Deciphering the role of protein kinase A in the control of FoxP3 expression in regulatory T cells in health and autoimmunity.

The molecular mechanisms that govern differential T cell development from CD4+CD25-conventional T (Tconv) into CD4+CD25+ forkhead-box-P3+ (FoxP3+) inducible regulatory T (iTreg) cells remain unclear. Herein, we investigated the relative contribution of protein kinase A (PKA) in this process. Mechanistically, we found that PKA controlled the efficiency of human iTreg cell generation through the expression of different FoxP3 splicing variants containing or not the exon 2. We found that transient PKA inhibition reduced the recruitment of cAMP-responsive element-binding protein (CREB) on regulatory regions of the FoxP3 gene, a condition that is associated with an impaired acquisition of their suppressive capacity in vitro. To corroborate our findings in a human model of autoimmunity, we measured CREB phosphorylation and FoxP3 levels in iTreg cells from treatment-naïve relapsing-remitting (RR)-multiple sclerosis (MS) subjects. Interestingly, both phospho-CREB and FoxP3 induction directly correlated and were significantly reduced in RR-MS patients, suggesting a previously unknown mechanism involved in the induction and function of human iTreg cells.

Ocrelizumab Alters Cytotoxic Lymphocyte Function While Reducing EBV-Specific CD8+ T-Cell Proliferation in Patients With Multiple Sclerosis.

BACKGROUND AND OBJECTIVES: The role of B cells in the pathogenic events leading to relapsing multiple sclerosis (R-MS) has only been recently elucidated. A pivotal step in defining this role has been provided by therapeutic efficacy of anti-CD20 monoclonal antibodies. Indeed, treatment with anti-CD20 can also alter number and function of other immune cells not directly expressing CD20 on their cell surface, whose activities can contribute to unknown aspects influencing therapeutic efficacy. We examined the phenotype and function of cytotoxic lymphocytes and Epstein-Barr virus (EBV)-specific immune responses in people with R-MS before and after ocrelizumab treatment. METHODS: In this prospective study, we collected blood samples from people with R-MS (n = 41) before and 6 and 12 months after initiating ocrelizumab to assess the immune phenotype and the indirect impact on cytotoxic functions of CD8+ T and NK cells. In addition, we evaluated the specific anti-EBV proliferative responses of both CD8+ T and NK lymphocytes as surrogate markers of anti-EBV activity. RESULTS: We observed that while ocrelizumab depleted circulating B cells, it also reduced the expression of activation and migratory markers on both CD8+ T and NK cells as well as their in vitro cytotoxic activity. A comparable pattern in the modulation of immune molecules by ocrelizumab was observed in cytotoxic cells even when patients with R-MS were divided into groups based on their prior disease-modifying treatment. These effects were accompanied by a significant and selective reduction of CD8+ T-cell proliferation in response to EBV antigenic peptides. DISCUSSION: Taken together, our findings suggest that ocrelizumab-while depleting B cells-affects the cytotoxic function of CD8+ and NK cells, whose reduced cross-activity against myelin antigens might also contribute to its therapeutic efficacy during MS.

N6-isopentenyladenosine inhibits aerobic glycolysis in glioblastoma cells by targeting PKM2 expression and activity.

