Clinical Immunology

ObjectiveTo define cellular and molecular mechanisms distinguishing active systemic lupus erythematosus (SLE) from remission by profiling autoreactive antinuclear antigen- positive (ANA+) and non-autoreactive B cells subsets in three cohorts: active disease (SLE-A), long-term, drug free remission (SLE-R), and healthy controls (HC). MethodsPeripheral blood B cells were phenotyped by flow cytometry, including ANA reactivity. Single-cell RNA sequencing (scRNA-seq) was performed on sorted ANA+ and ANA- subsets. ResultsSeven transcriptomic B cell clusters were resolved: quiescent (Naive 1, Marginal Zone B cells [MZB], IgG Memory 1) and activated (Age-Associated B cells [ABCs], Naive 2, IgM Memory, IgG Memory 2). SLE-A showed expansion of activated clusters, MZB contraction, and a higher IgG:IgM B cell ratio. SLE-R exhibited an "immunological reset," distinct from healthy homeostasis, with reduced ABCs and IgG Memory 2, persistence of Naive 2, and partial restoration of MZB and Naive 1. Interferon- (IFNa) signaling was elevated across clusters in SLE-A (SLE-A > SLE-R > HC), whereas TNF signaling was enriched in activated clusters across cohorts, with minimal differences between SLE-R and SLE-A. IFNa and TNF scores were inversely correlated. B cells predominantly expressed TNFR2, suggesting immunomodulatory TNF effects in remission. ANA+ cells in HC and SLE-R showed enriched Fc{gamma}RIIb inhibitory and IL-4/STAT6 signaling, suggesting reinstated regulatory control. DiscussionCompared to SLE-A, SLE-R was characterized by partial reversion to HC homeostasis with residual activation. These findings delineate immunologic features of remission and suggest therapeutic opportunities, including modulation of TNFR2, Fc{gamma}RIIb, and IL-4 to help sustain remission. What is already known on this topicSome patients with SLE achieve complete clinical remission without treatment, referred as immune reset; the mechanisms that underlie this state have not been well characterized. Healthy individuals and patients with Systemic Lupus Erythematosus (SLE) normally harbor similar frequencies of autoreactive B cells; the checkpoints that regulate activation of these cells are not fully defined. What this study addsB cells, stratified by their reactivity to nuclear antigens (ANA), from active SLE (SLE-A), drug-free remission (SLE-R), and healthy controls (HC) were analyzed using single cell sequencing and flow cytometry. We identified B cells states associated with disease activity; SLE-R displayed a distinct profile that differed from SLE-A and HC. TNF signaling was present in activated B cell subsets in SLE-A and SLE-R. This persistence in SLE-R may reflect an immunomodulatory function of TNF on TNFR2, which is expressed on B cells. ANA+ cells in SLE-R and HC were enriched for inhibitory Fc{gamma}RIIb and IL-4/STAT6 programs. How this study might affect research, practice or policyThe signatures identified help define the "immunological reset" state in patients with SLE-R. We also identified pathways, such as type I IFN, TNFR2, Fc{gamma}RIIb, IL-4/STAT6 as potential targets for maintaining remission.

Sarcoidosis is a heterogenous disease of unknown etiology characterized by non-caseating granulomas. Disease prevalence and presentation vary significantly by ancestry and ranges from acute, self-resolving disease to severe, chronic disease. Following previous reports suggesting B cells in the development and pathogenesis of sarcoidosis, we present here results of single-cell RNA sequencing, supporting B cell involvement in sarcoidosis through altered immediate early response, rewiring of MAPK signaling, and ancestry-specific preferential expansion of B cell receptors. Peripheral blood mononuclear cells were obtained from individuals of African or European Ancestry (AA and EA, respectively) including 48 healthy controls, 59 sarcoidosis patients, and 28 systemic lupus erythematosus (SLE) patients. SLE samples were used as a disease control. Differential expression analysis highlighted many differentially expressed genes (DEGs) with almost 5x more in the AA sarcoidosis versus AA control group compared to the EA sarcoidosis versus EA control group. B cells had the most DEGs of all cell types and expression patterns were similar between ancestries, however, sarcoidosis had an opposite transcription pattern than SLE, demonstrating an alternative immune response to acute activation than that seen in a prototypical autoinflammatory disease. This trend was maintained when examining specialized B cell subsets, with the most pronounced effect in the AA sarcoidosis versus AA control comparison. Our results strongly support further investigation of the role of humoral immune response in sarcoidosis and the potential to highlight patient groups likely to benefit from existing B cell therapies.

