 TamoxCre (PDK1 ) and PDK1 TamoxCre (WT) mice were treated ± 4OHT for 3 d to delete PDK1 and subjected to immunoblot analysis. For, A, B, and D–G, data are representative of at least three experiments. Molecular mass is indicated in kilodaltons. kinase PDK1 because this is known to be an essential regula- these results show that in CD8 T cells, mTORC1 activity tor of glucose metabolism in CD8 T cells (Macintyre et al., and HIF1 protein expression are controlled by a PI3K– 2011). We therefore examined whether PDK1 regulates Akt-independent pathway mediated by PDK1. mTORC1 activity and HIF1 expression in activated CD8 T cells. For these experiments, mice expressing floxed PDK1 DISCUSSION alleles were backcrossed with mice that express a tamoxi- The present study explores the molecular pathways that fen-regulated Cre recombinase (Macintyre et al., 2011). mediate mTORC1 control of effector CD8 T cell differen - o fl x/o fl x PDK1 TamoxCre CTLs were generated and treated with tiation. A key finding was that mTORC1 activity is required 4-hydroxytamoxifen (4OHT) to delete floxed PDK1 alleles. for immune-activated CD8 T cells to sustain high rates of PDK1 deletion was confirmed by analysis of PDK1 protein glucose uptake. mTORC1 activity is also necessary for CD8 expression and by the loss of RSK phosphorylation on its T cells to initiate and sustain a switch to a glycolytic metabo- PDK1 target site (S227; Fig. 7 E). Importantly, deletion lism. One way in which mTORC1 controls glucose uptake of PDK1 in CTLs prevented the phosphorylation of the in CD8 T cells is by controlling expression of the glucose mTORC1 substrates S6K1 and 4EBP1 on mTORC1 target transporter Glut1. CD8 T cells also show mTORC1 depen- residues (Fig. 7 F). PDK1 loss also resulted in loss of expression dence for the expression of hexokinase 2, a key enzyme which of HIF1 and the HIF1 target Glut1 (Fig. 7 G). Together phosphorylates glucose to produce glucose-6-phosphate, JEM Vol. 209, No. 13 2449 an essential intermediate in most pathways for glucose T cells thus affords the insight that mTORC1 regulation of metabolism. Importantly, mTORC1 controls expression of HIF1 controls expression of a subset of CTL effector mole - + + key rate-limiting glycolytic enzymes in CD8 T cells such as cules and controls CD8 T cell trafficking. These findings phosphofructokinase 1, lactate dehydrogenase, and pyruvate reveal a fundamental mechanism linking nutrient sensing and kinase M2. transcriptional control of CD8 T cell die ff rentiation. Although This ability of mTORC1 to coordinate and sustain the HIF1 complex evolved to function as a metabolic sensor expression of glucose transporters and essential glycolytic of cellular oxygen levels, the present study reveals a novel role enzymes during CD8 T cell differentiation stems from its for the HIF1 complex in coupling mTORC1 to the control ability to control expression of the HIF1 transcription factor of T cell differentiation. complex. The initial increase in glucose uptake and switch How does this mTORC1–HIF1 pathway coordinate to glycolysis that immediately follows TCR engagement is with other signaling pathways that control CD8 T cell dif- not HIF1 dependent. However, HIF1 is essential for antigen- ferentiation? A particular issue is that models of lymphocyte primed T cells to sustain high levels of glucose uptake and signal transduction invariably link mTORC1 to PI3K–Akt glycolytic enzyme expression as they differentiate to cytolytic signaling. As discussed, the biochemical experiments that effector cells. In this respect, CTL differentiation is controlled initially proposed this model are flawed because the pharma - by the strength and duration of signaling by inflammatory cological tools used have numerous off-target effects. Never - cytokines such as IL-2 (Kalia et al., 2010; Pipkin et al., 2010). theless, the concept of a linear model of PI3K–Akt–mTORC1 The present data now show that the ability of IL-2 to sus- signaling is still compelling because the phenotypes of T cells tain high levels of glucose uptake and maintain a glycolytic activated in the presence of PI3K–Akt or mTORC1 inhibitors metabolism is dependent on mTORC1 induction of HIF1 show similarities. For example, the expression of perforin is complexes. However, it was notable that the inhibition of positively regulated by both Akt (Macintyre et al., 2011) and mTORC1 in IL-2–maintained CTLs suppressed glucose mTORC1 (Fig. 4 G), and the expression of CD62L, CCR7, uptake and lactate output without having any impact on the and S1P is negatively regulated by both Akt and mTORC1 expression of c-myc (Fig. 3 E). This is relevant because it (Sinclair et al., 2008; Macintyre et al., 2011). In a linear model, has been shown recently that c-myc is required for the it would be assumed that PI3K and Akt activity are required initial TCR-induced glycolytic switch in naive T cells (Wang for mTORC1 activation. Moreover, there are models propos- et al., 2011). The present data thus reveal that c-myc ex- ing mTORC1 control of Akt activity. This latter idea stems pression alone is not sufficient to maintain glucose uptake from experiments showing that in some cell systems, pro- and glycolysis in CD8 T cells. Moreover the coordination longed inhibition of mTORC1 with rapamycin can disrupt of c-myc and HIF1 expression must be required for T cells the mTORC2 serine/threonine kinase complex (Sarbassov to sustain glucose uptake and glycolysis dur ing CD8 et al., 2006). mTORC2 phosphorylates Akt on S473, and it T cell differentiation. has been shown that prolonged rapamycin treatment reduces The ability of HIF1 to link mTORC1 to the control of Akt S473 phosphorylation in CD4 T cells activated with CD3 glucose metabolism in CD8 T cells made us question and CD28 antibodies (Lee et al., 2010; Delgoffe et al., 2011). whether any other actions of mTORC1 in CD8 T cell are It is frequently assumed that loss of Akt S473 phosphorylation directed by the HIF1 pathway. In particular, it is well docu- will block Akt catalytic activity. However, the key rate-limiting mented that mTORC1 activity is required for expression phosphorylation on Akt is the PDK1-mediated phosphoryla- of CTL effector molecules and for effector CD8 T cells to tion of T308, and the impact of the reduced Akt S473 phos- switch off expression of the chemokine receptors and adhe - phorylation on Akt catalytic function in CD4 T cells has not sion molecules that direct naive and memory T cell entry been studied in detail. Moreover, does rapamycin treatment and egress from secondary lymphoid tissues. For example, actually disrupt mTORC2 complexes in all T cells? The cur- mTORC1 signaling causes down-regulation of the adhesion rent study addresses these issues and shows unequivocally that molecule CD62L and the chemokine receptors CCR7 and PI3K and Akt activity are not essential for mTORC1 activa- S1P (Sinclair et al., 2008). In this regard, the phenotype of tion or for HIF1 expression in CD8 T cells. Moreover, rapa- the immune-activated HIF1-null T cells is intriguing. They mycin treatment, even long term, does not prevent Akt have many features of effector CD8 T cells such as normal phosphorylation or activity, as judged by the normal phos- IFN- production and expression of effector molecules such phorylation and nuclear exclusion of the Foxo transcription as Fas ligand and lymphotoxin. Also, they are normal in cell factors in rapamycin-treated T cells. There is a well-documented size and can rapidly proliferate. However, the HIF1-null cells Akt–Foxo-mediated pathway to control T cell trafficking in lack perforin and granzyme expression. Moreover, although CD8 T cells, but the mTORC1–HIF1 pathway that controls they acquire the expression of effector CTL chemokine T cell trafficking described herein is independent of Akt and receptors such as CXCR3 and CCR5, they retain the expres- Foxos. The present study thus indicates that PI3K–Akt and sion of naive T cell lymph node–homing receptors CD62L, mTORC1 signaling are not linked in a linear pathway. These CCR7, and S1P . They also retain the secondary lymphoid molecules are activated independently but converge to control tissue migratory program of naive T cells in vivo. The tran- the expression of key molecules required for effector CTL scriptional profiling of HIF1 -null immune-activated CD8 function. An essential requirement for these two independent 2450 mTOR/HIF1 controls CTL metabolism and migration | Finlay et al. A r t i c l e P-40, 1 mM PMSF, 1 mM Na VO , and 1 mM DTT) and centrifuged at 4°C signaling pathways to control T cell differentiation highlights 3 4 for 1 min at 10,000 g. Cytosolic fractions were removed, and nuclear mem- the importance of integrating lymphocyte signal transduction brane fractions were suspended in the aforementioned Tris lysis buffer. for appropriate immune responses. Immunoblot analysis of IB and Smc1 was used to confirm the purity of cytoplasmic and nuclear fractions, respectively. MATERIALS AND METHODS Mice. All mice used in this study were approved by the University of Dundee Glucose uptake. 