Jeffrey Rathmell - US grants

DESCRIPTION (provided by applicant): Social control of cell survival and homeostasis by extrinsic signals is critical in maintaining cell survival and preventing cancer. It remains uncertain, however, why ATP production and substrate uptake fail to match intracellular demand in the absence of extrinsic signals. To determine how extrinsic signals from multiple receptor/ligand families, such as TCR/MHC, gp130/IL-6, and gamma[c]/IL-7, and growth-promoting oncogenic signaling pathways, such as Akt and c-Myc, control cellular trophic state and test the hypothesis that extrinsic signals promote survival by stimulating nutrient uptake and utilization, I propose the following aims: 1. Determine the role of extrinsic signals in T cell m e t a b o l ism, trophic state and apoptosis. A. Identify metabolic characteristics of primary T cells immediately upon isolation and after 24 hours neglect or treatment with anti-TCR, IL-6, or IL-7 as representative ligands for three distinct receptor families by measuring glycolysis, oxygen usage, glucose uptake and metabolic intermediates. B. Modulate specific metabolic pathways to determine their importance in survival and trophic state of neglected or cytokine-treated primary T cells. 2. Examine Akt and c-Myc regulation of T cell survival and trophic state. A. Measure the activation of Akt and c-Myc by TCR, IL-6R or IL-7R signaling. B. Express Akt and c-Myc and determine metabolic characteristics and trophic state in primary T cells through the generation of transgenic mice. C. Express Glut1 to determine if upregulation of Glut1 RNA is sufficient to support T cell metabolism and trophic state in the absence of extrinsic signals.

[unreadable] DESCRIPTION (provided by applicant): Maintenance of lymphoid homeostasis is critical to avert diseases such as autoimmunity, lymphoma, and immunodeficiency. We propose to study a novel mechanism of T cell homeostasis - control of nutrient usage, or trophic level, by regulation of glucose uptake and the glucose transporter, Glut1. Lymphocytes require dynamic regulation of glucose uptake to allow both cell survival in a resting state and robust growth and proliferation in immune responses. We have shown that glucose uptake and metabolism are regulated events that can have a profound impact on T cell survival and function. We show that glucose uptake in both resting and activated T cells is tightly regulated in vitro by the cytokines interleukin 7 (IL7) and interleukin 2 (IL2), respectively. The in vivo signals and signaling mechanisms required for regulation of Glut1 and the effects of altered glucose uptake on T cell size, survival, and function, however, remain uncertain. In this application, we propose to address these issues to bridge the fields of immunology and cellular metabolism. We will: (1) Determine the in vivo role of IL7 in regulation of Glut1 expression and intracellular trafficking by adoptive transfer of T cells into IL7-/- hosts and conditional deletion of IL7R in mature T cells. (2) Identify signaling mechanisms that regulate Glut1 expression and cell surface localization. The PI3K/Akt and Jak/STAT signaling pathways are activated by IL7 and IL2 and these pathways will be analyzed for their role in Glut1 regulation in cell line and primary T cell models using RNAi, transgenic, and knockout approaches. (3) Examine the role of Glut1 expression in T cell development and homeostasis using a T cell specific Glut1 transgenic mouse model and in vitro RNAi of Glut1. The ability of Glut1 expression to modulate lymphoma and autoimmunity observed in Akt transgenic mice will be investigated to determine how altered glucose uptake may affect disease. Together, these experiments will identify signals and signaling mechanisms that regulate T cell glucose uptake and determine the role of glucose uptake in T cell survival, activation, and diseases of the immune system. [unreadable] [unreadable] [unreadable]

A key barrier that leukemic cells overcome in cancer progression is dependence on cytokine growth factors for survival. We have shown that prior to commitment to cell death, growth factor-deprivation of normal lymphoid cells results in cellular atrophy with decreased glucose metabolism, activation of autophagy, and proteolytic degradation of the anti-apoptotic Bcl-2 family member, Mcl1. In contrast, leukemic cells or cells with activated forms of the oncogenic kinase, Akt/PKB, resist atrophy and cell death, are highly glycolytic, and maintain Mcl1 even in the absence of growth factors. The role of this increased glucose metabolism is unknown. We show that increased glucose metabolism characteristic of cancer activates an anti-apoptotic nutrient signaling pathway. This glucose-stimulated signaling pathway involves inhibitory phosphorylation of glycogen synthase kinase-3ff//? (GSK3) by protein kinase C (PKC), which prevents degradation of Mcl1. McM stabilization appears critical as enhanced glucose metabolism failed to provide a survival