Immunology

BackgroundImmunoglobulin E (IgE) plays a fundamental role in the pathogenesis of allergic disease, including asthma. The IgE-producing plasma cells (PCs) are thought to persist indefinitely, providing a sustained source of allergen-specific IgE. Although these cells can accumulate in the bone marrow (BM), after prolonged allergen exposure, their frequency remains remarkably low, and the mechanisms that regulate their migration are poorly understood. ObjectiveTo investigate the chemokine receptor profile and the migration potential of the human IgE-producing cells. MethodsTonsil B cells were stimulated with IL-4 and anti-CD40 to induce class switching to IgE and IgG1. The chemokine receptor profile of IgE+ and IgG1+ switched cells was determined using flow cytometry and migration towards relevant chemokines was quantified using transwell chemotaxis assays. Chemokine expression was also validated by re-analysis of a published single cell RNA sequencing (scRNAseq) dataset of PCs isolated from nasal polyps (NP) of patients with allergic fungal rhinosinusitis. ResultsIgE PCs exhibit significantly reduced expression of the BM-homing chemokine receptor CXCR4 and impaired migration towards its ligand, CXCL12. While IgE+ PCs can upregulate CCR10 and respond to its ligand, CCL28, this behaviour is similar to IgG1+ PCs. Strikingly, however, IgE PCs selectively upregulate CCR2 and migrate robustly towards its ligand CCL2. Re-analysis of NP scRNAseq data confirmed that IgE PCs express significantly higher levels of CCR2 compared with PCs of all other isotypes. ConclusionsThese findings identify CCR2 as a key regulator of IgE PC migration and provide insights into their homing preferences that may shape the nature of the IgE responses.

Background: Short-chain fatty acids (SCFAs) are microbial metabolites with immunoregulatory properties. Human milk contains SCFAs which have been proposed as potential modulators of infant immune development. We aimed to examine associations between human milk SCFA concentrations and infant allergic disease outcomes in a high-risk cohort of infants of atopic mothers. Methods: SCFAs were measured by targeted liquid chromatography-mass spectrometry in human milk samples collected at 3 and 6 months postpartum from atopic mothers enrolled in the Infant Fish Oil Supplementation (IFOS) Study (n=147). Associations between milk SCFA concentrations and early childhood allergic disease outcomes (atopic dermatitis, food allergy, allergic rhinitis, and allergen sensitisation at 1 and 2-3 years) were examined using logistic regression. Results: Human milk SCFA concentrations were broadly stable between 3 and 6 months postpartum, except for acetate which was significantly elevated at 6 months. No significant associations were observed between human milk SCFA concentrations and any allergic disease outcome after correction for multiple comparisons (all p>0.05). Conclusions: Human milk SCFA concentrations are not associated with allergic disease outcomes up to 4 years of age. These findings suggest that oral SCFA exposure via human milk is insufficient to reduce infant allergy risk, and that gut SCFA production may be a more relevant target for future allergy prevention research.

BackgroundSevere eosinophilic asthma (SEA), eosinophilic granulomatosis with polyangiitis (EGPA) and nasal polyposis (NP) are immune-mediated diseases characterised by eosinophilic inflammation. However, there is also increasing interest in the potential pathological roles of autoantibodies in these diseases. Understanding their B cell receptor (BCR) repertoires may provide valuable insights into disease mechanisms, and potential role of B cells in their pathology. MethodsWe conducted BCR repertoire sequencing using peripheral blood from 43 patients, comprising SEA with nasal polyps (SEA+NP), SEA without nasal polyps (SEA-NP), and EGPA, along with 16 healthy controls (HCs). ResultsCompared to HCs, patients with EGPA exhibited increased relative proportions of IgA1, IgG1, IgG2, and IgG4 subclasses. Similarly, SEA-NP patients demonstrated significantly high proportion of IgG2 sequences. Notably, the IgG4 subclass was significantly elevated across all patient groups compared to HCs. Patients receiving anti-IL-5/5R biologic treatments showed increased relative proportions of IgA2 and IgG2 subclasses compared to untreated patients. Some variation across participant groups in mean somatic hypermutation and mutation frequency was evident. 1,508 clones shared across patients, but not healthy controls, were evident though the majority showed low clonal expansion. Nevertheless, a few shared clones did show either high prevalence across patients and/or higher clonal expansion. ConclusionChanges in BCR repertoires in SEA/EGPA are consistent with a pattern of a more mature B cell component in the periphery and with the T2 inflammatory response observed in SEA and EGPA. BCR clonotypes shared across patients were evident, however, whether such clonotypes are pathological in SEA/EGPA requires further investigation.

