SUMMARY Activated T cells differentiate into functional subsets with distinct metabolic programs. Glutaminase (GLS) converts glutamine to glutamate to support the tricarboxylic acid cycle and redox and epigenetic reactions. Here, we identify a key role for GLS in T cell activation and specification. Though GLS deficiency diminished initial T cell activation and proliferation and impaired differentiation of Th17 cells, loss of GLS also increased Tbet to promote differentiation and effector function of CD4 Th1 and CD8 CTL cells. This was associated with altered chromatin accessibility and gene expression, including decreased PIK3IP1 in Th1 cells that sensitized to IL-2-mediated mTORC1 signaling. In vivo, GLS null T cells failed to drive Th17-inflammatory diseases, and Th1 cells had initially elevated function but exhausted over time. Transient GLS inhibition, however, led to increased Th1 and CTL T cell numbers. Glutamine metabolism thus has distinct roles to promote Th17 but constrain Th1 and CTL effector cell differentiation.

Proliferating cells depend on glucose uptake more than quiescent cells for their growth. While glucose transport in keratinocytes is mediated largely by the Glut1 facilitative transporter, the keratinocyte-specific ablation of Glut1 did not compromise mouse skin development and homeostasis. Ex vivo metabolic profiling revealed altered sphingolipid, hexose, amino acid, and nucleotide metabolism in Glut1-deficient keratinocytes, suggesting metabolic adaptation. On the other hand, Glut1-deficient keratinocytes in culture displayed metabolic and oxidative stress and impaired proliferation. Similarly, Glut1 deficiency impaired in vivo keratinocyte proliferation and migration within wounded or UV-damaged mouse skin. Notably, both genetic and pharmacological Glut1 inactivation reduced hyperplasia in mouse models of psoriasis-like disease. Topical application of a Glut1 inhibitor also reduced inflammation in these models. Glut1 inhibition decreased expression of pathology-associated genes in human psoriatic skin organoids. Thus, Glut1 is selectively required for injury- and inflammation-associated keratinocyte proliferation, and its inhibition offers a novel treatment strategy for psoriasis.

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Helicobacter pylori colonization of the human stomach can lead to adverse clinical outcomes including gastritis, peptic ulcers, or gastric cancer. Current data suggest that in addition to bacterial virulence factors, the magnitude and types of immune responses influence the outcome of colonization. Specifically, CD4+ T cell responses impact the pathology elicited in response to H. pylori. Because gastritis is believed to be the initiating host response to more detrimental pathological outcomes, there has been a significant interest in pro-inflammatory T cell cytokines, including the cytokines produced by T helper 17 cells. Th17 cells produce IL-17A, IL-17F, IL-21 and IL-22. While these cytokines have been linked to inflammation, IL-17A and IL-22 are also associated with anti-microbial responses and control of bacterial colonization. The goal of this research was to determine the role of IL-22 in activation of antimicrobial responses in models of H. pylori infection using human gastric epithelial cell lines and the mouse model of H. pylori infection. Our data indicate that IL-17A and IL-22 work synergistically to induce antimicrobials and chemokines such as IL-8, components of calprotectin (CP), lipocalin (LCN) and some β-defensins in both human and primary mouse gastric epithelial cells (GEC) and gastroids. Moreover, IL-22 and IL-17A-activated GECs were capable of inhibiting growth of H. pylori in vitro. While antimicrobials were activated by IL-17A and IL-22 in vitro, using a mouse model of H. pylori infection, the data herein indicate that IL-22 deficiency alone does not render mice more susceptible to infection, change their antimicrobial gene transcription, or significantly change their inflammatory response.

T cells have dramatic functional and proliferative shifts in the course of maintaining immune protection from pathogens and cancer. To support these changes, T cells undergo metabolic reprogramming upon stimulation and again after antigen clearance. Depending on the extrinsic cell signals, T cells can differentiate into functionally distinct subsets that utilize and require diverse metabolic programs. Effector T cells (Teff) enhance glucose and glutamine uptake, whereas regulatory T cells (Treg) do not rely on significant rates of glycolysis. The dependence of these subsets on specific metabolic programs makes T cells reliant on these signaling pathways and nutrients. Metabolic pathways, such as those regulated by mTOR and Myc, augment T cell glycolysis and glutaminolysis programs to promote T cell activity. These pathways respond to signals and control metabolism through both transcriptional or post-transcriptional mechanisms. Epigenetic modifications also play an important role by stabilizing the transcription factors that define subset specific reprogramming. In addition, circadian rhythm cycling may also influence energy use, immune surveillance, and function of T cells. In this review, we focus on the metabolic and nutrient requirements of T cells, and how canonical pathways of growth and metabolism regulate nutrients that are essential for T cell function.

Women have increased prevalence of Th17-mediated autoimmune diseases, including lupus and multiple sclerosis, and severe asthma. While estradiol and progesterone increased IL-17A production in Th17 cells by inhibiting Let7f miRNA expression and increasing IL-23 receptor (IL-23R) expression, it remained unclear how estrogen signaling through the canonical nuclear receptors, estrogen receptor α (ERα) and/or ERβ, regulated this pathway. We hypothesized that estrogen signaling through ERα increased IL-23R expression and IL-17A production from Th17 cells. To test this hypothesis, naïve T cells from WT female, WT male, Esr1−/− and Esr2−/− female mice were differentiated into Th17 cells. IL-17A production and IL-23R expression were significantly increased in Th17 cells from WT female mice compared to Th17 cells from WT male mice. Deletion of ERα (Esr1−/−), but not ERβ (Esr2−/−), significantly decreased IL-17A production and IL-23R expression in Th17 cells by limiting IL-23R expression in a Let-7f dependent manner. ERα deficiency also decreased Th17 cell proliferation as well as decreased T cell metabolism as measured by ATP-linked oxygen consumption rate and proton leakage. Further, we found that Cox20 expression, a protein involved in mitochondrial respiration through assembly of cytochrome c oxidase in the electron transport chain, was increased in Th17 cells from WT female mice compared to Th17 cells from WT male and Esr1−/− female mice. Inhibition of Cox20 decreased IL-17 production in Th17 cells from WT female mice. Combined these studies showed that ERα signaling increased IL-17A production in Th17 cells by upregulating IL-23R expression and promoting mitochondrial respiration and proliferation.

Mass cytometry permits high-dimensional analysis of diverse aspects of cellular behavior. Here, we adapted this platform to simultaneously profile metabolism, signaling, cell cycle, and effector function with single-cell resolution. Using this approach, we measured enzymes characterizing glycolysis, the TCA cycle, fatty acid oxidation, oxidative phosphorylation, and nutrient transport.In conjunction, we measured downstream targets of TCR signaling regulating translation, proliferation and cytotoxicity as well as surface markers and transcription factors delineating CD8 T cell differentiation. High-dimensional single-cell metabolic analysis by mass cytometry permits identification of unique metabolic and differentiation states of extremely rare cell populations, such as antigen-specific T cells. We interrogated antigen-specific CD8 T cell activation in vitro as well as the trajectory of CD8 T cells responding to Listeria monocytogenes infection, a well-characterized model for studies of T cell differentiation. This integrated, highdimensional approach revealed a novel, activated, and highly metabolically active transitional T cell subset emerging at four days post-infection. These cells simultaneously exhibit glycolytic and oxidative functional programs, which we propose represents a key metabolic inflection point in CD8+ T cell differentiation. This approach should be useful for mechanistic investigations of metabolic regulation of immune responses in vivo.Recombinant human IL-2 (IL-2; TECIN (Teceleukin)) was provided by the National Cancer Institute.

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