ObjectivesThe aetiopathogenesis of SLE encompasses genetic, environmental and epigenetic factors. We investigated associations between an SLE methylation risk score (MRS), HLA-DRB1*03:01, a non-HLA polygenic risk score (PRS) and clinical and immunological phenotypes. MethodsDNA methylation in whole blood from patients fulfilling [&ge;]4 ACR-82 criteria and controls were investigated using the Illumina HM450K array. The discovery cohort included 311 patients and 400 controls, and the replication cohort comprised 175 patients and 187 controls. Seventeen independent, top differentially methylated CpG sites ({Delta}{beta} of [&ge;]0.1) from case-control comparisons, were used to calculate the MRS. Genotyping was performed using the Immunochip, and the PRS included 57 non-HLA SLE SNVs. Clinical data were collected from patient charts, and serum IFN-2 was measured using Simoa. ResultsHigher MRS was strongly associated with serum IFN-2 levels (p=1.04x10-14). In both cohorts, higher MRS associated with discoid lupus, immunologic involvement, and anti-SSA/SSB/RNP/Sm autoantibodies (all p<0.05), and with higher disease activity in the discovery cohort (p=1.50x10-). MRS was also elevated in patients with multiple autoantibodies (p<1.0x10-15) and in HLA-DRB1*03:01 carriers (p<1.0x10-3). In contrast, higher PRS was associated with nephritis, anti-dsDNA positivity, and lower prevalence of anti-SSB antibodies (all p<0.05). No correlation was observed between the MRS and the PRS (p=0.35). ConclusionThe MRS defines an interferon-high, HLA-DRB1*03:01-linked SLE subset with multiple autoantibodies, partly distinct from PRS-associated nephritis risk, highlighting potentially divergent pathogenic pathways. These findings underscore the value of integrating genetic and epigenetic data to better understand underlying disease mechanisms in SLE. Key MessagesO_LIHigher MRS, but not PRS, correlated with increased levels of serum IFN-. C_LIO_LIThe MRS was associated with discoid rash, hematologic disorder, hypocomplementemia, antibodies including anti-SSA and HLA-DRB1*03:01. C_LIO_LIHigher PRS was linked to nephritis and anti-dsDNA positivity, and did not associate with the MRS. C_LI

Purpose: Aicardi-Goutieres syndrome (AGS) is a type I interferonopathy presently associated with nine genes. PTPN1 is a negative regulator of the interferon pathway previously associated with chronic inflammation and recently type 1 IFN autoinflammation. Methods: Genomic data from undiagnosed individuals with suspected AGS were interrogated for PTPN1 variants, and predicted loss-of-function (pLOF) and damaging missense variants in PTPN1 were sought in two additional academic databases as well as the All of Us database. Results: We identified 13 cases with ultra-rare heterozygous pLOF or highly damaging missense variants in PTPN1. Nine cases were identified in a cohort of 53 individuals (~ 17%) with clinical, imaging and persistent biochemical features of AGS. Median age of onset is 1.75 years (IQR 0.67), significantly later (p< 0.0001) than other AGS genotypes. Four additional cases were identified in academic datasets with variable clinical features suggestive of autoinflammation. Additionally, 49 individuals with ultra-rare, damaging PTPN1 variants were identified in the All of Us database, none had features suggestive of AGS, but autoimmunity was highly prevalent (~21.6%). Conclusion: Our data implicate PTPN1 as a cause of later-onset presentations of AGS within a broader spectrum of autoinflammatory phenotypes. Segregation and biobank data demonstrate reduced penetrance, with carriers being enriched for autoimmune disorders.