10 cells were suspended in 400 µl glucose-free media ethical review committee and maintained in compliance with UK Home 3 3 containing 0.5 µCi/ml 2-deoxy-d-[1- H]glucose ([ H] 2-DG; GE Health- flox/flox Office Animals (Scientific Procedures) Act 1986 guidelines. PDK1 care) and incubated for 3 (CTLs) or 10 min (TCR-simulated CD8 T cells). tm9(CreEsr1)Arte TamoxCre mice were generated by breeding C57BL/6GT(ROSA)26 Cells were pelleted, washed, and lysed overnight with 200 µl of 1 M NaOH, (TamoxCre), purchased from Taconic, to mice carrying PDK1 floxed alleles and the incorporated H radioactivity was quantified via liquid scintillation flox/flox (Mora et al., 2003). HIF1 mice (Geng et al., 2006) were backcrossed counting. Measurements were performed in triplicates per condition. with mice expressing cre recombinase under the control of the CD4 pro- flox/flox D910A/D910A moter, generating HIF1 CD4Cre mice. PI3K p110 mice Quantitative real-time PCR. RNA was extracted using the RNeasy carry a point mutation that switches Asp →Ala (D910A) in the p110 RNA puric fi ation mini kit (QIAGEN) according to the manufacturer’s pro - subunit to inactivate catalytic activity (Okkenhaug et al., 2002). P14-LCMV tocol. Purie fi d RNA was reverse transcribed using the qScript cDNA syn - and OTI transgenic mice have been described previously (Pircher et al., thesis kit (Quanta). Real-time PCR was performed in triplicates in 96-well 1989; Kurts et al., 1996). plates using iQ SYBR Green–based detection on an iCycler (Bio-Rad Labo- ratories). For the analysis of mRNA levels, the derived values were averaged Cell culture. CTLs were generated as described previously (Waugh et al., and normalized to HPRT mRNA levels. 2009). In brief, lymphocytes isolated from spleens or lymph nodes of P14- LCMV or OTI transgenic mice or nontransgenic mice were activated for Primers. Primers used are as follows: HPRT forward, 5-TGATCAGTCA- 48 h with either 100 ng/ml of soluble LCMV or OTI-specific peptide, gp33- ACGGGGGACA-3; HPRT reverse, 5-TTCGAGAGGTCCTTTTCA- 41 and SIINFEKL, respectively, or 0.5 µg/ml anti-CD3 antibody (2c11). Cells CCA-3; CD62L forward, 5-ACGGGCCCCAGTGTCAGTATGTG-3; were then cultured in 20 ng/ml IL-2 (Proleukin) at 37°C for an additional CD62L reverse, 5-TGAGAAATGCCAGCCCCGAGAA-3; CCR7 forward, 6 d. For conditional deletion of PDK1, CTLs were prepared by activating 5-CAGCCTTCCTGTGTGATTTCTACA-3; CCR7 reverse, 5-ACC- flox/flox PDK1 TamoxCre splenocytes with 2c11 for 2 d followed by culturing ACCAGCACGTTTTTCCT-3; Perforin forward, 5-CGTCTTGGTGG- in IL-2 for 5 d, with 0.6 µM of 4OHT (Sigma-Aldrich) being added for the GACTTCAG-3; Perforin reverse, 5-GCATTCTGACCGAGGGCAG-3; final 72 h of culture. Where indicated, cells were treated with various inhibi - IFN- forward, 5-TTACTGCCACGGCACAGTC-3; and IFN- reverse, tors: 1 µM Akti1/2 (EMD Millipore), 20 nM rapamycin (EMD Millipore), 5-AGATAATCTGGCTCTGGCTCTGCGG-3. 10 µM IC87114 (synthesized in-house), and 10 µM LY294002 (Promega). For short-term (20 h) TCR stimulations, naive CD8 T cells were puri- Lactate measurement. 2 × 10 /ml CTLs were cultured for 4 h in RPMI flox/flox WT/WT fied from lymph nodes of HIF1 CD4Cre and HIF1 CD4Cre 1640 containing 10% dialyzed fetal calf serum, and then the cells were spun nontransgenic mice or alternatively from P14-LCMV or OTI transgenic and the supernatants were collected. Lactate concentration in the supernatant mice by magnetic cell sorting (Miltenyi Biotec). P14-LCMV and OTI naive was quantified using an LDH (lactate dehydrogenase)-based enzyme assay, CD8 T cells were activated with gp33-41 plus 3 ng/ml anti-CD28 (clone monitoring the emergence of NADH through increased absorption at 340 nm 37.51; eBioscience) and SIINFEKL, respectively. Nontransgenic naive CD8 (the reaction contained 320 mM glycine, 320 mM hydrazine, 2.4 mM NAD , T cells were activated with 2c11 plus anti-CD28. and 3 U/ml LDH). A standard curve was generated, and the concentration of lactate in the supernatant added to this reaction was calculated. Flow cytometric analysis. Cells were labeled with APC-efluor780 CD8 flox/flox (53-6.7), FITC CD25 (7D4), APC CD44 (IM7), PE CD71 (C2), APC Adoptive transfer. CTLs were generated from the spleens of HIF1 flox/flox CD62L (MEL-14), Alexa Fluor 700 CD45.1 (104), and V450 Horizon CD4Cre mice or HIF1 mice (as described in Cell culture but with CD45.2 (A20) purchased from eBioscience or BD. Live cells were gated 4 d in IL-2). Cells were then labeled with either CFSE (Invitrogen) or Cell- according to their forward scatter and side scatter. Data were acquired on Tracker Orange (CMTMR; Invitrogen) for 15 min at 37°C, washed, mixed either a FACSCalibur or an LSRFortessa (BD) and analyzed using FlowJo at 1:1 ratio, and suspended in sterile PBS. 5 × 10 mixed cells were injected software (Tree Star). into the tail vein of C57BL/6 host mice. After 4 h, the mice were sacrificed, and lymph nodes, spleen, and blood were analyzed and the ratio of CFSE- and flox/flox Western blot analysis and cytoplasmic nuclear fractionation. Cells CMTMR-labeled T cells quantified. Values indicate recovery of HIF1  flox/flox were lysed (2 × 10 /ml) in Tris lysis buffer containing 10 mM Tris, pH 7.05, CD4Cre cells versus HIF1 cells as a percentage of the total recovered 50 mM NaCl, 30 mM Na pyrophosphate, 50 mM NaF, 5 µM ZnCl , 10% transferred cells. glycerol, 0.5% Triton, 1 µM DTT, and protease inhibitors (Roche). Lysates were centrifuged (4°C, 16,000 g for 10 min) and separated by SDS-PAGE Chromatin immunoprecipitation (ChIP). Real-time PCR-based ChIP and transferred to nitrocellulose membrane. Blots were probed with antibodies analysis to measure Pol II binding to the Perforin locus was performed as T24/34 S235/236 T389 T421/S424 T308 recognizing pFoxo1/3a , pS6 , pS6K , pS6K , pAkt , described previously (Cruz-Guilloty et al., 2009) with minor modifications. S473 S37/46 S65 pAkt , p4EBP1 , and p4EBP1 (Cell Signaling Technology); In brief, chromatin was immune precipitated with anti–Pol II (Santa Cruz S227 pRSK (Santa Cruz Biotechnology, Inc.); and unphosphorylated S6, S6K, Biotechnology, Inc.) or normal rabbit IgG (Cell Signaling Technology) Akt, and c-Myc (Cell Signaling Technology); RSK2, IB, PTEN, and from 5 × 10 cells in the presence of 0.2 mg/ml BSA. ChIP-grade protein HIF1 (Santa Cruz Biotechnology, Inc.); HIF1 (R&D Systems); PDK1 G magnetic beads (Cell Signaling Technology) were used to collect the (EMD Millipore); Glut1 (a gift from G. Holman, University of Bath, Bath, immune complexes. Chromatin was purified via a NucleoSpin Extract II kit England, UK; Holman et al., 1990); Perforin (a gift from G. Griffith, Cam - (Macherey-Nagel) and resuspended in TE buffer. Real-time PCR was bridge Institute for Medical Research, Cambridge, England, UK); and Foxo1 performed in an iQ5 (Bio-Rad Laboratories) with Perfecta SYBR green and Foxo3a (generated in-house). Immunoblots were probed with an SmcI FastMix for iQ (Quanta BioSciences). Primers are as follows: Perforin TSS antibody as a loading control (Bethyl Laboratories, Inc.). forward, 5-CAGGGCAGGAAGTAGTAATGATATG-3; Perforin TSS To obtain cytoplasmic and nuclear fractions, cells were lysed in Hepes reverse, 5-CTTCCTCCTCCTTACCTGAAGTC-3; Perfor in Exon2 hypotonic lysis buffer (20 × 10 cells/ml; 10 mM Hepes, pH 7.9, 4 mM forward, 5-CCAGAGTTTATGACTACTGTG-3; and Perforin Exon2 MgCl , 15 mM KCl, 10 mM NaF, 0.1 mM EDTA, 0.15% [vol/vol] Nonidet reverse, 5-GTGCTTCTGCTTGCATTCTG-3. JEM Vol. 209, No. 13 2451 SILAC. P14-LCMV CTLs were cultured in SILAC medium as described 2008. FOXO1 regulates L-Selectin and a network of human T cell previously (Navarro et al., 2011) with some minor modifications. In brief, homing molecules downstream of phosphatidylinositol 3-kinase. J. Immunol. 181:2980–2989. cells were combined and a Thermo Fisher Scientific subcellular protein frac - Fox, C.J., P.S. Hammerman, and C.B. Thompson. 2005. Fuel feeds function: tionation kit was used to fractionate CTLs according to the manufacturer’s energy metabolism and the T-cell response. Nat. Rev. Immunol. 5:844– instructions. Fractions were chloroform methanol precipitated and further 852. http://dx.doi.org/10.1038/nri1710 separated by molecular weight using denaturing size exclusion chromatog- Geng, S., A. Mezentsev, S. Kalachikov, K. Raith, D.R. Roop, and A.A. raphy before digestion and LC-MS/MS analysis as described previously Panteleyev. 2006. Targeted ablation of Arnt in mouse epidermis results in (Larance et al., 2012). The peptide mixture was separated by nanoscale C18 profound defects in desquamation and epidermal barrier function.J . Cell reverse phase liquid chromatography (Ultimate 3000 nLC; Dionex) coupled Sci. 119:4901–4912. http://dx.doi.org/10.1242/jcs.03282 to a LTQ-Orbitrap Velos (Thermo Fisher Scientific). MaxQuant version Holman, G.D., I.J. Kozka, A.E. Clark, C.J. Flower, J. Saltis, A.D. Habberfield, 1.3.0.5 (Cox and Mann, 2008) was used to process raw MS spectra, using the I.A. Simpson, and S.W. Cushman. 1990. Cell surface labeling of glucose default settings against the mice Uniprot database (July 2012). transporter isoform GLUT4 by bis-mannose photolabel. Correlation with stimulation of glucose transport in rat adipose cells by insulin and Online supplemental material. Table S1 lists microarray analysis showing phorbol ester. J. Biol. Chem. 265:18172–18179. / decreased gene expression in HIF1 versus HIF1 WT CTLs. Table S2 lists Huang, W., B.T. Sherman, and R.A. Lempicki. 