mTOR, metabolism, and the regulation of T-cell differentiation and function

The two signal model, mTOR, and metabolism

As a critical component of the adaptive immune response T cells are characterized by their large diversity of antigen specific receptors and the ability to rapidly respond in a recall response. While T-cell receptor (TCR) engagement heralds recognition, the ultimate outcome of antigen recognition is guided by the integration of multiple signals derived from the immune microenvironment. In this regard, the two signal model provides a framework to understand how a particular antigenic encounter can lead to the appropriate activating or tolerogenic response (1, 2). In this paradigm, antigen recognition in the absence of costimulatory signals can lead to tolerance while antigen recognition in the context of costimulation leads to robust immune responses. TCR engagement in the absence of costimulation leads to activation-induced cell death and anergy (3). The addition of costimulation, for example in the form of CD28 activation, leads to interleukin-2 (IL-2) production and proliferation (4, 5). However, in the past decade, the increased complexity of the response has been better appreciated. For CD4⁺ T cells, signals from the immune microenvironment dictate the nature of the effector response. Indeed, depending on the cytokine milieu, full activation (Signal 1+2) can lead to a T-helper 1 (Th1), Th2, Th9, or Th17 response (6-8). Alternatively, suboptimal Signal 1 or full stimulation in the presence of transforming growth factor-β (TGF-β) can lead to the induction of regulatory T cells (9).

Our group has proposed that the mammalian target of rapamycin (mTOR) is a central integrator of diverse signals derived from the immune microenvironment that make up ‘Signal 2’ (10, 11). mTOR is an evolutionarily conserved serine/threonine kinase that is a member of the phosphorinositol 3-kinase (PI3K) family. From yeast to mammalian cells, TOR integrates cues from the environment to regulate a diversity of cellular functions including growth, apoptosis, metabolism, actin reorganization, and ribosome biogenesis (12, 13). Early studies demonstrated that by inhibiting mTOR, TCR engagement in the presence of costimulation (Signal 1+2) led to tolerance in the form of T-cell anergy (14). Subsequent studies revealed that in the absence of mTOR signaling, CD4⁺ T cells fail to differentiate into effector cells under appropriate skewing conditions. Rather, TCR stimulation in the absence of mTOR leads to the generation of forkhead box protein 3 (Foxp3) ⁺ regulatory T cells (15). Recent studies have demonstrated that mTOR inhibition promotes the generation of memory CD8⁺ T cells (16-18). Complementing these observations in T cells are a number of studies demonstrating a critical role for mTOR in integrating signals necessary for antigen-presenting cell (APC) and B-cell differentiation and function (19-23).

The stochastic generation of a diverse array of antigen receptors is an essential component of the ability of T cells to promote immunity. The clonal selection theory provides a model whereby a low frequency of naïve antigen-specific T cells can be expanded to protect the host. Critical to development of T-cell immunity is the extraordinary clonal expansion that accompanies antigen recognition. Such expansion requires energy and the de novo synthesis of proteins, nucleic acids, and lipids rivaling that of cancer cells. As such, it has been appreciated that upon activation, T cells employ metabolic pathways similar to cancer cells (24, 25). Specifically, even in the presence of oxygen, T cells metabolize glucose via glycolytic and mitochondrial pathways, thus not only generating energy but also generating substrates necessary for cellular growth and division (25).

While the metabolic changes associated with T-cell activation have been known for some time, it is only relatively recently that the precise mechanisms by which T cells support these changes have begun to be elucidated. Studies by Frauwirth et al. (26, 27) demonstrated that costimulation in the form of CD28 stimulation leads to the upregulation of the metabolic machinery necessary to support increased glycolysis and full T-cell activation. These observations make a critical link between signal 2 and the metabolic function necessary to support T-cell activation. Other studies have revealed the ability of T-cell activation to regulate glutamine metabolism (28). Importantly, these observations suggest that the upregulation of the metabolic machinery is not merely a consequence of T-cell activation but rather play a role in promoting T-cell activation (24). In line with this concept is the observation that blocking leucine metabolism or glucose metabolism can promote T-cell anergy in antigen-stimulated Th1 CD4⁺ T cells (29). Furthermore, recent studies have demonstrated critical differences between the metabolic requirements and pathways employed by CD8⁺ effector and memory T cells (30, 31).