Monocytes are critical regulators of allergic inflammation, whose functions are modified by IgE-driven processes. Monocytes are heterogeneous; comprised of multiple subsets which implies differential functions. In allergic inflammation, this heterogeneity is likely influenced by IgE-mediated effects. We sought to identify phenotypically distinct monocyte subsets related to allergic disease and then further delineate functional differences in cytokine release and antiviral responses. Using high dimensional spectral flow cytometry, we identified monocyte surface phenotypes directly related to surface levels of the high affinity IgE receptor (Fc{epsilon}RI) and surface-bound IgE. Fc{epsilon}RI+IgE+ monocytes, or FIMs, correlated with allergic disease and the level of atopy (i.e. serum IgE levels) of individual subjects. The FIM population also had differential surface expression of other molecules of monocyte maturation, which closely resembled a type 2 conventional dendritic cell (cDC2) phenotype. Functionally, FIMs had enhanced antiviral responses and IgE-driven IL-10 cytokine release. Finally, we showed that FIMs could be identified at higher levels in lung tissue from individuals with asthma. This study supports that atopic disease drives differential monocyte phenotypes, with the FIM population, specifically, as a more mature cell population closely related to dendritic cells with enhanced antiviral responses. The presence of monocytes in lung tissue during lethal asthma exacerbation further supports a role in regulating tissue inflammatory responses in allergic airway disease.

Cross-reactive antibodies against dengue virus are known to cause antibody-dependent enhancement (ADE) of infection or disease severity under specific conditions. In our previous study, we showed that primary immunization with the COVID-19 vaccine induces induces cross-reactive IgG causing ADE against dengue. In the present study, we investigated the influence of IgG Fc-glycosylation (analyzed by LC-MS/MS) on ADE mediated by cross-reactive IgG against dengue from IgG against SARS-CoV-2. We found a clear correlation between anti-DENV2 E IgG2 galactosylation and the ADE capacity of cross-reactive IgG against dengue in individuals vaccinated against COVID-19. IgG2 sialylation increased over time; however, it was not correlated with ADE capacity. This phenomenon was restricted to IgG2, whereas anti-DENV2 E IgG1 Fc-glycosylation remained stable after COVID-19 vaccination.

Background Growing evidence suggests that the gut microbiome influences nasal and ocular allergic inflammation through gut-mucosal immune interactions. Yet, its association with Allergic rhinitis (AR) and allergic eye symptoms (AES) remains incompletely understood in large population-based cohorts. Objective To examine associations between the gut microbiome and self-reported AR and AES in Finnish adults. Methods Shallow metagenomic sequencing was performed on stool samples from a population-based cohort (FINRISK02; n = 7,231). Microbial taxonomic and functional profiles were compared between individuals with AR (n = 1,950), AES (n = 1,554), combined allergies (AR and/or AES; n = 2,305), and controls without reported symptoms (n = 3,175). Results Allergic groups exhibited lower microbial richness and phylogenetic diversity than controls. Shared microbial and functional signatures were observed across AR and AES, consistent with their high co-occurrence (N = 1,199). Compared with controls, allergic groups showed enrichment of 17 bacterial species, predominantly from the Clostridia class, including taxa previously associated with asthma, chronic obstructive pulmonary disease, and atopic dermatitis. Allergic individuals also exhibited enrichment of pathways related to mucosal carbohydrate processing, shikimate metabolism, histidine turnover, and broader amino acid metabolism. Concurrent enrichment of histidine biosynthesis and degradation suggested altered microbial histidine metabolism. Conclusions Adult allergic symptoms are associated with gut microbiome taxonomic and functional alterations linked to mucosal barrier function and immune-related metabolism, supporting a shared gut-mucosal immune axis across allergic phenotypes.