BackgroundDisorders affecting the spinal cord (myelopathies) can cause severe disability. Despite diagnostic advances, approximately 12-18% of myelopathy cases continue to elude an etiological diagnosis, hampering effective treatment. MethodsThis retrospective, multicenter, tertiary care cohort study conducted from 2014 to 2025 evaluated archived biofluids from patients with IM, known autoimmune myelitis, or other neurological diseases (ONDs). Proteome-wide phage display was used to discover novel autoantibodies. Targeted immunoassays were used to screen for a candidate autoantibody. Downstream metabolites were measured in the cerebrospinal fluid (CSF). ResultsAutoantibodies targeting the transcobalamin receptor (CD320) responsible for cellular transport of vitamin B12 were identified in 18 out of 32 IM patients (56%) in a discovery cohort. Bioactive B12 concentration was decreased in the CSF of anti-CD320 positive patients compared to OND controls (P = 0.0273), indicative of autoimmune B12 central deficiency (ABCD). Compared to anti-CD320 negative IM cases, anti-CD320 positive IM cases demonstrated a higher frequency of subacute time course (56% vs 7%, P = 0.008), normal CSF profile (83% vs 50%, P = 0.044), and dorsolateral spinal cord abnormalities on magnetic resonance imaging (MRI) (61% vs 7%, P = 0.003). In two independent validation cohorts comprising 94 and 25 patients with IM, anti-CD320 was detected in 43 (46%) and 12 (48%) patients, respectively. Comorbid anti-CD320 was detected in a smaller proportion of patients with other known autoimmune etiologies of myelopathy. Five anti-CD320 positive IM patients received B12 supplementation with or without concurrent immunosuppression, and four out of five clinically improved. ConclusionsABCD is associated with a substantial proportion of IM. Screening for anti-CD320 followed by metabolic confirmation of a CNS-restricted B12 deficiency may be considered in the diagnostic evaluation of myelopathy.

ObjectivesTransformative observations demonstrate unprecedented success of B cell-depleting interventions in many human autoimmune diseases, calling for a deeper understanding of the triggers leading to B cell-mediated autoimmunity and its perpetuation in human disease. Here, we investigated whether the autoreactive B cell response targeting human topoisomerase 1 (TOP1), a hallmark of systemic sclerosis, could cross-react with TOP1 of microbial origin. MethodsHomologies between human and microbial TOP1 were analyzed using Foldseek. TOP1-reactive monoclonal antibodies from patient-derived, human TOP1-reactive B cell receptors were generated and assessed for reactivity against human TOP1 and TOP1 from a prototypic yeast, Saccharomyces cerevisiae (S. cerevisiae). Reactivity of polyclonal serum IgG from anti-TOP1 autoantibody (ATA)+, anti-centromere autoantibody (ACA)+ SSc patients and healthy donors (HDs) was tested. Finally, B cell lines were generated expressing human ATA to study B cell activation upon antigenic stimulation. ResultsStructural homologues of human TOP1 were found in many microbes, particularly in fungi. Taking TOP1 from S. cerevisiae as a prototype, microbial TOP1 was recognized by polyclonal patient IgG and by several monoclonal ATAs. Importantly, S. cerevisiae TOP1 also activated B cells expressing a patient-derived, human TOP1-reactive B cell receptor. Patients affected by interstitial lung disease most frequently showed recognition of microbial TOP1. ConclusionsThese findings identify fungi as potential drivers of immune dysregulation in human autoimmunity, specifically in SSc, highlighting microbial antigen cross-reactive cells as important therapeutic targets. Moreover, these data provide first functional evidence for a breach of B cell tolerance against human TOP1 triggered by cross-reactivity to fungal TOP1.

