Given that inflammatory T-cells have a highly glycolytic metabolism, whereas regulatory T-cells rely more on oxidative glucose metabolism, there is growing interest in understanding how T-cell metabolism relates to T-cell function. The mTORC1 (mammalian target of rapamycin complex 1) has a crucial role to determine the balance between effector and regulatory T-cell differentiation, but is also described as a key regulator of metabolism in non-immune cell systems. The present review explores the relationship between these diverse functions of mTORC1 with regard to T-cell function. In many cell systems, mTORC1 couples PI3K (phosphoinositide 3-kinase) and PKB (protein kinase B), also known as Akt, with the control of glucose uptake and glycolysis. However, this is not the case in activated CD8+ CTLs (cytotoxic T-lymphocytes) where PI3K/PKB signalling is dispensable for the elevated levels of glycolysis that is characteristic of activated T-cells. Nevertheless, mTORC1 is still essential for glycolytic metabolism in CD8+ T-cells, and this reflects the fact that mTORC1 does not lie downstream of PI3K/PKB signalling in CD8+ T-cells, as is the case in many other cell systems. mTORC1 regulates glucose metabolism in CTLs through regulating the expression of the transcription factor HIF1α (hypoxia-inducible factor 1α). Strikingly, HIF1α functions to couple mTORC1 with a diverse transcriptional programme that extends beyond the control of glucose metabolism to the regulation of multiple key T-cell functions. The present review discusses the idea that mTORC1/HIF1α signalling integrates the control of T-cell metabolism and T-cell function.
- cytotoxic T-lymphocyte
- hypoxia-inducible factor 1α (HIF1α)
- mammalian target of rapamycin complex 1 (mTORC1)
- protein kinase B (PKB)
mTORC (mammalian target of rapamycin complex) 1 signalling and function
mTOR (mammalian target of rapamycin) is a serine/threonine kinase with important functions for a range of cellular processes. mTOR forms two distinct multiprotein complexes, mTORC1 and mTORC2, with only mTORC1 being acutely sensitive to the inhibitor rapamycin . mTORC1 integrates inputs from major cellular signals including growth factors, energy status, oxygen and amino acids to promote cell growth and function. One of the dominant mechanisms through which these signals control mTORC1 activity is through modulating the function of the TSC2 (tuberous sclerosis complex 2), which acts as a GTPase-activating protein for the small GTPase Rheb. TSC2 regulates mTORC1 negatively by promoting the conversion of Rheb to its inactive GDP-bound state. Therefore, when the cellular energy status is compromised, AMPK (AMP-activated protein kinase) is active to phosphorylate and activate TSC2, thus inhibiting mTORC1 activity. Conversely, growth factors induce the activity of multiple kinases, including PKB (protein kinase B), also known as Akt, which phosphorylate and inhibit TSC2 to promote mTORC1 activity. PKB, activated downstream of PI3K (phosphoinositide 3-kinase), also regulates mTORC1 in a TSC2-independent manner by phosphorylating the mTORC1 inhibitor PRAS40 (proline-rich Akt substrate of 40 kDa), inducing its dissociation from mTORC1 . Whereas in lymphocytes PI3K/PKB signalling is frequently considered an upstream obligatory regulator of mTORC1 activity in response to diverse stimuli [2–4], the present review describes how this is not always the case.
Multiple roles for mTORC1 have been described in the regulation of various aspects of cellular metabolism. mTORC1 activity promotes various anabolic processes including protein synthesis, glycolysis and lipid synthesis while inhibiting catabolic processes such as autophagy. Although mTORC1 regulation of these processes appears to be rather complex, certain key mTORC1 effectors have been identified. mTORC1 is linked to the control of glycolysis through two distinct transcription factors, i.e. c-Myc and HIF (hypoxia-inducible factor) 1α, each of which promote the expression of multiple glycolytic enzymes and glucose transporters. mTORC1 activity has also been linked to the control of lipid synthesis through the SREBP (sterol-regulatory-element-binding protein) 1 and 2 transcription factors that control the expression of enzymes required for fatty acid and cholesterol synthesis. Additionally, mTORC1 activity also regulates autophagy negatively through the phosphorylation and inactivation of Ulk1, a kinase that promotes autophagosome formation .
