Innate immune responses depend on diverse cell subsets whose frequencies and functions can shift rapidly during infection or inflammation. We developed a high-parameter flow cytometry platform that uses <1 mL of fresh whole blood to identify multiple circulating innate cell subsets and to measure, in each subset, intracellular cytokines and chemokines after stimulation with lipopolysaccharide (LPS; bacterial mimic) or R848 (resiquimod; viral mimic). The platform operates on less than 1 mL of fresh whole blood, preserving mononuclear phagocytes and avoiding density-gradient cell separation, with phenotypic and functional readouts integrated into a single acquisition. Because several canonical lineage markers, most notably CD16, are rapidly downmodulated upon activation, the gating strategy relies instead on markers whose expression remains stable under stimulation, allowing nonclassical monocytes and other subsets to be tracked reliably across resting and activated conditions. Combining lineage exclusion with these stable markers, we resolve plasmacytoid and conventional dendritic cells, classical, intermediate, and nonclassical monocytes, and additional minor subsets including inflammatory dendritic cells. LPS elicits broad TNF, IL-6, and IL-1β responses with subset-specific magnitudes, whereas R848 preferentially induces type I interferon programs alongside pro-inflammatory cytokines in selected lineages. The same gating framework was further validated in PBMCs challenged with intact UV-inactivated bacterial and fungal preparations, demonstrating portability across sample matrices. This compact, scalable assay enables precise monitoring of both subset composition and effector functions from scarce clinical samples, supporting translational studies in infection, autoimmunity, and immunotherapy where blood volume and turnaround time are limited.

Progressive supranuclear palsy (PSP) is a movement neurodegenerative disorder characterized by the primary aggregation of Tau. Although several studies using experimental models of tauopathies have significantly contributed to our understanding of Tau pathology, no animal models currently recapitulate the key cytopathological features of the disease. Primary dermal fibroblasts from 7 PSP patients and 4 healthy donors were collected, and several features were investigated to find promising biological patterns. We found that fibroblasts from PSP patients showed a slower proliferation rate, an increased expression of p16/p21, two cell cycle checkpoints, and an increase in the number of G3BP1-positive stress granules’ association with Tau. Furthermore, we observed an altered mitochondrial homeostasis and a clear differential clustering of their proteome. Our findings suggest that cell cycle dynamics are altered in PSP and highlight, at least in primary fibroblasts, an increase in mitochondrial function, which could be interpreted as a coping strategy for a diseased cell. In line with other studies, these findings reinforce fibroblasts as a suitable platform for investigating neurodegenerative diseases and a reliable patient-derived model for studying PSP pathological features and testing druggable compounds. Furthermore, they pinpoint cell cycle alterations and senescence as a facet that needs extra consideration in PSP.

OBJECTIVE:Multiple sclerosis (MS) is a chronic autoimmune disease where B cells play a central pathogenic role. Cladribine, an oral therapy, provides durable benefits by reshaping lymphocyte populations, yet its specific long-term impact on distinct B-cell subsets is not fully understood. This study aimed to define cladribine's longitudinal effects on B-cell subsets and characterize their inflammatory properties. METHODS:We conducted a 48-month longitudinal study of 36 persons with relapsing-remitting MS treated with cladribine who had completed 2 annual treatment cycles, using high-parameter flow cytometry to track B-cell subset dynamics. In a parallel cross-sectional analysis, B cells from 16 untreated patients and 16 healthy donors were profiled for polyfunctional cytokine production granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and key surface markers. RESULTS:Cladribine induced profound, sustained depletion of memory B cells, with marginal zone-like (MZ-like) cells showing the deepest and most persistent loss, alongside reconstitution of transitional and naïve populations. Functionally, MZ-like B cells were the most potent polyfunctional producers of proinflammatory cytokines and immunoglobulin M (IgM), a capacity significantly enhanced in persons with MS compared to healthy donors, and expressed the highest levels of CD1c and CD1d, consistent with specialization in lipid antigen presentation. INTERPRETATION:MZ-like B cells are a dominant, polyfunctional inflammatory subset in MS, equipped for non-canonical lipid antigen presentation and IgM secretion. They represent a preferential pharmacological target of cladribine. Sustained reduction of this pathogenic memory pool, followed by repopulation with less inflammatory B cells, offers a mechanistic basis for cladribine's durable clinical benefit and nominates MZ-like cells as biomarkers of response and therapeutic targets. ANN NEUROL 2026.