2009a. Bioinformatics en- / microarray analysis showing increased gene expression in HIF1 versus richment tools: paths toward the comprehensive functional analysis HIF1 WT CTLs. Online supplemental material is available at http://www of large gene lists. Nucleic Acids Res. 37:1–13. http://dx.doi.org/10 .jem.org/cgi/content/full/jem.20112607/DC1. .1093/nar/gkn923 Huang, W., B.T. Sherman, and R.A. Lempicki. 2009b. Systematic and integra- We thank members of the Biological Services Unit, R. Clarke of the Flow Cytometry tive analysis of large gene lists using DAVID bioinformatics resources. Facility, members of the “Fingerprints” Proteomics Facility, and members of the Nat. Protoc. 4:44–57. http://dx.doi.org/10.1038/nprot.2008.211 D.A. Cantrell laboratory for critical reading of the manuscript. We thank D. Alessi at Kalia, V., S. Sarkar, S. Subramaniam, W.N. Haining, K.A. Smith, and R. Ahmed. the University of Dundee for providing mice carrying PDK1 floxed alleles and the 2010. Prolonged interleukin-2Ralpha expression on virus-specic fi CD8+ Finnish DNA Microarray Centre at the Centre for Biotechnology (Turku, Finland) for T cells favors terminal-effector differentiation in vivo. Immunity. 32: the microarray analysis. 91–103. http://dx.doi.org/10.1016/j.immuni.2009.11.010 This work was supported by a Wellcome Trust Principal Research Fellowship Kerdiles, Y.M., D.R. Beisner, R. Tinoco, A.S. Dejean, D.H. Castrillon, R.A. and Program Grant (065975/Z/01/A). E. Rosenzweig and J.L. Hukelmann were DePinho, and S.M. Hedrick. 2009. Foxo1 links homing and survival of supported by Wellcome Trust PhD Studentships. naive T cells by regulating L-selectin, CCR7 and interleukin 7 receptor. The authors have no conflicting financial interests. Nat. Immunol. 10:176–184. http://dx.doi.org/10.1038/ni.1689 Kurts, C., W.R. Heath, F.R. 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PDK1 regulation of mTOR and hypoxia-inducible factor 1 integrate metabolism and migration of CD8+ T cells

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Abstract

A r t i c l e PDK1 regulation of mTOR and hypoxia-inducible factor 1 integrate metabolism and migration of CD8 T cells 1,2 3 3 David K. Finlay, Ella Rosenzweig, Linda V. Sinclair, 3 3 3 Carmen Feijoo-Carnero, Jens L. Hukelmann, Julia Rolf, 4 5 3 Andrey A. Panteleyev, Klaus Okkenhaug, and Doreen A. Cantrell 1 2 School of Biochemistry and Immunology and School of Pharmacy and Pharmaceutical Sciences, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin 2, Ireland 3 4 Division of Cell Signalling and Immunology, College of Life Sciences; and Division of Cancer Research, Medical Research Institute, College of Medicine, Dentistry, and Nursing; University of Dundee, Dundee DD1 4HN, Scotland, UK Laboratory of Lymphocyte Signalling and Development, Babraham Institute, Cambridge CB22 3AT, England, UK 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 glycoly- sis. 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 demon- strates 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. The differentiation of effector CTLs requires expression of the glucose transporter Glut1. CORRESPONDENCE Doreen A. Cantrell: that naive T cells undergo clonal expansion and In this context, it has been reported that rela- [email protected] reprogram their transcriptome to express the key tively high levels of exogenous glucose are OR cytolytic effector molecules that mediate the required to sustain the transcriptional program David K. Finlay: [email protected] CD8 T cell immune response. Moreover, a of CTLs (Cham and Gajewski, 2005; Cham striking feature of CD8 T cells is that they et al., 2008). Abbreviations used: 4OHT, massively increase glucose uptake as they respond During CD8 T cell die ff rentiation, the gly - 4-hydroxytamoxifen; AHR, to an immune challenge and differentiate to colytic switch is initiated by antigen receptors Aryl hydrocarbon receptor; ChIP, chromatin immunopre- cytolytic effectors (Fox et al., 2005; Maciver and co-stimulatory molecules but is then sus- cipitation; PI3K, phosphati- et al., 2008). They also switch from metaboliz- tained by inflammatory cytokines such as IL-2. dylinositol-3 kinase; Pol II, ing glucose primarily through oxidative phos- This cytokine controls the transcriptional pro- RNA polymerase II. phorylation to using the glycolytic pathway. gram that determines CD8 T cell differentia - Glycolysis requires that T cells switch on and tion and promotes ee ff ctor CTL die ff rentiation sustain expression of rate-limiting glycolytic at the expense of memory cell formation (Kalia enzymes such as hexokinase 2, phosphofructo- et al., 2010; Pipkin et al., 2010). In many cells, kinase 1, pyruvate kinases, and lactate dehydrog- growth factors and cytokines control glucose enase and also requires that T cells can sustain © 2012 Finlay et al. This article is distributed under the terms of an Attribution– high levels of glucose uptake by maintaining Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share D.K. Finlay and E. Rosenzweig contributed equally to Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/ this paper. by-nc-sa/3.0/). The Rockefeller University Press $30.00 J. Exp. Med. 2012 Vol. 209 No. 13 2441-2453 www.jem.org/cgi/doi/10.1084/jem.20112607 The Journal of Experimental Medicine metabolism via signaling pathways controlled by phosphati- dylinositol-3 kinase (PI3K) signals and the serine/threonine kinase Akt (also called protein kinase B). However, although PI3K and Akt direct the transcriptional program of CTLs, they are not required for the TCR-mediated initiation of glucose uptake nor are they required for IL-2 to sustain glucose uptake and glycolysis (Macintyre et al., 2011). Rather, this role is controlled by a PI3K-independent mechanism inv olving PDK1 (phosphoinositide-dependent kinase 1; Macintyre et al., 2011). In this context, in CD4 T cells, the serine kinase mTORC1 (mammalian target of rapamycin complex 1) can control glucose metabolism via regulation of HIF1 (hypoxia-inducible factor 1) complexes (Shi et al., 2011). In CD8 T cells, it has been recently reported that the initial glycolytic switch induced in response to antigen receptor triggering is mediated by c-myc and is independent of HIF1 (Wang et al., 2011). It thus remains to be determined whether the mTORC1–HIF1 pathway plays any role in controlling CD8 T cell metabolism. Never- theless, mTORC1 does play an essential role in CD8 T cells to integrate inputs from nutrients, antigen, and cytokine receptors to control T cell die ff rentiation (Powell and Delgoffe, 2010). For example, inhibition of mTORC1 activity in effec - tor CD8 T cells can divert these cells to a memory fate (Araki et al., 2009). Moreover, mTORC1 signaling controls expression of cytolytic effector molecules in CTLs (Rao et al., 2010) and dictates the tissue-homing properties of these cells by regulating the expression of chemokine and adhesion receptors (Sinclair et al., 2008). However, the molecular Figure 1. mTORC1 regulates glucose uptake and glycolysis in TCR- mechanisms used by mTORC1 to control CD8 T cell diff - stimulated CD8 T cells. (A) Immunoblot analysis of Glut1 expression in erentiation are not fully understood; neither are the signaling 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 processes that activate mTORC1. Here it is pertinent that assayed for glucose uptake (B) and lactate production (C). (D) Immunoblot mTORC1 activity in CD8 T cells is proposed to be con- analysis of naive OTI CD8 T cells ± TCR (SIINFEKL) stimulation for 20 h. trolled by PI3K and Akt (Rao et al., 2010). If this model were mTORC1 activity was determined by analyzing the phosphorylation of correct, then the PI3K–Akt independence of glucose metab- target sequences on S6K1 (T389 and S241/242) and phosphorylation of olism in CD8 T cells would argue against a role for mTORC1 the S6K1 substrate S6 ribosomal protein. PTEN was used as a loading in CD8 T cell metabolism. The caveat is that models propos- control. (E–G) Immunoblot analysis (E) and analysis of glucose uptake ing PI3K control of mTORC1 activity in T cells are based (F) and lactate production (G) for naive P14-LCMV CD8 T cells ± TCR on experiments with the PI3K inhibitors wortmannin and (gp33-41/anti-CD28) stimulation with or without rapamycin for 20 h. LY294002, drugs which have very well-documented o- ff target For all panels, data are mean ± SEM or representative of at least three experiments. All metabolic assays were preformed in triplicate (**, P < 0.01; effects; of most concern is that they can directly inhibit ***, P < 0.001). Molecular mass is indicated in kilodaltons. mTOR catalytic function (Brunn et al., 1996). The possibility thus remains that mTORC1 is a key regulator of glucose metabolism in CD8 T cells but is activated via PI3K–Akt- RESULTS mTORC1 regulates glucose uptake and glycolysis independent pathways. in TCR- and IL-2–stimulated CD8 T cells Accordingly, the focus of the present study is the regulation + + and role of mTORC1 in CD8 T cells. The data establish that TCR triggering of naive CD8 T cells with peptide–MHC PI3K- and Akt-independent mechanisms mediated by PDK1 complexes induced expression of the glucose transporter Glut1 and a concomitant increase in glucose uptake and lactate control mTORC1 activity in CD8 T cells. They also expose that a PI3K- and Akt-independent pathway mediated by PDK1 output (Fig. 1, A–C). TCR triggering also activated mTORC1 and mTORC1 controls expression of HIF1 transcriptional com- activity (Fig. 1 D), as judged by