Understanding T-cell differentiation and function necessitates understanding the signals that regulate T-cell metabolism. Along these lines, since mTOR plays such a central role in regulating the expression of proteins involved in glycolysis, glucose uptake, lipid and amino acid metabolism, it stands to reason that the ability of mTOR to guide T-cell differentiation and function will be in part through the regulation of metabolism. In this review, we highlight our current knowledge regarding the ability of mTOR to regulate T-cell differentiation and function and the ability of mTOR to regulate metabolism (Fig. 1). In this context, some of the connections between mTOR and metabolism in T cells are well established. Alternatively, we also present data demonstrating the ability of mTOR to regulate metabolic pathways in other systems and in doing so speculate that such pathways may also exist in T cells and thus regulate T-cell function.

A primer on mTOR signaling

mTOR forms two functionally distinct signaling complexes: mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) (12). mTORC1 is comprised of the regulatory-associated protein of mTOR (Raptor), mammalian lethal with Sec13 protein 8 (mLST8), the proline-rich Akt substrate of 40 kDa (PRAS40), and DEP-domain-containing mTOR-interacting protein (DEPTOR). While the mTORC2 complex also contains mLST8 and Deptor, it additionally binds with the rapamycin-insensitive companion of TOR (RICTOR), mSIN1 proteins, and the Protein Observed with RICTOR (PROTOR)(12)

The kinase activity of the mTORC1 complex is positively regulated by the small GTPase Ras homolog enriched in brain (Rheb), whose activity is in turn inhibited by tuberous sclerosis complex 1 (TSC-1) and TSC-2 (32, 33). Phosphorylation of TSC-1/2 by the kinase AKT, whose activity is dependent on PI3K-mediated PDK-1 activation, inhibits the complexes’ GAP activity, thereby facilitating mTORC1 activation by GTP-bound Rheb (34). The kinase AKT can also target PRAS40, which under conditions of low AKT activity inhibits mTOR activity by directly binding with Raptor (35, 36). The activity of the mTORC1 complex is canonically measured by the phosphorylation of its substrates p70 S6-kinase and 4E-BP1 (37). The phosphorylation of 4E-BP1 results in its release of the translation-initiation factor eIF-4e leading to increased protein synthesis. Interestingly, while it has long been thought that mTOR broadly regulates cellular protein translation, detailed proteomic analysis has revealed that a fairly limited number of gene products are directly sensitive to mTOR inhibition. Notably, this includes mRNAs possessing a ‘5′-terminal oligopyrimidine tract’ (5′TOP) sequence (38). Interestingly, the pattern of protein translation inhibition seen in cells treated with allosteric inhibitors of mTOR activity (such as rapamycin) and those treated with selective mTOR kinase inhibitors (such as PP242, Torin1, and INK128) differ significantly (39).

While the upstream signals that regulate mTORC1 activity have been very well defined, identification of the precise signals regulating mTORC2 activity is still an active area of investigation. Recent studies have shown that mTORC2 is strongly and specifically activated following association with ribosomes, while its kinase activity is inhibited by ER stress and the glycogen synthetase kinase-3 β (GSK-3 β) (40, 41). Downstream targets of mTORC2 include Akt, serum and glucocorticoid-inducible kinase 1 (SGK-1), and protein kinase C (PKC) (42, 43). Akt acts as both an upstream regulator of mTORC1 activity (as indicated by the PI3K/PDK-1 dependent phosphorylation at the T308 residue) as well as a downstream target of mTORC2 (as indicated by phosphorylation at S473 residue). Akt-dependent inhibition of TSC2 (upstream of mTORC1) does not require mTORC2 in T cells (43-45).

