The folate way to T cell fate.

The role of folate-dependent one carbon (1C) metabolism in CD4+ T cell polarization is incompletely understood. In this issue of Immunity, Sugiura et al. (2021) provide evidence that blocking the 1C metabolic enzyme MTHFD2 may curb pro-inflammatory CD4+ T cells, while redirecting them toward a regulatory T cell phenotype.

Regulatory T cells as metabolic sensors.

Compelling experimental evidence links immunity and metabolism. In this perspective, we propose forkhead-box-P3 (FoxP3)+CD4+CD25+ regulatory T (Treg) cells as key metabolic sensors controlling the immunological state in response to their intrinsic capacity to perceive nutritional changes. Treg cell high anabolic state in vivo, residency in metabolically crucial districts, and recirculation between lymphoid and non-lymphoid sites enable them to recognize the metabolic cues and adapt their intracellular metabolism and anti-inflammatory function at the paracrine and systemic levels. As privileged regulators at the interface between neuroendocrine and immune systems, the role of Treg cells in maintaining metabolic homeostasis makes these cells promising targets of therapeutic strategies aimed at restoring organismal homeostasis not only in autoimmune but also metabolic disorders.

Signals of pseudo-starvation unveil the amino acid transporter SLC7A11 as key determinant in the control of Treg cell proliferative potential.

Human CD4+CD25hiFOXP3+ regulatory T (Treg) cells are key players in the control of immunological self-tolerance and homeostasis. Here, we report that signals of pseudo-starvation reversed human Treg cell in vitro anergy through an integrated transcriptional response, pertaining to proliferation, metabolism, and transmembrane solute carrier transport. At the molecular level, the Treg cell proliferative response was dependent on the induction of the cystine/glutamate antiporter solute carrier (SLC)7A11, whose expression was controlled by the nuclear factor erythroid 2-related factor 2 (NRF2). SLC7A11 induction in Treg cells was impaired in subjects with relapsing-remitting multiple sclerosis (RRMS), an autoimmune disorder associated with reduced Treg cell proliferative capacity. Treatment of RRMS subjects with dimethyl fumarate (DMF) rescued SLC7A11 induction and fully recovered Treg cell expansion. These results suggest a previously unrecognized mechanism that may account for the progressive loss of Treg cells in autoimmunity and unveil SLC7A11 as major target for the rescue of Treg cell proliferation.

Metabolic pressure and the breach of immunological self-tolerance.

The prevalence of autoimmune disorders in affluent countries has reached epidemic proportions. Over the past 50 years, a reverse trend between the frequency of infectious diseases and the incidence of autoimmune and allergic diseases led to the so-called 'hygiene hypothesis'. Given the epidemiological evidence and recent experimental data, we propose that this concept should also include metabolic pressure secondary to exposure to excessive daily caloric intake and overnutrition. We discuss how metabolic workload can modulate immunological tolerance and review the molecular mechanisms and the state of the art of the field. We also critically evaluate possibilities for restoring immunological homeostasis under conditions of metabolic pressure.

Glycolysis controls the induction of human regulatory T cells by modulating the expression of FOXP3 exon 2 splicing variants.

Human regulatory T cells (T(reg) cells) that develop from conventional T cells (T(conv) cells) following suboptimal stimulation via the T cell antigen receptor (TCR) (induced T(reg) cells (iT(reg) cells)) express the transcription factor Foxp3, are suppressive, and display an active proliferative and metabolic state. Here we found that the induction and suppressive function of iT(reg) cells tightly depended on glycolysis, which controlled Foxp3 splicing variants containing exon 2 (Foxp3-E2) through the glycolytic enzyme enolase-1. The Foxp3-E2-related suppressive activity of iT(reg) cells was altered in human autoimmune diseases, including multiple sclerosis and type 1 diabetes, and was associated with impaired glycolysis and signaling via interleukin 2. This link between glycolysis and Foxp3-E2 variants via enolase-1 shows a previously unknown mechanism for controlling the induction and function of T(reg) cells in health and in autoimmunity.

Regulatory T Cell Migration Is Dependent on Glucokinase-Mediated Glycolysis.

