T cell differentiation and fate are orchestrated by signaling events involving the T cell receptor (TCR), co-stimulatory or co-inhibitory receptor stimulation, and cytokines. In addition, a variety of other environmental factors can also contribute to this decision. T cells switch between highly proliferative states (i.e., developing thymocytes and activated proliferating T cells) and quiescent states (i.e., naive, memory, and anergic T cells), characterized by the activation of different intracellular metabolic pathways. T cells use glucose as their primary fuel source for generation of adenosine triphosphate (ATP) and it is necessary for cell survival, growth, activation, proliferation, and cytokine production.
T cell receptor stimulation is accompanied by signals from growth factors and cytokines such as interleukin (IL)-2 or IL-7, and co-stimulatory molecules, such as CD28, which lead to an increase in glucose uptake and glycolysis through induction of phosphoinositide-3-kinase (PI3K)-dependent activation of Akt. Akt induces glucose metabolism by facilitating glucose uptake via the upregulation of glucose transporter 1 (Glut1) on the T cell membrane. Failure of T cells to up-regulate glucose metabolism results in decreased cytokine production, proliferation, and ultimately to apoptosis or anergy. An increase in the rate of protein synthesis also occurs following T cell activation and is regulated via Akt, which controls the activation of the mammalian target of rapamycin (mTOR), which is a key regulator of protein synthesis in T cells.
Naturally occurring regulatory T cells (T reg), defined as CD4+CD25+Foxp3+ T cells, play a non-redundant role in the maintenance of physiological tolerance to self-antigens and prevention of autoimmune responses. T reg generation in the thymus is promoted by recognition of self-peptides with intermediate affinity. T reg cells are characterized by a specific metabolic signature regulating their responsiveness to antigenic stimulations when compared to other CD4+ T cell subsets. Specifically, Th1, Th2, and Th17 cells express high surface levels of Glut1 and are highly glycolytic. T reg, in contrast, express low levels of Glut1 and have high lipid oxidation rates in vitro. Furthermore, blocking glycolysis promotes T reg cell generation through the transcription factor hypoxia-inducible factor 1α (HIF1α), whose induction required mTOR pathway activation. In turn, HIF1α enhances Th17 development through direct transcriptional activation of RORγt, and concurrently, it attenuates T reg development, by binding FoxP3 and targeting it for proteasomal degradation.
Collectively, these observations underscore the key role of metabolic cues and regulatory pathways in defining T cell differentiation and function.
Signaling through the TCR is accompanied by cytokine signals from IL-2, IL-7, and leptin as well as co-stimulation via CD28. Together, these signals cause increased glycolysis through the induction of PI3K-dependent activation of Akt, which in turn increases glucose transport via the upregulation of Glut1 on the T cell surface. Activated pAkt increases the rate of protein synthesis via activation of mTORC1. Activated mTORC1 activates HIF1α, regulates lipid metabolism and glycolysis.
The mTOR is a key regulator of T cell metabolism, that serves to integrate nutrient sensing pathways with signaling pathways involved in differentiation, growth, survival, and proliferation. TCR and co-stimulatory signals along with cytokines tweak the mTOR pathway via the upstream PI3K/Akt signaling networks to match the energy requirements associated with T cell activation. Conventional CD4+ and CD8+ T cells, upon stimulation, utilize the mTOR pathway to meet the increased metabolic demands of T cell activation by switching from primarily oxidative phosphorylation, seen in resting T cells, toward a state of enhanced aerobic glycolysis, a phenomenon popularly described as the Warburg effect. The importance of this phenomenon in determining T cell fate was first noticed using the selective inhibitor of mTOR, rapamycin, which prevented the generation of T eff responses and promoted the generation of T reg cells. Additionally, T cell-specific mTOR knockouts were shown to have poor T eff responses and defaulted toward a more T reg phenotype. These studies not only revealed the importance of mTOR as a critical regulator in the differentiation of T reg, but also highlighted the importance of the metabolic pathways that predominate within functionally different T cell subsets.
