Ryan D. Michalek

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.

The Glucose Transporter Glut1 Is Selectively Essential for CD4 T Cell Activation and Effector Function

CD4 T cell activation leads to proliferation and differentiation into effector (Teff) or regulatory (Treg) cells that mediate or control immunity. While each subset prefers distinct glycolytic or oxidative metabolic programs in vitro, requirements and mechanisms that control T cell glucose uptake and metabolism in vivo are uncertain. Despite expression of multiple glucose transporters, Glut1 deficiency selectively impaired metabolism and function of thymocytes and Teff. Resting T cells were normal until activated, when Glut1 deficiency prevented increased glucose uptake and glycolysis, growth, proliferation, and decreased Teff survival and differentiation. Importantly, Glut1 deficiency decreased Teff expansion and the ability to induce inflammatory disease in vivo. Treg cells, in contrast, were enriched in vivo and appeared functionally unaffected and able to suppress Teff, irrespective of Glut1 expression. These data show a selective in vivo requirement for Glut1 in metabolic reprogramming of CD4 T cell activation and Teff expansion and survival.

Apoptosis Regulators Bim and Fas Function Concurrently to Control Autoimmunity and CD8+ T Cell Contraction

Throughout most of adult life, lymphocyte number remains constant because of a balance of proliferation and apoptosis. Mutation of Bim, a proapoptotic protein in the intrinsic death pathway, or Fas, a tumor necrosis factor receptor (TNFR) superfamily member of the extrinsic pathway, results in late-onset autoimmunity and increased antigen-specific CD8(+) T cell responses during viral infection. However, virus-specific immune responses eventually return to amounts comparable to those for nonmutant mice. Here, we show that loss of both Bim and Fas function resulted in a synergistic disruption of lymphoid homeostasis, rapid-onset autoimmunity, and organ-specific blocks on contraction of antiviral immune responses. When lymphocytic choriomeningitis virus (LCMV)-specific immune responses were quantitated, double-mutant mice had 100-fold more antigen-specific memory CD8(+) T cells in their lymph nodes than wild-type mice. Our results demonstrate that multiple death pathways function concurrently to prevent autoimmunity and downsize T cell responses.

Estrogen-related receptor-α is a metabolic regulator of effector T-cell activation and differentiation

Stimulation of resting CD4+ T lymphocytes leads to rapid proliferation and differentiation into effector (Teff) or inducible regulatory (Treg) subsets with specific functions to promote or suppress immunity. Importantly, Teff and Treg use distinct metabolic programs to support subset specification, survival, and function. Here, we describe that the orphan nuclear receptor estrogen-related receptor-α (ERRα) regulates metabolic pathways critical for Teff. Resting CD4+ T cells expressed low levels of ERRα protein that increased on activation. ERRα deficiency reduced activated T-cell numbers in vivo and cytokine production in vitro but did not seem to modulate immunity through inhibition of activating signals or viability. Rather, ERRα broadly affected metabolic gene expression and glucose metabolism essential for Teff. In particular, up-regulation of Glut1 protein, glucose uptake, and mitochondrial processes were suppressed in activated ERRα−/− T cells and T cells treated with two chemically independent ERRα inhibitors or by shRNAi. Acute ERRα inhibition also blocked T-cell growth and proliferation. This defect appeared as a result of inadequate glucose metabolism, because provision of lipids, but not increased glucose uptake or pyruvate, rescued ATP levels and cell division. Additionally, we have shown that Treg requires lipid oxidation, whereas Teff uses glucose metabolism, and lipid addition selectively restored Treg—but not Teff—generation after acute ERRα inhibition. Furthermore, in vivo inhibition of ERRα reduced T-cell proliferation and Teff generation in both immunization and experimental autoimmune encephalomyelitis models. Thus, ERRα is a selective transcriptional regulator of Teff metabolism that may provide a metabolic means to modulate immunity.

The metabolic life and times of a T‐cell

Summary:  The regulation of lymphocyte homeostasis is critical for the development and formation of productive immune responses. Cell numbers must be maintained to allow sufficient numbers of lymphocytes to combat foreign pathogens but prevent the accumulation of excess lymphocytes that may increase the risk of developing autoimmunity or neoplasia. Cell extrinsic growth factors are essential to maintain homeostasis and cell survival, and it has become increasingly apparent that a key mechanism of this control is through regulation of cell metabolism. The metabolic state of T cells can have profound influences on cell growth and survival and even differentiation. In particular, resting T cells utilize an energy efficient oxidative metabolism but shift to a highly glycolytic metabolism when stimulated to grow and proliferate by pathogen encounter. After antigen clearance, T cells must return to a more quiescent oxidative metabolism to support T‐cell memory. This review highlights how these metabolic changes may be intricately involved with both T‐cell growth and death in the control of homeostasis and immunity.

Akt Inhibition Enhances Expansion of Potent Tumor-Specific Lymphocytes with Memory Cell Characteristics

Adoptive cell therapy (ACT) using autologous tumor-infiltrating lymphocytes (TIL) results in complete regression of advanced cancer in some patients, but the efficacy of this potentially curative therapy may be limited by poor persistence of TIL after adoptive transfer. Pharmacologic inhibition of the serine/threonine kinase Akt has recently been shown to promote immunologic memory in virus-specific murine models, but whether this approach enhances features of memory (e.g., long-term persistence) in TIL that are characteristically exhausted and senescent is not established. Here, we show that pharmacologic inhibition of Akt enables expansion of TIL with the transcriptional, metabolic, and functional properties characteristic of memory T cells. Consequently, Akt inhibition results in enhanced persistence of TIL after adoptive transfer into an immunodeficient animal model and augments antitumor immunity of CD8 T cells in a mouse model of cell-based immunotherapy. Pharmacologic inhibition of Akt represents a novel immunometabolomic approach to enhance the persistence of antitumor T cells and improve the efficacy of cell-based immunotherapy for metastatic cancer.