advantage in Mel 1-deficient cells. The means by which glucose hydrolysis promotes PKC activity and regulates of McM remain uncertain. Glucose metabolism is also required for oncogenic Akt to prevent cell death in the absence of growth factor and the pentose phosphate pathway (PPP), in particular, may be important. In contrast, we show that Bcl-xL supports growth factor-independent survival in the absence of glucose and instead must rely on autophagy to both maintain mitochondrial metabolites and attenuate cell death. We hypothesize that the increased glucose utilization of cancer cells initiates cell metabolism and survival pathways that impact both mitochondrial and alternative cell death pathways and may play important roles in cancer cell resistance to death. We propose to: (1) Identify the mechanism of anti-apoptotic glucose-mediated signal transduction to activate PKC and stabilize McM;(2) Examine the role of glucose metabolism in cells expressing oncogenic Akt to determine the role that the PPP or alternative metabolic pathways play in regulation of Mcl1 and cell death;and (3) Establish the role of increased glucose metabolism on autophagy as a source of cell metabolism and survival in cytokine withdrawal. These studies will identify mechanisms by which cell metabolism may regulate cell death and how the highly glycolytic nature of cancer cells may affect these pathways to better understand cancer cell survival mechanisms.

DESCRIPTION (provided by applicant): Asthma is a chronic inflammatory disease of the lung that results in airway remodeling and acute pulmonary allergic responses that can be debilitating or fatal. Many individuals with severe asthma do not respond or are poorly responsive to current therapies and alternative methods to treat or manage this disease are essential. One approach that may allow specific control over immunity in asthma is to influence differentiation pathways of mature CD4 T cells into effector (Teff) or inducible regulatory (Treg) subsets. Asthma has been associated with an imbalance of these subsets marked by increased frequency of Teff and decreased Treg cells, yet mechanisms that control this balance are poorly understood. We propose that modulation of T cell metabolism may provide a new approach to manipulate CD4 T cell differentiation and treat asthma. We have shown that T cell stimulation promotes a switch from an oxidative to a predominantly glycolytic metabolism in which glucose-derived pyruvate is converted to lactate or used to support biosynthesis rather than mitochondrial oxidation. Cell metabolism must, however, be tuned to specific cellular demands and consistent with the distinct roles and activities of Teff and Treg CD4 T cells, we found that differentiated CD4 T cell subsets had distinctly different metabolic patterns and requirements. Specifically, Teff cells were highly glycolytic and required glucose for metabolism and survival while Treg cells remained oxidative and required lipids for mitochondrial oxidation. Further, we found that the nuclear hormone receptor Estrogen Related Receptor-alpha (ERRa) was activated following T cell stimulation and essential to promote increased glucose metabolism of Teff. Importantly, inhibition of ERRa selectively suppressed Teff generation whereas treatment of mice with activators of AMP-protein kinase (AMPK) to directly promote oxidative metabolism increased Treg generation in vivo. Together these findings have led to the hypothesis that T cell metabolism is a critical factor in T cell differentiation into effector or regulatory populations with glycolytic metabolism favoring inflammatory effector and oxidative metabolism favoring regulatory T cells and that manipulation of T cell metabolism through ERRa and AMPK may allow for selective Treg generation to suppress allergic asthma. To test this hypothesis we propose to: (1) Determine how metabolism influences CD4 T cell differentiation into Treg or Teff populations;(2) Examine the role of ERRa in regulation of T cell metabolism and differentiation;and (3) Establish how modulation of T cell metabolism in vivo impacts a model of allergic asthma. Together these aims will define the regulation and role of Teff and Treg metabolic programs and point towards metabolic regulatory mechanisms that can be targeted in novel therapies to treat or control asthma. PUBLIC HEALTH RELEVANCE: Asthma is a chronic inflammatory disease of the lung that results in airway remodeling and acute pulmonary allergic responses that can be debilitating or fatal and many individuals with severe asthma respond poorly to current therapies. Modulation of CD4 T cell differentiation into inflammatory effector or suppressive regulatory subtypes may prevent or treat asthma and we have found that control of cell metabolism can have a strong influence on these CD4 T cell fates. This study will examine the role and regulation of metabolism in T cell differentiation and asthma to explore new directions in the treatment of allergic asthma.

