Tregs maintain immune tolerance through mechanisms tightly coupled to cellular metabolism. Whereas glycolysis supports migration of Tregs, lipid metabolism sustains their suppressive phenotype. Here, we identify SREBP1c as a central regulator of Treg immunobiology. Tregs from Srebp1c-deficient mice displayed impaired suppressive function, reduced frequencies in circulation and lymphoid tissues, and diminished expression of functional markers. These defects stemmed from intrinsic metabolic rewiring rather than systemic alterations, as both ex vivo Tregs (CD4+CD25hiFoxP3+) and in vitro-derived Tregs lacking Srebp1c were shifted toward glycolysis. Integrated transcriptomic and lipidomic analyses revealed that Srebp1c-deficient Tregs exhibited defective phospholipid remodeling, with an accumulation of lysophosphatidylcholines over phosphatidylcholines, which we attributed to enhanced cytosolic phospholipase A2 (cPLA2α) activity and disruption of the Lands cycle. Altered lipid composition impaired adenosine-mediated immunosuppression by reducing CD73 expression and extracellular adenosine generation. Accordingly, pharmacological inhibition of cPLA2α restored adenosine signaling, CD73 expression, and Treg suppressive capacity. Thus, by preserving phospholipid homeostasis, SREBP1c functions as an immunometabolic checkpoint that links lipid metabolism to adenosine-dependent Treg suppression.

BACKGROUND AND OBJECTIVES:Fc gamma receptor 3A (FCGR3A) V158F polymorphism has been shown to modify the response to anti-CD20 therapy across several autoimmune diseases. Ocrelizumab (OCR), an anti-CD20 agent, suppresses inflammatory activity in multiple sclerosis (MS), yet whether FCGR3A V158F polymorphism affects its efficacy in MS remains unclear. Here, we tested whether this genetic variant influences B-cell repopulation and disease activity in MS participants treated with OCR and assessed genotype-dependent differences in OCR binding to FcγRIIIa-expressing natural killer (NK) cells. METHODS:In this observational cohort study, we enrolled people with MS treated with OCR consecutively between May 2022 and August 2025. FCGR3A V158F genotyping was performed by pyrosequencing. The primary outcome was preinfusion CD19+ B-cell repopulation, defined as CD19+ B cells ≥1%. Secondary outcomes included clinical and MRI inflammatory activity and composite disease activity/disability-worsening measures. In a parallel mechanistic ex vivo substudy, OCR or rituximab (RTX) binding to NK cells was evaluated in genotype-selected donors by flow cytometry. Cycle-based repeated measures were analyzed using mixed-effects logistic regression. RESULTS:In 101 participants, 500 interinfusion intervals were analyzed. The odds of B-cell repopulation decreased with higher cycle number (odds ratio [OR] per cycle 0.77; 95% CI 0.65-0.91; p = 0.002) and increased with longer infusion intervals (OR per +30 days, 2.02; 95% CI 1.27-3.21; p = 0.0029). FCGR3A F-carrier status significantly modified the effect of interval length (interaction OR, 2.47; 95% CI 1.04-5.89; p = 0.042). Specifically, the odds of B-cell repopulation increased with longer intervals in MS participants carrying the FCGR3A-F allele (OR, 3.67; 95% CI 1.84-7.34; p = 0.00023) but not in FCGR3A-VV individuals (OR, 1.49; 95% CI 0.87-2.54; p = 0.146). FCGR3A genotype was not associated with clinical or MRI activity outcomes. In ex vivo assays, NK cells from FCGR3A-FF donors exhibited significantly lower binding of OCR (p = 4.34 × 10-4) and RTX (p = 0.00172) as compared with VV donors. DISCUSSION:Longer OCR infusion intervals were associated with higher odds of B-cell repopulation. The FCGR3A V158F polymorphism modified this interval-dependent repopulation, possibly by affecting OCR binding to NK cells. Prospective studies are needed to determine whether FCGR3A V158F polymorphism and B-cell repletion kinetics can inform optimized interval-based OCR dosing in MS.

Physical exercise (PE) exerts beneficial effects in people with multiple sclerosis (pwMS). Preclinical studies in mice with experimental autoimmune encephalomyelitis (EAE), an animal model of MS, indicate that PE may attenuate key pathological features of the disease, including immune dysregulation and inflammation-driven synaptotoxicity, although the underlying mechanisms remain unclear. Clinical evidence, however, is still limited and fragmented, leaving the disease-modifying potential of PE in MS largely unresolved. Here, we investigated the impact of PE on T cell immunometabolic function and its downstream consequences on synaptotoxicity in both EAE mice and progressive MS (PMS) subjects, also assessing the contribution of vagal innervation to PE-mediated effects in the EAE model. Specifically, we found that PE improved EAE clinical course, by mitigating neuronal damage and modulating peripheral T cell proliferation, activation, and metabolic activity. These beneficial effects were partially blunted by preventive cervical vagotomy, suggesting a role for vagal integrity in mediating PE-driven neuroimmune modulation. In PMS subjects, a structured PE program improved clinical functional outcomes and enhanced mitochondrial respiration in peripheral T cells. Moreover, patch-clamp recordings revealed that glutamatergic synaptotoxicity induced by PMS-derived T cells was abolished following PE. Together, these findings highlight the therapeutic potential and disease-modifying value of PE in MS and suggest the vagal pathway as a key modulator of exercise-induced neuroimmune benefits.

