The glutamate/aspartate transporter EAAT1 is crucial for T-cell acute lymphoblastic leukemia proliferation and survival.

T-cell acute lymphoblastic leukemia (T-ALL) is a cancer of the immune system. Approximately 20% of paediatric and 50% of adult T-ALL patients have refractory disease or relapse and die from the disease. To improve patient outcome new therapeutics are needed. With the aim to identify new therapeutic targets, we combined the analysis of T-ALL gene expression and metabolism to identify the metabolic adaptations that T-ALL cells exhibit. We found that glutamine uptake is essential for T-ALL proliferation. Isotope tracing experiments showed that glutamine fuels aspartate synthesis through the TCA cycle and that glutamine and glutamine-derived aspartate together supply three nitrogen atoms in purines and all but one atom in pyrimidine rings. We show that the glutamate-aspartate transporter EAAT1 (SLC1A3), which is normally expressed in the central nervous system, is crucial for glutamine conversion to aspartate and nucleotides and that T-ALL cell proliferation depends on EAAT1 function. Through this work, we identify EAAT1 as a novel therapeutic target for T-ALL treatment.

TNF-α signals through ITK-Akt-mTOR to drive CD4+ T cell metabolic reprogramming, which is dysregulated in rheumatoid arthritis.

Upon activation, T cells undergo metabolic reprogramming to meet the bioenergetic demands of clonal expansion and effector function. Because dysregulated T cell cytokine production and metabolic phenotypes coexist in chronic inflammatory disease, including rheumatoid arthritis (RA), we investigated whether inflammatory cytokines released by differentiating T cells amplified their metabolic changes. We found that tumor necrosis factor-α (TNF-α) released by human naïve CD4+ T cells upon activation stimulated the expression of a metabolic transcriptome and increased glycolysis, amino acid uptake, mitochondrial oxidation of glutamine, and mitochondrial biogenesis. The effects of TNF-α were mediated by activation of Akt-mTOR signaling by the kinase ITK and did not require the NF-κB pathway. TNF-α stimulated the differentiation of naïve cells into proinflammatory T helper 1 (TH1) and TH17 cells, but not that of regulatory T cells. CD4+ T cells from patients with RA showed increased TNF-α production and consequent Akt phosphorylation upon activation. These cells also exhibited increased mitochondrial mass, particularly within proinflammatory T cell subsets implicated in disease. Together, these findings suggest that T cell-derived TNF-α drives their metabolic reprogramming by promoting signaling through ITK, Akt, and mTOR, which is dysregulated in autoinflammatory disease.

Uptake of long-chain fatty acids from the bone marrow suppresses CD8+ T-cell metabolism and function in multiple myeloma.

T cells demonstrate impaired function in multiple myeloma (MM) but suppressive mechanisms in the bone marrow microenvironment remain poorly defined. We observe that bone marrow CD8+ T-cell function is decreased in MM compared with controls, and is also consistently lower within bone marrow samples than in matched peripheral blood samples. These changes are accompanied by decreased mitochondrial mass and markedly elevated long-chain fatty acid uptake. In vitro modeling confirmed that uptake of bone marrow lipids suppresses CD8+ T function, which is impaired in autologous bone marrow plasma but rescued by lipid removal. Analysis of single-cell RNA-sequencing data identified expression of fatty acid transport protein 1 (FATP1) in bone marrow CD8+ T cells in MM, and FATP1 blockade also rescued CD8+ T-cell function, thereby identifying this as a novel target to augment T-cell activity in MM. Finally, analysis of samples from cohorts of patients who had received treatment identified that CD8+ T-cell metabolic dysfunction resolves in patients with MM who are responsive to treatment but not in patients with relapsed MM, and is associated with substantial T-cell functional restoration.

Canagliflozin impairs T cell effector function via metabolic suppression in autoimmunity.

Augmented T cell function leading to host damage in autoimmunity is supported by metabolic dysregulation, making targeting immunometabolism an attractive therapeutic avenue. Canagliflozin, a type 2 diabetes drug, is a sodium glucose co-transporter 2 (SGLT2) inhibitor with known off-target effects on glutamate dehydrogenase and complex I. However, the effects of SGLT2 inhibitors on human T cell function have not been extensively explored. Here, we show that canagliflozin-treated T cells are compromised in their ability to activate, proliferate, and initiate effector functions. Canagliflozin inhibits T cell receptor signaling, impacting on ERK and mTORC1 activity, concomitantly associated with reduced c-Myc. Compromised c-Myc levels were encapsulated by a failure to engage translational machinery resulting in impaired metabolic protein and solute carrier production among others. Importantly, canagliflozin-treated T cells derived from patients with autoimmune disorders impaired their effector function. Taken together, our work highlights a potential therapeutic avenue for repurposing canagliflozin as an intervention for T cell-mediated autoimmunity.