BACKGROUND AND AIMS:Autoimmune hepatitis (AIH) and primary biliary cholangitis (PBC) are distinct autoimmune liver diseases (AILDs) that differ in pathogenesis and response to therapy. Moreover, some patients exhibit features of both, known as AIH/PBC overlap. Although AILDs are traditionally considered T cell-driven, B cells have emerged as key contributors to disease progression. In this study, we investigated CD11c+T-bet+ B cells, implicated in different autoimmune diseases and never explored in AILDs. METHODS:Circulating peripheral blood mononuclear cells (PBMC) were isolated from whole blood obtained from patients with AIH (n = 15), PBC (n = 8), AIH/PBC overlap syndrome (n = 11), and healthy donors (HD, n = 12). B and T cell subsets were analysed by conventional and spectral flow-cytometry. Clinical and laboratory parameters were recorded at the time of sample collection, and serum IL-21, sCD40L, and BAFF levels were measured by ELISA and by flow cytometry. RESULTS:Total CD19+ B-cell frequencies were comparable across groups. However, the canonical atypical B-cell subset defined as CD11c+T-bet+CD21- was significantly expanded in AIH and AIH/PBC patients but not in PBC. Phenotypic characterisation showed that these cells were enriched in double-negative and naïve-like B-cell subsets and displayed the expected chemokine receptor profile of atypical B cells, with higher CXCR3 and lower CXCR5 expression compared with conventional B cells. Circulating follicular helper T cells (cTfh) were increased in AIH and AIH/PBC and correlated with the overall T-bet+ B-cell compartment. Serum IL-21 levels were higher in patients with AILDs, particularly in treatment non-responders. CONCLUSIONS:Expansion of atypical CD11c+T-bet+ B cells, together with increased cTfh frequencies, hallmarks AIH and AIH/PBC. These findings support disease-specific B-cell alterations and distinct immune signatures in AIH and AIH/PBC compared with PBC, highlighting potential therapeutic implications for targeting pathogenic B cell subsets in AIH.

Smaller midbrain volumes predict Alzheimer’s Disease (AD) progression and faster conversion from Mild Cognitive Impairment (MCI) to dementia. Along with this, various midbrain-target areas are characterized by neuroinflammation since the MCI stage. The concomitance of neuroinflammation, Αβ and tau appears to be a strong predictor for conversion from MCI to dementia. Yet, how midbrain degeneration could cause disease progression, and what mechanisms are involved in triggering neuroinflammation in midbrain-target areas such as the hippocampus remain unexplored. Using adult C57BL/6N mice we generated a new mouse model carrying lesions in three midbrain nuclei, the dopaminergic Ventral Tegmental Area (VTA) and Substantia Nigra pars compacta (SNpc) and the serotonergic Interpeduncular Nucleus (IPN), to evaluate the consequences of dopamine and serotonin deprivation in midbrain-target areas. We characterized this model by performing stereological cell counts, analysis of monoaminergic fibers, monoamine levels, electrophysiology and behavioral tests. We then assessed hippocampal neuroinflammation by analyzing glia cell count, changes in morphology, NLRP3 inflammasome activation and cytokine levels, and microglia transcriptional profiling. In a separate set of experiments, we induced experimental midbrain lesion in Tg2576 transgenic mice overexpressing the Swedish mutant amyloid precursor protein, to evaluate the effect of monoamine deprivation on the hippocampus in concomitance with amyloid-β (Aβ) accumulation. The lesion performed in Tg2576 mice, as opposed to that in C57BL/6N mice, provides valuable insights into how neuroinflammation is influenced by Aβ accumulation versus the exclusive impact of impaired monoaminergic signaling. The concomitant depletion of dopaminergic and serotonergic inputs within the hippocampus of C57BL/6N mice provokes a pronounced activation of microglia via the NLRP3-inflammasome pathway, accompanied by increased IL-1β expression. Pharmacological intervention with either dopaminergic (L-DOPA or A68930) or serotonergic (fluoxetine) agents abrogates this neuroinflammatory response. In the Tg2576 transgenic mouse model of amyloid pathology, which exhibits progressive Aβ deposition, superimposed midbrain degeneration markedly amplifies AD-like neuropathology. This includes exacerbation of microglial reactivity, robust astrocyte response, precocious Aβ plaque burden, and induction of pathological tau hyperphosphorylation. Notably, administration of L-DOPA or fluoxetine significantly attenuates both the astrocyte reactivity and tau hyperphosphorylation in the lesioned Tg2576 cohort. These results highlight the pivotal role of midbrain damage for the amplification of neuroinflammatory cascades and AD pathology. Moreover, they offer mechanistic insight into the faster progression to dementia in patients with midbrain deficits. By translating these findings into clinical practice, we can advance towards a precision medicine approach in disease management.