Interleukin-33 (IL-33) is a key cytokine in mast cell mediated immunity, promoting inflammatory cytokine production without inducing degranulation. Here, we compared IL-33 induced proteomic responses across three mast cell culture systems, Foetal Liver derived Mast Cells (FLMCs), Bone Marrow derived Mast Cells (BMMCs), and Peritoneal Mast Cells (PMCs), using quantitative data-independent acquisition mass spectrometry. Although baseline proteomes were largely conserved across all mast cell types, clear differences were observed between culture systems. PMCs exhibited a more mature phenotype, characterised by higher abundance of granule-associated proteins and lower levels of proteins involved in metabolism and translation. In contrast, FLMCs and BMMCs displayed higher levels of biosynthetic and metabolic machinery, consistent with a less differentiated state. IL-33 stimulation induced a conserved proteomic programme across all mast cell types, enriched for inflammatory signalling pathways, cytokine production, and enzymes involved in prostaglandin and biogenic amine biosynthesis. Pathway analysis demonstrated robust activation of nuclear factor {kappa}B (NF{kappa}B) associated signalling, with a relative enrichment of components linked to non-canonical NF{kappa}B signalling and tumour necrosis factor (TNF) receptor associated pathways. Mechanistically, IL-33 driven proteomic remodelling was strongly regulated by mitogen-activated protein kinase (MAPK) signalling. p38 MAPK emerged as the dominant regulator of the IL-33 response, with ERK1/2 contributing to a subset of induced proteins. These pathways differentially regulated key effector outputs, including IL-6, IL-9, IL-1 family cytokines, and enzymes required for prostaglandin, serotonin, and histamine biosynthesis. Together, these data define conserved IL-33 dependent inflammatory programmes across mast cell differentiation states and demonstrate how MAPK signalling pathways shape the composition of mast cell effector responses.

T cell activation is associated with, and dependent upon, the upregulation of amino acid uptake from the extracellular environment. Uptake of the non-essential amino acid asparagine (Asn) is mediated via amino transporters such as Slc1a5 whilst Asn can be synthesized within cells that express asparagine synthetase (ASNS). Previous work demonstrated that initial activation of CD8+ T cells is perturbed in the absence of Asn, whereas effector cytotoxic T cells cells upregulate ASNS and lose their dependence on Asn uptake. By contrast, less is known of the role of Asn uptake and ASNS in CD4+ T cell responses. Here we demonstrate that CD4+ T cells are more reliant than CD8+ T cells on Asn uptake for initial activation, differentiation, metabolic reprogramming and regulation of autophagy. These phenotypes are associated with enhanced expression of ASNS in CD8+ as compared to CD4+ effector T cells.

NK cells can form clonal populations demonstrating features of adaptive immunity, including long-term memory and at least partial antigenic specificity. Given the limited individual diversity of activating receptors, the nature of NK cell antigenic specificity remains elusive. To explore this riddle, we combined scRNA-Seq of ex vivo FACS-sorted NK cell subsets expressing specific KIR receptors, single-cell cloning and bulk RNA-Seq of in vitro cultured KIR2DS4 NK cell clones, transcriptomic profiling of antigen-stimulated NK cells, and in silico modeling of glycosylated KIR2DS4-peptide-HLA complexes. scRNA-Seq resolved 12-15 clusters per KIR subset with highly heterogeneous KIR, KLRC and NCR expression patterns, consistent with clonal lineages. Notably, those clusters demonstrated over 30 differentially expressed glycosyltransferase genes, potentially involved in post-translational modification of NK cell receptors. Single-cell-derived KIR2DS4 cultures exhibited clone-specific cytotoxic, chemokine and KIR receptor genes, and transcriptional differences in > 40 glycosyltransferases. In peptide culturing autologous assays, SARS-CoV-2 (KTFPPTEPK) and EBV (CRAKFKHLL) peptides elicited NK cell proliferation and distinct transcriptional programs linking cytotoxicity genes, KIR2DS4 and glycosyltransferases. Structural modeling revealed that N-linked glycosyl residues in specific regions of KIR2DS4 may alter its contacts and interaction with MHCI and the presented peptide. We conclude that KIR human NK cells comprise clonally imprinted populations with distinct glycosyltransferase expression profiles, and site-specific KIR2DS4 glycosylation may modulate interaction with peptide-MHCI complexes, suggesting a post-translational layer of clonal NK cell diversification as a clue to their antigenic specificity.