Since the onset of the COVID-19 pandemic, the World Health Organization has recommended vaccination for people with multiple sclerosis (pwMS). However, vaccine-induced immune responses in pwMS receiving immunomodulatory therapies are currently poorly characterized. Herein, we evaluated humoral and T cell-mediated immune responses against BNT162b2 mRNA vaccine in pwMS treated with cladribine (n = 30), focusing on how the time interval between drug administration and vaccination affects the magnitude and dynamics of vaccine-induced immune response. We found that cladribine influenced both B and T cell frequencies with different kinetics over time. Accordingly, vaccination after cladribine administration resulted in reduced antibody production but preserved T cell responses, whereas pwMS receiving vaccine before cladribine exhibited an effective humoral immunity but hampered SARS-CoV-2 Spike-specific T cell response. Our findings may inform the clinical decision-making process by providing evidence to support the identification of an appropriate time window for vaccination during cladribine treatment.

CD4+CD25hiFoxP3+ regulatory T cells (Treg cells) are key controllers of immune self-tolerance, and their suppressive function is impaired in people with relapsing-remitting multiple sclerosis (pwRR-MS). Because the mechanisms underlying this condition are still ill-defined, we investigated the role of Treg cell-derived extracellular vesicles (Treg-EVs) in Treg cell dysfunction observed in pwRR-MS. We found that Treg-EVs from healthy individuals inhibit CD4+ conventional T (Tconv) cells by shuttling miR-142-3p from the Treg cell to the Tconv cell. There, miR-142-3p down-regulated mRNAs necessary for Tconv cell growth and effector functions, such as the redox controller cystine carrier SLC7A11. However, Treg cells from pwRR-MS released EVs containing reduced amounts of miR-142-3p, resulting in impaired suppressive function. Furthermore, Treg-EV miR-142-3p inversely correlated with the disability score and gadolinium-enhancing lesions in pwRR-MS. Together, our results elucidate a molecular mechanism involving miR-142-3p shuttled by Treg-EVs in the control of immune self-tolerance and unveil its pathogenetic implications in human autoimmunity.

Autophagy is a highly conserved cellular catabolic process recognized as an essential pathway for the maintenance of cellular homeostasis. Growing evidence implicates autophagic dysfunction in the pathogenesis of several neurodegenerative disorders, including Alzheimer's disease (AD), thus its modulation might represent an interesting therapeutic tool. Searching for a compound that stimulates autophagic pathway, led us to identify the inhibitor of RPSA receptor, NSC47924. In this study, we show that, NSC47924 down-modulated Akt-mTOR-axis pathway, the master regulator of autophagy, which was abnormally hyperactivated in fibroblasts from genetic AD-affected patients. Consistently, by monitoring the conversion of LC3, we found that inhibition of RPSA enhanced and restored the compromised autophagic flux. Moreover, by qRT-PCR analysis we found that inhibitor treatment upregulated the expression of autophagy-linked genes. Importantly, AD-affected fibroblasts exhibited massive mitochondrial network fragmentation and mitophagy defects, which were restored through the stimulation of autophagy induced by RPSA inhibition. Consistent with an efficient elimination of dysfunctional mitochondria, we found that the turnover of both the mitophagy regulators PINK1 and Parkin and the autophagic receptors p62, NDP52, OPTN, was modulated, thus restoring a highly interconnected organelle's network. In addition, the improvement of mitochondrial morphology correlated with a functional recovery, as assessed by Seahorse analysis and mitochondrial ROS production evaluation. Collectively, our findings suggest that RPSA inhibition stimulates an autophagic pathway promoting the efficient removal of damaged mitochondria, favouring the recovery of cellular homeostasis, and counteracting crucial AD pathogenic mechanisms.

Maturation of hematopoietic stem and progenitor cells (HSPCs) in the bone marrow (BM) and of T lymphocytes in the thymus occurs within stromal regions innervated by noradrenergic fibers of the sympathetic nervous system (SNS). However, the neuronal pathways governing noradrenergic activity in lymphoid organs remain largely unexplored. In experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis (MS), we demonstrated that noradrenergic signals promote myeloid hematopoiesis in the BM and regulate the intra-thymic frequency of regulatory T lymphocytes via β-3 adrenergic receptors (B3ARs). We further showed that B3ARs in the BM and thymus are controlled by hypothalamic neurons expressing agouti-related protein (AgRP), which are dysfunctional in EAE. Notably, elevated serum levels of AgRP correlate with disease severity and magnetic resonance imaging markers of neuroinflammation in people with MS. These findings reveal a mechanism of immune regulation mediated by noradrenergic transmission, offering potential therapeutic targets for immune-mediated diseases.