IntroductionAcute COVID-19 infection causes significant alterations in the innate and adaptive immune systems. While most individuals recover naturally, some develop long COVID (LC) syndrome, marked by persistent or new symptoms weeks to months after SARS-CoV-2 infection. Despite its prevalence, there are no clinical tests to distinguish LC patients from those fully recovered. Understanding the immunological basis of LC is essential for improving diagnostic and treatment approaches.MethodsWe performed deep immunophenotyping and functional assays to examine the immunological profiles of LC patients, individuals with active COVID-19, recovered patients, and healthy donors. This analysis assessed both innate and adaptive immune features, identifying potential biomarkers for LC syndrome. A Binomial Generalized Linear Model (BGLM) was used to pinpoint immune features characterizing LC.ResultsCOVID-19 patients exhibited depletion of innate immune cell subsets, including plasmacytoid and conventional dendritic cells, classical, non-classical, and intermediate monocytes, and monocyte-derived inflammatory dendritic cells. Elevated basal inflammation was observed in COVID-19 patients compared to LC patients, whose immune profiles were closer to those of healthy donors and recovered individuals. However, LC patients displayed persistent immune alterations, including reduced T cell subsets (CD4, CD8, Tregs) and switched memory B cells, similar to COVID-19 patients. Through BGLM, a unique adaptive immune signature for LC was identified, featuring memory CD8 and gd T cells with low proliferative capacity and diminished expression of activation and homing receptors.DiscussionThe findings highlight a unique immunological signature associated with LC syndrome, characterized by persistent adaptive immune dysregulation. While LC patients displayed recovery in innate immune profiles comparable to healthy and Recovered individuals, deficits in T cell and memory B cell populations were evident, differentiating LC from full recovery. These findings provide insights into LC pathogenesis and may support the development of diagnostic tools and targeted therapies.