assessing the impact of TCR ligation on phosphorylation of mTORC1 substrate sequences plexes in CD8 T cells. This mTORC1–HIF1 pathway is re- quired to sustain glucose metabolism and glycolysis in ee ff ctor on S6K1 (T389 and S421/424) and the phosphorylation of CTLs and strikingly functions to couple mTORC1 to a diverse the S6K1 substrate S6 ribosomal protein (S235/236). The in- hibition of mTORC1 activity with rapamycin blocked TCR- transcriptional program that controls expression of cytolytic induced increases in Glut1 expression and glucose uptake and ee ff ctor molecules and essential chemokine and adhesion recep - tors that regulate T cell trac ffi king. reduced lactate production (Fig. 1, E–G). TCR-primed CD8 2442 mTOR/HIF1 controls CTL metabolism and migration | Finlay et al. A r t i c l e 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 expres- sion 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 dehy- drogenase (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. mTORC1 controls glucose uptake and glycolysis via HIF1 The expression of Glut1 can be controlled by the HIF1 and HIF1 (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 HIF1 and HIF1. Furthermore, as CD8 T cells differ - entiated to CTLs, they increased and sustained high levels of HIF1 and HIF1 (Fig. 3 A) in a response that requires sustained IL-2 signaling and mTORC1 activity (Fig. 3 B). TCR-induced HIF1 expression in CD8 T cells was thus dependent on mTORC1 activity (Fig. 3 C). Similarly, HIF1 protein expression in IL-2–maintained CTLs was dependent on continuous mTORC1 activation (Fig. 3 D). The mTORC1 Figure 2. mTORC1 regulates glucose uptake and glycolysis in IL-2– dependence of HIF1 expression correlates with the require- maintained CTLs. (A) Immunoblot analysis for Glut1 expression in naive ment for sustained mTORC1 and cytokine signaling to control + OTI CD8 T cells ± TCR (SIINFEKL) stimulation for 20 h and also mature OTI Glut1 expression and a glycolytic metabolism in CD8 T cells CTLs. The black line indicates that intervening lanes have been spliced out. (Figs. 1 and 2). In this respect, a previous study has implicated (B) Immunoblot analysis for Glut1 expression in CTLs treated with or with- out IL-2 for 20 h. 4EBP1 was used as a loading control. (C and D) Analysis of c-myc as a major regulator of glucose metabolism in T cells glucose uptake (C) and lactate production (D) in P14-LCMV CTLs treated (Wang et al., 2011). However, the expression of c-myc in IL- with or without IL-2 for 20 h. (E and F) Analysis of lactate production 2–sustained CTLs was not dependent on mTORC1 (Fig. 3 E). (E) and glucose uptake (F) in P14-LCMV CTLs treated with or without rapa- The inhibition of mTORC1 with rapamycin thus inhibits mycin for 20 h. (G) Immunoblot analysis for Glut1 expression in P14-LCMV Glut1 expression, glucose uptake, and glycolysis in CTLs CTLs treated with or without rapamycin for 20 h. 4EBP1 was used as a load- independently of any effect on c-myc expression. ing control. (A–G) Data are mean ± SEM or representative of at least three experiments. All metabolic assays were preformed in triplicate (**, P < 0.01; The HIF1 complex does not initiate but sustains ***, P < 0.001). Molecular mass is indicated in kilodaltons. (H) SILAC-based proteomic analysis of P14-LCMV CTLs treated with and without rapamycin glycolytic metabolism in CD8 T cells for 48 h. Shown is the relative expression of rate-limiting glycolytic en- To explore a causal link between mTORC1, HIF1, and gly- zymes (rapamycin/untreated). Data are mean ± SEM for three experiments colysis, we deleted functional HIF1 transcriptional complexes and were analyzed by ANOVA (**, P < 0.01; ***, P < 0.001). CD8 was used in CD8 T cells. In these experiments, mice with floxed as a control protein with unchanged expression. HIF1 alleles were backcrossed to transgenic mice expressing Cre recombinase under the control of the CD4 promoter to TCR and IL-2 triggering, as judged by their ability to nor- mally up-regulate expression of CD25, CD71, and CD44 and (CD4Cre). These mice produced a normal complement of undergo blastogenesis (Fig. 3 F and not depicted). The deletion peripheral / T cells in the thymus, lymph nodes, and spleen (not depicted). HIF1-null CD8 T cells activated in response of HIF1 was confirmed by immunoblot analysis (Fig. 3 G). JEM Vol. 209, No. 13 2443 Figure 3. mTORC1 controls glucose uptake and glycolysis via HIF1. (A–D) Immunoblot analysis of HIF1 and HIF1 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 WT/WT flox/flox / + HIF1 CD4Cre (WT) and HIF1 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. 2444 mTOR/HIF1 controls CTL metabolism and migration | Finlay et al. A r t i c l e A previous study has suggested that the TCR-induced glyco - of mTORC1 with rapamycin, which down-regulates HIF1 lytic switch is regulated by c-myc and is not HIF1 dependent complexes and severely impairs glucose metabolism and gly- (Wang et al., 2011). The present data confirm this and show colysis in CD8 T cells, does not block the proliferation of that TCR-induced glucose uptake and lactate output were CTLs (Fig. 4 B). normal in HIF1-null cells (Fig. 3, H and I). However, strik- A key role for mTORC1 in CD8 T cells is to promote ingly, activated HIF1-null CD8 T cells could not sustain effector differentiation of CTLs by driving expression of high levels of glucose uptake as they differentiated to become genes encoding cytolytic effector molecules such as perforin, cytolytic effectors in response to IL-2. HIF1 -null immune- granzymes, and IFN-. We therefore interrogated the data to activated CD8 T cells maintained in the presence of IL-2 determine whether HIF1 transcriptional complexes mediate thus had greatly decreased Glut1 expression and glucose uptake mTORC1 control of any of the key molecules that control and produced significantly less lactate compared with control CTL die ff rentiation. It was thus striking that immune-activated IL-2–maintained CTLs (Fig. 3, J–L). The ability of IL-2 to HIF1-null CD8 T cells had lost expression of perforin and sustain glucose uptake and glycolysis is thus strictly depen- multiple granzymes (Fig. 4 C). This was not a global block in dent on HIF1. In this context, HIF1 can complex with CD8 T cell differentiation as HIF1 complexes were not AHR. However, CTLs do not express high levels of the AHR, required for expression of other CD8 effector molecules such and there is little evidence of functional AHR signaling. More- as IFN-, Fas ligand, or lymphotoxin (Fig. 4, C and D). More- over, AHR-null CTLs have normal glucose uptake and lactate over, activated HIF1-null CD8 T cells retained expression output (not depicted). of the transcription factors T-bet and Blimp-1, which drive mTORC1 can regulate glycolysis by controlling expres- T cell die ff rentiation (Fig. 4 E and Tables S1 and S2). The failure sion of Glut1 and the expression of key glycolytic enzymes of immune-activated HIF1-null CD8 T cells to express (Fig. 2, G and H). Is the role of HIF1 in CD8 T cells perforin was confirmed independently by quantitative PCR restricted to control of Glut1 expression? To explore this issue, analysis of perforin mRNA levels and Western blot analysis we used Affymetrix microarray analysis to transcriptionally of perforin protein expression (Fig. 4, F and G). Consistent profile HIF1 -null CTLs. Approximately 11,517 annotated with a role for mTORC1–HIF1 in controlling perforin ex- genes were expressed in CTLs, and the impact of HIF1 loss pression, protein levels for perforin were also decreased after was a decrease in the expression of <5% of these genes and an rapamycin treatment in WT CTLs (Fig. 4 G). We also assessed increase in the expression of another 6%. The full list of genes whether the expression of HIF1 complexes was rate limiting changing in expression is detailed in Tables S1 and S2. We for perforin gene expression by examining the impact of then used the functional annotation tools within DAVID hypoxia on the expression of perforin in WT CTLs. In these Bioinformatics Resources 6.7 (Huang et al., 2009a,b) to per- experiments, CTLs were switched from normoxic (21%) to form gene annotation enrichment analysis and KEGG path- hypoxic (1%) oxygen for 24 h. The switch to hypoxia strik- way mapping of the significantly changed genes in the ingly increased expression of HIF1 and also expression of HIF1-null cells. This analysis indicated that one significant Glut1, a direct HIF1 transcriptional target (Fig. 4 H). Impor- impact caused by the loss of HIF1 was down-regulation of tantly hypoxia also induced expression of perforin (Fig. 4 H). multiple genes encoding proteins that control glycolysis and These data may explain earlier observations that CTLs cul- pyruvate metabolism (Fig. 3 M). HIF1-null cells thus cannot tured under hypoxic conditions display increased cytotoxic sustain expression of key rate-limiting glycolytic enzymes: function (Caldwell et al., 2001). hexokinase 2, pyruvate kinase 2, phosphofructokinase, and What is the mechanism for HIF1 control of perforin gene lactate dehydrogenase. The ability of HIF1 complexes to sus- expression? HIF1 regulates the expression of Glut1 by direct tain the T cell glycolytic switch thus extends beyond a simple binding to the Glut1 promoter. Similarly, HIF1-regulated gly- model of HIF1 regulation of Glut1 and glucose