Regulation of mTOR activity

The activity of the mTOR kinase signaling complex is regulated by a wide array of extrinsic immunological and metabolic cues, as well as cell intrinsic stress and danger signals. Ligation of the TCR can induce mTOR activation via the activation of PI3K; however, this activation is potently enhanced by stimulation by the costimulatory receptors CD28, ICOS, and OX-40 (46-52). The T-cell-restricted inhibitory receptors CTLA-4 and PD-1 can antagonize mTOR activity via AKT and PI3K inhibition (53-55). Many soluble factors such as the interleukins, interferons, chemokines, the lysophospholipid S₁P₁, and the adipokine leptin all possess the ability to induce mTOR activity in T cells expressing the appropriate receptors (56-68). Under conditions of metabolic stress, a decrease in the normally high intracellular ATP:AMP ratio induces the activation of AMP-kinase, which facilitates the inhibition of mTOR by phosphorylating and activating the mTORC1 inhibitory TSC1/2 complex as well as directly phosphorylating the mTORC1 scaffolding protein Raptor (69, 70). Low oxygen tension can also facilitate the inhibition of mTOR activity via the protein REDD1, which facilitates the phosphorylation and stabilization of the TSC1/2 complex (71, 72). Furthermore, cellular stress can induce the activation of the classic tumor suppressor gene p53 in a AMPK-dependent fashion, resulting in enhanced expression of many mTOR-inhibitory proteins including TSC2, PTEN, REDD1, and AMPK (73-75)

mTOR is an important regulator of cellular metabolism

In the presence of oxygen, most mammalian cells rely on the TCA cycle and oxidative phosphorylation to generate ATP. However, as previously mentioned, newly activated T cells (like cancer cells) primarily rely on the processes of aerobic glycolysis for their energetic needs (24). This phenomenon of eschewing the largely efficient process of oxidative phosphorylation following glycolysis (which can produce a total of 30 to 35 molecules of ATP per molecule of glucose) in favor of the much less efficient processes of aerobic glycolysis (a net gain of 2 ATP per glucose) was initially described by Otto Warburg in cancer cells (76). It has been proposed that although such metabolism appears inefficient in its ability to produce energy in the form of ATP, it provides intermediate metabolites that can lead to the generation of structural components necessary for rapidly dividing cells (77). The process of aerobic glycolysis is directly regulated by mTORC1 signaling by the transcriptional regulation of many key glycolytic enzymes (78). Indeed, mTOR activation regulates (in part through HIF and Myc as described below) the transcription of enzymes involved in nearly every step of glucose metabolism from its transport into the cell to the conversion of pyruvate to lactate (79-81).

The pentose phosphate pathway (PPP) is an anabolic program employed in many cells (including activated T cells) that utilizes glucose-6-phosphate generated during glycolysis but yields a different array of metabolic intermediates (82). The primary substrate for the PPP is glucose-6-phosphate produced during the first stage of glycolysis by the enzyme hexokinase 1, but unlike glycolysis, the PPP does not directly generate ATP. During the oxidative phase of the reaction, 2 NADP+ are reduced to NADPH by the enzymes glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. NADPH provides reducing equivalents necessary for lipid and nucleotide synthesis and prevents oxidative stress. This anabolic program finally results in the generation of ribose-5 phosphate and erythrose-4-phosphate, which can be utilized by the cell for the generation of nucleic acids or amino acids, respectively. Alternately, these products can feed back into glycolysis, providing more metabolic substrates for the cell. mTORC1 activity directly regulates the expression of enzymes in the PPP, while at the same time facilitates the uptake of glucose via the Glut1 membrane transporter (79). Conversely, inhibition of mTOR activity by rapamycin treatment greatly decreases the expression of these genes (79).

The process of glutaminolysis, the degradation of glutamine to generate substrates for the TCA cycle, is another pathway found in activated T cells. Inhibition of glutaminolysis significantly decreases T-cell proliferation, a phenomenon that can be partially reversed by the addition of supplementary nucleotides and polyamines, two products of glutamine degradation (80). Interestingly, the cellular importation of glutamine via the SLC1A1 Na+ dependent symporter can not only feed into the catabolic glutaminolysis pathway but can also facilitate the induction of mTOR activity. Amino acid availability, especially the branched-chain amino acid leucine, is essential for the induction of mTORC1 activity. Indeed, the lack of plasma-membrane leucine transporters can completely abolish mTORC1 activity, which can be overcome by cytoplasmic injection of this amino acid (83). In part, this phenomenon is thought to be due to the ability of leucine to activate heterodimers of Rag GTPases, which can deliver the mTOR kinase complex to GTP-bound Rheb (84, 85). The import of extracellular leucine occurs via a glutamine antiporter, SLC7A5-SLC3A2. Intracellular glutamine does not itself possess any ability to directly induce mTORC1 activity, but without intracellular glutamine, lecuine influx is mitigated and, therefore, so is mTORC1 activity (86, 87).