In T-lymphocytes, the best characterized role for mTORC1 is in the regulation of T-cell fate and function. mTORC1 promotes the differentiation of effector CD4+ T-cell subsets over regulatory CD4+ T-cells and also promotes effector CD8+ CTL (cytotoxic T-lymphocyte) development over CD8+ memory T-cells . However, until recently, a role for mTORC1 in controlling T-cell metabolism has not been appreciated. Neither has the relationship between these apparently distinct roles for mTORC1 in controlling metabolism and cellular function been considered.
Although naive T-cells only require energy to prevent atrophy and for survival and migration, activated T-cell subsets have a greatly increased metabolic demand as they engage in rapid growth and proliferation, and the production of cytokines and other effector molecules. It is crucial that activated T-cells increase their metabolism to meet the biosynthetic needs of the T-cell as it responds either to developmental or pathogenic cues. To achieve this, T-cells respond to extrinsic signals from antigen receptors and cytokines to up-regulate the surface expression of key nutrient receptors: amino acid transporters, the transferrin receptor and glucose transporters [5–7]. Additionally, T-cells switch their glucose metabolism from oxidative phosphorylation to aerobic glycolysis, i.e. glucose is metabolized to produce lactate even though oxygen is readily available . Aerobic glycolysis is an inefficient route to generating ATP, producing only two molecules of ATP/glucose compared with >30 molecules of ATP/glucose generated by oxidative phosphorylation. Therefore cells must be able to sustain high levels of glucose uptake and an elevated glycolytic flux to generate a sufficient amount of ATP (Figure 1). This is achieved by increasing the expression of Glut1 (glucose transporter 1) and certain rate-limiting enzymes within the glycolytic pathway [9,10]. However, the real advantage of switching from oxidative phosphorylation to glycolysis is that it allows glucose to be used as a source of carbon to generate nucleic acids, amino acids and phospholipids . The generation of these biosynthetic precursors is critical for cells engaging in rapid growth, proliferation and the synthesis of effector molecules. Therefore, to facilitate their differentiation and function, activated T-cells up-regulate the expression of Glut1, increase glucose uptake and activate the switch to aerobic glycolysis [8,11–13].
The metabolic switch to aerobic glycolysis is initiated by TCR (T-cell receptor) activation and then maintained in certain T-cell subsets by cytokines such as IL-2 (interleukin-2) and is crucial for normal T-cell activation and differentiation in the periphery. Thus limiting glucose availability in activating T-cells compromises TCR-induced growth and proliferation and also the expression of certain effector molecules such as perforin and IFNγ (interferon γ) [5,14]. Therefore it appears that a highly glycolytic metabolic profile is associated with inflammatory T-cell subsets, CD4+ Th1, Th2 and Th17 T-cell subsets and CD8+ CTLs, but not CD4+ Tregs (regulatory T-cells) or CD8+ memory T-cells [15–18]. The transcription factor c-Myc is crucial for the metabolic switch in glucose metabolism that accompanies the activation of naive T-cells . Accordingly, deletion of c-Myc in naive T-cells prevents TCR-induced glucose uptake and glycolysis, and activated c-Myc-null T-cells completely fail to grow or proliferate [19–22]. However, in activated CD8+ CTLs, elevated glucose uptake and glycolysis is maintained by IL-2 independently of c-Myc . In CTLs, the critical transcription factor that is required to promote glucose uptake and glycolysis is the HIF1α–HIF1β transcriptional complex. Thus deletion of HIF1β alleles results in decreased Glut1 expression and glycolysis in CD8+ CTLs .