Glioblastoma (GBM) is a primary tumor in the central nervous system with poor prognosis. It exhibits elevated glucose uptake and lactate production. This metabolic state of aerobic glycolysis is known as the Warburg effect. N6-isopentenyladenosine (iPA), a natural cytokine modified with an isopentenyl moiety derived from the mevalonate pathway, has well-established anti-tumor activity. It inhibits cell proliferation in glioma cells, inducing cell death by apoptosis and/or necroptosis. In the present study, we found that iPA inhibits aerobic glycolysis in unmodified U87MG cells and in the same cell line engineered to over-express wild-type epidermal growth factor receptor (EGFR) or EGFR variant III (vIII), as well as in a primary GBM4 patient-derived cell line. The detection of glycolysis showed that iPA treatment suppressed ATP and lactate production. We also evaluated the response of iPA treatment in normal human astrocyte primary cells, healthy counterpart cells of the brain. Aerobic glycolysis in treated normal human astrocyte cells did not show significant changes compared to GBM cells. To determine the mechanism of iPA action on aerobic glycolysis, we investigated the expression of certain enzymes involved in this metabolic pathway. We observed that iPA reduced the expression of pyruvate kinase M2 (PKM2), which plays a key role in the regulation of aerobic glycolysis, promoting tumor cell proliferation. The reduction of PKM2 expression is a result of the inhibition of the inhibitor of nuclear factor kappa-B kinase subunit, beta/nuclear factor-kappa B pathway upon iPA treatment. In conclusion, these experimental results show that iPA may inhibit aerobic glycolysis of GBM in stabilized cell lines and primary GBM cells by targeting the expression and activity of PKM2.

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.

Autoantibody and T cell responses to oxidative post-translationally modified insulin neoantigenic peptides in type 1 diabetes.

AIMS/HYPOTHESIS: Antibodies specific to oxidative post-translational modifications (oxPTM) of insulin (oxPTM-INS) are present in most individuals with type 1 diabetes, even before the clinical onset. However, the antigenic determinants of such response are still unknown. In this study, we investigated the antibody response to oxPTM-INS neoepitope peptides (oxPTM-INSPs) and evaluated their ability to stimulate humoral and T cell responses in type 1 diabetes. We also assessed the concordance between antibody and T cell responses to the oxPTM-INS neoantigenic peptides. METHODS: oxPTM-INS was generated by exposing insulin to various reactive oxidants. The insulin fragments resulting from oxPTM were fractionated by size-exclusion chromatography further to ELISA and LC-MS/MS analysis to identify the oxidised peptide neoepitopes. Immunogenic peptide candidates were produced and then modified in house or designed to incorporate in silico-oxidised amino acids during synthesis. Autoantibodies to the oxPTM-INSPs were tested by ELISA using sera from 63 participants with new-onset type 1 diabetes and 30 control participants. An additional 18 fresh blood samples from participants with recently diagnosed type 1 diabetes, five with established disease, and from 11 control participants were used to evaluate, in parallel, CD4+ and CD8+ T cell activation by oxPTM-INSPs. RESULTS: We observed antibody and T cell responses to three out of six LC-MS/MS-identified insulin peptide candidates: A:12-21 (SLYQLENYCN, native insulin peptide 3 [Nt-INSP-3]), B:11-30 (LVEALYLVCGERGFFYTPKT, Nt-INSP-4) and B:21-30 (ERGFFYTPKT, Nt-INSP-6). For Nt-INSP-4 and Nt-INSP-6, serum antibody binding was stronger in type 1 diabetes compared with healthy control participants (p≤0.02), with oxidised forms of ERGFFYTPKT, oxPTM-INSP-6 conferring the highest antibody binding (83% binders to peptide modified in house by hydroxyl radical [●OH] and >88% to in silico-oxidised peptide; p≤0.001 vs control participants). Nt-INSP-4 induced the strongest T cell stimulation in type 1 diabetes compared with control participants for both CD4+ (p<0.001) and CD8+ (p=0.049). CD4+ response to oxPTM-INSP-6 was also commoner in type 1 diabetes than in control participants (66.7% vs 27.3%; p=0.039). Among individuals with type 1 diabetes, the CD4+ response to oxPTM-INSP-6 was more frequent than to Nt-INSP-6 (66.7% vs 27.8%; p=0.045). Overall, 44.4% of patients showed a concordant autoimmune response to oxPTM-INSP involving simultaneously CD4+ and CD8+ T cells and autoantibodies. CONCLUSIONS/INTERPRETATION: Our findings support the concept that oxidative stress, and neoantigenic epitopes of insulin, may be involved in the immunopathogenesis of type 1 diabetes.

