Nancie J. MacIver

Cutting Edge: Distinct Glycolytic and Lipid Oxidative Metabolic Programs Are Essential for Effector and Regulatory CD4+ T Cell Subsets

Stimulated CD4+ T lymphocytes can differentiate into effector T cell (Teff) or inducible regulatory T cell (Treg) subsets with specific immunological roles. We show that Teff and Treg require distinct metabolic programs to support these functions. Th1, Th2, and Th17 cells expressed high surface levels of the glucose transporter Glut1 and were highly glycolytic. Treg, in contrast, expressed low levels of Glut1 and had high lipid oxidation rates. Consistent with glycolysis and lipid oxidation promoting Teff and Treg, respectively, Teff were selectively increased in Glut1 transgenic mice and reliant on glucose metabolism, whereas Treg had activated AMP-activated protein kinase and were dependent on lipid oxidation. Importantly, AMP-activated protein kinase stimulation was sufficient to decrease Glut1 and increase Treg generation in an asthma model. These data demonstrate that CD4+ T cell subsets require distinct metabolic programs that can be manipulated in vivo to control Treg and Teff development in inflammatory diseases.

Metabolic Regulation of T Lymphocytes

T cell activation leads to dramatic shifts in cell metabolism to protect against pathogens and to orchestrate the action of other immune cells. Quiescent T cells require predominantly ATP-generating processes, whereas proliferating effector T cells require high metabolic flux through growth-promoting pathways. Further, functionally distinct T cell subsets require distinct energetic and biosynthetic pathways to support their specific functional needs. Pathways that control immune cell function and metabolism are intimately linked, and changes in cell metabolism at both the cell and system levels have been shown to enhance or suppress specific T cell functions. As a result of these findings, cell metabolism is now appreciated as a key regulator of T cell function specification and fate. This review discusses the role of cellular metabolism in T cell development, activation, differentiation, and function to highlight the clinical relevance and opportunities for therapeutic interventions that may be used to disrupt immune pathogenesis.

Glucose Uptake Is Limiting in T Cell Activation and Requires CD28-Mediated Akt-Dependent and Independent Pathways

T cell activation potently stimulates cellular metabolism to support the elevated energetic and biosynthetic demands of growth, proliferation, and effector function. We show that glucose uptake is limiting in T cell activation and that CD28 costimulation is required to allow maximal glucose uptake following TCR stimulation by up-regulating expression and promoting the cell surface trafficking of the glucose transporter Glut1. Regulation of T cell glucose uptake and Glut1 was critical, as low glucose prevented appropriate T cell responses. Additionally, transgenic expression of Glut1 augmented T cell activation, and led to accumulation of readily activated memory-phenotype T cells with signs of autoimmunity in aged mice. To further examine the regulation of glucose uptake, we analyzed CD28 activation of Akt, which appeared necessary for maximal glucose uptake of stimulated cells and which we have shown can promote Glut1 cell surface trafficking. Consistent with a role for Akt in Glut1 trafficking, transgenic expression of constitutively active myristoylated Akt increased glucose uptake of resting T cells, but did not alter Glut1 protein levels. Therefore, CD28 appeared to promote Akt-independent up-regulation of Glut1 and Akt-dependent Glut1 cell surface trafficking. In support of this model, coexpression of Glut1 and myristoylated Akt transgenes resulted in a synergistic increase in glucose uptake and accumulation of activated T cells in vivo that were largely independent of CD28. Induction of Glut1 protein and Akt regulation of Glut1 trafficking are therefore separable functions of CD28 costimulation that cooperate to promote glucose metabolism for T cell activation and proliferation.

Glucose metabolism in lymphocytes is a regulated process with significant effects on immune cell function and survival

Lymphocytes require glucose uptake and metabolism for normal survival and function. The signals that regulate the expression and localization of glucose transporter 1 (Glut1) to allow glucose uptake in T cells are now beginning to be understood. Resting T cells require extracellular signals, such as cytokines, hormones, and growth factors, or low‐level TCR stimulation to take up adequate glucose to maintain housekeeping functions. In the absence of extrinsic signals, resting T cells internalize and degrade Glut1 and cannot maintain viability. Activated T cells have dramatically increased metabolic requirements to support the energy and biosynthetic needs necessary for growth, proliferation, and effector function. In particular, glucose metabolism and aerobic glycolysis fuel this demand. Therefore, activation of T cells causes a large increase in Glut1 expression and surface localization. If glucose uptake is limited, glycolytic flux decreases to a level that no longer sustains viability, and proapoptotic Bcl‐2 family members become activated, promoting cell death. However, excessive glucose uptake can promote hyperactive immune responses and possible immune pathology. Tight regulation of glucose uptake is required to maintain immune homeostasis, and understanding of these metabolic pathways may lead to therapeutic strategies to target some forms of cancer or autoimmunity.