BackgroundIntrauterine inflammation, commonly presenting as chorioamnionitis, is variably linked to preterm birth, neonatal infections and postnatal chronic inflammatory disorders. However, the effects of systemic maternal inflammation on exposed fetuses and offspring are less clear. We previously reported inflammatory responses in murine pups born after brief gestational exposure to experimental maternal inflammation. These findings led us to hypothesize that fetal exposure to maternal inflammation could lead to persistent alterations in postnatal immunity. ObjectiveTo test our hypothesis, we examined immune responses to vaccination, a useful measure of immune status, in young adult offspring with late gestational exposure to maternal LPS. Design/MethodsLate-gestation pregnant dams were treated with LPS or saline. Offspring (LPS-exposed or saline controls) were either immunized with the Tdap vaccine or remained unimmunized (naive mice), and were subsequently infected with Bordetella pertussis. Lung and spleen immune responses were assessed by multi-parameter flow cytometry, protein microarray and RT-PCR. ResultsWe observed that young adult (7 week old) mice exposed to maternal LPS during gestation, vaccinated with TDaP, and subsequently infected with pertussis exhibited lower lung neutrophil but higher CD4+ lymphocyte proportions relative to unexposed controls. In splenic studies, LPS-exposed mice had lower frequencies of CD4+IFN{psi}+ (Th1) and CD4+IL-17+ (Th17) cell populations. In vitro studies of post-vaccination responses to heat-killed B. pertussis showed variable levels of IL-2 and IL-4 in splenic cultures from LPS-exposed vs. control mice. Vaccinated, LPS-exposed mice showed variable splenic Stat3 and NFkb gene expression levels relative to those of naive LPS-exposed mice. ConclusionOur present murine studies show that experimental maternal inflammation during late gestation can alter immune response patterns to secondary challenge in young adult offspring. However, whether such intrauterine inflammatory exposure might also influence protective immune function remains to be determined. Our findings lead us to speculate that fetal exposure to systemic maternal inflammation in humans could have long-term implications for protective immunity.

Dimeric immunoglobulin A (dIgA) is generated from IgA monomers (mIgA) via JCHAIN-dependent polymerization. DIgA is transported across epithelial barriers by the poly Ig receptor (PIGR) and confers mucosal protection, while serum contains substantial amounts of IgA monomers. Distinct plasma cell subsets have been proposed to produce either monomeric or dimeric IgA, with bone marrow plasma cells as a primary source of mIgA. Here, we addressed whether IgA plasma cell populations segregate based on mIgA or dIgA production. Flow cytometric analysis of antibody-secreting cells from bone marrow, lymphoid and mucosal tissues revealed universal intracellular JCHAIN expression across isotypes and failed to identify a discrete JCHAIN-negative IgA plasma cell population. To detect polymeric IgA, we generated a recombinant soluble PIGR that selectively bound JCHAIN-containing dIgA in Western blot, ELISA, and flow cytometry. Soluble PIGR binding was detected in all IgA plasma cells irrespective of tissue origin, arguing against a dedicated mIgA-producing plasma cell subset incapable of dIgA formation. Ex vivo cultures and single-cell DropMap secretion assays demonstrated that bone marrow and lamina propria IgA antibody-secreting cells co-secrete mIgA and dIgA. These findings suggest that dIgA assembly and secretion are general properties of IgA plasma cells and disfavor a dedicated mIgA-producing population.

Vaccine-induced immune responses are the result of an intricate interplay between different cell populations of the innate and adaptive immune system, which is so far only partly understood. In particular, the role of polymorphonuclear neutrophils (PMNs) has long been neglected. Here, we studied the effects of a whole inactivated virus influenza vaccine (WIV) in an in vitro system consisting of freshly isolated human PMNs alone or PMNs combined with autologous peripheral blood mononuclear cells (PBMCs). Isolated PMNs showed minimal responses to the vaccine with respect to apoptosis, gene expression, cytokine production, and reactive oxygen species production. However, in WIV-stimulated PMN/PBMC co-cultures, PMNs particularly enhanced monocyte dynamics, CD14-CD11c+ cell activation, effector T cell differentiation, and B cell antibody production. On the other hand, PMNs decreased T follicular helper cell frequencies. Without vaccine stimulation, PMN presence resulted in enhanced levels of baseline inflammatory cytokines in PMN/PBMC co-cultures. However, with vaccine stimulation, PMNs dampened the vaccine-induced cytokine secretion of PBMCs. These findings reveal PMNs as regulators of vaccine responses whose effects depend on crosstalk with other immune cells, balancing pro-inflammatory and adaptive immune activation. Author summaryPolymorphonuclear neutrophils (PMNs) are essential and predominant cells of the human innate immune system. Growing evidence implicates that PMNs are involved in vaccine-induced immune activation, but their exact role is so far poorly defined. In our study, human PMNs were tested alone to observe their response to whole inactivated virus influenza vaccine (WIV), or combined with autologous peripheral blood mononuclear cells (PBMCs) to investigate how their presence influences vaccine responses of various cell populations within PBMCs. Our results show that WIV had little direct effect on isolated PMNs. However, when PMNs were combined with other immune cells, PMNs acted as crucial regulators: they enhanced the activity of innate immune cells, regulated the responses to the vaccine of T and B cells, and helped control the overall level of inflammation. Our study forms the groundwork for a more comprehensive understanding of human immune cell interactions under vaccine stimulation.