Migration of activated regulatory T (Treg) cells to inflamed tissue is crucial for their immune-modulatory function. While metabolic reprogramming during Treg cell differentiation has been extensively studied, the bioenergetics of Treg cell trafficking remains undefined. We have investigated the metabolic demands of migrating Treg cells in vitro and in vivo. We show that glycolysis was instrumental for their migration and was initiated by pro-migratory stimuli via a PI3K-mTORC2-mediated pathway culminating in induction of the enzyme glucokinase (GCK). Subsequently, GCK promoted cytoskeletal rearrangements by associating with actin. Treg cells lacking this pathway were functionally suppressive but failed to migrate to skin allografts and inhibit rejection. Similarly, human carriers of a loss-of-function GCK regulatory protein gene-leading to increased GCK activity-had reduced numbers of circulating Treg cells. These cells displayed enhanced migratory activity but similar suppressive function, while conventional T cells were unaffected. Thus, GCK-dependent glycolysis regulates Treg cell migration.

Metabolic Plasticity of Regulatory T Cells in Health and Autoimmunity.

Immunometabolism has been demonstrated to control immune tolerance and the pathogenic events leading to autoimmunity. Compelling experimental evidence also suggests that intracellular metabolic programs influence differentiation, phenotype, proliferation, and effector functions of anti-inflammatory CD4+CD25+Foxp3+ regulatory T (Treg) cells. Indeed, alterations in intracellular metabolism associate with quantitative and qualitative impairments of Treg cells in several pathological conditions. In this review, we summarize the most recent advances linking how metabolic pathways control Treg cell homeostasis and their alterations occurring in autoimmunity. Also, we analyze how metabolic manipulations could be employed to restore Treg cell frequency and function with the aim to create novel therapeutic opportunities to halt immune-mediated disorders.

Cellular and molecular signaling towards T cell immunological self-tolerance.

The binding of a cognate antigen to T cell receptor (TCR) complex triggers a series of intracellular events controlling T cell activation, proliferation, and differentiation. Upon TCR engagement, different negative regulatory feedback mechanisms are rapidly activated to counterbalance T cell activation, thus preventing excessive signal propagation and promoting the induction of immunological self-tolerance. Both positive and negative regulatory processes are tightly controlled to ensure the effective elimination of foreign antigens while limiting surrounding tissue damage and autoimmunity. In this context, signals deriving from co-stimulatory molecules (i.e., CD80, CD86), co-inhibitory receptors (PD-1, CTLA-4), the tyrosine phosphatase CD45 and cytokines such as IL-2 synergize with TCR-derived signals to guide T cell fate and differentiation. The balance of these mechanisms is also crucial for the generation of CD4+ Foxp3+ regulatory T cells, a cellular subset involved in the control of immunological self-tolerance. This review provides an overview of the most relevant pathways induced by TCR activation combined with those derived from co-stimulatory and co-inhibitory molecules implicated in the cell-intrinsic modulation of T cell activation. In addition to the latter, we dissected mechanisms responsible for T cell-mediated suppression of immune cell activation through regulatory T cell generation, homeostasis, and effector functions. We also discuss how imbalanced signaling derived from TCR and accessory molecules can contribute to autoimmune disease pathogenesis.

The Proteomic Landscape of Human Ex Vivo Regulatory and Conventional T Cells Reveals Specific Metabolic Requirements.

Human CD4(+)CD25(hi)Foxp3(+)CD127(-) Treg and CD4(+)CD25(-)Foxp3(-) Tconv cell functions are governed by their metabolic requirements. Here we report a comprehensive comparative analysis between ex vivo human Treg and Tconv cells that comprises analyses of the proteomic networks in subcellular compartments. We identified a dominant proteomic signature at the metabolic level that primarily impacted the highly-tuned balance between glucose and fatty-acid oxidation in the two cell types. Ex vivo Treg cells were highly glycolytic while Tconv cells used predominantly fatty-acid oxidation (FAO). When cultured in vitro, Treg cells engaged both glycolysis and FAO to proliferate, while Tconv cell proliferation mainly relied on glucose metabolism. Our unbiased proteomic analysis provides a molecular picture of the impact of metabolism on ex vivo human Treg versus Tconv cell functions that might be relevant for therapeutic manipulations of these cells.