Metabolic programming and PDHK1 control CD4+ T cell subsets and inflammation

Activation of CD4+ T cells results in rapid proliferation and differentiation into effector and regulatory subsets. CD4+ effector T cell (Teff) (Th1 and Th17) and Treg subsets are metabolically distinct, yet the specific metabolic differences that modify T cell populations are uncertain. Here, we evaluated CD4+ T cell populations in murine models and determined that inflammatory Teffs maintain high expression of glycolytic genes and rely on high glycolytic rates, while Tregs are oxidative and require mitochondrial electron transport to proliferate, differentiate, and survive. Metabolic profiling revealed that pyruvate dehydrogenase (PDH) is a key bifurcation point between T cell glycolytic and oxidative metabolism. PDH function is inhibited by PDH kinases (PDHKs). PDHK1 was expressed in Th17 cells, but not Th1 cells, and at low levels in Tregs, and inhibition or knockdown of PDHK1 selectively suppressed Th17 cells and increased Tregs. This alteration in the CD4+ T cell populations was mediated in part through ROS, as N-acetyl cysteine (NAC) treatment restored Th17 cell generation. Moreover, inhibition of PDHK1 modulated immunity and protected animals against experimental autoimmune encephalomyelitis, decreasing Th17 cells and increasing Tregs. Together, these data show that CD4+ subsets utilize and require distinct metabolic programs that can be targeted to control specific T cell populations in autoimmune and inflammatory diseases.

The Liver Kinase B1 Is a Central Regulator of T Cell Development, Activation, and Metabolism

T cell activation leads to engagement of cellular metabolic pathways necessary to support cell proliferation and function. However, our understanding of the signal transduction pathways that regulate metabolism and their impact on T cell function remains limited. The liver kinase B1 (LKB1) is a serine/threonine kinase that links cellular metabolism with cell growth and proliferation. In this study, we demonstrate that LKB1 is a critical regulator of T cell development, viability, activation, and metabolism. T cell-specific ablation of the gene that encodes LKB1 resulted in blocked thymocyte development and a reduction in peripheral T cells. LKB1-deficient T cells exhibited defects in cell proliferation and viability and altered glycolytic and lipid metabolism. Interestingly, loss of LKB1 promoted increased T cell activation and inflammatory cytokine production by both CD4+ and CD8+ T cells. Activation of the AMP-activated protein kinase (AMPK) was decreased in LKB1-deficient T cells. AMPK was found to mediate a subset of LKB1 functions in T lymphocytes, as mice lacking the α1 subunit of AMPK displayed similar defects in T cell activation, metabolism, and inflammatory cytokine production, but normal T cell development and peripheral T cell homeostasis. LKB1- and AMPKα1-deficient T cells each displayed elevated mammalian target of rapamycin complex 1 signaling and IFN-γ production that could be reversed by rapamycin treatment. Our data highlight a central role for LKB1 in T cell activation, viability, and metabolism and suggest that LKB1–AMPK signaling negatively regulates T cell effector function through regulation of mammalian target of rapamycin activity.

Foxp3 and Toll-like receptor signaling balance Treg cell anabolic metabolism for suppression

CD4+ effector T cells (Teff cells) and regulatory T cells (Treg cells) undergo metabolic reprogramming to support proliferation and immunological function. Although signaling via the lipid kinase PI(3)K (phosphatidylinositol-3-OH kinase), the serine-threonine kinase Akt and the metabolic checkpoint kinase complex mTORC1 induces both expression of the glucose transporter Glut1 and aerobic glycolysis for Teff cell proliferation and inflammatory function, the mechanisms that regulate Treg cell metabolism and function remain unclear. We found that Toll-like receptor (TLR) signals that promote Treg cell proliferation increased PI(3)K-Akt-mTORC1 signaling, glycolysis and expression of Glut1. However, TLR-induced mTORC1 signaling also impaired Treg cell suppressive capacity. Conversely, the transcription factor Foxp3 opposed PI(3)K-Akt-mTORC1 signaling to diminish glycolysis and anabolic metabolism while increasing oxidative and catabolic metabolism. Notably, Glut1 expression was sufficient to increase the number of Treg cells, but it reduced their suppressive capacity and Foxp3 expression. Thus, inflammatory signals and Foxp3 balance mTORC1 signaling and glucose metabolism to control the proliferation and suppressive function of Treg cells.