Succinate uptake by T cells suppresses their effector function via inhibition of mitochondrial glucose oxidation.

Succinate dehydrogenase (SDH) loss-of-function mutations drive succinate accumulation in tumor microenvironments, for example in the neuroendocrine tumors pheochromocytoma (PC) and paraganglioma (PG). Control of innate immune cell activity by succinate is described, but effects on T cells have not been interrogated. Here we report that exposure of human CD4+ and CD8+ T cells to tumor-associated succinate concentrations suppresses degranulation and cytokine secretion, including of the key anti-tumor cytokine interferon-γ (IFN-γ). Mechanistically, this is associated with succinate uptake-partly via the monocarboxylate transporter 1 (MCT1)-inhibition of succinyl coenzyme A synthetase activity and impaired glucose flux through the tricarboxylic acid cycle. Consistently, pharmacological and genetic interventions restoring glucose oxidation rescue T cell function. Tumor RNA-sequencing data from patients with PC and PG reveal profound suppression of IFN-γ-induced genes in SDH-deficient tumors compared with those with other mutations, supporting a role for succinate in modulating the anti-tumor immune response in vivo.

1,25-Dihydroxyvitamin D3 suppresses CD4+ T-cell effector functionality by inhibition of glycolysis.

In CD4+ T helper cells, the active form of vitamin D3 , 1,25-dihydroxyvitamin D3 (1,25D) suppresses production of inflammatory cytokines, including interferon-gamma (IFN-γ), but the mechanisms for this are not yet fully defined. In innate immune cells, response to 1,25D has been linked to metabolic reprogramming. It is unclear whether 1,25D has similar effects on CD4+ T cells, although it is known that antigen stimulation of these cells promotes an anabolic metabolic phenotype, characterized by high rates of aerobic glycolysis to support clonal expansion and effector cytokine expression. Here, we performed in-depth analysis of metabolic capacity and pathway usage, employing extracellular flux and stable isotope-based tracing approaches, in CD4+ T cells treated with 1,25D. We report that 1,25D significantly decreases rates of aerobic glycolysis in activated CD4+ T cells, whilst exerting a lesser effect on mitochondrial glucose oxidation. This is associated with transcriptional repression of Myc, but not repression of mTOR activity under these conditions. Consistent with the modest effect of 1,25D on mitochondrial activity, it also did not impact CD4+ T-cell mitochondrial mass or membrane potential. Finally, we demonstrate that inhibition of aerobic glycolysis by 1,25D substantially contributes to its immune-regulatory capacity in CD4+ T cells, since the suppression of IFN-γ expression was significantly blunted in the absence of aerobic glycolysis. 1,25-Dihydroxyvitamin D3 (1,25D) suppresses the production of inflammatory cytokines such as interferon-gamma (IFN-γ) by CD4+ T cells, but the underpinning mechanisms are not yet fully defined. Here, we identify that 1,25D inhibits aerobic glycolysis in activated CD4+ T cells, associated with decreased c-Myc expression. This mechanism appears to substantially contribute to the suppression of IFN-γ by 1,25D, since this is significantly blunted in the absence of aerobic glycolysis.

Autoimmune disease and interconnections with vitamin D.

Vitamin D has well-documented effects on calcium homeostasis and bone metabolism but recent studies suggest a much broader role for this secosteroid in human health. Key components of the vitamin D system, notably the vitamin D receptor (VDR) and the vitamin D-activating enzyme (1α-hydroxylase), are present in a wide array of tissues, notably macrophages, dendritic cells and T lymphocytes (T cells) from the immune system. Thus, serum 25-hydroxyvitamin D (25D) can be converted to hormonal 1,25-dihydroxyvitamin D (1,25D) within immune cells, and then interact with VDR and promote transcriptional and epigenomic responses in the same or neighbouring cells. These intracrine and paracrine effects of 1,25D have been shown to drive antibacterial or antiviral innate responses, as well as to attenuate inflammatory T cell adaptive immunity. Beyond these mechanistic observations, association studies have reported the correlation between low serum 25D levels and the risk and severity of human immune disorders including autoimmune diseases such as inflammatory bowel disease, multiple sclerosis, type 1 diabetes and rheumatoid arthritis. The proposed explanation for this is that decreased availability of 25D compromises immune cell synthesis of 1,25D leading to impaired innate immunity and over-exuberant inflammatory adaptive immunity. The aim of the current review is to explore the mechanistic basis for immunomodulatory effects of 25D and 1,25D in greater detail with specific emphasis on how vitamin D-deficiency (low serum levels of 25D) may lead to dysregulation of macrophage, dendritic cell and T cell function and increase the risk of inflammatory autoimmune disease.