mTORC1, but not PI3K or PKB, controls T-cell glucose metabolism
What are the signalling pathways that control glucose metabolism in activated T-cells? Antigen receptor-induced c-Myc expression and glucose uptake was originally attributed to PI3K and PKB signalling [5,11,19,23,24]. However, one criticism of these studies is that they rely on experiments involving the use of the PI3K inhibitor LY294002, which also inhibits various other kinases including mTORC1 [25–27]. A more thorough analysis of PI3K/PKB signalling using pharmacological inhibitors of PI3Kδ (IC87114) and PKB (Akti1/2) with substantially greater selectivity than LY294002 [28–30] has shown that PI3K/PKB activities are dispensable for increased glucose uptake and glycolysis in TCR-activated T-cells [17,28]. Once activated, T-cells differentiate into various different effector T-cell subsets depending on the local environment and cytokine availability. In CD8+ CTLs responding to the cytokine IL-2, it is also apparent that glucose uptake and glycolysis is maintained independently of PI3K and PKB . Instead, mTORC1 is the key regulator of both TCR- and IL-2-induced glucose metabolism in CD8+ T-cells. Thus mTORC1 activity is required for TCR-induced c-Myc expression and the switch to increased glycolysis [17,19] (Figure 1). Likewise, inhibition of mTORC1 in IL-2-maintained CTLs results in decreased glycolysis, in this instance, as a result of the loss of HIF1α expression  (Figure 1). Indeed, inhibition of mTORC1 also decreases glycolysis in CD4+ T-cells activated under Th17-polarizing conditions , thus emphasizing the importance of this kinase in promoting glycolysis in T-cells.
However, as stated above, lymphocyte signalling models position PI3K/PKB signalling as an upstream obligatory regulator of mTORC1 activity [2–4], which is inconsistent with the observation that mTORC1, but not PI3K/PKB, signalling promotes glycolysis in T-cells [17,28]. This apparent contradiction is redressed by the recent revelation that in CD8+ T-cells, mTORC1 activity is in fact maintained independently of PI3K and PKB . Thus mTORC1 activity is unaffected when PI3K or PKB activity is disrupted by either pharmacological or genetic perturbations . If not PI3K/PKB, what signalling pathways are responsible for mTORC1 activation in CTLs? A key role has been identified for PDK1 (phosphoinositide-dependent kinase 1), a kinase responsible for the phosphorylation and activation of multiple members of the AGC family [protein kinase A/protein kinase G/PKC (protein kinase C) family] kinases including PKB, PKC, S6K (70 kDa ribosomal S6 kinase), RSK (90 kDa ribosomal S6 kinase) and SGK (serum/glucocorticoid-regulated kinase) . As members of this kinase family have overlapping substrate specificity [32–35] (Figure 2), it is likely that PDK1-dependent and PKB-independent regulation of mTORC1 and glucose metabolism reflects functional redundancy within the AGC family of protein kinases with regard to the activation of mTORC1, although the exact mechanism has yet to be determined. Indeed, consistent with this role for PDK1 in the activation of mTORC1, PDK1-deficient CTLs have reduced HIF1α and Glut1 expression, and decreased levels of glucose uptake and glycolysis [17,28].
mTORC1 and HIF1 integrate T-cell metabolism and function
IL-2 sustains elevated levels of glucose metabolism and glycolysis in CD8+ CTLs by maintaining mTORC1 activity and the protein expression of HIF1α . In fact, HIF1 functions to couple mTORC1 to a diverse transcriptional programme that extends beyond the control of glucose metabolism . Thus, although mTORC1/HIF1 controls expression of glucose transporters and multiple rate-limiting glycolytic enzymes, this signalling axis also regulates the expression of cytolytic effector molecules and essential chemokine and adhesion receptors that regulate T-cell trafficking . CTLs deficient for HIF1α–HIF1β transcriptional complexes fail to express a discrete subset of effector cytolytic molecules including granzymes and perforin while maintaining normal expression of IFNγ, TNFα and lymphotoxins . HIF1-null CTLs also retain the expression of key lymph node homing receptors CD62L (also called L-selectin) and CCR7 (CC chemokine receptor 7) and have an altered trafficking profile relative to wild-type CTLs, homing preferentially to lymph nodes rather than sites of inflammation .