uptake. colytic enzymes are direct HIF1 target genes. This raises the question of whether or not HIF1 regulation of perforin gene The HIF1 complex regulates the CD8 T cell transcriptional expression is direct or indirect. There was no evidence for program but is not essential for T cell proliferation HIF1-binding sites in the perforin promoter. Moreover, there The massive up-regulation of glucose metabolism and the was no change in the recruitment of RNA polymerase II switch to glycolysis under normoxia that accompanies the (Pol II) to the transcription start site and distal exons of the immune activation of CD8 T cells are thought to be essential perforin gene in HIF1-null CD8 T cells (Fig. 4 I). Hence the to meet the metabolic demands caused by the rapid prolifera - loss of perforin mRNA in HIF1-null CD8 T cells (Fig. 4 F) + + tion of clonally expanding CD8 T cells. However, CD8 was not a consequence of the failure to recruit the appro- T cells deleted of HIF1 transcriptional complexes proliferated priate transcription factors to the perforin locus. These data and clonally expanded normally (Fig. 4 A). Moreover, tran- argue that the HIF1 effect on perforin expression is indirect. scriptional profiling of HIF1 -null CTLs using Affymetrix In this regard, one possibility we considered was that the loss microarray analysis did not reveal any negative impact of HIF1 of perforin expression might be an indirect consequence of deletion on cell cycle progression, cell survival, or mitosis failed glucose uptake. Here it is relevant that a previous study (Tables S1 and S2). The failure to see proliferative defects in has shown that glucose deprivation prevents perforin expres- HIF1-null T cells is consistent with observations that inhibition sion in immune-activated CD8 T cells (Cham et al., 2008). JEM Vol. 209, No. 13 2445 + Figure 4. The HIF1 complex regulates the CD8 T cell transcriptional program but is not essential for T cell proliferation. (A and B) Proliferation analysis / of WT and HIF1 CTLs (A) and P14-LCMV CTLs treated with and without rapamycin (B). Cells were seeded at 0.3 × 10 /ml, and CTL numbers were counted after 24 h. Data are mean ± SEM of five experiments. (C) Heat map showing the relative normalized expression of selected genes that are significantly different in expression in / WT versus HIF1 CTLs, as determined by microarray. (D) Real-time PCR analysis of IFN- expression in WT / and HIF1 CTLs. Data are mean ± SEM of three experi- ments in triplicate. (E) Immunoblot analysis of T-bet and / Blimp1 expression in WT and HIF1 CTLs. Data are representative of two experiments. (F) Real-time PCR / analysis of Perforin mRNA expression in WT and HIF1 CTLs. Data are mean ± SEM of three experiments in trip- licate (**, P < 0.01). (G) Immunoblot analysis of Perforin / protein expression in WT and HIF1 CTLs (top) and P14-LCMV CTLs treated ± rapamycin for 20 h (bottom). Data are representative of at least three experiments. (H) IL-2–maintained CTLs were placed in either hypoxic (1%) or normoxic (20%) oxygen for 24 h before being subjected to immunoblot analysis for HIF1, Glut1, and perforin expression. Data are representative of three experiments. (I) ChIP was performed with anti–Pol II, and the changes in Pol II binding to the Perforin transcription start site and the second exon were quantified by real- time PCR. Data were normalized to input DNA amounts and plotted as fold over the values for Pol II binding to the HPRT proximal promoter. Data are mean ± SEM of three experiments performed in duplicate. (J) P14-LCMV T cells were activated for 2 d with gp33-41 and then cultured for a further 4 d with IL-2 in different glucose concentrations. Cells were then subjected to immunoblot analysis for perforin expression. Data are representative of two experiments. Molecular mass is indicated in kilo- daltons. Black lines (solid or dotted) indicate that inter- vening lanes have been spliced out. The present data (Fig. 4 J) show that CTLs maintained in immune-activated CTLs (Fig. 5 A). Moreover, the expression 2 mM glucose express substantially lower levels of perforin of the gene encoding the cell adhesion molecule CD62L compared with the control cells maintained in 10 mM glucose. (L-selectin) was increased in HIF1-null cells (Fig. 5, A and B). This could explain why an early study noted that glucose Immune-activated CD8 T cells down-regulate CD62L gene deprivation can limit the cytolytic function of effector CTLs transcription, and thus CTLs normally express low levels of (MacDonald and Koch, 1977). this adhesion molecule (Fig. 5, B and C; Sinclair et al., 2008). However, CD62L mRNA and protein levels were high in HIF1 regulation of chemokines and chemokine receptors activated HIF1-null CD8 T cells (Fig. 5, B and C). CCR7 One unexpected outcome from the microarray experiment is a chemokine receptor that coordinates the migration of came from the bioinformatic pathway analysis of the genes T cells into lymph nodes and is normally down-regulated in whose expression was increased in HIF1-null T cells. This CTLs. PCR analysis confirmed the microarray data: immune- analysis indicated that immune-activated HIF1-null CD8 activated HIF1-null T cells retained high levels of CCR7 T cells up-regulated the expression of genes involved in cyto- mRNA compared with normal CTLs (Fig. 5 D). kine/cytokine receptor interactions (Fig. 5 A). Closer data CD62L and CCR7 are expressed at high levels in naive interrogation revealed that these HIF1-regulated genes enco- and memory T cells and are essential for lymphocyte trans- ded chemokines, chemokine receptors, and adhesion mole - migration from the blood into secondary lymphoid tissue. cules. For example, loss of HIF1 transcriptional complexes CD62L and CCR7 loss is thus part of the program that redi- increased expression of mRNA encoding the chemokine rects effector T cell trafficking away from lymphoid tissue receptors S1P , CXCR4, CXCR3, CCR5, and CCR7 in toward sites of inflammation. In this respect, inhibition of 2446 mTOR/HIF1 controls CTL metabolism and migration | Finlay et al. A r t i c l e T cells not only retained expression of the secondary lym- phoid organ-homing receptors but also increased expression of inflammatory chemokine receptors such as CXCR3 and CCR5 (Fig. 5 A). These pleiotropic effects make it more dif - ficult to predict the impact of HIF1 loss on T cell trafficking in vivo. Accordingly, the in vivo lymph node–homing ability of activated control or HIF1-null CD8 T cells was com- pared. Strikingly, activated CD8 T cells that lacked HIF1- mediated transcription retained the capacity to home to secondary lymphoid organs and accumulated in the lymph nodes (Fig. 5 E). There is therefore a dominant requirement for HIF1 transcriptional complexes for the normal program- ming of effector CD8 T cell trafficking. Is HIF1 regulation of CD62L gene expression direct, or is the impact of HIF1 on CD62L expression an indirect consequence of the inability of HIF1-null cells to maintain glucose uptake? The experi- ment in Fig. 5 F addresses this issue and shows that CD8 T cells stimulated by antigen and IL-2 in culture media with low glucose levels fail to down-regulate CD62L expression. PI3K- and Akt-independent control of mTORC1 activity and HIF1 expression in CD8 T cells The insight that HIF1 transcription factor complexes mediate mTORC1 control of the expression of CD62L, CCR7, and S1P raises a question. How does the mTORC1–HIF1 pathway that controls T cell trafficking connect to a well- documented PI3K–Akt–Foxo pathway that also controls the expression of these key trafficking molecules? The retention Figure 5. HIF1 regulation of chemokines and chemokine receptors. / (A) A comparison of the transcriptional profile of WT versus HIF1 CTLs of high levels of CCR7 and CD62L expression by immune- was performed by microarray. Shown here are KEGG pathway analysis of activated HIF1-null CD8 T cells thus phenocopies the / genes up-regulated in HIF1 CTLs (top) and a heat map showing the impact of inhibiting PI3K–Akt signaling in activated CD8 relative normalized expression of selected genes that are significantly T cells (Sinclair et al., 2008; Waugh et al., 2009; Macintyre / different in expression in WT versus HIF1 CTLs, as determined by et al., 2011). PI3K–Akt control of CD62L, CCR7, and S1P microarray. (B) Real-time PCR analysis of CD62L expression in WT and expression reflects that the expression of these molecules is / / HIF1 CTLs. (C) Analysis of CD62L surface expression on WT and HIF1 regulated by the Foxo1 transcription factor (Fabre et al., 2008; CTLs by flow cytometry. (D) Real-time PCR analysis of CCR7 expression in / / Kerdiles et al., 2009). Foxo1 is inactivated by Akt, which WT and HIF1 CTLs. (E) WT and HIF1 CTLs were labeled with CFSE or phosphorylates Foxo1, resulting in its nuclear exclusion and CellTracker orange (CMTMR) and mixed at a ratio of 1:1 before being in- / jected into C57BL/6 host mice. Values indicate recovery of WT or HIF1 retention in the cytosol. Akt inhibition results in dephosphor- cells as a percentage of the total recovered transferred cells from the ylation of Foxo1 and restores its nuclear location and tran- blood and lymph nodes 4 h after transfer. Each dot indicates a mouse; scriptional activity and restores expression of CD62L and horizontal bars indicate mean. (F) P14 T