DESCRIPTION (provided by applicant): Lymphocyte activation must be controlled to allow proper immunity while preventing inappropriate inflammatory immune responses. One element that we have found critical to support T cell growth, proliferation, and effector function is glucoe metabolism. In particular, the glucose transporter Glut1 and glycolysis are upregulated upon activation and differentiation of CD4 T cells into effectors (Teff; Th1, Th2, and Th17 are considered here). Regulatory T cells (Treg), however, express lower levels of Glut1 and utilize lipid oxidation rather than glycolysis as a primary metabolic program. Importantly, cell metabolism must match the demands of each cell type and we have shown that the inhibition of glucose metabolism prevents specification and function of Teff, while Treg are preferentially generated if glucose is limiting or glycolysis is inhibited. Manipulation of CD4 T cell metabolism, therefore, may provide a new approach to modulate immunity and reduce Teff function in inflammatory and autoimmune diseases. It is unclear, however, how T cell metabolism is regulated and what impact disruption of glucose metabolism may have in vivo, where a wide variety of alternate nutrients are available to potentially replace glucose. To address this question we have studied the role and regulation of Glut1 using a unique set of animal models. Our preliminary data show that Glut1 overexpression leads to selective lymphoproliferation of Teff while conditional deletion of Glut1 in T cells reduces peripheral T cell numbers and effector function. Regulatory mechanisms that control Glut1 may therefore provide potential targets for immune suppression. Indeed, we have recently shown that the orphan nuclear hormone receptor Estrogen Related Receptor-? (ERR?) plays a key role in the glucose metabolism and function of Teff and may mediate the effects of the Aryl- hydrocarbon Receptor (AhR) on CD4 specification into Teff or Treg via regulation of Glut1. We hypothesize that glucose uptake and metabolism are central regulators of effector T cell generation and function and that Glut1 regulation through AhR and ERR? may provide a novel avenue for therapy of immune diseases. We propose to: (1) Determine the role of Glut1 in T cell metabolism, survival, and effector function; (2) Establish regulatory pathways that control Glut1 expression and cell surface trafficking in murine and human T cell activation and in CD4 subsets; and (3) Examine the regulation and role of Glut1 in Teff generation and function in experimental autoimmune encephalomyelitis and graft-vs.-host disease. These studies will apply our unique set of animal models in normal activation and in two inflammatory diseases to directly establish the role of Glut1 and glucose metabolism in immune function and regulation of Glut1 and glucose metabolism as potential modulators of immunological disease.