The observation that mTORC1/HIF1 controls T-cell trafficking through regulating the expression of CD62L and CCR7 raises the question of how this mTORC1/HIF1 pathway connects to a well-documented PI3K/PKB/FOXO (forkhead box O) pathway that also controls the expression of these key trafficking molecules . The retention of high levels of CCR7 and CD62L expression by immune-activated HIF1-null CD8+ T-cells phenocopies the impact of inhibiting PI3K/PKB signalling in activated CD8+ T-cells [28,37,38]. PI3K/PKB-regulated CD62L and CCR7 reflects that the expression of these molecules is regulated by the FOXO1 transcription factor [39,40]. FOXO1 is phosphorylated by PKB on multiple residues resulting in FOXO1 nuclear to cytoplasmic translocation, thus inhibiting FOXO1 transcriptional activity. Inhibition of PKB in CTLs results in the dephosphorylation of FOXO1, restoring FOXO1 nuclear localization, FOXO1 transcriptional activity and the expression of CD62L and CCR7 [28,37]. To date, the effect of mTORC1 inhibition on T-cell trafficking has been attributed to the observation that, in some cells, long-term rapamcyin treatment can result in PKB inhibition via disruption of mTORC2, which would be predicted to result in nuclear translocation of FOXO1 and the expression of CD62L and CCR7. However, this is not the case and FOXO1 cellular localization is unaffected in rapamycin-treated CTLs or indeed in HIF1-null CTLs .
Although HIF1 directly regulates the transcription of glucose transporters and glycolytic enzymes, it appears likely that mTORC1/HIF1 regulation of CTL effector molecules is indirect and potentially a secondary event to altered glucose metabolism. In support of this idea, CTLs cultured in limiting glucose concentrations or treated with 2-deoxyglucose lose the expression of perforin molecules [14,17], whereas CTLs deprived of glucose also maintain high levels of CD62L expression .
mTORC1 and PKB are activated independently, but have convergent functions
mTORC1/HIF1 regulates CTL trafficking through a mechanism that is independent of FOXO transcription factors. Therefore PKB and mTORC1 are activated independently in CTLs, but converge to regulate T-cell trafficking through distinct signalling pathways (Figure 3). Indeed, PKB/FOXO and mTORC1 signalling also converge to regulate other key T-cell functions. PI3K/PKB and mTORC1 signalling both promote IFNγ expression through distinct mechanisms that differ in the requirement for FOXO transcription factors [28,41]. Additionally, both PKB and mTORC1 signalling are required for the expression of the cytolytic molecules perforin and granzyme in CD8+ CTLs [17,28] (Figure 3).
Therefore PKB and mTORC1 are activated independently, but converge to control the expression of key molecules required for effector CTL function. An essential requirement for these two independent signalling pathways to control T-cell differentiation highlights the importance of integrating lymphocyte signal transduction for appropriate immune responses.
D.K.F. is funded by a Marie Curie Career Integration Grant [grant number 321603].
Regulation of Metabolism in Cancer and Immune Cells: Irish Area Section Meeting held at Trinity Biomedical Sciences Institute, Dublin, Ireland, 8–9 November 2012. Organized and Edited by Richard Porter (Trinity College Dublin, Ireland).
Abbreviations: AGC, family, protein kinase A/protein kinase G/protein kinase C family; CCR7, CC chemokine receptor 7; CTL, cytotoxic T-lymphocyte; FOXO, forkhead box O; Glut1, glucose transporter 1; HIF1, hypoxia-inducible factor 1; IFNγ, interferon γ; IL-2, interleukin-2; mTOR, mammalian target of rapamycin; mTORC, mTOR complex; PDK1, phosphoinositide-dependent kinase 1; PI3K, phosphoinositide 3-kinase; PKB, protein kinase B; PKC, protein kinase C; RSK, 90 kDa ribosomal S6 kinase; S6K, 70 kDa ribosomal S6 kinase; SGK, serum/glucocorticoid-regulated kinase; TCR, T-cell receptor; TSC2, tuberous sclerosis complex 2
- © The Authors Journal compilation © 2013 Biochemical Society