N6-Isopentenyladenosine Impairs Mitochondrial Metabolism through Inhibition of EGFR Translocation on Mitochondria in Glioblastoma Cells.

Glioblastoma multiforme (GBM) is the most aggressive malignant brain tumor and is poorly susceptible to cytotoxic therapies. Amplification of the epidermal growth factor receptor (EGFR) and deletion of exons 2 to 7, which generates EGFR variant III (vIII), are the most common molecular alterations of GBMs that contribute to the aggressiveness of the disease. Recently, it has been shown that EGFR/EGFRvIII-targeted inhibitors enhance mitochondrial translocation by causing mitochondrial accumulation of these receptors, promoting the tumor drug resistance; moreover, they negatively modulate intrinsic mitochondria-mediated apoptosis by sequestering PUMA, leading to impaired apoptotic response in GBM cells. N6-isopentenyladenosine (i6A or iPA), a cytokinin consisting of an adenosine linked to an isopentenyl group deriving from the mevalonate pathway, has antiproliferative effects on numerous tumor cells, including GBM cells, by inducing cell death in vitro and in vivo. Here, we observed that iPA inhibits the mitochondrial respiration in GBM cells by preventing the translocation of EGFR/EGFRvIII to the mitochondria and allowing PUMA to interact with them by promoting changes in mitochondrial activity, thus playing a critical role in cell death. Our findings clearly demonstrate that iPA interferes with mitochondrial bioenergetic capacity, providing a rationale for an effective strategy for treating GBM.

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.

Metabolomics, Lipidomics, and Immunometabolism.

Metabolomics, lipidomics, and the study of cellular metabolism are gaining increasing interest particularly in the field of immunology, since the activation and effector functions of immune cells are profoundly controlled by changes in cellular metabolic asset. Among the different techniques that can be used for the evaluation of cellular metabolism, the Seahorse Extracellular Flux Analyzer allows the real time measurement of both glycolytic and mitochondrial respiration pathways in cells of interest, through the assessment of extracellular acidification and oxygen consumption rate. Metabolomics, on the other hand, is the high-throughput analysis of metabolites, i.e., the substrates, intermediates, and products of cellular metabolism, starting from biofluids, cells or tissues. The metabolome does not include lipids as their properties are different from water-soluble metabolites and are classified under the lipidome. Lipidomics analysis allows the identification and quantification of lipid species. Metabolomics and lipidomics are currently performed with mass-spectrometry coupled with liquid or gas chromatography (LC-MS or GC-MS) and/or nuclear-magnetic resonance (NMR). Here we describe the protocol for the evaluation of metabolic rate, metabolomics, and lipidomics in T cells, examining the detailed experimental approaches.

Lipid homeostasis and mevalonate pathway in COVID-19: Basic concepts and potential therapeutic targets.

Despite encouraging progresses achieved in the management of viral diseases, efficient strategies to counteract infections are still required. The current global challenge highlighted the need to develop a rapid and cost-effective strategy to counteract the SARS-CoV-2 pandemic. Lipid metabolism plays a crucial role in viral infections. Viruses can use the host lipid machinery to support their life cycle and to impair the host immune response. The altered expression of mevalonate pathway-related genes, induced by several viruses, assures survival and spread in host tissue. In some infections, statins, HMG-CoA-reductase inhibitors, reduce cholesterol in the plasma membrane of permissive cells resulting in lower viral titers and failure to internalize the virus. Statins can also counteract viral infections through their immunomodulatory, anti-inflammatory and anti-thrombotic effects. Beyond statins, interfering with the mevalonate pathway could have an adjuvant effect in therapies aimed at mitigating endothelial dysfunction and deregulated inflammation in viral infection. In this review we depicted the historical and current evidence highlighting how lipid homeostasis and mevalonate pathway targeting represents a valid approach to rapidly neutralize viruses, focusing our attention to their potential use as effective targets to hinder SARS-CoV-2 morbidity and mortality. Pros and cons of statins and Mevalonate-pathway inhibitors have been also dissected.