DESCRIPTION (provided by applicant): Immunological diseases such as Systemic Lupus Erythematosus (SLE) are driven by disruption of regulatory mechanisms in both T and B lymphocytes leading to autoantibody production and inflammation. As autoantibodies promote a variety of SLE-associated pathologies, including immune complex formation and deposition responsible for vasculitis and glomerulonephritis, many therapeutic approaches to treat SLE have targeted B cell survival or function. However, effectiveness can be limited and secondary toxicities can be severe. A new approach may be to interfere with the basic metabolic processes necessary for lymphocyte growth and function. We have shown that cellular metabolism is highly regulated in lymphocyte activation and that while resting T cells rely on an oxidative metabolism, activation leads to a metabolic reprogramming to greatly increase glycolysis. Inhibition of glycolysis in T cells can prevent their effector function. It is likely that B cells lso undergo metabolic reprogramming that is essential for activation and effector function that may allow targeting of B cell metabolism to suppress autoantibody production. To test this new direction for treatment of rheumatic diseases, it is essential to understand B cell metabolism and how changes in metabolism impact B cell tolerance and autoimmunity. In our preliminary studies, we show that B cells do undergo a metabolic reprogramming upon activation to increase glycolysis and lactate production and inhibition of this metabolic transition prevents antibody production. Importantly, B cells from SLE-prone BAFF-transgenic mice were highly glycolytic immediately upon isolation, suggesting that this metabolic transition occurs in vivo coincident with autoantibody production and disease. A key regulator of glucose metabolism is Pyruvate Dehydrogenase Kinase 1 (PDHK1), which provides an inhibitory phosphorylation of Pyruvate Dehydrogenase (PDH) and directs pyruvate conversion into lactate to elevate glycolysis. We show that inhibition of PDHK1 can reduce B cell glycolysis and antibody production. B cell metabolism has not the focus of previous immunological studies and we propose to evaluate the potential of targeting this process to suppress autoantibody production and disease in SLE. We hypothesize that high rates of glycolysis are essential for B cell autoantibody production and that PDHK1 will provide a new metabolic target to suppress B cell proliferation and autoreactivity to treat inflammatory and autoantibody-mediated diseases, such as SLE. To test this hypothesis we will: (1) Establish if metabolic reprogramming of B cells to become highly glycolytic is required for antibody production; and (2) Test if modulation of glucose metabolism by PDHK1 inhibition impacts B cell autoreactivity in vitro and in vivo. Together, these studies are the first to directly focus on mechanisms of B cell metabolism and also to test the potential of pharmacological manipulation of PDHK1 to target metabolism and suppress B cell autoreactivity and autoantibody production in SLE.

? DESCRIPTION (provided by applicant): Inflammatory diseases are often caused by inappropriate responses of effector CD4 T cells (Teff). Th1 and Th17 Teff are recognized to drive a variety of immune pathologies, including Inflammatory Bowel Disease (IBD) and Multiple Sclerosis (MS). Regulatory T cells (Treg), in contrast, suppress Teff to protect from disease. A key therapeutic objective in efforts to shift the immunologic balance towards tolerance, therefore, is to selectively inhibit Teff and promote Treg. We show here that Teff and Treg utilize fundamentally different metabolic programs and propose that identifying specific requirements of each subset will provide a new approach to selectively modulate CD4 T cells in inflammatory disease. We have found Th1 and Th17 cells have high expression of the glucose transporter Glut1, and Th17 cells in particular have increased Pyruvate Dehydrogenase Kinase 1 (PDHK1), an enrichment of glycolytic intermediates up to Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH), and elevated rates of glycolytic flux to lactate. Treg, in contrast, have low expression of Glut1 and PDHK1, limited glycolytic flux, and are instead enriched for mitochondrial oxidative gene expression and metabolites. Importantly, our analysis of T cell specific Glut1 conditional deletion or targeting of PDHK1 showed that a glycolytic program is selectively required for Teff function in vivo. Here we propose to test additional metabolic events that were identified by high- resolution metabolomics mass spectrometry as selectively regulated in Teff and that may provide vulnerabilities for Th1 and Th17. The regulation and requirements of Treg metabolism, in contrast, have been poorly understood. However, our data show that the transcription factor FoxP3 promotes Treg oxidative metabolism and suppresses the Phosphoinositide-3-kinase (PI3K)/Akt/mTOR pathway to lower Glut1 expression and glycolysis. Surprisingly, high rates of glycolysis reduced Treg suppressive capacity, as we found Glut1 transgenic Treg are functionally impaired and could not fully protect from IBD. Based on the distinct metabolic requirements of Teff and Treg, we hypothesize that key glucose-dependent metabolites are selectively essential for Teff while glycolysis is a Treg vulnerability that FoxP3 restrains to optimize suppressive capacity. To test this model we will: (1) Identify and characterize metabolites and metabolic pathways selectively required for Teff specification and function; (2) Determine how FoxP3 regulates metabolism and the role of glycolysis in Treg expansion and protection from IBD; (3) Establish how inhibition of glycolysis alters the Teff and Treg balance in a model of MS using selective PDHK1 inhibitors and targeting of Teff metabolic vulnerabilities. These studies will establish specific and selective metabolic demands of Teff and Treg physiology and identify pathways to modulate the Teff and Treg balance in inflammatory diseases.