UNC93B1 is a crucial chaperone protein for the trafficking of TLRs and regulates antigen presentation in dendritic cells (DCs), which activates downstream immune responses. Here, we identified a novel homozygous gain-of-function (GOF) UNC93B1 variant in an early-onset lupus patient. The patient presented with elevated level of inflammation and auto-antibody, and organ damage. The Unc93b1R95L/R95L transgenic mice also exhibited with autoimmune and autoinflammatory phenotypes. The transcriptional analysis revealed increased inflammation and elevated activation of DCs in the patients PBMCs and bone marrow-derived DCs (BMDCs) from Unc93b1R95L/R95L mice. In addition to the selected TLR7/8 activation in previously reported UNC93B1 GOF variants, the single-cell transcriptome and flow cytometry of splenocytes from Unc93b1R95L/R95L mice demonstrated increased phagocytosis activity and T helper cell differentiation with altered ICAM and MHC signaling in DCs and T cells, respectively. These results suggest UNC93B1 GOF variant enhances antigen presentation from DCs to T cells in the pathogenesis of immune dysregulation. Our study expands the pathogenic variants spectrum of UNC93B1 and offers insight into the underlying mechanism of antigen presentation in immune dysregulation caused by UNC93B1 beyond its trafficking function of TLRs.

An overlap syndrome of myositis and/or myocarditis associated with myasthenia gravis (MG) has emerged as a life-threatening immune-related adverse event (irAE) in cancer patients treated with immune checkpoint inhibitors (ICIs). This syndrome closely resembles a rare form of idiopathic inflammatory myopathy (IIM) seen in a subset of MG patients. In this systematic review, we searched PubMed for reports of concurrent MG and IIM as well as ICI-related overlap syndromes. By integrating clinical, serological, and pathological observations, we delineated a previously unrecognized clinicopathological subtype of myositis that overlaps with MG. This entity is defined by a strong association with striational antibodies (StrAbs) and frequent co- occurrence with thymoma as a paraneoplastic process, and we classify it as StrAb-associated myositis. The idiopathic and ICI-induced forms share similar, though not identical, clinical, serological, and histopathological characteristics. We found that AChR antibody positivity, independent of established clinical risk factors such as respiratory or cardiac involvement, predicted more severe ICI-myotoxicity. Together with supporting evidence, our findings suggest a pathogenic model in which thymoma-driven cytotoxic T-cell responses trigger secondary AChR autoimmunity. These results highlight the potential utility of StrAbs and anti-AChR antibodies as practical biomarkers for diagnosis, risk stratification, and early intervention in patients at risk for severe neuromuscular irAEs.

Red blood cell (RBC) alloimmunization is a clinically significant complication in transfused patients whose immunological determinants remain incompletely understood. Type I interferon (IFN-I) signaling drives RBC alloimmunization in murine models, and systemic lupus erythematosus (SLE) is characterized by constitutive IFN-I hyperactivation alongside elevated alloimmunization rates. We analyzed three publicly available SLE RNA-seq cohorts (GSE72509, GSE112087, GSE122459; whole blood and PBMC; total n = 150 SLE) in a pre-specified discovery-replication-validation design. A 14-gene IFN-I signature score was computed per sample; differential expression, gene set enrichment analysis, and Spearman correlation were performed independently per cohort. IFN-I scores were significantly elevated in SLE versus healthy controls in all three cohorts (p < 0.01 each). IFN-high SLE patients showed 665 differentially expressed genes, with enrichment of alloimmunization-associated and plasmablast differentiation gene sets confirmed by GSEA. The alloimmunization signature score correlated significantly with IFN-I score across all three independent cohorts ({rho} = +0.77, +0.51, +0.60; all FDR q < 0.05); Tfh differentiation showed no association in any cohort. To our knowledge, this represents the first human transcriptomic evidence that IFN-I pathway activity in SLE is coupled to alloimmunization-associated immune programs in vivo. These findings identify IFN-I score as a candidate biomarker of alloimmunization susceptibility in SLE and provide translational rationale for prospective studies incorporating transfusion outcome data.