cells were activated for 2 d with CCR7 in CTLs (Waugh et al., 2009; Macintyre et al., 2011). cognate peptide and then cultured for a further 4 d with IL-2 in different Accordingly, a key question is whether mTORC1 and/or glucose concentrations. Cells were then analyzed for the surface expres- expression of HIF1 complexes regulate Akt activity and Foxo sion of CD62L by flow cytometry. In B and D, mean ± SEM of three phosphorylation in T cells. It is also pertinent to question experiments performed in triplicate is shown; in C and F, data are whether PI3K and Akt control mTORC1 activity in CD8 representative of at least three experiments (*, P < 0.05; **, P < 0.01). T cells. Lymphocyte signaling models frequently position PI3K–Akt signaling as an upstream obligatory regulator of mTORC1 with rapamycin can restore expression of CD62L mTORC1 activity. However, the present results showing that and CCR7 in effector T cells and reprogram their trafficking mTORC1 controls glucose metabolism in T cells are incon- such that they regain the ability to home to secondary lym- sistent with these models because they are discrepant with phoid organs (Sinclair et al., 2008). The retention of CD62L observations that PI3K and Akt do not control glucose uptake + + and CCR7 on immune-activated HIF1-null CD8 T cells in CD8 T cells (Macintyre et al., 2011). Moreover, the present thus raises the possibility that these cells may retain the migra- data herein show that the mTORC1–HIF1 pathway controls tory properties of naive T cells and preferentially home to expression of the rate-limiting glycolytic enzymes (Figs. 2 H secondary lymphoid tissues. However, HIF1-null CD8 and 3 M). In contrast, microarray analysis of Akt-regulated JEM Vol. 209, No. 13 2447 genes in T cells failed to find any evidence that Akt controlled glucose metabolism or glycolysis (Macintyre et al., 2011). To explore the links between PI3K, Akt, mTORC1, and HIF1, we addressed two questions. Do mTORC1 and HIF1 regulate Akt activity and Foxo phosphorylation? Is PI3K and Akt activity required for mTORC1 activation and HIF1 expression? In the context of the first question, long-term inhibition of mTORC1 can destabilize mTORC2 complexes in some cell systems and suppress Akt function (Sarbassov et al., 2006). Indeed, rapamycin treatment of CD4 T cells activated with CD3 and CD28 antibodies does diminish Akt S473 phosphorylation, although the impact of this on Akt activity has not been fully assessed (Lee et al., 2010; Delgoffe et al., 2011). If this were true in CTLs, then long-term treat- ment of T cells with rapamycin would result in the loss of Foxo phosphorylation and cause cells to regain Foxo tran- scriptional activity and thus CD62L and CCR7 expression. We therefore examined the impact of long-term inhibition of mTORC1 with rapamycin on Foxo phosphorylation and localization in CTLs. We also assessed Akt–Foxo and mTORC1 signaling in IL-2–maintained HIF1-null T cells to assess whether loss of HIF1 complexes compromised Foxo phosphorylation/inactivation. Fig. 6 (A and B) shows that inhibition of Akt decreased Foxo phosphorylation (Fig. 6 A) and restored nuclear localization of Foxos in CTLs (Fig. 6 B). Figure 6. mTORC1 and HIF1 do not regulate Akt activity or Foxo In contrast, Akt remained active, i.e., phosphorylated on T308 phosphorylation. (A) Immunoblot analysis of phosphorylated AKT and and S473, and Foxos remained highly phosphorylated and Foxos in P14-LCMV CTLs treated with and without Akti1/2 or rapamycin excluded from the nucleus of rapamycin-treated CTLs (Fig. 6, for 24 h. (B) P14-LCMV CTLs were treated with and without rapamycin or A and B). Additionally, Akt phosphorylation (T308) and the Akti1/2 for 24 h and subjected to nuclear/cytoplasmic fractionation before immunoblot analysis for Foxo1 and Foxo3a expression. Purity of phosphorylation of Foxo transcription factors on Akt sub- cytoplasmic and nuclear fractions was confirmed by I B and Smc1 strate sites (T24/32) were unaffected in HIF1 -null CTLs, / expression. (C) Immunoblot analysis of WT or HIF1 CTLs for Akt–Foxo demonstrating that Akt signaling is not altered by HIF1 dele- / and mTORC1 signaling. HIF1 CTLs were treated with rapamycin as a tion (Fig. 6 C). It was also evident that the phosphorylation negative control for mTORC1 activity. For all panels, data are representa- of mTORC1 substrates S6K1 (p70 S6-kinase 1) and 4EBP1 tive of at least three experiments. Molecular mass is indicated in kilodal- (eIF4E-binding protein 1), on T389 and S65, respectively, tons. Dotted lines indicate that intervening lanes have been spliced out. and downstream signaling to S6 ribosomal protein (an S6K1 substrate) were normal in HIF1-null CD8 T cells (Fig. 6 C). Therefore, mTORC1 and HIF1 regulate the expression of inhibitor, Akti1/2, or a p110 inhibitor, IC87114, potently CD62L and CCR7 and T cell trafficking but do not control inhibited Akt activity in CTLs as judged by the loss of phos- Akt–Foxo phosphorylation and localization. phorylated Akt on T308 and S473 and inhibition of the phos- What about the second question? Are PI3K and Akt phorylation of the Akt substrates, Foxo transcription factors activity required for mTORC1 activation and HIF1 expres- (Fig. 7, A and B). However, neither Akti1/2 nor IC87114 + + sion in CD8 T cells? To address this issue, we used comple- prevented mTORC1 activity in CD8 T cells as neither com- mentary genetic and pharmacological strategies to block PI3K pound blocked the phosphorylation of mTORC1 substrates S6K1 and 4EBP1 or S6 phosphorylation in CTLs (Fig. 7 A). and Akt and then monitored the impact of these perturba- tions on mTORC1 activity by assessing the phosphoryla- Moreover, CTLs that had WT PI3K p110 catalytic subunits D910A tion of mTORC1 substrate sequences on S6K1 (T389 and substituted with a catalytically inactive mutant (p110 ) S421/424) and 4EBP1 (S35/47) and the phosphorylation did not activate Akt in response to IL-2 but showed normal of the S6K1 substrate S6 ribosomal protein (S235/236). The rapamycin-sensitive phosphorylation of S6K1 and S6 (Fig. 7 C). data show that IL-2–maintained CTLs contain high levels Further evidence that mTORC1 activity is independent of of active Akt phosphorylated on threonine 308 and high PI3K and Akt is that the expression of HIF1 was not regu- levels of mTORC1 signaling (Fig. 7 A). mTORC1 inhibition lated by Akt and PI3K inhibitors (Fig. 7 D). These data reveal with rapamycin abolished the phosphorylation of S6K1 on that PI3K–Akt activity is dispensable for mTORC1 activity T389 and S421/424 and blocked S6 phosphorylation. In and HIF1 expression in CTLs. If mTORC1 activity is not mediated by Akt, then what is CD8 T cells, Akt is activated via a PI3K complex containing the p110 catalytic subunit (Macintyre et al., 2011). The Akt the alternative pathway? One candidate is the serine/threonine 2448 mTOR/HIF1 controls CTL metabolism and migration | Finlay et al. A r t i c l e Figure 7. PI3K and Akt do not regulate mTORC1 activity. (A and B) CTLs were cultured in the presence or absence of Akti1/2, IC87114, rapamycin, or LY294002 for 60 min (A and B) or 24 h (A) and subjected to immunoblot analysis with the indicated antibodies. (C) CTLs generated from WT or D910A p110 mice were subjected to immunoblot analysis with or without rapamycin treatment (30 min). Data are representative of two experiments. (D) CTLs were cultured in the presence or absence of Akti1/2, IC87114, or rapamycin for 24 h and subjected to immunoblot analysis with the indicated flox/flox Flox WT/WT antibodies. (E–G) CTLs generated from PDK1 TamoxCre (PDK1 ) and PDK1 TamoxCre (WT) mice were treated ± 4OHT for 3 d to delete PDK1 and subjected to immunoblot analysis. For, A, B, and D–G, data are representative of at least three experiments. Molecular mass is indicated in kilodaltons. kinase PDK1 because this is known to be an essential regula- these results show that in CD8 T cells, mTORC1 activity tor of glucose metabolism in CD8 T cells (Macintyre et al., and HIF1 protein expression are controlled by a PI3K– 2011). We therefore examined whether PDK1 regulates Akt-independent pathway mediated by PDK1. mTORC1 activity and HIF1 expression in activated CD8 T cells. For these experiments, mice expressing floxed PDK1 DISCUSSION alleles were backcrossed with mice that express a tamoxi- The present study explores the molecular pathways that fen-regulated Cre recombinase (Macintyre et al., 2011). mediate mTORC1 control of effector CD8 T cell differen - o fl x/o fl x PDK1 TamoxCre CTLs were generated and treated with tiation. A key finding was that mTORC1 activity is required 4-hydroxytamoxifen (4OHT) to delete floxed PDK1 alleles. for immune-activated CD8 T cells to sustain high rates of PDK1 deletion was confirmed by analysis of PDK1 protein glucose uptake. mTORC1 activity is also necessary for CD8 expression and by the loss of RSK phosphorylation on its T cells to initiate and sustain a switch to a glycolytic metabo- PDK1 target site (S227; Fig. 7 E). Importantly, deletion lism. One way in which mTORC1 controls glucose uptake of PDK1 in CTLs prevented the phosphorylation of the in CD8 T cells is by controlling expression of the glucose mTORC1 substrates S6K1 and 4EBP1 on mTORC1 target transporter Glut1. CD8 T cells also show mTORC1 depen- residues (Fig. 7 F). PDK1 loss also resulted in loss of expression