Caloric Restriction Promotes Immunometabolic Reprogramming Leading to Protection from Tuberculosis.

There is a strong relationship between metabolic state and susceptibility to Mycobacterium tuberculosis (MTB) infection, with energy metabolism setting the basis for an exaggerated immuno-inflammatory response, which concurs with MTB pathogenesis. Herein, we show that controlled caloric restriction (CR), not leading to malnutrition, protects susceptible DBA/2 mice against pulmonary MTB infection by reducing bacterial load, lung immunopathology, and generation of foam cells, an MTB reservoir in lung granulomas. Mechanistically, CR induced a metabolic shift toward glycolysis, and decreased both fatty acid oxidation and mTOR activity associated with induction of autophagy in immune cells. An integrated multi-omics approach revealed a specific CR-induced metabolomic, transcriptomic, and proteomic signature leading to reduced lung damage and protective remodeling of lung interstitial tightness able to limit MTB spreading. Our data propose CR as a feasible immunometabolic manipulation to control MTB infection, and this approach offers an unexpected strategy to boost immunity against MTB.

Immunometabolism of regulatory T cells in cancer.

Regulatory T (Treg) cells are known to orchestrate the regulatory mechanisms aimed at suppressing pathological auto-reactive immune responses and are thus key in ensuring the maintenance of immune homeostasis. On the other hand, the presence of Treg cells with enhanced suppressive capability in a plethora of human cancers represents a major obstacle to an effective anti-cancer immune response. A relevant research effort has thus been dedicated to comprehend Treg cell biology, leading to a continuously refining characterization of their phenotype and function and unveiling the central role of metabolism in ensuring Treg cell fitness in cancer. Here we focus on how the peculiar biochemical characteristics of the tumor microenvironment actually support Treg cell metabolic activation and favor their selective survival and proliferation. Moreover, we examine the key metabolic pathways that may become useful targets of novel treatments directed at hampering tumor resident Treg cell proficiency, thus representing the next research frontier in cancer immunotherapy.

CD4+ T Cell Defects in a Mulibrey Patient With Specific TRIM37 Mutations.

Mulibrey (muscle-liver-brain-eye) syndrome (MUL) is an autosomal recessive disorder caused by mutations in the TRIpartite motif (TRIM)37 gene, encoding for TRIM37 a member of the TRIM E3 ubiquitin ligase protein family. MUL patients are characterized by growth retardation, dysmorphic features, and a wide range of abnormalities affecting different organs. However, T-cell abnormalities have not been observed in MUL subjects, to date. Here we described the immunological features of a MUL child carrying recently identified TRIM37 mutations, a 17q22 deletion of maternal origin combined with a TRIM37 variant of paternal origin. Here we found quantitative and functional defects in CD4+ T cells from this MUL case. Low levels of TRIM37 protein were specifically detected in CD4+ T cells of MUL patient and associated with their altered proliferation and cytokine production. Of note, both CD4+ and CD8+ T lymphocytes of MUL child displayed an effector memory phenotype compared with healthy children. This clinical case research highlighted the possible role of TRIM37 in the control of immune cell number and function, especially in CD4+ T cells. Finally, this study may contribute to the novel mechanistic studies aim of identifying, in depth, the role of the TRIM37 protein in the immune system.

Immunometabolism and autoimmunity.

Over the last few years, immune cell metabolism has become one of the most stimulating areas of investigation in the field of immunology. Compelling evidence has revealed that metabolic pathways are closely associated to cell functions and immune cells adopt defined metabolic programs to sustain their activity and respond to micro-environmental demands. It is now clear that alterations in cell metabolism can favour dysregulation typical of autoreactive immune cells, thus sustaining loss of immunological self-tolerance. In this short review, we highlight the main metabolic alterations associated with both innate and adaptive immune cells in autoimmune conditions, such as multiple sclerosis (MS) and type 1 diabetes (T1D). We also summarize recent findings reporting the use of pharmacological agents, which modulate the immunometabolism to possibly control immune responses during autoimmune disorders.