BACKGROUND AND OBJECTIVES:Immune reconstitution therapies for multiple sclerosis (MS) are based on selective lymphocyte reduction, followed by repopulation and rescue of immune tolerance. Among these therapies, cladribine is an adenosine analog that interferes with cell division and depletes several lymphocyte subtypes. Regulatory T cells (Tregs), physiologically devoted to immune suppression, are dysfunctional in the context of MS. In this study, we explored the effects of cladribine on Treg dynamics and phenotype. METHODS:In vivo, deep immunophenotyping was conducted on peripheral blood of patients with MS (n = 11), longitudinally collected before and after 6 and 12 months of cladribine therapy. In vitro, expanded Tregs were treated with cladribine and analyzed for their phenotypic, molecular, and metabolic profiles. RESULTS:In vivo, Tregs were overall less sensitive than conventional T cells (Tconvs) to the depleting effects of cladribine. This phenomenon was particularly evident in the subset of the resting (rest) Tregs. At baseline, while activated (act) Tregs presented markers of proliferation, senescence, and survival, restTregs highly expressed the antiapoptotic protein Bcl2 and the quiescence marker Bach2. In vitro, cladribine strongly reduced Treg viability while inducing a program of senescence and dysfunction and compromising their metabolic fitness. When Treg dynamics were analyzed ex vivo in relation to neuroinflammation and response to therapy, restTregs exhibited resistance to depletion in nonresponders, in association with increasing expression of Bcl2. DISCUSSION:These results indicate that the efficacy of cladribine therapy may require reduction and repopulation of the Treg compartment, an event that may be hindered by restTreg resistance, which is supported by antiapoptotic signals.

ABSTRACT Objective Despite the availability of effective therapies for Multiple Sclerosis (MS), the unpredictable nature of disease progression and the variability in individual treatment outcomes call for reliable biomarkers. This pilot study aims to investigate the potential of plasma circulating microRNAs (miRNAs) as predictive biomarkers for clinical responses to dimethyl fumarate (DMF), a widely used oral treatment for MS. Methods Peripheral blood samples were collected from nineteen treatment‐naïve people with relapsing–remitting MS (pwRRMS) before and after 3, 6, 12, and 24 months of DMF administration, as well as from nineteen healthy individuals. MiRNAs were quantified by RT‐qPCR after plasma RNA extraction, and peripheral blood immune cells were analyzed by flow cytometry. Pathway enrichment and protein–protein interaction analyses were performed to identify the biological processes and molecular networks associated with mRNAs targeted by the specific DMF‐modulated miRNAs. Results We identified a DMF‐modulated miRNA signature with significant changes occurring at early treatment stages. Notably, specific miRNAs were correlated with both clinical and immunological outcomes upon DMF treatment, including lymphocyte count reduction (let‐7b‐5p and miR‐223‐3p) and disease progression over 2 years (miR‐223‐3p, miR‐23a‐3p, miR‐23b‐3p, miR‐27a‐3p, and miR‐27b‐3p), suggesting their potential as predictive biomarkers for treatment response. Moreover, the validated mRNA targets of DMF‐modulated miRNAs were enriched for IL‐6 signaling and NRF2‐dependent antioxidant pathways, highlighting the potential molecular mechanisms underpinning DMF efficacy. Interpretation This small exploratory study underscores the potential of plasma circulating miRNAs as candidate biomarkers for predicting therapeutic outcomes in MS and it calls for validation in larger studies, which may enhance our understanding of disease pathophysiology and offer a promising tool for personalized treatment strategies.

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.

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.

AbstractExtracellular vesicles (EVs) are released by all cells and contribute to cell‐to‐cell communication. The capacity of EVs to target specific cells and to efficiently deliver a composite profile of functional molecules have led researchers around the world to hypothesize their potential as therapeutics. While studies of EV treatment in animal models are numerous, their actual clinical benefit in humans has more slowly started to be tested. In this scoping review, we searched PubMed and other databases up to 31 December 2023 and, starting from 13,567 records, we selected 40 pertinent published studies testing EVs as therapeutics in humans.The analysis of those 40 studies shows that they are all small pilot trials with a large heterogeneity in terms of administration route and target disease. Moreover, the absence of a placebo control in most of the studies, the predominant local application of EV formulations and the inconsistent administration dose metric still impede comparison across studies and firm conclusions about EV safety and efficacy. On the other hand, the recording of some promising outcomes strongly calls out for well‐designed larger studies to test EVs as an alternative approach to treat human diseases with no or few therapeutic options.

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.