Survival Analysis of Training Methodologies and Other Risk Factors for Musculoskeletal Injury in 2-Year-Old Thoroughbred Racehorses in Queensland, Australia.

Musculoskeletal injuries remain a global problem for the Thoroughbred racing industry and there is conflicting evidence regarding the effect of age on the incidence of injuries. The ideal time to commence race training is strongly debated, with limited supporting literature. There is also conflicting evidence regarding the effect of high-speed exercise on musculoskeletal injuries. There is a strong interest in developing training and management strategies to reduce the frequency of injuries. The types of musculoskeletal injuries vary between 2-year-old and older horses, with dorsal metacarpal disease the most common injury in 2-year-old horses. It is likely that risk factors for injury in 2-year-old horses are different than those for older horses. It is also likely that the risk factors may vary between types of injury. This study aimed to determine the risk factors for musculoskeletal injuries and dorsal metacarpal disease. We report the findings of a large scale, prospective observational study of 2-year-old horses in Queensland, Australia. Data were collected weekly for 56-weeks, from 26 trainers, involving 535 2-year-old Thoroughbred racehorses, 1, 258 training preparations and 7, 512-weeks of exercise data. A causal approach was used to develop our statistical models, to build on the existing literature surrounding injury risk, by incorporating the previously established causal links into our analyses. Where previous data were not available, industry experts were consulted. Survival analyses were performed using Cox proportional hazards or Weibull regression models. Analysis of musculoskeletal injuries overall revealed the hazard was reduced with increased exposure to high-speed exercise [Hazard ratio (HR) 0.89, 95% Confidence Interval (CI) 0.84, 0.94, p < 0.001], increased number of training preparations (HR 0.58, 95% CI 0.50, 0.67, p < 0.001), increased rest before the training preparation (HR 0.89, 95% CI 0.83, 0.96, p = 0.003) and increased dam parity (HR 0.86, 95% CI 0.77, 0.97, p = 0.01). The hazard of injury was increased with increasing age that training commenced (HR 1.13, 95% CI 1.06, 1.19, p < 0.001). Analyses were then repeated with the outcome of interest dorsal metacarpal disease. Factors that were protective against dorsal metacarpal disease and musculoskeletal injuries overall included: increased total cumulative distance (HR 0.89, 95% CI 0.82, 0.97, p = 0.001) and total cumulative days exercised as a gallop (HR 0.96, 95% CI 0.92, 0.99, p = 0.03), the number of the training preparations (HR 0.43, 95% CI 0.30, 0.61, p < 0.001). The age that training commenced was harmful for both dorsal metacarpal disease (HR 1.17, 95% CI 1.07, 1.28, p < 0.001 and overall musculoskeletal injuries.). The use of non-ridden training modalities was protective for dorsal metacarpal disease (HR 0.89, 95% CI 0.81, 0.97, p = 0.008), but not musculoskeletal injuries overall. The male sex increased the hazard of DMD compared to females (HR 2.58, 95% CI 1.20, 5.56, p = 0.02), but not MSI overall. In summary, the hazard of musculoskeletal injury is greatest for 2-year-old horses that are born from uniparous mares, commence training at a later age, are in their first training preparation, have undertaken little high-speed exercise or had limited rest before their training preparation. The hazard of dorsal metacarpal disease is greatest for 2-year-old horses that are males, commence training at a later age, are in their first training preparation, have undertaken little high-speed exercise or had limited use of non-ridden training modalities. Close monitoring of these high-risk horses during their training program could substantially reduce the impact of MSI. Furthermore, an understanding of how training methodologies affect the hazard of MSI facilitates modification of training programs to mitigate the risk impact of injury. The strengths of this study include a large sample size, a well-defined study protocol and direct trainer interviews. The main limitation is the inherent susceptibility to survival bias.

A Prospective Study of Training Methods for Two-Year-Old Thoroughbred Racehorses in Queensland, Australia, and Analysis of the Differences in Training Methods between Trainers of Varying Stable Sizes.