Consistent with the above findings, T reg display higher levels of AMP kinase activity and preferential lipid oxidation for their energy requirements. The AMP-activated kinase acts as a sensor of the AMP/ATP ratio, which is increased during hypoxia and inhibits mTOR kinase to promote mitochondrial oxidative metabolism rather than glycolysis. Interestingly, activation of AMP kinase via Metformin, a drug used to treat diabetes mellitus, increased the T reg population in the CD4+ T cell compartment in an in vivo murine model of asthma. In this study, mice sensitized by aerosol to ovalbumin in the presence of metformin, and challenged 21 days later showed an increase in the frequency and number of CD4+Foxp3+ T cells in the draining lymph nodes as compared to mice immunized in the absence of metformin. However, no change in airway responsiveness was noted even though there were fewer lymphocytes recovered in the bronchial alveolar lavage in the metformin treated animals. Additionally, inhibition of mitochondrial lipid uptake and oxidation pathways by Etomoxir, an inhibitor that prevents long chain fatty acid uptake to the inner mitochondrial membrane for beta oxidation, abrogated the generation of T reg without altering T eff differentiation. Furthermore, T reg were shown to express lower levels of the glucose transporter Glut1 as compared to T eff, and transgenic CD4+ T cells overexpressing Glut1 were shown to develop fewer T reg. Overall, these studies indicate that fatty acid oxidation is the dominant metabolic process utilized for the generation of energy in T reg.
While inhibition of mTOR enhances T reg generation during an immune response, mTOR activity is known to be required to maintain their suppressive capabilities. In this section, we review recent findings that investigated this apparent dichotomy in the function of mTOR in T reg biology. mTOR exists as two structurally distinct complexes (mTORC1 and mTORC2). Both complexes localize within different subcellular compartments and have different functions in the cell; rapamycin-sensitive mTORC1 forms the fundamental nutrient sensing complex that is activated by Akt kinase downstream of PI3K signaling induction (via the TCR, co-stimulatory receptors, and cytokines) whereas the rapamycin-insensitive mTORC2 controls spatial aspects of cell growth through activation of cytoskeletal components. The mTORC2 complex also, in turn, activates the kinase Akt. Thus, Akt lies both upstream and downstream of mTOR. In mice, CD4+ T cells lacking both mTORC1 and mTORC2 complexes fail to differentiate into any T eff lineage (Th1, Th2, or Th17) and instead differentiate toward the T reg cell phenotype, consistent with the CD4+ population of mTOR null mice. However, recent findings by Hu Zheng et al. indicate a crucial role of the mTORC1 complex to the suppressive activity of T reg. Indeed, mTORC1 activity was shown to be higher in T reg than naive T cells under steady state conditions. Impairment of the mTORC1 pathway in T reg via selective genetic deletion of Raptor, an obligatory component of mTORC1, in the CD4+ FOXP3+ compartment, led to the early onset of a fatal autoimmune disease in mice. Moreover, the disease mimicked the autoimmune disease seen in Scurfy mice that bear a loss-of-function mutation in the FoxP3 transcription factor, indicating impaired T reg function. Mechanistically, the mTORC1 pathway in T reg was shown to be necessary to initiate the upregulation of surface CTLA-4 and ICOS, key intrinsic receptors for T reg-mediated suppression. In addition, mTORC1 was shown to induce cholesterol and lipid metabolism as well as proliferation in the T reg population. Finally, recent investigations have revealed a non-redundant role of mTORC1 in mitochondrial metabolism. Collectively, these investigations imply a differential use of mTOR in T reg as compared to conventional effector cells.
From the aforementioned studies, it is clear that the metabolic cues from the environment and subsequent mTOR activity play a key role in T reg differentiation. Powell et al. have proposed a model of T reg differentiation based on mTOR activity that mimics that seen in conventional T cell differentiation. Briefly, naïve T cells, receiving strong mTOR activation upon antigen recognition (through environmental cues, TCR, cytokine, and co-stimulatory stimulation), differentiated into short-lived T eff cells exhibiting high glycolytic activity, while those receiving weak mTOR activation developed into long-lived memory T cells dependent on oxidative phosphorylation to meet their energy needs. One can suggest that the high level of mTOR activity in T eff cells would be necessary to sustain higher demand for energy via glycolytic pathways while the opposite would hold true for quiescent memory T cells. A similar model can be applied to induced T reg where naïve T cells in the presence of TGF-β receiving either high or low mTOR activating signals could result in the differentiation of “effector” and “memory” Foxp3+ T reg respectively. As such, CD4+ Foxp3+ T cells that traffic to activating lymph nodes and become robustly stimulated (mTOR hi) generate short-lived “effector” T reg. These effector T reg would then home to the tissues and control immune responses. This model can explain why T cel