Fatty acid oxidation, specifically the process of β-oxidation of free fatty acids (FFA) in the mitochondria, provides an abundant source of ATP for cellular respiration in a process that is highly dependent on the presence of oxygen. The inhibition of mTOR activity by rapamycin treatment decreases the rate of glycolysis, but causes a corresponding increase in the rate of fatty acid oxidation (88). Furthermore, rapamycin-mediated mTOR inhibition blocks the process of fatty acid and complex lipid synthesis by downregulating the expression of acetyl-coenzyme A carboxylase I and mitochondrial glycerol phosphate acyltransferase (89). Fatty acid oxidation appears to play an important role in the metabolic program of a naive resting T cells, while it is significantly down regulated following T-cell activation (80, 90).

The reduced coenzyme NADH/NADPH generated by aerobic glycolysis, the PPP, and the activity of the TCA cycle are utilized by the mitochondrial electron transport chain under aerobic conditions to generate ATP. mTORC1 activity facilitates this process by inducing mitochondrial biogenesis. Inhibition of mTOR activity by rapamycin dramatically reduces mitochondrial oxygen utilization in a process that appears to be independent of S6kinase, while mTORC1 appears to directly control the expression of many mitochondrial genes (91). Recently, it has been shown that mTORC1 regulates the activity of the Yin Yang 1 (YY1) zinc-finger GLI-kruppell class transcription factor, as well as its transcriptional co-activator peroxisome-proliferator-activated receptor coactivator (PGC)-1a, both of which are known to regulate the expression of many mitochondrial genes (92). Furthermore, the inhibition of mTOR activity, either by starvation or pharmacological intervention, can result in a dramatic reduction in cellular mitochondrial load as a result of the induction of autophagy. Therefore, high levels of mTOR activity promote both the generation, as well as the stability, of mitochondria.

The metabolic programs described thus far play an important role during the tremendous growth phase that characterizes T-cell activation. Alternatively, autophagy is a tightly controlled catabolic metabolic process during which a nutrient-deprived or stressed cell degrades its own organelles, cytoplasm, lipids, or proteins to derive energy. Not surprisingly, the process of autophagy is a finely balanced calculation, weighing the benefits of degrading critical cellular components to insure continued cell survival during nutrient deprivation against the possibility of inducing cell death. In addition, the process of autophagy provides a cell with a pathway by which to dispose of damaged organelles and aggregation-prone proteins independently of nutrient availability (93). Therefore, autophagy is both a pro- and anti- survival process, depending on the context of initiation. While there are several autophagic pathways and programs, including macroautophagy, microautophagy, and chaperone-mediated autophagy, the process is commonly quantified by the accumulation of intracellular LC3B coated vesicles (94). LC3 is ubiquitously expressed in mammalian cells during all metabolic states, but it rapidly accumulates on autophagic vesicles following nutrient withdrawal (95).

Autophagy and the formation of autophagic vesicles are directly regulated by mTOR signaling in a process primarily involving the mTORC1 complex (96, 97). The addition of rapamycin to a cell culture rapidly induces autophagy, while the constitutive activation of mTORC1 by the deletion of the negative regulator protein TSC-2 renders a cell resistant to autophagy induction (98). The initiation of autophagy in mammalian cells is dependent on the assembly and activity of a complex containing the kinase ULK1, the adapter protein autophagy-related gene-13 (Atg13), and the 200 kDa focal adhesion kinase family-interacting protein (FIP200) (97). Unlike their yeast homologs, the ULK1:Atg13:Fip200 complex in mammalian cells does not appear to disassemble under condition of high mTOR signaling and abundant nutrient availability (99). Rather, the mTOR kinase complex appears to directly phosphorylate inhibitory sites on the kinase ULK1 and the adapter protein Atg13, thereby inhibiting the induction of autophagy. In contrast, under conditions of low mTOR activity, ULK1 appears to autophosphorylate as well as directly phosphorylate Atg13 and FIP200, facilitating the activation of the complex and the induction of autophagy (100, 101). Inhibition of this process by siRNA-mediated knockdown of ULK1 renders cells resistant to rapamycin-induced autophagy (102).