Worldwide, musculoskeletal injuries remain a major problem for the Thoroughbred racing industry. There is a strong interest in developing training and management strategies to reduce the impact of musculoskeletal injuries, however, progress has been limited by studies reporting conflicting findings, and a limited understanding of the role of different training methods in preventing injury. There is little data on patterns of rest periods and exercise data and how these vary between trainers. This prospective study of two-year-old racehorses was conducted in Queensland, Australia and involved weekly personal structured interviews with 26 trainers over 56 weeks. Detailed daily exercise data for 535 horses providing 1258 training preparations and 7512 weeks at risk were collected. Trainers were categorised into three groups by the mean number of two-year-old horses that they had in work each week over the study duration: (1) Small stables with five or less, (2) Medium stables with 6 to 15 and (3) Large stables with greater than 15 horses in training. Differences between trainers with small, medium and large stable sizes were evaluated using linear regression, Kruskal-Wallis equality-of-populations rank test if linear models were mis-specified or Chi-squared tests for categorical variables. Significant differences were observed between trainers, with horses from larger stables accumulating a greater high-speed exercise volume (p < 0.001), attaining training milestones more frequently (p = 0.01) and taking less time to reach their training milestones (p = 0.001). This study provides detailed data to which training practices from other locations can be compared. Presenting actual training data rather than trainers' estimation of a typical program provides a more accurate assessment of training practices. Understanding how training practices vary between regions improves comparability of studies investigating risk factors and is an important step towards reducing the impact of musculoskeletal injuries.

Control of T Cell Metabolism by Cytokines and Hormones.

Dynamic, coordinated changes in metabolic pathway activity underpin the protective and inflammatory activity of T cells, through provision of energy and biosynthetic precursors for effector functions, as well as direct effects of metabolic enzymes, intermediates and end-products on signaling pathways and transcriptional mechanisms. Consequently, it has become increasingly clear that the metabolic status of the tissue microenvironment directly influences T cell activity, with changes in nutrient and/or metabolite abundance leading to dysfunctional T cell metabolism and interlinked immune function. Emerging evidence now indicates that additional signals are integrated by T cells to determine their overall metabolic phenotype, including those arising from interaction with cytokines and hormones in their environment. The impact of these on T cell metabolism, the mechanisms involved and the pathological implications are discussed in this review article.

Tolerogenic effects of 1,25-dihydroxyvitamin D on dendritic cells involve induction of fatty acid synthesis.

The active form of vitamin D, 1,25-dihydroxyvitamin D (1,25D) is a potent regulator of immune function, promoting anti-inflammatory, tolerogenic T cell responses by modulating antigen presentation by dendritic cells (DC). Transcriptomic analyses indicate that DC responses to 1,25D involve changes in glycolysis, oxidative phosphorylation, electron transport and the TCA cycle. To determine the functional impact of 1,25D-mediated metabolic remodelling, human monocyte-derived DC were differentiated to immature (+vehicle, iDC), mature (+LPS, mDC), and immature tolerogenic DC (+1,25D, itolDC) and characterised for metabolic function. In contrast to mDC which showed no change in respiration, itolDC showed increased basal and ATP-linked respiration relative to iDC. Tracer metabolite analyses using 13C -labeled glucose showed increased lactate and TCA cycle metabolites. Analysis of lipophilic metabolites of 13C-glucose revealed significant incorporation of label in palmitate and palmitoleate, indicating that 1,25D promotes metabolic fatty acid synthesis in itolDC. Inhibition of fatty acid synthesis in itolDC altered itolDC morphology and suppressed expression of CD14 and IL-10 by these cells. These data indicate that the ability of 1,25D to induce tolerogenic DC involves metabolic remodelling leading to synthesis of fatty acids.

Peripheral lymph nodes contain migratory and resident innate lymphoid cell populations.

Tissue residency is considered a defining feature of the innate lymphoid cell (ILC) populations located within mucosal and adipose tissues. ILCs are also present within all lymphoid tissues, but whether ILCs migrate between lymphoid and nonlymphoid sites and in what context is poorly understood. To determine whether migratory ILCs exist within peripheral lymph nodes (LNs), we labeled all cells within the brachial LN (bLN) of transgenic mice expressing a photoconvertible fluorescent protein by direct exposure to light. Tracking of cellular changes in the labeled LN revealed the gradual migration of new ILCs into the tissue, balanced by egress of ILCs dependent on sphingosine-1-phosphate receptors. Most of the migratory ILCs were ILC1s, entering LNs directly from the circulation in a CD62L- and CCR7-dependent manner and thus behaving like conventional natural killer (cNK) cells. Upon egress, both ILC1s and cNK cells were found to recirculate through peripheral LNs. A distinct population of migratory ILC2s were detected in the LN, but most of the ILC3s were tissue resident. Functionally, both migratory and resident ILC1s within LNs were able to rapidly produce IFN-γ to support the generation of robust TH1 T cell responses after immunization. Thus, migratory and resident ILC populations exist within peripheral LNs, with ILC1s, akin to cNK cells, able to traffic into these tissues where they can contribute to the initiation of adaptive immunity.