Progression of type 1 diabetes is associated with high levels of soluble PD-1 in islet autoantibody-positive children.

AIMS/HYPOTHESIS: Type 1 diabetes is an autoimmune disorder that is characterised by destruction of pancreatic beta cells by autoreactive T lymphocytes. Although islet autoantibodies (AAb) are an indicator of disease progression, specific immune biomarkers that can be used as target molecules to halt development of type 1 diabetes have not been discovered. Soluble immune checkpoint molecules (sICM) play a pivotal role in counteracting excessive lymphocyte responses, but their role in type 1 diabetes is unexplored. In this longitudinal study, we measured sICM levels in AAb-positive (AAb+) children to identify molecules related to type 1 diabetes progression. METHODS: We measured the levels of 14 sICM in the sera of AAb+ children (n=57) compared to those with recent-onset type 1 diabetes (n=79) and healthy children (n=44), obtained from two cohorts. AAb+ children were followed up and divided based on their progression to type 1 diabetes (AAbP) or not (AAbNP) (if they lost islet autoimmunity and did not develop disease in subsequent years). sICM were also measured in the sample taken at the visit closest to disease onset in AAbP children. RESULTS: We found that AAb+ children had a distinct sICM profile compared with healthy children and those with recent-onset type 1 diabetes. In addition, AAb+ children who progressed to type 1 diabetes (AAbP) had higher sICM concentrations than non-progressors (AAbNP). Further, sICM levels decreased in AAbP children close to disease onset. Application of Cox regression models highlighted that high concentrations of soluble programmed cell death protein 1 (sPD-1) are associated with type 1 diabetes progression (HR 1.71; 95% CI 1.16, 2.51; p=0.007). CONCLUSIONS/INTERPRETATION: This study reveals an sICM profile that is dysregulated during the preclinical stage of type 1 diabetes, and identifies sPD-1 as a pathophysiologically-relevant molecule that is associated with disease progression, offering a potential target for early interventions in autoimmune diabetes.

T Cells: Warriors of SARS-CoV-2 Infection.

Severe infection with severe acute respiratory syndrome coronavirus (SARS-CoV)-2 is characterized by massive cytokine release and T cell loss. The exaggerated host immune response, incapable of viral clearance, instead aggravates respiratory distress, as well as cardiac, and/or damage to other organs. The mortality pattern of SARS-CoV-2 infection, higher in older versus younger adults and almost absent in children, is possibly caused by the effects of age and pre-existing comorbidities on innate and adaptive immunity. Here, we speculate that the abnormal and excessive immune response to SARS-CoV-2 infection partly depends on T cell immunological memory, which is more pronounced in adults compared with children, and may significantly contribute to immunopathology and massive collateral damage in coronavirus disease 2019 (COVID-19) patients.

The Cellular and Molecular Basis of Translational Immunometabolism.

The immune response requires major changes to metabolic processes, and indeed, energy metabolism and functional activation are fully integrated in immune cells to determine their ability to divide, differentiate, and carry out effector functions. Immune cell metabolism has therefore become an attractive target area for therapeutic purposes. A neglected aspect in the translation of immunometabolism is the critical connection between systemic and cellular metabolism. Here, we discuss the importance of understanding and manipulating the integration of systemic and immune cell metabolism through in-depth analysis of immune cell phenotype and function in human metabolic diseases and, in parallel, of the effects of conventional metabolic drugs on immune cell differentiation and function. We examine how the recent identification of selective metabolic programs operating in distinct immune cell subsets and functions has the potential to deliver tools for cell- and function-specific immunometabolic targeting.

Bifidobacteria modulate immune response in pediatric patients with cow's milk protein allergy.