mTOR-mediated transcription intersects with T-cell transcriptional programs

The ability of mTOR to regulate metabolic programs is mediated through the regulation of key transcriptional regulators. These same factors implicated in the regulation of metabolism also play roles in T-cell function. Thus, in this fashion, mTOR plays a role in coordinating the cellular immune response with metabolic demands (Fig. 2).

HIF-1α

HIF-1 is a hetrodimeric protein consisting of the constitutively expressed β subunit and an inducible α subunit. The α subunit undergoes rapid proteasomal degradation under normoxic conditions due to prolyl 4-hydroxylase dependent modification of the Pro-402/564 residues, resulting in its being targeted for destruction by E3 ubiquitin ligases (103). However, under conditions of low oxygen tension, this protein degradation pathway is strongly inhibited, thereby allowing the accumulation of HIF-1α and the formation of a transcriptionally active HIF-1 dimer (104, 105). As would be expected in light of its mode of activation, HIF-1 controls the expression of many genes critical for cellular adaptation to a low-oxygen metabolic environment. As glycolysis is the primary metabolic program utilized by oxygen-starved cells, HIF-1 directly upregulates the expression of gene products along the entire glycolytic pathway, including the plasma membrane glucose transporter Glut1 and lactate dehydrogenase A (LDH-A) (81, 106). Furthermore, HIF-1 indirectly inhibits the ability of pyruvate produced as the product of glycolysis to enter into the TCA cycle by inducing the activity of pyruvate dehydrogenase kinase 1 (PDK1) (107). This enzyme antagonizes the activity of pyruvate dehydrogenase, which converts pyruvate to acetyl-CoA and CO₂, thereby further enforcing an anaerobic and glycolytic metabolic program.

In addition to the turnover rate of HIF-1α being regulated by the availability of oxygen, HIF-1α transcription is enhanced by mTORC1 activity (108). This effect is readily apparent under low-oxygen conditions, where the addition of rapamycin can abrogate many of the pro-survival effects mediated by HIF-1 expression (109). In T cells, HIF-1α, expression is induced by a number of immunologic cues even in the presence of normoxia. This includes TCR stimulation as well as the presence of IGF-1 and glucose availability (110-112). This control can be quite complex. For example, as mentioned previously the HIF-1α protein is stabilized under hypoxic conditions. However, mTOR activity, and therefore HIF-1α transcription, is inhibited under conditions of low oxygen tension via the protein REDD-1 (71, 72, 113). Thus, hypoxic conditions simultaneously stabilize the HIF protein and inhibit the transcriptional expression of HIF-1α.

Myc

The oncogenic transcription factor Myc regulate many key metabolic pathways critical for cellular growth and proliferation (80, 114, 115). These pathways include glycolysis, glutaminolysis, and fatty acid oxidation (80). T cells rapidly induce Myc expression following TCR stimulation to facilitate the marked metabolic shift required for rapid proliferation (80). Additionally, Myc is among the most commonly overexpressed genes in many tumors due to its pro-survival/growth activity (116, 117). The kinase activity of mTORC1 appears to facilitate Myc translation by directly phosphorylating the translational repressor 4E-BP, which results in the release of eIF-4e. This effect is in part due to the tertiary structure of the 5′-UTR of the Myc mRNA, which has been shown to contain an internal ribosome entry site (118-122). Additionally, there is evidence that AKT/mTOR activity can regulate Myc expression at the transcriptional level via AIP4/ITCH-mediated degradation of the transcriptional repressor JunB (123). Interestingly, Myc is a transcriptional repressor of TSC2 gene expression, meaning that high levels of Myc expression facilitates a feed-forward loop resulting in less TSC2 protein, higher mTOR activity, and even more Myc expression (124). In addition to directly regulating the expression of Myc protein, high levels of mTOR activity synergizes with Myc expression by cooperatively enhancing protein translation and elongation (124). Indeed, many tumor cells that exhibit resistance to rapamycin show a compensatory increase in Myc expression, and Myc null cells are significantly more sensitive to rapamycin-induced inhibition of proliferation (124, 125).