The requirement of reversible cysteine sulfenic acid formation for T cell activation and function

Reactive oxygen intermediates (ROI) generated in response to receptor stimulation play an important role in mediating cellular responses. We have examined the importance of reversible cysteine sulfenic acid formation in naive CD8+ T cell activation and proliferation. We observed that, within minutes of T cell activation, naive CD8+ T cells increased ROI levels in a manner dependent upon Ag concentration. Increased ROI resulted in elevated levels of cysteine sulfenic acid in the total proteome. Analysis of specific proteins revealed that the protein tyrosine phosphatases SHP-1 and SHP-2, as well as actin, underwent increased sulfenic acid modification following stimulation. To examine the contribution of reversible cysteine sulfenic acid formation to T cell activation, increasing concentrations of 5,5-dimethyl-1,3-cyclohexanedione (dimedone), which covalently binds to cysteine sulfenic acid, were added to cultures. Subsequent experiments demonstrated that the reversible formation of cysteine sulfenic acid was critical for ERK1/2 phosphorylation, calcium flux, cell growth, and proliferation of naive CD8+ and CD4+ T cells. We also found that TNF-α production by effector and memory CD8+ T cells was more sensitive to the inhibition of reversible cysteine sulfenic acid formation than IFN-γ. Together, these results demonstrate that reversible cysteine sulfenic acid formation is an important regulatory mechanism by which CD8+ T cells are able to modulate signaling, proliferation, and function.

IL-7 Is Essential for Homeostatic Control of T Cell Metabolism In Vivo

It has become apparent that T cells require growth signals to maintain function and viability necessary to maintain proper immune homeostasis. One means by which cell extrinsic signals may mediate these effects is by sustaining sufficient basal cell metabolism to prevent cell atrophy. The role of metabolism and the specific growth factors essential to maintain metabolism of mature T cells in vivo, however, are poorly defined. As IL-7 is a nonredundant cytokine required for T cell development and survival and can regulate T cell metabolism in vitro, we hypothesized it may be essential to sustain metabolism of resting T cells in vivo. Thus, we generated a model for conditional expression of IL-7R in mature T cells. After IL-7R deletion in a generally normal lymphoid environment, T cells had reduced responses to IL-7, including abrogated signaling and maintenance of antiapoptotic Bcl-2 family expression that corresponded to decreased survival in vitro. T cell survival in vivo was also reduced after loss of the IL-7R in a T cell-intrinsic manner. Additionally, IL-7R deletion resulted in delayed growth and proliferation following stimulation. Importantly, in vivo excision of IL-7R led to T cell atrophy that was characterized by delayed mitogenesis and reduced glycolytic flux. These data are the first to identify an in vivo requirement for a specific cell extrinsic signal to sustain lymphocyte metabolism and suggest that control of glycolysis by IL-7R may contribute to the well-described roles of IL-7 in T cell development, homeostatic proliferation, and survival.

Autophagy is essential to suppress cell stress and to allow BCR-Abl-mediated leukemogenesis.

Hematopoietic cells normally require cell extrinsic signals to maintain metabolism and survival. In contrast, cancer cells can express constitutively active oncogenic kinases such as BCR-Abl that promote these processes independent of extrinsic growth factors. When cells receive insufficient growth signals or when oncogenic kinases are inhibited, glucose metabolism decreases and the self-digestive process of autophagy is elevated to degrade bulk cytoplasm and organelles. Although autophagy has been proposed to provide a cell-intrinsic nutrient supply for mitochondrial oxidative metabolism and to maintain cellular homeostasis through degradation of damaged organelles or protein aggregates, its acute role in growth factor deprivation or inhibition of oncogenic kinases remains poorly understood. We therefore developed a growth factor-dependent hematopoietic cell culture model in which autophagy can be acutely disrupted through conditional Cre-mediated excision of the autophagy-essential gene Atg3. Treated cells rapidly lost their ability to perform autophagy and underwent cell cycle arrest and apoptosis. Although Atg3 was essential for optimal upregulation of mitochondrial oxidative pathways in growth factor withdrawal, this metabolic contribution of autophagy did not appear critical for cell survival, as provision of exogenous pyruvate or lipids could not completely rescue Atg3 deficiency. Instead, autophagy suppressed a stress response that otherwise led to p53 phosphorylation and upregulation of p21 and the pro-apoptotic Bcl-2 family protein Puma. Importantly, BCR-Abl-expressing cells had low basal levels of autophagy, but were highly dependent on this process, and rapidly underwent apoptosis upon disruption of autophagy through Atg3 deletion or treatment with chemical autophagy inhibitors. This dependence on autophagy extended in vivo, as Atg3 deletion also prevented BCR-Abl-mediated leukemogenesis in a cell transfer model. Together these data demonstrate a critical role for autophagy to mitigate cell stress, and that cells expressing the oncogenic kinase BCR-Abl appear particularly dependent on autophagy for cell survival and leukemogenesis.