To understand the molecular and cellular mechanisms beyond B-cell depletion with the anti-CD20 monoclonal antibody ocrelizumab, we used comprehensive muti-modal flow cytometry and functional assays in a prospective longitudinal multiple sclerosis (MS) cohort. Ocrelizumab depleted the vast majority of B cells and showed selective effects on different B cells subsets. Analysis of residual/replenished B cells revealed relative enrichment of regulatory B cells like CD27+CD43+ B1 and CD24hiCD38hi transitional B cells, and reduction of CD27+ memory B cell subsets and CD19+IgD+CD27-naive B cells at early time points (1-3 month) and before subsequent infusions at 4-7 months, 11-14 months, and >18 months. CD20+ T cells and peripheral helper T-cells (Tph) were also reduced. RNA sequencing analysis showed B1 cells have significantly higher expression of LGALS1, KCNN4, ITGB1, and IL2RB. Compared to transitional B cells, B1 cells also displayed significantly higher expression of tissue homing molecules ITGAX (CD11c), S100A4, ITGB1, and CXCR3. IL10 signaling pathway is increased in these B cells. Ex vivo B cell functional assays indicated the residual/replenishing B cells were anergic following ocrelizumab, with increased IL10/TNF and IL10/IL6 ratios under BCR stimulation. Ocrelizumab treatment may create a self-reinforcing regulatory circuit: the reduction of Tph cells could alleviate suppression of regulatory B cells, which subsequently expand and further promote regulatory T cell networks via IL2RB, LGALS1, and an increased IL-10 signaling pathway.

ObjectivesMosaic chromosomal alterations (mCAs) increase with age and are associated with many diseases, including autoimmune diseases. The associations between mCAs and systemic sclerosis (SSc) and its clinical subtypes have not been explored. MethodsWe recruited study subjects from two independent datasets (Set 1: 635 SSc, 4,401 controls; Set 2: 347 SSc, 2,170 controls) and detected mCAs (Loss, LOH, Gain, and mLOX) from their peripheral blood samples. Logistic regression analyses were conducted with covariates in each cohort, and the results were meta-analyzed. We also conducted stratified analyses by age groups, the age at disease onset, clinical phenotypes based on the skin lesions, autoantibody profiles, the presence of complications. ResultsWe observed a trend of increased Loss in SSc, especially in old age (P=0.0063). The association of Loss was strengthened in certain subtypes of SSc, including lcSSc (OR=2.22, P=0.019) and SSc with vascular complications (digital ulcers, pulmonary hypertension, or renal crisis, OR=3.30, P=0.0054). The effect sizes of Loss increased in patients with high cell fractions (CFs). We also observed that mLOX was significantly associated with SSc, lcSSc, and ACA-SSc only for subjects with high CFs. mLOX was significantly associated with lcSSc and ACA-SSc even compared with dcSSc and ATA-SSc, respectively. These associations were consistently observed in each of the two data sets. Finally, we identified majority of the associations of Loss were mainly driven by SSc with late age at onset. ConclusionsLoss and mLOX were significantly and differentially associated with SSc and its subtypes, underscoring potential phenotype-specific contributions of mCAs. WHAT IS ALREADY KNOWN ON THIS TOPICO_LISystemic sclerosis (SSc) is a heterogeneous disease, with its phenotypes and disease outcomes varying among patients. C_LIO_LIAge-related mosaic chromosomal alterations (mCAs) in blood and subsequent clonal haematopoiesis are associated with various adverse health outcomes. C_LIO_LImCAs have also been linked to several immune-mediated diseases, such as LORA, and hence may influence immune cells and their functions. C_LI WHAT THIS STUDY ADDSO_LIAutosomal copy-number loss (Loss) is increased in SSc in aged subjects. C_LIO_LILoss was associated with lcSSc, ACA-SSc. ILD-SSc, and VC-SSc in a dose-dependent manner of cell fraction. C_LIO_LImLOX was associated with SSc and its subtypes only in patients with high cell fraction. C_LIO_LILate-onset SSc and its subtypes show stronger associations with Loss with higher effect sizes compared to non-late onset SSc. C_LI HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICYO_LIOur study facilitates further research to recapitulate the current findings in independent cohorts as well as in different ancestries. C_LIO_LIIncorporating profiles of Loss and mLOX in blood into conventional clinical information may enable a better stratification of SSc patients and the development of a better management strategy. C_LIO_LIFurther experimental approaches, such as whole genome sequences and single-cell C_LI RNA sequences, that investigate the underlying molecular mechanisms of phenotypic heterogeneity of SSc driven by Loss and mLOX are also warranted.