? DESCRIPTION (provided by applicant): Altered metabolism with increased glycolysis in a metabolic program termed aerobic glycolysis has been established as a fundamental mechanism to support biosynthesis of cancer cells. It is now apparent, however, that normal cells can also use this pathway to support growth and proliferation. We have shown that normal T cells induce aerobic glycolysis upon activation as they transition from quiescence to become highly proliferative but short-lived effectors. Comparing metabolic and signaling events in T cell Acute Lymphoblastic Leukemia (T-ALL) with normal T cell activation led to the surprising finding that while cancer cells are considered highly glycolytic and T-ALL cells use aerobic glycolysis; they do so at a much lower rate than normal stimulated T cells. Thus, T-ALL cells utilize aerobic glycolysis at a sufficient rate to support proliferation, but do not achieve the rates of short-livd effector T cells. We propose that T-ALL cells balance the biosynthetic benefits of aerobic glycolysis against the stress of overly deregulated metabolism that leads to apoptosis of activated T cells. The Phosphotidylinositide 3-Kinase (PI3K)/Akt/mTOR and 5'AMP Protein Kinase (AMPK) signaling pathways may control this metabolic balance, as the PI3K/Akt/mTOR pathway promotes aerobic glycolysis and is oncogenic while AMPK suppresses this pathway and can suppress tumorigenesis. Paradoxically, however, constitutive activation of PI3K was recently associated with reduced T cell numbers and immunodeficiency. Further, we show here that constitutively high Akt activity increases ROS and cell death in leukemia. Unexpectedly, we found AMPK to be activated in T-ALL cells and this may restrain excessive PI3K/Akt/mTOR and aerobic glycolysis. Thus while slowing cell growth, AMPK may also promote T-ALL cell survival. Inactivation of AMPK, however, may release PI3K/Akt/mTOR and excessive glycolytic metabolism to prevent control of ROS and lead to apoptosis. Our long-term objectives are to better understand metabolic vulnerabilities of T-ALL and identify mechanisms that may allow metabolic reprogramming of T-ALL cells to enter short-lived states similar to activated T cells. To achieve these goals we will test the hypothesis that elevated glycolysis via Akt promotes ROS and a short-lifespan for leukemic cells, but is held in check by AMPK. We propose to (1) Test the effects of direct modulation of glucose metabolism on the leukemic progression and cell lifespan of primary T-ALL and (2) Determine how AMPK regulates T- ALL progression and metabolism to test if AMPK provides a metabolic brake that prevents T-ALL apoptosis. Together these studies based on comparison of normal and leukemic T cell metabolism will provide new understanding of T-ALL metabolism and test a new model of T-ALL cell fate that may be applicable to a wide range of glycolytic cancers.