Plasma circulating miR-23~27~24 clusters correlate with the immunometabolic derangement and predict C-peptide loss in children with type 1 diabetes.

AIMS/HYPOTHESIS: We aimed to analyse the association between plasma circulating microRNAs (miRNAs) and the immunometabolic profile in children with type 1 diabetes and to identify a composite signature of miRNAs/immunometabolic factors able to predict type 1 diabetes progression. METHODS: Plasma samples were obtained from children at diagnosis of type 1 diabetes (n = 88) and at 12 (n = 32) and 24 (n = 30) months after disease onset and from healthy control children with similar sex and age distribution (n = 47). We quantified 60 robustly expressed plasma circulating miRNAs by quantitative RT-PCR and nine plasma immunometabolic factors with a recognised role at the interface of metabolic and immune alterations in type 1 diabetes. Based on fasting C-peptide loss over time, children with type 1 diabetes were stratified into the following groups: those who had lost >90% of C-peptide compared with diagnosis level; those who had lost <10% of C-peptide; those showing an intermediate C-peptide loss. To evaluate the modulation of plasma circulating miRNAs during the course of type 1 diabetes, logistic regression models were implemented and the correlation between miRNAs and immunometabolic factors was also assessed. Results were then validated in an independent cohort of children with recent-onset type 1 diabetes (n = 18). The prognostic value of the identified plasma signature was tested by a neural network-based model. RESULTS: Plasma circulating miR-23~27~24 clusters (miR-23a-3p, miR-23b-3p, miR-24-3p, miR-27a-3p and miR-27b-3p) were upmodulated upon type 1 diabetes progression, showed positive correlation with osteoprotegerin (OPG) and were negatively correlated with soluble CD40 ligand, resistin, myeloperoxidase and soluble TNF receptor in children with type 1 diabetes but not in healthy children. The combination of plasma circulating miR-23a-3p, miR-23b-3p, miR-24-3p, miR-27b-3p and OPG, quantified at disease onset, showed a significant capability to predict the decline in insulin secretion 12 months after disease diagnosis in two independent cohorts of children with type 1 diabetes. CONCLUSIONS/INTERPRETATIONS: We have pinpointed a novel miR-23a-3p/miR-23b-3p/miR-24-3p/miR-27b-3p/OPG plasma signature that may be developed into a novel blood-based method to better stratify patients with type 1 diabetes and predict C-peptide loss.

T1D progression is associated with loss of CD3+CD56+ regulatory T cells that control CD8+ T cell effector functions.

An unresolved issue in autoimmunity is the lack of surrogate biomarkers of immunological self-tolerance for disease monitoring. Here, we show that peripheral frequency of a regulatory T cell population, characterized by the co-expression of CD3 and CD56 molecules (TR3-56), is reduced in subjects with new-onset type 1 diabetes (T1D). In three independent T1D cohorts, we find that low frequency of circulating TR3-56 cells is associated with reduced ß-cell function and with the presence of diabetic ketoacidosis. As autoreactive CD8+ T cells mediate disruption of insulin-producing ß-cells1-3, we demonstrate that TR3-56 cells can suppress CD8+ T cell functions in vitro by reducing levels of intracellular reactive oxygen species. The suppressive function, phenotype and transcriptional signature of TR3-56 cells are also altered in T1D children. Together, our findings indicate that TR3-56 cells constitute a regulatory cell population that controls CD8+ effector functions, whose peripheral frequency may represent a traceable biomarker for monitoring immunological self-tolerance in T1D.

Correction: miR-27a is a master regulator of metabolic reprogramming and chemoresistance in colorectal cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.