dence for the expression of hexokinase 2, a key enzyme which of HIF1 and the HIF1 target Glut1 (Fig. 7 G). Together phosphorylates glucose to produce glucose-6-phosphate, JEM Vol. 209, No. 13 2449 an essential intermediate in most pathways for glucose T cells thus affords the insight that mTORC1 regulation of metabolism. Importantly, mTORC1 controls expression of HIF1 controls expression of a subset of CTL effector mole - + + key rate-limiting glycolytic enzymes in CD8 T cells such as cules and controls CD8 T cell trafficking. These findings phosphofructokinase 1, lactate dehydrogenase, and pyruvate reveal a fundamental mechanism linking nutrient sensing and kinase M2. transcriptional control of CD8 T cell die ff rentiation. Although This ability of mTORC1 to coordinate and sustain the HIF1 complex evolved to function as a metabolic sensor expression of glucose transporters and essential glycolytic of cellular oxygen levels, the present study reveals a novel role enzymes during CD8 T cell differentiation stems from its for the HIF1 complex in coupling mTORC1 to the control ability to control expression of the HIF1 transcription factor of T cell differentiation. complex. The initial increase in glucose uptake and switch How does this mTORC1–HIF1 pathway coordinate to glycolysis that immediately follows TCR engagement is with other signaling pathways that control CD8 T cell dif- not HIF1 dependent. However, HIF1 is essential for antigen- ferentiation? A particular issue is that models of lymphocyte primed T cells to sustain high levels of glucose uptake and signal transduction invariably link mTORC1 to PI3K–Akt glycolytic enzyme expression as they differentiate to cytolytic signaling. As discussed, the biochemical experiments that effector cells. In this respect, CTL differentiation is controlled initially proposed this model are flawed because the pharma - by the strength and duration of signaling by inflammatory cological tools used have numerous off-target effects. Never - cytokines such as IL-2 (Kalia et al., 2010; Pipkin et al., 2010). theless, the concept of a linear model of PI3K–Akt–mTORC1 The present data now show that the ability of IL-2 to sus- signaling is still compelling because the phenotypes of T cells tain high levels of glucose uptake and maintain a glycolytic activated in the presence of PI3K–Akt or mTORC1 inhibitors metabolism is dependent on mTORC1 induction of HIF1 show similarities. For example, the expression of perforin is complexes. However, it was notable that the inhibition of positively regulated by both Akt (Macintyre et al., 2011) and mTORC1 in IL-2–maintained CTLs suppressed glucose mTORC1 (Fig. 4 G), and the expression of CD62L, CCR7, uptake and lactate output without having any impact on the and S1P is negatively regulated by both Akt and mTORC1 expression of c-myc (Fig. 3 E). This is relevant because it (Sinclair et al., 2008; Macintyre et al., 2011). In a linear model, has been shown recently that c-myc is required for the it would be assumed that PI3K and Akt activity are required initial TCR-induced glycolytic switch in naive T cells (Wang for mTORC1 activation. Moreover, there are models propos- et al., 2011). The present data thus reveal that c-myc ex- ing mTORC1 control of Akt activity. This latter idea stems pression alone is not sufficient to maintain glucose uptake from experiments showing that in some cell systems, pro- and glycolysis in CD8 T cells. Moreover the coordination longed inhibition of mTORC1 with rapamycin can disrupt of c-myc and HIF1 expression must be required for T cells the mTORC2 serine/threonine kinase complex (Sarbassov to sustain glucose uptake and glycolysis dur ing CD8 et al., 2006). mTORC2 phosphorylates Akt on S473, and it T cell differentiation. has been shown that prolonged rapamycin treatment reduces The ability of HIF1 to link mTORC1 to the control of Akt S473 phosphorylation in CD4 T cells activated with CD3 glucose metabolism in CD8 T cells made us question and CD28 antibodies (Lee et al., 2010; Delgoffe et al., 2011). whether any other actions of mTORC1 in CD8 T cell are It is frequently assumed that loss of Akt S473 phosphorylation directed by the HIF1 pathway. In particular, it is well docu- will block Akt catalytic activity. However, the key rate-limiting mented that mTORC1 activity is required for expression phosphorylation on Akt is the PDK1-mediated phosphoryla- of CTL effector molecules and for effector CD8 T cells to tion of T308, and the impact of the reduced Akt S473 phos- switch off expression of the chemokine receptors and adhe - phorylation on Akt catalytic function in CD4 T cells has not sion molecules that direct naive and memory T cell entry been studied in detail. Moreover, does rapamycin treatment and egress from secondary lymphoid tissues. For example, actually disrupt mTORC2 complexes in all T cells? The cur- mTORC1 signaling causes down-regulation of the adhesion rent study addresses these issues and shows unequivocally that molecule CD62L and the chemokine receptors CCR7 and PI3K and Akt activity are not essential for mTORC1 activa- S1P (Sinclair et al., 2008). In this regard, the phenotype of tion or for HIF1 expression in CD8 T cells. Moreover, rapa- the immune-activated HIF1-null T cells is intriguing. They mycin treatment, even long term, does not prevent Akt have many features of effector CD8 T cells such as normal phosphorylation or activity, as judged by the normal phos- IFN- production and expression of effector molecules such phorylation and nuclear exclusion of the Foxo transcription as Fas ligand and lymphotoxin. Also, they are normal in cell factors in rapamycin-treated T cells. There is a well-documented size and can rapidly proliferate. However, the HIF1-null cells Akt–Foxo-mediated pathway to control T cell trafficking in lack perforin and granzyme expression. Moreover, although CD8 T cells, but the mTORC1–HIF1 pathway that controls they acquire the expression of effector CTL chemokine T cell trafficking described herein is independent of Akt and receptors such as CXCR3 and CCR5, they retain the expres- Foxos. The present study thus indicates that PI3K–Akt and sion of naive T cell lymph node–homing receptors CD62L, mTORC1 signaling are not linked in a linear pathway. These CCR7, and S1P . They also retain the secondary lymphoid molecules are activated independently but converge to control tissue migratory program of naive T cells in vivo. The tran- the expression of key molecules required for effector CTL scriptional profiling of HIF1 -null immune-activated CD8 function. An essential requirement for these two independent 2450 mTOR/HIF1 controls CTL metabolism and migration | Finlay et al. A r t i c l e P-40, 1 mM PMSF, 1 mM Na VO , and 1 mM DTT) and centrifuged at 4°C signaling pathways to control T cell differentiation highlights 3 4 for 1 min at 10,000 g. Cytosolic fractions were removed, and nuclear mem- the importance of integrating lymphocyte signal transduction brane fractions were suspended in the aforementioned Tris lysis buffer. for appropriate immune responses. Immunoblot analysis of IB and Smc1 was used to confirm the purity of cytoplasmic and nuclear fractions, respectively. MATERIALS AND METHODS Mice. All mice used in this study were approved by the University of Dundee Glucose uptake. 10 cells were suspended in 400 µl glucose-free media ethical review committee and maintained in compliance with UK Home 3 3 containing 0.5 µCi/ml 2-deoxy-d-[1- H]glucose ([ H] 2-DG; GE Health- flox/flox Office Animals (Scientific Procedures) Act 1986 guidelines. PDK1 care) and incubated for 3 (CTLs) or 10 min (TCR-simulated CD8 T cells). tm9(CreEsr1)Arte TamoxCre mice were generated by breeding C57BL/6GT(ROSA)26 Cells were pelleted, washed, and lysed overnight with 200 µl of 1 M NaOH, (TamoxCre), purchased from Taconic, to mice carrying PDK1 floxed alleles and the incorporated H radioactivity was quantified via liquid scintillation flox/flox (Mora et al., 2003). HIF1 mice (Geng et al., 2006) were backcrossed counting. Measurements were performed in triplicates per condition. with mice expressing cre recombinase under the control of the CD4 pro- flox/flox D910A/D910A moter, generating HIF1 CD4Cre mice. PI3K p110 mice Quantitative real-time PCR. RNA was extracted using the RNeasy carry a point mutation that switches Asp →Ala (D910A) in the p110 RNA puric fi ation mini kit (QIAGEN) according to the manufacturer’s pro - subunit to inactivate catalytic activity (Okkenhaug et al., 2002). P14-LCMV tocol. Purie fi d RNA was reverse transcribed using the qScript cDNA syn - and OTI transgenic mice have been described previously (Pircher et al., thesis kit (Quanta). Real-time PCR was performed in triplicates in 96-well 1989; Kurts et al., 1996). plates using iQ SYBR Green–based detection on an iCycler (Bio-Rad Labo- ratories). For the analysis of mRNA levels, the derived values were averaged Cell culture. CTLs were generated as described previously (Waugh et al., and normalized to HPRT mRNA levels. 