Abstract Background Immune-inflammatory mechanisms are promising targets for antidepressant pharmacology [1-3]. Based on reported immune cell abnormalities, we defined an antidepressant potentiation treatment with add-on low-dose interleukin 2 (IL-2), a T-cell growth factor of proven anti-inflammatory efficacy in autoimmune conditions, increasing thymic production of naï ve CD4+ T cells [4-6], and possibly correcting the partial T cell defect observed in mood disorders [7, 8]. Aims & Objectives We performed a single-center, randomised, double-blind, placebo-controlled phase II trial in depressed patients with major depressive or bipolar disorder administering low-dose IL-2 (aldesleukin). The primary endpoint was the change in the relative concentration of peripheral blood Tregs (CD3+CD4+FoxP3+CD127loCD25hi Treg). Secondary endpoints were changes in Th1 and Th2, Naï ve T cells, Central memory T cells; safety and antidepressant efficacy of aldesleukin at day 36 (end of treatment) and at day 60 (end of follow up). Methods Thirty-six consecutively recruited inpatients at the Mood Disorder Unit at San Raffaele Hospital in Milan, Italy, were randomised in a 2:1 ratio to receive either aldesleukin (12 MDD and 12 BD) or placebo (6 MDD and 6 BD). The treatment (either IL-2 or placebo) was administered daily for the first 5 days (induction phase) and weekly for the following 4 weeks (maintenance phase). Clinical evaluations (Montgomery-Å sberg Depression Rating Scale (MADRS), Inventory for Depressive Symptomatology Self-Rated (IDS-SR)) have been performed concurrently with each injection; blood samples have been collected at baseline, after 5 days and at week 60 and analysed through flow cytometry with a 28-color flow cytometry panel. Results Twenty-eight out of 36 enrolled patients, concluded the study (10 Placebo, 6 BD and 4 MDD) whereas all patients completed the induction phase. Low-dose IL-2 significantly potentiated antidepressant response to ongoing SSRI/SNRI treatment in both diagnostic groups as measured by a reduction in both MDRS and IDS-SR scores, and expanded the population of Treg, Th2, and Naive CD4+/CD8+ immune cell counts. Changes in cell counts were rapidly induced in the first five days of treatment, and predicted the later improvement of depression severity. No serious adverse effect was observed. Discussion & Conclusion This is the first RCT evidence supporting the hypothesis that a treatment able to strengthen the T cell system could be a successful way to correct the immuno-inflammatory abnormalities associated with mood disorders, and potentiate antidepressant response. Indeed, the main effect observed of IL-2 was to promote Tregs expansion and enhance response at 5 weeks of treatment preventing the subsequent relapse, thus promoting and consolidating improvement over time. References 1.Benedetti, F. and B. Vai, New biomarkers in mood disorders: Insights from immunopsychiatry and neuroimaging. European Neuropsychopharmacology: the Journal of the European College of Neuropsychopharmacology, 2023. 69: p. 56-57. 2.Benedetti, F., R. Zanardi, and M.G. Mazza, Antidepressant psychopharmacology: is inflammation a future target? International clinical psychopharmacology, 2022. 37(3): p. 79-81. 3.Branchi, I., et al., Brain-immune crosstalk in the treatment of major depressive disorder. European Neuropsychopharmacology, 2021. 45: p. 89-107. 4.Klatzmann, D. and A.K. Abbas, The promise of low-dose interleukin-2 therapy for autoimmune and inflammatory diseases. Nat Rev Immunol, 2015. 15(5): p. 283-94. 5.Rosenzwajg, M., et al., Immunological and clinical effects of low-dose interleukin-2 across 11 autoimmune diseases in a single, open clinical trial. Annals of the rheumatic diseases, 2019. 78(2): p. 209-217. 6.Graß hoff, H., et al., Low-dose IL-2 therapy in autoimmune and rheumatic diseases. Frontiers in immunology, 2021. 12: p. 648408. 7.Simon, M.S., et al., Monocyte mitochondrial dysfunction, inflammaging, and inflammatory pyroptosis in major depression. Progress in Neuro-Psychopharmacology and Biological Psychiatry, 2021. 111: p. 110391. 8.Simon, M.S., et al., Premature T cell aging in major depression: A double hit by the state of disease and cytomegalovirus infection. Brain, Behavior, &Immunity-Health, 2023. 29: p. 100608.

Introduction Affective symptoms are prevalent features in people with multiple sclerosis (PwMS). Structural brain changes, as well as cytokine-driven synaptic and network alterations, might contribute to the development of these clinical features. In the present study, we evaluated the association between the cerebrospinal fluid (CSF) levels of the pro-inflammatory cytokine interferon-gamma (IFN-gamma) at multiple sclerosis (MS) diagnosis and the subsequent development of depression and anxiety. Methods PwMS and a negative history for psychiatric disorders were recruited at MS onset. CSF IFN-gamma concentrations were measured by Single Molecule Array. Socio-demographic and clinical information was collected, and all subjects underwent brain magnetic resonance imaging (MRI). After a mean follow-up time of 3 years, psychometric assessment was performed. Depressive symptoms were evaluated with the Beck Depression Inventory II, while the State and Trait Anxiety Inventory Y was used to assess anxiety symptoms. Results In our sample (n = 28), 21.4 % subjects suffered from clinically significant depression at follow-up, while the prevalence raised at 51.7 % for both state and trait anxiety. Subjects with clinically relevant depression presented greater disability levels at baseline (p = 0.020), as well as more severe state (p = 0.010) and trait (p < 0.001) anxiety at follow-up. Baseline IFN-gamma concentrations were significantly higher in subjects who displayed clinically significant depression at follow-up (p = 0.037), an association that was not replicated for state and trait anxiety. No significant associations were found between brain MRI measures and psychiatric symptoms at follow-up. Conclusions Proinflammatory cytokines might play a relevant role in the development of affective symptoms, particularly depressive, in PwMS. Their possible predictive value deserves further attention, possibly helping the early detection of at-risk subjects and the implementation of tailored treatments.