PPARα

The peroxisome proliferator-activated receptor α (PPARα) is a nuclear hormone receptor that is thought to regulate fatty acid metabolism as well as glucose homeostasis by acting as a intracellular sensor of endogenous fatty acids (126). The precise endogenous ligand for PPARα is still an active area of investigation, but it has been shown that mono/polyunsaturated fatty acids, long-chain fatty acyl-CoAs, and saturated fatty acids can all bind and activate the receptor (127-130). The binding of these natural ligands to PPARα facilitates the nuclear translocation of the protein, displacement of transcriptional repressors from target genes, and the recruitment of transcriptional activators. PPARα possesses the ability to regulate a wide range of metabolic processes, including mitochondrial fatty acid β-oxidation (131, 132), fatty acid uptake (133), and glucose homeostasis (134). However, PPARα also possess the ability to directly repress the expression of several pro-inflammatory transcription factors, including AP1 and NF-κB (135-137). While the transcriptional activity of PPARα has long been thought to be primarily dictated by nuclear translocation, it has recently been shown that PPARα protein levels and transcriptional activity is inhibited by mTORC1 activity downstream of TSC1/2 (138). This was determined by the observation that the livers of fasted mice show very low levels of mTORC1 activity relative to fed controls but exhibit complementarily high level of PPARα and PPARα-target gene expression. This phenomenon could be reversed by the genetic deletion of TSC-1, which eliminated the fasting-induced upregulation of PPARα expression and transcriptional activity (138).

PPARγ

Like its homolog PPARα, PPARγ is a nuclear hormone receptor that plays a critical role in regulating adipogenesis, as well as lipid metabolism and glucose homeostasis in cells. There are three known mRNA isoforms of PPARγ, with isoform 1 showing widespread tissue expression, while isoforms 2 and 3 appear to be exclusively expressed in adipose tissue (139-142). It has been shown that polyunsaturated acids (such as linoleic acid and aracidonic acid), eicosanoids, and many synthetic ligands (such as TZDs and tyrosine agonists) can bind to PPARγ with high affinity and induce nuclear translocation (128, 143, 144). Interestingly, unlike PPARα, PPARγ transcriptional activity appears to be enhanced by mTOR activity. The addition of rapamycin to cultured 3T3-L1 pre-adipocytes profoundly represses the rate of adipogenisis in a process that appears to be dependent on the disruption of a feed-forward loop between PPARγ and another metabolically important transcription factor C/EBPα (145). Furthermore, the development of mature adipocytes in vitro is dependent on the availability of extracellular amino acids. Stimulation of AKT/mTOR activity by the addition of amino acids to the culture conditions significantly enhances PPARγ activity in an mTOR dependent fashion, thereby increasing the rate of adipogensis. The genetic deletion of the mTORC1 inhibitory protein TSC in mouse embryonic fibroblasts and 3T3-L1 adipocytes additionally results in a significantly enhanced rate of adipogenesis in an mTORC1 and PPARγ dependent fashion (146).

SREBP

The sterol regulatory element binding proteins (SREBPs) family of basic helix-loop-helix transcription factors are master regulators of cellular lipogenesis, facilitating the transcription of anabolic enzymes involved in fatty acid and cholesterol synthesis (147). The activity of SREBP-1, the canonical member of the transcription factor family which is expressed in T cells, is regulated by several mechanism, including cellular localization, post-translational modification, degradation, and transcription in a process that is dependent on mTORC1 activity downstream of AKT (148, 149). The transcription of SREBP is suppressed by rapamycin-mediated mTORC1 inhibition, inducing a feed-forward process due to the fact that SREBP binds to its own promoter and facilitates its own expression (150, 151). Furthermore, mTORC1 signaling facilitates SREBP transcriptional activity by inducing the ER to Golgi translocation of the immature membrane-bound form as well as the cleavage of the immature factor (79, 150). Lastly, mTORC1 can facilitate the DNA binding activity of SREBP by the direct phosphorylation and subsequent nuclear-exclusion of the phosphatidic acid phosphatase Lipin-1, which under conditions of low-mTOR activity antagonizes the nuclear accumulation of SREBP (152).