BACKGROUND: In children with an allergy to cow's milk proteins (CMA), the altered composition of intestinal microbiota influences the immune tolerance to milk proteins (CMP). This study aims to investigate the effect of probiotics on the phenotype and activation status of peripheral basophils and lymphocytes in a pediatric CMA cohort. METHODS: CMA children underwent 45 days of treatment with Bifidobacteria. The basophil degranulation and the immune phenotype of B cells, T helper cells, and regulatory T cells were analyzed in peripheral blood at diagnosis (T0), after a 45-day probiotic treatment (T1), and 45 days after the probiotic wash-out (T2). RESULTS: We observed in probiotic-treated CMA patients a decrease in naive T lymphocytes. Among the CD3+ cell subsets, both naive and activated CD4+ cells resulted markedly reduced after taking probiotics, with the lowest percentages at T2. A decreased basophil degranulation was observed in response to all analyzed CMP at T1 compared to T0. CONCLUSIONS: The probiotic treatment resulted in a decrease of circulating naive and activated CD4+ T cells, as well as degranulating basophils. These data suggest that the Bifidobacteria could have a beneficial effect in the modulation of oral tolerance to CMP. TRIAL REGISTRATION: ISRCTN69069358. URL of registration: https://www.isrctn.com/ISRCTN69069358 . IMPACT: Probiotic treatment with Bifidobacteria induces a reduction of both naive and activated circulating CD4+ T cells in pediatric patients with cow's milk allergy (CMA). The probiotic supplementation induces a decreased basophil degranulation. The immunological tolerance persists even after 45 days of the probiotic wash-out. Bifidobacteria in vivo supplementation down-modulates the activation of innate and adaptive immunity in pediatric patients with cow's milk allergy. Bifidobacteria contribute to the development of immune tolerance in CMA patients.

Antibody Profile, Gene Expression and Serum Cytokines in At-Risk Infants before the Onset of Celiac Disease.

Immunological events that precede the development of villous atrophy in celiac disease (CeD) are still not completely understood. We aimed to explore CeD-associated antibody production (anti-native gliadin (AGA), anti-deamidated gliadin (DGP) and anti-tissue transglutaminase (anti-tTG)) in infants at genetic risk for CeD from the Italian cohorts of the PREVENT-CD and Neocel projects, as well as the relationship between antibody production and systemic inflammation. HLA DQ2 and/or DQ8 infants from families with a CeD case were followed from birth. Out of 220 at-risk children, 182 had not developed CeD by 6 years of age (CTRLs), and 38 developed celiac disease (CeD). The profiles of serum cytokines (INFγ, IL1ß, IL2, IL4, IL6, IL10, IL12p70, IL17A and TNFα) and the expression of selected genes (FoxP3, IL10, TGFß, INFγ, IL4 and IL2) were evaluated in 46 children (20 CeD and 26 CTRLs). Among the 182 healthy CTRLs, 28 (15.3%) produced high levels of AGA-IgA (AGA+CTRLs), and none developed anti-tTG-IgA or DGP-IgA, compared to 2/38 (5.3%) CeD infants (Chi Sq. 5.97, p = 0.0014). AGAs appeared earlier in CTRLs than in those who developed CeD (19 vs. 28 months). Additionally, the production of AGAs in CeD overlapped with the production of DGP and anti-tTG. In addition, gene expression as well as serum cytokine levels discriminated children who developed CeD from CTRLs. In conclusion, these findings suggest that the early and isolated production of AGA-IgA antibodies is a CeD-tolerogenic marker and that changes in gene expression and cytokine patterns precede the appearance of anti-tTG antibodies.

High levels of blood circulating immune checkpoint molecules in children with new-onset type 1 diabetes are associated with the risk of developing an additional autoimmune disease.