2009). In brief, lymphocytes isolated from spleens or lymph nodes of P14- LCMV or OTI transgenic mice or nontransgenic mice were activated for Primers. Primers used are as follows: HPRT forward, 5-TGATCAGTCA- 48 h with either 100 ng/ml of soluble LCMV or OTI-specific peptide, gp33- ACGGGGGACA-3; HPRT reverse, 5-TTCGAGAGGTCCTTTTCA- 41 and SIINFEKL, respectively, or 0.5 µg/ml anti-CD3 antibody (2c11). Cells CCA-3; CD62L forward, 5-ACGGGCCCCAGTGTCAGTATGTG-3; were then cultured in 20 ng/ml IL-2 (Proleukin) at 37°C for an additional CD62L reverse, 5-TGAGAAATGCCAGCCCCGAGAA-3; CCR7 forward, 6 d. For conditional deletion of PDK1, CTLs were prepared by activating 5-CAGCCTTCCTGTGTGATTTCTACA-3; CCR7 reverse, 5-ACC- flox/flox PDK1 TamoxCre splenocytes with 2c11 for 2 d followed by culturing ACCAGCACGTTTTTCCT-3; Perforin forward, 5-CGTCTTGGTGG- in IL-2 for 5 d, with 0.6 µM of 4OHT (Sigma-Aldrich) being added for the GACTTCAG-3; Perforin reverse, 5-GCATTCTGACCGAGGGCAG-3; final 72 h of culture. Where indicated, cells were treated with various inhibi - IFN- forward, 5-TTACTGCCACGGCACAGTC-3; and IFN- reverse, tors: 1 µM Akti1/2 (EMD Millipore), 20 nM rapamycin (EMD Millipore), 5-AGATAATCTGGCTCTGGCTCTGCGG-3. 10 µM IC87114 (synthesized in-house), and 10 µM LY294002 (Promega). For short-term (20 h) TCR stimulations, naive CD8 T cells were puri- Lactate measurement. 2 × 10 /ml CTLs were cultured for 4 h in RPMI flox/flox WT/WT fied from lymph nodes of HIF1 CD4Cre and HIF1 CD4Cre 1640 containing 10% dialyzed fetal calf serum, and then the cells were spun nontransgenic mice or alternatively from P14-LCMV or OTI transgenic and the supernatants were collected. Lactate concentration in the supernatant mice by magnetic cell sorting (Miltenyi Biotec). P14-LCMV and OTI naive was quantified using an LDH (lactate dehydrogenase)-based enzyme assay, CD8 T cells were activated with gp33-41 plus 3 ng/ml anti-CD28 (clone monitoring the emergence of NADH through increased absorption at 340 nm 37.51; eBioscience) and SIINFEKL, respectively. Nontransgenic naive CD8 (the reaction contained 320 mM glycine, 320 mM hydrazine, 2.4 mM NAD , T cells were activated with 2c11 plus anti-CD28. and 3 U/ml LDH). A standard curve was generated, and the concentration of lactate in the supernatant added to this reaction was calculated. Flow cytometric analysis. Cells were labeled with APC-efluor780 CD8 flox/flox (53-6.7), FITC CD25 (7D4), APC CD44 (IM7), PE CD71 (C2), APC Adoptive transfer. CTLs were generated from the spleens of HIF1 flox/flox CD62L (MEL-14), Alexa Fluor 700 CD45.1 (104), and V450 Horizon CD4Cre mice or HIF1 mice (as described in Cell culture but with CD45.2 (A20) purchased from eBioscience or BD. Live cells were gated 4 d in IL-2). Cells were then labeled with either CFSE (Invitrogen) or Cell- according to their forward scatter and side scatter. Data were acquired on Tracker Orange (CMTMR; Invitrogen) for 15 min at 37°C, washed, mixed either a FACSCalibur or an LSRFortessa (BD) and analyzed using FlowJo at 1:1 ratio, and suspended in sterile PBS. 5 × 10 mixed cells were injected software (Tree Star). into the tail vein of C57BL/6 host mice. After 4 h, the mice were sacrificed, and lymph nodes, spleen, and blood were analyzed and the ratio of CFSE- and flox/flox Western blot analysis and cytoplasmic nuclear fractionation. Cells CMTMR-labeled T cells quantified. Values indicate recovery of HIF1  flox/flox were lysed (2 × 10 /ml) in Tris lysis buffer containing 10 mM Tris, pH 7.05, CD4Cre cells versus HIF1 cells as a percentage of the total recovered 50 mM NaCl, 30 mM Na pyrophosphate, 50 mM NaF, 5 µM ZnCl , 10% transferred cells. glycerol, 0.5% Triton, 1 µM DTT, and protease inhibitors (Roche). Lysates were centrifuged (4°C, 16,000 g for 10 min) and separated by SDS-PAGE Chromatin immunoprecipitation (ChIP). Real-time PCR-based ChIP and transferred to nitrocellulose membrane. Blots were probed with antibodies analysis to measure Pol II binding to the Perforin locus was performed as T24/34 S235/236 T389 T421/S424 T308 recognizing pFoxo1/3a , pS6 , pS6K , pS6K , pAkt , described previously (Cruz-Guilloty et al., 2009) with minor modifications. S473 S37/46 S65 pAkt , p4EBP1 , and p4EBP1 (Cell Signaling Technology); In brief, chromatin was immune precipitated with anti–Pol II (Santa Cruz S227 pRSK (Santa Cruz Biotechnology, Inc.); and unphosphorylated S6, S6K, Biotechnology, Inc.) or normal rabbit IgG (Cell Signaling Technology) Akt, and c-Myc (Cell Signaling Technology); RSK2, IB, PTEN, and from 5 × 10 cells in the presence of 0.2 mg/ml BSA. ChIP-grade protein HIF1 (Santa Cruz Biotechnology, Inc.); HIF1 (R&D Systems); PDK1 G magnetic beads (Cell Signaling Technology) were used to collect the (EMD Millipore); Glut1 (a gift from G. Holman, University of Bath, Bath, immune complexes. Chromatin was purified via a NucleoSpin Extract II kit England, UK; Holman et al., 1990); Perforin (a gift from G. Griffith, Cam - (Macherey-Nagel) and resuspended in TE buffer. Real-time PCR was bridge Institute for Medical Research, Cambridge, England, UK); and Foxo1 performed in an iQ5 (Bio-Rad Laboratories) with Perfecta SYBR green and Foxo3a (generated in-house). Immunoblots were probed with an SmcI FastMix for iQ (Quanta BioSciences). Primers are as follows: Perforin TSS antibody as a loading control (Bethyl Laboratories, Inc.). forward, 5-CAGGGCAGGAAGTAGTAATGATATG-3; Perforin TSS To obtain cytoplasmic and nuclear fractions, cells were lysed in Hepes reverse, 5-CTTCCTCCTCCTTACCTGAAGTC-3; Perfor in Exon2 hypotonic lysis buffer (20 × 10 cells/ml; 10 mM Hepes, pH 7.9, 4 mM forward, 5-CCAGAGTTTATGACTACTGTG-3; and Perforin Exon2 MgCl , 15 mM KCl, 10 mM NaF, 0.1 mM EDTA, 0.15% [vol/vol] Nonidet reverse, 5-GTGCTTCTGCTTGCATTCTG-3. JEM Vol. 209, No. 13 2451 SILAC. P14-LCMV CTLs were cultured in SILAC medium as described 2008. FOXO1 regulates L-Selectin and a network of human T cell previously (Navarro et al., 2011) with some minor modifications. In brief, homing molecules downstream of phosphatidylinositol 3-kinase. J. Immunol. 181:2980–2989. cells were combined and a Thermo Fisher Scientific subcellular protein frac - Fox, C.J., P.S. Hammerman, and C.B. Thompson. 2005. Fuel feeds function: tionation kit was used to fractionate CTLs according to the manufacturer’s energy metabolism and the T-cell response. Nat. Rev. Immunol. 5:844– instructions. Fractions were chloroform methanol precipitated and further 852. http://dx.doi.org/10.1038/nri1710 separated by molecular weight using denaturing size exclusion chromatog- Geng, S., A. Mezentsev, S. Kalachikov, K. Raith, D.R. Roop, and A.A. raphy before digestion and LC-MS/MS analysis as described previously Panteleyev. 2006. Targeted ablation of Arnt in mouse epidermis results in (Larance et al., 2012). The peptide mixture was separated by nanoscale C18 profound defects in desquamation and epidermal barrier function.J . Cell reverse phase liquid chromatography (Ultimate 3000 nLC; Dionex) coupled Sci. 119:4901–4912. http://dx.doi.org/10.1242/jcs.03282 to a LTQ-Orbitrap Velos (Thermo Fisher Scientific). MaxQuant version Holman, G.D., I.J. Kozka, A.E. Clark, C.J. Flower, J. Saltis, A.D. Habberfield, 1.3.0.5 (Cox and Mann, 2008) was used to process raw MS spectra, using the I.A. Simpson, and S.W. Cushman. 1990. Cell surface labeling of glucose default settings against the mice Uniprot database (July 2012). transporter isoform GLUT4 by bis-mannose photolabel. Correlation with stimulation of glucose transport in rat adipose cells by insulin and Online supplemental material. Table S1 lists microarray analysis showing phorbol ester. J. Biol. Chem. 265:18172–18179. / decreased gene expression in HIF1 versus HIF1 WT CTLs. Table S2 lists Huang, W., B.T. Sherman, and R.A. Lempicki. 2009a. Bioinformatics en- / microarray analysis showing increased gene expression in HIF1 versus richment tools: paths toward the comprehensive functional analysis HIF1 WT CTLs. Online supplemental material is available at http://www of large gene lists. Nucleic Acids Res. 37:1–13. http://dx.doi.org/10 .jem.org/cgi/content/full/jem.20112607/DC1. .1093/nar/gkn923 Huang, W., B.T. Sherman, and R.A. Lempicki. 2009b. Systematic and integra- We thank members of the Biological Services Unit, R. Clarke of the Flow Cytometry tive analysis of large gene lists using DAVID bioinformatics resources. Facility, members of the “Fingerprints” Proteomics Facility, and members of the Nat. Protoc. 4:44–57. http://dx.doi.org/10.1038/nprot.2008.211 D.A. Cantrell laboratory for critical reading of the manuscript. We thank D. Alessi at Kalia, V., S. Sarkar, S. Subramaniam, W.N. Haining, K.A. Smith, and R. Ahmed. the University of Dundee for providing mice carrying PDK1 floxed alleles and the 2010. Prolonged interleukin-2Ralpha expression on virus-specic fi CD8+ Finnish DNA Microarray Centre at the Centre for Biotechnology (Turku, Finland) for T cells favors terminal-effector differentiation in vivo. Immunity. 32: the microarray analysis. 91–103. http://dx.doi.org/10.1016/j.immuni.2009.11.010 This work was supported by a Wellcome Trust Principal Research Fellowship Kerdiles, Y.M., D.R. Beisner, R. Tinoco, A.S. Dejean, D.H. Castrillon, R.A. and Program Grant (065975/Z/01/A). E. Rosenzweig and J.L. Hukelmann were DePinho, and S.M. Hedrick. 2009. Foxo1 links homing and survival of supported by Wellcome Trust PhD Studentships. naive T cells by regulating L-selectin, CCR7 and interleukin 7 receptor. The authors have no conflicting financial interests. Nat. Immunol. 10:176–184. http://dx.doi.org/10.1038/ni.1689 Kurts, C., W.R. Heath, F.R. 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Published: Dec 17, 2012

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