Neoantigens are cancer-specific antigens arising from genomic alterations. Single Amino Acid Variants (SAAVs) represent a primary class of these neoantigens. To evaluate the therapeutic potential of Neurofibromin 1 (NF1)-derived SAAVs - given that NF1 is frequently mutated in malignant brain tumors - we prioritized the 40 NF1 SAAVs determined to be HLA-A*02:01 binders using computational prediction coupled with experimental validation. To validate these predicted neoepitopes, we employed a two-tiered experimental approach in HLA-A*02:01 homozygous U87-MG cells. We first synthesized minigene constructs encoding the predicted neoepitopes, introduced them via lentiviral transfection and confirmed their expression by mass spectrometry (MS). Subsequently, we performed endogenous validation using pan-HLA immunoprecipitation mass spectrometry (IP-MS), confirming 4 (10 neoepitopes) of the 40 candidate SAAVs. We observed a discrepancy between in silico predictions and the observed sequences. Our endogenous peptidomics further revealed conserved peptide motifs and demonstrated that peptide selection for HLA presentation is transient. While our study substantiates the therapeutic feasibility of T-cell immunotherapies targeting NF1 mutations, these results underscore a limitation in current computational prediction. Our study highlights the necessity of experimental validation to refine neoantigen prioritization strategies.

Avian influenza A viruses (IAV) pose a constant pandemic threat, with the recent 2.3.4.4b clade of the H5 subtype causing high pathogenicity and spreading across animal species and geographic locations. Understanding human pre-existing immunity to avian H5 IAV can inform on population susceptibility, a critical aspect of pandemic preparedness. To that end, we analysed the IAV HA-specific antibodies across individuals born between 1928-1999 with different early life exposures to IAV subtypes. Individuals born prior to 1957 had the highest pre-existing serum antibodies to group 1 HA antigens, including the 2.3.4.4b H5 and a group 1 HA stem antigen. These birth-year-specific patterns were not reflected in the limited pre-existing serum neutralising antibodies detectable against a 2.3.4.4b H5 IAV or in H5-specific memory B cell populations. They were however evident in pre-existing nasal IgG and IgA titres to H5, which were greater in individuals born prior to 1957. Our findings demonstrate that the immunological biases afforded by early life exposure extend to antibodies detected in the nasal mucosa, the site of IAV replication. ImportanceUnderstating pre-existing immunity to influenza A viruses of pandemic potential is an important aspect of pandemic preparedness. This includes an understanding the heterogeneity of pre-existing immunity across the population. Here, we demonstrate that pre-existing antibodies to H5 IAV vary according to year of birth and childhood imprinting. We demonstrate that this is the case for both systemic and nasal antibodies, highlighting the importance of understanding pre-existing mucosal immunity at the sites of influenza virus replication.

PRV-101 is a multivalent formalin-inactivated Coxsackievirus B (CVB) vaccine developed to prevent CVB infections, which are associated with increased risk of islet autoimmunity. While PRV-101 induces robust neutralizing antibody responses, its T-cell immunogenicity is unknown. We analyzed peripheral blood mononuclear cells from 25 healthy adults receiving three high or low PRV-101 doses or placebo in a Phase I randomized, placebo-controlled trial. CVB-reactive CD8 T-cell responses were assessed using HLA Class I multimers, and CD4 and T follicular helper (Tfh) responses were measured by activation-induced marker assays following stimulation with a CVB peptide library. PRV-101 elicited minimal CVB-reactive CD8 T-cell responses but robust CD4 and Tfh responses, peaking at week 12 and persisting through week 32. Responses were observed in both seronegative and seropositive individuals, consistent with effective immune priming and boosting. Tfh frequencies correlated with neutralizing antibody titers. Female participants exhibited higher peak Tfh responses than males. We conclude that PRV-101 elicits a CVB-protective immune profile, dominated by Tfh responses supporting durable humoral immunity and devoid of potentially diabetogenic cytotoxic T-cell responses. This profile invites further investigations in vaccine trials for type 1 diabetes prevention.