Previous exposure to Epstein–Barr virus (EBV) is strongly associated with the development of multiple sclerosis (MS). By contrast, past cytomegalovirus (CMV) infection may have no association, or be negatively associated with MS. This study aimed to investigate the associations of herpesvirus infections with MS in an Italian population. Serum samples (n = 200) from Italian people with multiple sclerosis (PwMS) classified as the relapsing-and-remitting clinical phenotype and (n = 137) healthy controls (HCs) were obtained from the CRESM Biobank, Orbassano, Italy. Both PwMS and HCs samples were selected according to age group (20–39 years, and 40 or more years) and sex. EBV virus capsid antigen (VCA) IgG, EBV nucleic acid-1 antigen (EBNA-1) IgG, CMV IgG, herpes simplex virus (HSV) IgG, and varicella zoster virus (VZV) IgG testing was undertaken using commercial ELISAs. EBV VCA IgG and EBNA-1 IgG seroprevalences were 100% in PwMS and 93.4% and 92.4%, respectively, in HCs. EBV VCA IgG and EBNA-1 IgG levels were higher (p < 0.001) in PwMS compared with HCs. For PwMS, the EBNA-1 IgG levels decreased with age, particularly in females. The CMV IgG seroprevalence was 58.7% in PwMS and 62.9% in HCs. CMV IgG seroprevalence increased with age. The HSV IgG seroprevalence was 71.2% in PwMS and 70.8% in HCs. HSV IgG levels were lower (p = 0.0005) in PwMS compared with HCs. VZV IgG seroprevalence was 97.5% in PwMS and 98.5% in HCs. In the population studied, several herpesvirus infections markers may have been influenced by the age and sex of the groups studied. The lack of a negative association of MS with CMV infection, and the observation of lower levels of HSV IgG in PwMS compared with HCs are findings worthy of further investigation.

Limited information exists regarding the impact of interferons (IFNs) on the information carried by extracellular vesicles (EVs). This study aimed at investigating whether IFN-α2b, IFN-β, IFN-γ, and IFN-λ1/2 modulate the content of EVs released by primary monocyte-derived macrophages (MDM).Small-EVs (sEVs) were purified by size exclusion chromatography from supernatants of MDM treated with IFNs. To characterize the concentration and dimensions of vesicles, Nanoparticle Tracking Analysis was used. SEVs surface markers were examined by flow cytometry.IFN treatments induced a significant down-regulation of the exosomal markers CD9, CD63, and CD81 on sEVs, and a significant modulation of some adhesion molecules, major histocompatibility complexes and pro-coagulant proteins, suggesting IFNs influence biogenesis and shape the immunological asset of sEVs. SEVs released by IFN-stimulated MDM also impact lymphocyte function, showing significant modulation of lymphocyte activation and IL-17 release.Altogether, our results show that sEVs composition and activity are affected by IFN treatment of MDM.

Immune-inflammatory mechanisms are promising targets for antidepressant pharmacology. Immune cell abnormalities have been reported in mood disorders showing a partial T cell defect. Following this line of reasoning we defined an antidepressant potentiation treatment with add-on low-dose interleukin 2 (IL-2). IL-2 is a T-cell growth factor which has proven anti-inflammatory efficacy in autoimmune conditions, increasing thymic production of naïve CD4 + T cells, and possibly correcting the partial T cell defect observed in mood disorders. We performed a single-center, randomised, double-blind, placebo-controlled phase II trial evaluating the safety, clinical efficacy and biological responses of low-dose IL-2 in depressed patients with major depressive (MDD) or bipolar disorder (BD). 36 consecutively recruited inpatients at the Mood Disorder Unit were randomised in a 2:1 ratio to receive either aldesleukin (12 MDD and 12 BD) or placebo (6 MDD and 6 BD). Active treatment significantly potentiated antidepressant response to ongoing SSRI/SNRI treatment in both diagnostic groups, and expanded the population of T regulatory, T helper 2, and percentage of Naive CD4+/CD8 + immune cells. Changes in cell frequences were rapidly induced in the first five days of treatment, and predicted the later improvement of depression severity. No serious adverse effect was observed. This is the first randomised control trial (RCT) evidence supporting the hypothesis that treatment to strengthen the T cell system could be a successful way to correct the immuno-inflammatory abnormalities associated with mood disorders, and potentiate antidepressant response.