ImportanceEmerging data show that B-cell depleting chemotherapies, which are increasingly used to treat autoimmune disorders and multiple sclerosis, can be associated with mucosal side effects such as inflammatory vaginitis. ObjectiveEvaluate the impact of rituximab treatment on vaginal mucosal immune markers, endocervical immune cell populations and vaginal microbiome. DesignCross-sectional observational study conducted between 2022 - 2024. SettingAcademic medical center, Boston Massachusetts. ParticipantsWe enrolled women aged >18 years who were either 1) receiving rituximab for autoimmune renal disease or were 2) healthy controls ExposureTreatment with rituximab, an anti CD20 monoclonal antibody. Main outcome and measureWe compared endocervical immune cell populations, vaginal fluid immune markers, vaginal fluid immunoglobulins and vaginal microbiome composition between individuals being treated with rituximab and healthy controls. ResultsWe enrolled 26 women treated with rituximab for autoimmune renal disease and 26 healthy controls. Median circulating and endocervical B-cell and plasma cell proportions were significantly lower in treated participants compared to controls. Median vaginal fluid IgA concentrations were significantly lower in participants treated with rituximab, while ILE, IgM, IgG1, IgG2, IgG3 and IgG4 were not different between groups. Total T cell frequencies were similar between groups, but the proportion of activated T cells (CD4+CD38+HLADR+) was significantly lower in people treated with rituximab. Concentrations of IL10, IL13, IL17, IL21, IL23, IL4, ITAC and TNFa were elevated in vaginal fluid from the rituximab group, while IL-8 was lower. A CST-IV-C, low-Lactobacillus pattern of vaginal microbiota was more common in the rituximab group. Conclusions and RelevanceSystemic B-cell depletion is associated with reduced vaginal fluid IgA, a more diverse microbiome composition, and increases in many vaginal fluid immune markers compared to healthy controls. The reduction in vaginal fluid IgA may provide opportunities for vaginal bacteria to induce inflammation. Key pointsO_ST_ABSQuestionC_ST_ABSHow does circulating B-cell depletion impact the vaginal microenvironment? FindingsIn this cross-sectional study of 52 women, B cell and plasma cell proportions were significantly lower in both blood and vaginal mucosa among rituximab-treated participants compared to healthy controls. Vaginal IgA concentrations, but not other immunoglobulins, were significantly lower in rituximab treated participants. In treated participants, vaginal cytokine concentrations were elevated, and microbiome composition shifted toward non-Lactobacillus-dominant communities. In six people with inflammatory vaginitis, both circulating and endocervical B cells were lowest in people with the most severe symptoms. MeaningSystemic B cell depletion is associated with alterations in vaginal mucosal immune markers and microbiome composition which increase local inflammation.

Anemia is one of the most debilitating and frequent complications of inflammatory bowel diseases (IBD) and is often treated with iron supplementation, which has limited efficacy. Damaged intestinal barrier function is a hallmark of IBD and causes the translocation of endotoxins from gut bacteria into the bloodstream. In a previous study in mice, we reported that endotoxin suppresses erythropoiesis by reprogramming erythroblastic island macrophages (EBI M{varphi}). Here, we show that IBD patients and mice with acute colitis developed endotoxemia associated with anemia. Endotoxemia in IBD patients was negatively correlated with blood erythrocyte counts. In line with this, mice with acute colitis caused by drinking water containing dextrin sodium sulphate (DSS) had endotoxemia together with anemia characterized by reduced red blood cell counts, hemoglobin content and hematocrit., and reduced medullary erythropoiesis which was in part compensated by increased extramedullary erythropoiesis. As the endotoxin receptor TLR4 is expressed by CD169+ gut-resident macrophages and erythroid island macrophages in the bone marrow, we tested the hypothesis that TLR4 expressed by these CD169+ macrophages mediate both inflammatory colitis and anemia. Indeed, mice with conditional deletion of the Tlr4 gene specifically in CD169+ tissue-resident macrophages were protected from DSS-induced anemia and colitis. In addition, treatment of DSS mice with the TLR4 inhibitor C34 abated inflammation and anemia. These results suggest that endotoxins leaking from the inflamed gut may play a crucial role in IBD and associated anemia and that drugs targeting TLR4 may protect against IBD-associated anemia. Key pointsO_LIPatients with IBD and mice with acute colitis are anemic with increased endotoxemia and inflammation. C_LIO_LIEndotoxemia is inversely correlated with blood erythrocyte counts in IBD patients. C_LIO_LIConditional deletion of endotoxin receptor gene Tlr4 specifically in CD169+ tissue-resident macrophages or administration of synthetic TLR4 inhibitor significantly reduced colitis-induced anemia in mice. C_LI