Summary Background Severe dengue reflects a temporally regulated interaction between viral burden, NS1 antigenaemia, cytokine and chemokine amplification, endothelial activation, glycocalyx injury, and organ stress. Although individual cytokines, endothelial markers, viral-burden measures, and clinical markers have been widely studied, the integrated pathogen-host evidence base remains fragmented. We synthesised evidence for cytokine, endothelial, and viral-burden signatures associated with severe dengue and assessed whether paired pathogen-host measurement provides a biologically coherent framework for severity assessment. Methods We searched MEDLINE, Embase, Scopus, Web of Science, Cochrane Library, Global Health, WHO Global Index Medicus, and medRxiv from database inception to 30 April 2026, without language restriction, for studies reporting viral burden, NS1 antigenaemia, cytokine, chemokine, endothelial, glycocalyx, inflammatory, or routine host-response markers in laboratory-confirmed dengue with severity outcomes. Eligible designs were prognostic-factor association studies, cross-sectional biomarker studies, and multivariable prediction-model studies. Risk of bias was assessed using QUIPS for prognostic-factor studies, PROBAST for prediction-model studies, and the relevant JBI critical appraisal checklist for cross-sectional biomarker studies, with the Newcastle-Ottawa Scale used selectively for cohort or case-control designs not amenable to QUIPS. Random-effects meta-analysis pooled standardised mean differences using restricted maximum likelihood with Hartung-Knapp adjustment. The protocol was registered with PROSPERO (CRD420261396923) before final extraction and synthesis. Findings Of 4,180 records identified, 79 studies including 47,612 participants met eligibility criteria. Forty-nine studies evaluated paired pathogen-host markers, 14 evaluated viral burden or NS1 antigenaemia alone, nine evaluated host biomarkers alone, and seven reported multivariable prediction models. Pathogen-side markers showed modest pooled severity associations whose magnitude depended on day of illness, immune status, and infecting serotype. Cytokine and chemokine markers, particularly IL-10, IL-6, IL-8, and CXCL10/IP-10, showed larger pooled effects favouring severe disease, while endothelial and glycocalyx markers, including angiopoietin-2 and syndecan-1, provided the most direct mechanistic link to plasma leakage. Routine clinical markers, especially platelet count, AST, ferritin, ALT, and lactate, retained substantial discriminatory value. Prediction models reported areas under the curve of up to 0{middle dot}96 in internal validation and 0{middle dot}97 in discovery analyses, but three had been externally validated, calibration was reported in two, and decision-curve analysis in none. Interpretation Current evidence supports severe dengue as an integrated pathogen-host injury syndrome in which viral burden and NS1 antigenaemia interact with cytokine amplification, endothelial dysfunction, glycocalyx injury, and routine markers of organ stress. The strongest translational direction is not a single biomarker but a parsimonious cytokine-endothelial-pathogen panel requiring prospective external validation across age groups, serotypes, immune-status strata, and endemic regions. Existing evidence supports candidate marker prioritisation and mechanistic synthesis, but not immediate routine clinical deployment.

Circulating proteins have been widely investigated as potential biomarkers in multiple sclerosis (MS), yet findings across studies are often inconsistent, likely reflecting differences in disease stage, treatment exposure, and cohort composition. Studying individuals at elevated risk of MS prior to disease onset offers a unique opportunity to identify immune alterations that precede clinical disease while minimizing confounders. Here, we investigated whether alterations in six previously MS-associated biomarkers are detectable and associate to underlying genetic susceptibility in two independent sample collections comprising people with MS (pwMS), healthy controls, and asymptomatic first-degree relatives of pwMS from the Genes & Environment in MS (GEMS) study cohort. The panel, representing complementary axes of MS immunopathology, included granzyme A (GZMA), MER tyrosine kinase (MERTK), interleukin-2 receptor alpha (IL2RA), osteopontin (SPP1), CD30 (TNFRSF8), and chitinase-3-like protein 1 (CHI3L1). None of the proteins demonstrated associations with MS. A composite score constructed from externally derived effect estimates was not associated with MS status in either collection or in meta-analysis. Among asymptomatic first-degree relatives, the composite score was not significantly associated with group status. In contrast, an inverse correlation between SPP1 and the MS genetic risk score among GEMS participants was found ({beta} = -0.246, p = 0.001). Together, these findings suggest that several circulating proteins recently proposed as MS biomarkers are not robust tools to distinguish MS from healthy individuals. However, SPP1 levels are highlighted for further evaluation among at-risk individuals, and further work is needed to determine whether circulating immune signatures can capture the earliest stages of MS in at-risk individuals.