Leptin Metabolically Licenses T Cells for Activation To Link Nutrition and Immunity

Immune responses are highly energy-dependent processes. Activated T cells increase glucose uptake and aerobic glycolysis to survive and function. Malnutrition and starvation limit nutrients and are associated with immune deficiency and increased susceptibility to infection. Although it is clear that immunity is suppressed in times of nutrient stress, mechanisms that link systemic nutrition to T cell function are poorly understood. We show in this study that fasting leads to persistent defects in T cell activation and metabolism, as T cells from fasted animals had low glucose uptake and decreased ability to produce inflammatory cytokines, even when stimulated in nutrient-rich media. To explore the mechanism of this long-lasting T cell metabolic defect, we examined leptin, an adipokine reduced in fasting that regulates systemic metabolism and promotes effector T cell function. We show that leptin is essential for activated T cells to upregulate glucose uptake and metabolism. This effect was cell intrinsic and specific to activated effector T cells, as naive T cells and regulatory T cells did not require leptin for metabolic regulation. Importantly, either leptin addition to cultured T cells from fasted animals or leptin injections to fasting animals was sufficient to rescue both T cell metabolic and functional defects. Leptin-mediated metabolic regulation was critical, as transgenic expression of the glucose transporter Glut1 rescued cytokine production of T cells from fasted mice. Together, these data demonstrate that induction of T cell metabolism upon activation is dependent on systemic nutritional status, and leptin links adipocytes to metabolically license activated T cells in states of nutritional sufficiency.

Role of T Cells in Malnutrition and Obesity

Nutritional status is critically important for immune cell function. While obesity is characterized by inflammation that promotes metabolic syndrome including cardiovascular disease and insulin resistance, malnutrition can result in immune cell defects and increased risk of mortality from infectious diseases. T cells play an important role in the immune adaptation to both obesity and malnutrition. T cells in obesity have been shown to have an early and critical role in inducing inflammation, accompanying the accumulation of inflammatory macrophages in obese adipose tissue, which are known to promote insulin resistance. How T cells are recruited to adipose tissue and activated in obesity is a topic of considerable interest. Conversely, T cell number is decreased in malnourished individuals, and T cells in the setting of malnutrition have decreased effector function and proliferative capacity. The adipokine leptin, which is secreted in proportion to adipocyte mass, may have a key role in mediating adipocyte-T cell interactions in both obesity and malnutrition, and has been shown to promote effector T cell function and metabolism while inhibiting regulatory T cell proliferation. Additionally, key molecular signals are involved in T cell metabolic adaptation during nutrient stress; among them, the metabolic regulator AMP kinase and the mammalian target of rapamycin have critical roles in regulating T cell number, function, and metabolism. In summary, understanding how T cell number and function are altered in obesity and malnutrition will lead to better understanding of and treatment for diseases where nutritional status determines clinical outcome.

Suppression of Glut1 and Glucose Metabolism by Decreased Akt/mTORC1 Signaling Drives T Cell Impairment in B Cell Leukemia

Leukemia can promote T cell dysfunction and exhaustion that contributes to increased susceptibility to infection and mortality. The treatment-independent mechanisms that mediate leukemia-associated T cell impairments are poorly understood, but metabolism tightly regulates T cell function and may contribute. In this study, we show that B cell leukemia causes T cells to become activated and hyporesponsive with increased PD-1 and TIM3 expression similar to exhausted T cells and that T cells from leukemic hosts become metabolically impaired. Metabolic defects included reduced Akt/mammalian target of rapamycin complex 1 (mTORC1) signaling, decreased expression of the glucose transporter Glut1 and hexokinase 2, and reduced glucose uptake. These metabolic changes correlated with increased regulatory T cell frequency and expression of PD-L1 and Gal-9 on both leukemic and stromal cells in the leukemic microenvironment. PD-1, however, was not sufficient to drive T cell impairment, as in vivo and in vitro anti–PD-1 blockade on its own only modestly improved T cell function. Importantly, impaired T cell metabolism directly contributed to dysfunction, as a rescue of T cell metabolism by genetically increasing Akt/mTORC1 signaling or expression of Glut1 partially restored T cell function. Enforced Akt/mTORC1 signaling also decreased expression of inhibitory receptors TIM3 and PD-1, as well as partially improved antileukemia immunity. Similar findings were obtained in T cells from patients with acute or chronic B cell leukemia, which were also metabolically exhausted and had defective Akt/mTORC1 signaling, reduced expression of Glut1 and hexokinase 2, and decreased glucose metabolism. Thus, B cell leukemia–induced inhibition of T cell Akt/mTORC1 signaling and glucose metabolism drives T cell dysfunction.