SUMMARY Inflammatory diseases are often characterized by imbalanced ratios or functions of T effector (Teff), such as elevated Th2 and Th17 that promote asthma, and regulatory (Treg) cells, that suppress inflammation. While many such diseases can be controlled with corticosteroids, some, including severe asthma and multiple sclerosis, can show steroid (glucocorticoid; GC)-resistance and lead to increasingly negative outcomes, including death. Understanding factors that promote steroid- resistance, therefore, is critical to establish new approaches to treat GC-resistant inflammatory diseases. It is now clear that while Th2 cells are GC sensitive, steroid-resistant asthma is often characterized by the presence of IL17 producing cells, including Th17 CD4 T cells that can have intrinsic GC resistance. GC are well known to inhibit metabolism and we propose that the unique metabolism of Th17 cells contributes to their selective GC resistance. We have shown that T cell metabolism is dynamic and the Phosphatidylinositide 3-Kinase (PI3K)/Akt/mTOR Complex 1 (mTORC1) pathway induces Th17 CD4 T cells to increase glycolysis and metabolism of glutamine to glutamate for mitochondrial oxidation (glutaminolysis). Consistent with this metabolic program, we show allergen challenge increases both lactate and glutamate in bronchalveolar lavage (BAL) fluid of asthmatics and that T cells from BAL of asthma patients express the glucose transporter Glut1 at high levels. Importantly, GC resistant Th17 cells have both higher levels of glucose metabolism and glutaminolysis than GC sensitive Th2 cells. Th17 cells also have higher mitochondrial respiratory capacity and ability to withstand mitochondrial inhibition than Th2 cells. In contrast, Treg use lipid oxidation and have high levels of active AMPK, which inhibits mTORC1. Further, we show Treg are functionally impaired by high rates of glycolysis. We also show that Th17 cells rely on Glutaminase (GLS) to support glutaminolysis and differentiation, and inhibition of GLS selectively impairs Th17 cells. Elevated levels of glucose and glutamine metabolism may directly protect Th17 cells from GC, as T cells with increased Glut1 were resistant to GC-induced reactive oxygen species and cell death. Here we will test the hypothesis that allergen-induced cytokines signal T cells to induce a flexible metabolism of glycolysis and glutaminolysis that promotes GC resistance in Th17 cells and impairs Treg. To test this hypothesis we will: (1) Determine T cell metabolic programs and regulatory signals in airway inflammation and (2) Test the contribution of T cell metabolism to GC resistance of Th17 cells. Together, these studies will address a poorly understood but potentially key contributor to the pathogenesis of GC-resistant inflammatory diseases, such as severe asthma, by establishing and targeting the unique metabolic program and requirements of Th17 cells.

SUMMARY Exploiting the immune system to eliminate cancer cells has been a goal for many years, but it has become apparent that tumors actively suppress immune cell functions. While inhibition of immunomodulatory receptors, such as through PD-1 checkpoint blockade therapy, holds tremendous promise, this treatment is effective in only a portion of patients. Factors that determine immune responsiveness against tumors remain largely uncertain. Our data show, however, that the metabolic demands of T cells may be a critical factor in the success of immunotherapy. We have shown that effector T cell (Teff) activation requires high rates of glucose and anabolic metabolism yet cancer cells and the tumor microenvironment can inhibit Teff metabolic pathways. This may represent a fundamental mechanism of tumor-mediated immune suppression. To better understand the influence of the tumor microenvironment on T cell metabolism and improve immunotherapies, we have examined tumor infiltrating lymphocytes (TIL) from surgically-excised human clear cell Renal Cell Carcinoma (ccRCC) tumor samples, a cancer responsive to PD-1 blockade and with a prognostic immune signature. ccRCC is highly associated with mutations and loss of the Von Hippel Lindau (VHL) tumor suppressor, which leads to stabilization of HIF1? and HIF2? and induction of a transcriptional pseudo-hypoxic response that alters the tumor to promote an immune suppressive microenvironment that can negatively impact ccRCC CD8 TIL function and anti-tumor immunity. We found CD8 TIL are abundant in ccRCC, yet these cells are uniformly PD-1high and functionally suppressed. In addition, CD8 TIL had multiple metabolic impairments and were unable to efficiently uptake glucose or perform glycolysis and had small, fragmented mitochondria that produced high levels of Reactive Oxygen Species (ROS). Importantly, neutralization of ROS or provision of the glycolytic end-product pyruvate could partially rescue ccRCC CD8 TIL function. Glutamine is also a key nutrient to support mitochondrial metabolism for T cells through glutaminolysis and we report here that inhibition or genetic deletion of the first enzyme in this pathway, Glutaminase 1 (GLS1), leads to a compensatory increase in glycolysis that can enhance cytotoxic CD8 function. This proposal will test the hypothesis that the ccRCC microenvironment impairs glycolysis and leads to accumulation of dysfunctional mitochondria in CD8 TIL and that rescue of TIL glycolysis will enhance T cell response to immunotherapy. We will study primary ccRCC tumors and mouse RCC models to: (1) Determine how mitochondria are dysregulated and impair activation and metabolism of ccRCC CD8 TIL; (2) Investigate if promoting glucose uptake or inhibiting GLS1 to enhance glucose metabolism can improve the metabolism and function of CD8 TIL; and (3) Test how PD-1 blockade therapy impacts T cell metabolism and functional populations in ccRCC. Together, these studies will establish the mechanism of metabolic dysfunction in ccRCC TIL and test if approaches to enhance T cell glycolysis can improve cancer immunotherapy.