Recent epidemiologic studies indicate that adult-onset type 1 diabetes (AOT1D) is more common than childhood-onset type 1 diabetes, yet it remains clinically underrecognized. Because little is known about the emergence of islet autoantibodies in AOT1D, we conducted a retrospective study using electronic medical records from the United States Military Health System and longitudinal serum samples from 169 individuals with AOT1D and 40 healthy controls obtained from the Department of Defense Serum Repository. Among 643 prediagnostic samples from individuals with AOT1D, IA-2 autoantibodies were the most prevalent (50%), followed by GADA (46%), IA-2{beta} (34%), ZnT8-R (27%), and ZnT8-W (15%). Overall, 85% (144/169) of subjects were seropositive for at least one autoantibody prior to diagnosis. Analysis of the earliest available sample from all of the AOT1D cases, grouped into 5-year intervals preceding diagnosis, demonstrated a progressive increase in seropositivity over time: 38% of subjects were seropositive more than 20 years before diagnosis, increasing to 44% at 20-15 years, 59% at 15-10 years, 73% at 10-5 years, and 91% within 5 years of diagnosis. Among the 144 seropositive individuals, positivity for two or more autoantibodies was the most common pattern, occurring in 50% (72/144) of cases. Isolated GADA positivity (22%) and isolated IA-2/IA-2{beta} positivity (24%) occurred at similar frequencies, whereas isolated ZnT8 positivity was uncommon (4%). Temporal analysis showed that isolated GADA positivity appeared earliest, with a median onset of 7.9 years before diagnosis, whereas multiple-autoantibody positivity, IA-2 positivity, and ZnT8 positivity emerged later, with median onsets of 4.6, 4.5, and 1.9 years before diagnosis, respectively. These findings extend observations from pediatric type 1 diabetes to adults and demonstrate that AOT1D-associated autoimmunity often begins decades before clinical diagnosis, highlighting a potentially important window for risk stratification and preventive intervention.

Background: Wide-spread mast cell (MC)-associated symptoms and MC activation syndrome (MCAS) are often reported in patients with hypermobile Ehlers-Danlos syndrome (hEDS) and hypermobility spectrum disorders (HSD). The goal of this study was to develop a novel MC score based on 11 self-reported MC-related conditions with clinical and research utility to better understand MC symptoms in hEDS and HSD patients. Methods: From November 1, 2019, to June 13, 2025, patients (n=2,141) filled out an Intake Questionnaire at the Mayo Clinic Florida EDS Clinic that included 11 self-reported questions related to categories of MC-related conditions for a MC score ranging from 0/11 to 11/11. Based on the MC score distribution in hEDS and HSD patients, a MC score of 0-1 was considered a low MC score and [&ge;]5 was considered a high MC score. Symptoms/comorbidities were compared between patients with high vs. low MC scores. Results: From the 2,141 hEDS/HSD patients, 535 (25.0%) had a MC score [&ge;]5 (Hi MC). MCAS-specific symptoms such as nausea and vomiting were reported more often in hEDS/HSD patients with a high vs. low MC score (p<0.0001). Random clinical blood tryptase and urinary MC markers were not elevated in patients with high MC scores (n=50/group), although high MC scores were found to significantly reduce urinary creatinine levels indicating that the protein used to normalize data was affected by MC activity. In contrast, random blood IgE, tryptase and major basic protein (MBP) by ELISA were increased in patients with high MC scores (e.g., IgE hEDS p=0.0004, HSD p=0.003). Of note, the percentage of patients reporting abuse or post-traumatic stress disorder was nearly doubled in patients with high vs. low MC scores (Abuse and PTSD: hEDS p < 0.0001; HSD p < 0.0001). Overall, 109/135 (80.7%) in hEDS and 129/135 (95.6%) in HSD reported more symptoms/comorbidities if they had a high MC score. Conclusions: We found that hEDS/HSD patients with high MC scores self-reported more widespread symptoms/comorbidities and higher MC-related blood markers than patients with low MC scores indicating the utility of this tool to evaluate the level of widespread MC activity in hEDS, HSD and other patients.