AIMS/HYPOTHESIS: We assessed the levels of blood circulating immune checkpoint molecules (ICMs) at diagnosis of type 1 diabetes, and determined their association with the risk of developing an additional autoimmune disorder over time. METHODS: Children with new-onset type 1 diabetes (n = 143), without biological and/or clinical signs of additional autoimmune disorders, and healthy children (n = 75) were enrolled, and blood circulating levels of 14 ICMs were measured. The children with type 1 diabetes were divided into two groups on the basis of the development of an additional autoimmune disease in the 5 years after diabetes onset. Differences in soluble ICM levels between the groups were assessed, and a Cox regression analysis was used to evaluate their association with the risk of development of an additional autoimmune disease over time. To validate the data, circulating ICMs were measured in an independent cohort of 60 children with new-onset type 1 diabetes stratified into two groups. RESULTS: We found that the levels of circulating ICMs were significantly higher in children with new-onset diabetes compared with healthy children. Further, we observed that children with type 1 diabetes who developed a second autoimmune disease over time (T1D-AAD+ children) had higher levels of soluble ICMs than children with type 1 diabetes who did not (T1D-AAD- children). Cox regression models revealed that high circulating levels of CD137/4-1BB and PD-1 molecules at diabetes diagnosis were associated with the risk of developing an additional autoimmune disease in both type 1 diabetes cohorts. CONCLUSIONS/INTERPRETATION: Our findings suggest that soluble CD137/4-1BB and PD-1 molecules may be used as prognostic biomarkers in children with type 1 diabetes, and may pave the way for novel immunological screening at diabetes onset, allowing early identification of children at higher risk of developing other autoimmune conditions over time.

MiR-142-3p regulates synaptopathy-driven disease progression in multiple sclerosis.

AIM: We recently proposed miR-142-3p as a molecular player in inflammatory synaptopathy, a new pathogenic hallmark of multiple sclerosis (MS) and of its mouse model experimental autoimmune encephalomyelitis (EAE), that leads to neuronal loss independently of demyelination. MiR-142-3p seems to be unique among potential biomarker candidates in MS, since it is an inflammatory miRNA playing a dual role in the immune and central nervous systems. Here, we aimed to verify the impact of miR-142-3p circulating in the cerebrospinal fluid (CSF) of MS patients on clinical parameters, neuronal excitability and its potential interaction with disease modifying therapies (DMTs). METHODS AND RESULTS: In a cohort of 151 MS patients, we found positive correlations between CSF miR-142-3p levels and clinical progression, IL-1ß signalling as well as synaptic excitability measured by transcranial magnetic stimulation. Furthermore, therapy response of patients with 'low miR-142-3p' to dimethyl fumarate (DMF), an established disease-modifying treatment (DMT), was superior to that of patients with 'high miR-142-3p' levels. Accordingly, the EAE clinical course of heterozygous miR-142 mice was ameliorated by peripheral DMF treatment with a greater impact relative to their wild type littermates. In addition, a central protective effect of this drug was observed following intracerebroventricular and ex vivo acute treatments of EAE wild type mice, showing a rescue of miR-142-3p-dependent glutamatergic alterations. By means of electrophysiology, molecular and biochemical analysis, we suggest miR-142-3p as a molecular target of DMF. CONCLUSION: MiR-142-3p is a novel and potential negative prognostic CSF marker of MS and a promising tool for identifying personalised therapies.

Regulation of mitochondrial complex III activity and assembly by TRAP1 in cancer cells.

BACKGROUND: Metabolic reprogramming is an important issue in tumor biology. A recently-identified actor in this regard is the molecular chaperone TRAP1, that is considered an oncogene in several cancers for its high expression but an oncosuppressor in others with predominant oxidative metabolism. TRAP1 is mainly localized in mitochondria, where it interacts with respiratory complexes, although alternative localizations have been described, particularly on the endoplasmic reticulum, where it interacts with the translational machinery with relevant roles in protein synthesis regulation. RESULTS: Herein we show that, inside mitochondria, TRAP1 binds the complex III core component UQCRC2 and regulates complex III activity. This decreases respiration rate during basal conditions but allows sustained oxidative phosphorylation when glucose is limiting, a condition in which the direct TRAP1-UQCRC2 binding is disrupted, but not TRAP1-complex III binding. Interestingly, several complex III components and assembly factors show an inverse correlation with survival and response to platinum-based therapy in high grade serous ovarian cancers, where TRAP1 inversely correlates with stage and grade and directly correlates with survival. Accordingly, drug-resistant ovarian cancer cells show high levels of complex III components and high sensitivity to complex III inhibitory drug antimycin A. CONCLUSIONS: These results shed new light on the molecular mechanisms involved in TRAP1-dependent regulation of cancer cell metabolism and point out a potential novel target for metabolic therapy in ovarian cancer.