mTOR, T cells, and metabolism

Following a 24-36 h period of activation, T cells are capable of undergoing a round of replication every 8 h (153). To facilitate this energy-intensive process, activated T cells utilize a wide range of metabolic processes to derive energy as well as metabolic intermediates required for cellular growth. However, it is clear that different types of T cells exhibit different metabolic demands. In this section, we focus on the intersection of these metabolic demands and the ability of mTOR to regulate function and metabolism.

CD8⁺ memory T-cell development

Inasmuch as the initiation phase of a T-cell-mediated immune response is accompanied by a dramatic upregulation in mTOR signaling, metabolic activity and glycolysis, the generation of a stable pool of memory T cells is reliant on a corresponding decrease in mTOR signaling and a transition to a non-glycolytic metabolic profile. The pharmacological inhibition of mTOR signaling by the administration of low-dose rapamycin during an active LCMV infection can markedly increase the number of antigen specific memory CD8⁺ T cells without inhibiting the ability of the infected animal to clear the pathogen (16). Furthermore, long-lived antigen-specific CD8⁺ T cells can be generated in vitro by the addition of rapamycin resulting in a stable pool of memory cells that persist when adoptively transferred into a recipient animal (17). The inclusion of rapamycin during T-cell stimulation facilitates the development of memory CD8⁺ T cells in part by inhibiting the expression of Tbet, while simultaneously stabilizing the expression of the related transcription factor Eomesodermin (18).

mTOR’s regulation of Tbet and eomesodermin expression has also been show to play a critical role in the process of homeostatic proliferation (HP)-induced memory CD8⁺ T-cell development (208). CD8⁺ T cells adoptively transferred into lymphopenic hosts rapidly acquire a memory cell phenotype in a process dependent on major histocompatibility complex (MHC) class I expressed self-peptide and the cytokines IL-7 and IL-15 (209, 210). Although not as potent as classically induced memory CD8⁺ T cells, these HP-induced memory CD8⁺ T cells are capable of rapid proliferation upon antigenic rechallenge, and possess potent anti-tumor capability (211, 212). Li et al. (208) have recently shown that the development of HP-derived memory CD8⁺ T cells is dependent on mTOR.

Recent work from the laboratory of Erika Pearce (30, 31) has highlighted the significant metabolic changes that a CD8⁺ T cell must undergo during the effector to memory cell transition. It was found that tumor necrosis factor receptor-associated factor 6 (TRAF6)-null T cells could become effector cells but failed to develop into memory T cells. Upon further analysis it was observed that the TRAF6-deficient CD8⁺ T cells exhibit a profound defect in their ability to up regulate genes associated with fatty acid oxidation. This was accompanied by an inability to induce mitochondrial fatty acid oxidation following growth factor withdrawal. Treatment with rapamycin or metformin rescued the memory defect in TRAF6-deficient CD8⁺ T cells and further enhanced the development of memory CD8⁺ T cells wildtype animals. Subsequently, this group went on to demonstrate that the effector to memory transition of CD8⁺ T cells is accompanied by a dramatic increase in the number of mitochondria in the cells. This results in an increase in the oxygen consumption of memory cells and the development of increased spare respiratory capacity (31).

Conclusion

In this review, we highlight the role of mTOR in regulating T-cell differentiation and function as well as the role of mTOR in regulating cellular metabolic function. Clearly, the metabolic demands of different subsets of T cells are quite diverse. To this end, the metabolic machinery plays an important role in regulating the outcome of T-cell activation. In this regard, we tried to accentuate the potential intersection between mTOR signaling and metabolic regulation and the ability of mTOR to guide the outcome of antigen recognition. In many cases, the details of this intersection have yet to be defined. However, the elucidation of these precise signaling pathways will provide the opportunity to specifically target mediators downstream of mTOR to selectively regulate T-cell responses. Likewise, pharmacologic inhibition of specific metabolic pathways might greatly facilitate the enhancement and/or inhibition of selective T-cell function.