SUMMARY The Vanderbilt University School of Medicine Immunological Mechanisms of Disease Training Program (IMDTP) will fill a much-needed gap in the training of basic immunologic mechanisms of human disease. The clinical translation of basic science immunology to benefit human health now requires the development of scientists with appreciation of both inflammation and the tissues in which it occurs. The primary focus of the IMDTP will be to provide pre- and postdoctoral trainees with the expertise necessary to make novel discoveries in basic immunology and to translate these discoveries to human disease. The IMDTP will provide trainees a skill set to address how the immune system is regulated and functions in complex tissues in human disease by integrating basic immunology and principles of pathology with a particular emphasis on chronic, inflammatory diseases, and cancer. A long-term goal of the IMDTP will be to not only train the next generation of research scientists in basic immunology, but to emphasize the importance of using acquired knowledge as a translational platform on which to develop new therapies and interventions with a high regard for responsible conduct of research and reproducibility. We plan to use an interdisciplinary training approach building upon the Vanderbilt University program in Molecular Pathology and Immunology. The training faculty has been specifically recruited from the greater Vanderbilt scientific community conducting immunology-based research of chronic inflammatory diseases and consists of twenty-six highly productive faculty with successful mentoring records. In order to also allow for the training of future mentors, three mentors are at the Assistant Professor level. All of the faculty mentors have successful histories training predoctoral and postdoctoral scientists and have primary interests in immune- and inflammatory-based mechanisms of disease. These research programs will provide highly developed, diverse, and interdisciplinary training opportunities. All trainees will acquire and expand their skills in critical thinking, oral and written communication and interrogation of the literature. These primary functions of the IMDTP will be facilitated through our bi-weekly immunology and pathology seminar series and annual research symposium. Training will include participation in courses and modules on both basic immunology and pathology of disease processes. In addition, we will provide each trainee with the necessary instruction on responsible conduct in science, rigor and reproducibility in research and information on alternative career options. Funds are requested to support three predoctoral and two postdoctoral trainees. Thoughtful mentoring of these trainees, made possible by the IMDTP, will ensure the future of translational research focused on immunologic mechanisms of disease.

PROJECT SUMMARY Systemic lupus erythematosus (SLE) is characterized by autoantibody production and immune complex deposition and affects five to seven million individuals worldwide. Atherosclerosis and cardiovascular disease are common causes of early mortality in SLE, but immune-mediated mechanisms leading to this and other disease sequelae are not well understood. Therefore, demand is high to identify targeted strategies to overcome the undesirable side-effects of overt immunosuppression. In this application, we propose that the cellular metabolism of follicular helper T cells (Tfh), critical in promoting autoreactive B cell responses, may provide novel SLE therapeutic targets. Conversely, regulatory T cells (Treg) may protect. Our group has demonstrated that activated T cells increase glucose and glutamine consumption as they proliferate and differentiate into specific functional subsets. Importantly, differentiation and biosynthesis following activation requires a distinct metabolic program. To date, Tfh metabolism remains poorly understood, but our data suggest that both glucose and glutamine are essential and that Tfh appear to have high rates of glutaminolysis and are limited by rates of glucose uptake. It is now clear that these metabolic pathways regulate chromatin accessibility and gene expression by providing substrates for epigenetic modifications. Our data suggest that Glutaminase (GLS) and ATP-Citrate Lyase (ACLY), which regulate glutamine-dependent production of ?