BackgroundHigh-titer glutamic acid decarboxylase 65 (GAD65) antibodies are found in patients with GAD65 antibody-associated neurological disorders, including stiff-person syndrome (SPS), GAD65 cerebellar ataxia (CA), and GAD65 epilepsy. Given the intracellular localization of the antigenic target, a direct pathogenic role for GAD65 autoantibodies is unlikely. Instead, the autoantibody may be a biomarker for the existence of pathogenic anti-GAD65 autoreactive CD8+ T cells. MethodsPBMC-derived dendritic cells (DCs) from 20 GAD65 antibody-positive patients with neurological manifestations (SPS, n=10; CA, n=7; epilepsy, n=3) and 15 healthy controls were pulsed with full-length GAD65 protein, full-length GAD67 protein, overlapping 15-mer peptide pools spanning GAD65 and GAD67, individual GAD65 15-mer peptides, or GAD65 9-mer peptides selected from predicted class I binding hotspots. T cell activation was quantified by flow cytometry-based activation-induced marker (AIM) assays using CD69 upregulation. Class I and class II HLA haplotypes were determined by high resolution typing. NetMHCpan v4.2c was used to generate residue-level peptide:HLA binding density maps across GAD65, and candidate 9-mers were validated for HLA binding by peptide:MHC monomer affinity testing. GAD65-peptide-HLA-restricted CD8+ T cells were identified by co-staining with two separately assembled tetramers carrying the same peptide:HLA complex on different fluorophores (APC and BV421), with double-positive events scored as antigen-specific. HLA-restricted cytotoxicity was measured by coculture of patient CD8+ T cells with GAD65-expressing HEK-293T cells reconstituted with defined HLA class I alleles using AAV-delivered Cre-dependent HLA-2A-eGFP cassettes. ResultsCD8+ T cells from GAD65 antibody-positive patients showed increased activation in response to DCs pulsed with full-length GAD65 relative to healthy controls (P=0.0157, Welchs t-test), whereas responses to GAD67 did not differ significantly between groups. CD4+ T cells responded to both GAD65 (P=0.0004) and GAD67 (P=0.0051). Peptide pool screening of GAD65 identified discrete CD8+ and CD4+ immunogenic regions, with refinement using individual 15-mers localizing CD8+ activity to multiple subdomains within GAD65(205-300), GAD65(316-435), and GAD65(447-520). HLA class I haplotyping in 16 non-Hispanic White GAD65+ patients revealed enrichment of HLA-B*08:01 (3.0-fold) and HLA-B*40:01 (4.1-fold) relative to USA NMDP European Caucasian reference frequencies (both BH q < 0.05), with suggestive enrichment of HLA-C*03:04 (2.9-fold; q = 0.09). Additionally, 44% of patients carried the HLA-A*01:01, HLA-B*08:01, and HLA-C*07:01 8.1 ancestral haplotype, approximately four-fold higher than the expected population frequency. Dual-fluorophore tetramer staining identified CD8+ T cells in GAD65+ subjects that bound a subset of HLA-A*11:01- and HLA-B*08:01-restricted GAD65 9-mers, with the clearest disease-skewed signals localized to GAD65(213-221), GAD65(257-265), and GAD65(529-537). In an HLA-reconstituted target-cell killing assay, CD8+ T cells from an HLA-B*08:01-positive GAD65+ patient mediated antigen- and HLA-restricted depletion of GAD65-expressing HEK-293T cells, with HLA-restricted target loss also observed in single-donor experiments across additional HLA-A, -B, and -C contexts. ConclusionsPatients with GAD65 antibody-associated neurological disorders harbor circulating CD8+ T cells that recognize discrete HLA class I-restricted GAD65 peptides and that are capable of cytotoxicity against GAD65-expressing HLA-matched target cells. We characterize the immunogenetic and cellular features of class I-restricted CD8+ T cell responses in GAD65 autoimmunity, including overrepresentation of the 8.1 ancestral haplotype, complementing the historical focus on antibodies and CD4+ T cell help, and we provide a panel of validated GAD65 peptide:HLA tetramers for prospective isolation, clonotypic analysis, and longitudinal monitoring of candidate pathogenic CD8+ T cell populations across the GAD65 antibody-associated neurological disease spectrum.