- ketoglutarate (?KG) and conversion of glucose-derived citrate to acetyl-CoA, respectively, regulate epigenetic marks, gene expression and differentiation essential for Tfh function. These observations build on our previous work demonstrating that GLS-inhibition led to reduced ?KG and differential alterations to histone methylation and chromatin accessibility in CD4 Th1 and Th17 cells. Importantly, both GLS and ACLY-deficient T cells failed to generate or maintain Tfh in an in vivo model of chronic inflammation. We have also used a model for SLE- accelerated atherosclerosis and shown that T cells in atherosclerosis have increased rates of metabolism. Further, Treg had reduced function and Tfh frequencies were increased. The current proposal will test the hypothesis that Tfh cells require glutamine and citrate metabolism to regulate epigenetic marks and chromatin accessibility to allow gene expression for germinal centers and autoantibody production in SLE and that targeting GLS or ACLY will disrupt epigenetic regulation of Tfh differentiation to treat disease. We will: (1) Establish the role of GLS and ACLY in differentiation, epigenetic regulation and gene expression, and metabolism of Tfh cells, and (2) Test inhibition of GLS or ACLY to decrease autoantibody production in murine SLE and impair circulating Tfh from SLE patients, and (3) determine the effect of manipulating Tfh metabolism on SLE-accelerated atherosclerosis. Our proposal to test the metabolic regulators of epigenetic marks and Tfh differentiation will leverage two targets that are currently under investigation as anti-cancer metabolism therapeutics and will determine if repurposing these drugs may offer new opportunities in SLE.

SUMMARY Asthma is a debilitating disease of airway inflammation with increasing worldwide occurrence. With higher incidence in females, asthma is exacerbated by obesity and metabolic disease. Underlying asthma is activation of innate immune and T cells that drive inflammation. Airway inflammation in severe asthma is caused in part by an imbalance between effector CD4 T cells (including Th2 and Th17) and suppressive regulatory (Treg) T cells. This imbalance is particularly heightened in obese females and leads to increased eosinophils and/or neutrophils in the airway, increased airway hyperresponsiveness, and increased mucus production. It is now clear that T cell metabolism plays a critical role to regulate immune function and the distinct metabolic programs of each T cell subset can influence inflammation and play roles in asthma. In addition to a requirement of T cells for glycolysis, our data show that glutamine metabolism plays a key role to support inflammatory T cells in asthma. Metabolomics data from bronchoalveolar fluid and mass cytometry of T cells from donors with severe asthma showed markers of elevated glutaminolysis. While IL17 producing cells both in vitro and from lungs had the highest expression of metabolic markers, both Th17 and Th2 cells relied on glutamine for differentiation and cytokine production. Glutamine metabolism is regulated through uptake by the transporter ASCT2 and Glutaminase (GLS)-dependent conversion to glutamate and ?-ketoglutarate that supports mitochondrial electron transport and regulates epigenetic marks that control chromatin and gene expression. We have now shown through inhibition, gene targeting, and in vivo primary T cell CRISPR screening in lung inflammation that ASCT2 and GLS are essential for Th17 and Th2 cells. These genes are dispensable for Treg and blocking glutamine uptake can instead enhance Treg differentiation. Importantly, we also show in an animal model that high fat diet fed female mice have selectively increased frequencies of Th17 cells. This effect is sex-specific, as high fat diet fed female, but not male, mice developed increased IL17 production and we show that the Estrogen Receptor (ER?) plays a key role in females to elevate CD4 T cell glycolysis and mitochondrial metabolism. We hypothesize that airway allergen exposure and inflammatory cytokines induce T cell glutamine metabolism, driving Th2 and Th17 cells that contribute to asthma incidence and severity in obese females. To test this hypothesis, we will: (1) Determine the role and mechanism of GLS and ASCT2-dependent glutamine metabolism to regulate the balance of Th2, Th17 and Treg cells in a mixed model of allergic airway inflammation and (2) Test if obesity- induced exacerbation of asthma severity in females is dependent on enhanced T cell glutamine metabolism in animal models and from peripheral blood and excised lungs of healthy and severe asthma normal weight or obese donors. These studies will establish metabolic mechanisms and if inhibiting glutamine uptake or metabolism through ASCT2 or GLS can shift the balance of Th17 and Th2 cells to instead favor Treg and protect against asthma to help alleviate this health disparity observed in females with metabolic disease.