BackgroundGroup 2 innate lymphoid cells (ILC2s) are key effector cells of type-2 immunity. A subset of ILC2s expresses KIT (CD117), which display increased phenotypic plasticity and were previously linked to severe asthma and psoriasis. However, the molecular mechanisms promoting a KIT+ ILC2 state remain poorly understood. ObjectiveDefine the molecular basis for the enhanced plasticity of KIT+ ILC2s and identify signals that induce this phenotype, including links with immune disease susceptibility. MethodsWe combine bulk as well as single-cell transcriptome (RNA-seq) and epigenome (ATAC-seq) with in vitro culture assays using primary human KIT+ or KITneg ILC2s and multipotent ILC precursors (ILCPs). Epigenomic data were integrated with genetic risk variants for major human immune diseases. ResultsMulti-omics analyses revealed that KIT+ ILC2s maintain a unique hybrid character marked by expression and open chromatin of genes linked to both ILCP and ILC2 biology. KIT+ ILC2s showed extensive epigenomic priming at gene loci related to naive lymphocyte biology, tissue homing, and ILC3 effector functions, including IL17 and IL23R - explaining why KIT+ ILC2s are poised to adopt an ILC3-like phenotype. Genetic risk variants for asthma and autoimmunity are enriched in the poised epigenome of KIT+ ILC2s. Common {gamma}-chain cytokines IL-2 and IL-7 induced a KIT+ phenotype in KITneg ILC2s through STAT5 activation. ConclusionsOur study defines KIT+ ILC2s as a developmentally immature state carrying a precursor-like epigenome that promotes phenotypic plasticity and is linked to immune disease susceptibility. Importantly, we identify STAT5-mediated cytokine signals as candidates for therapeutic targeting of KIT+ ILC2s.

DNA methylation is a stable epigenetic mark that critically influences the phenotype of immune cells. Identifying differentially methylated regions within immune cell lineages supports their phenotypic and functional characterization, leading to a better understanding of lineage-specific transcriptional regulation. Here, we performed a genome-wide methylation analysis of human innate lymphoid cells (ILCs), which allowed us to define specific epigenetic marker regions for ILC1, ILC2, and ILC3. These regions were associated with genes that have well-described functions in ILCs, such as TBX21 in ILC1, GATA3 and MAF in ILC2, RORC and IL23R in ILC3, but were also found in genetic loci that have not been previously associated with ILCs. In-depth analysis of ILC2-related marker regions within the HPGDS and NRROS gene loci confirmed their critical role in transcriptional regulation and suggested a novel role for NRROS in ILC2. Genome-wide methylation analysis of ILC2, derived from the blood of juvenile donors with atopy or asthma led to the identification of several disease-specific epigenetic regions associated with genes such as GIMAP4 and PTGS2. Together, our study not only provides novel epigenetic marker regions in human ILCs and confirms the functional role of ILC2-related markers, but also identifies promising markers for studying allergies in humans.

Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system affecting 2.8 million people worldwide. Both genetic and environmental factors contribute to MS risk, with Epstein-Barr virus (EBV) infection being an important environmental factor. To better clarify the role of EBV in MS, we examined its impact on gene expression, chromatin accessibility, and transcription factor binding in primary B cells and EBV-transformed B cells derived from patients with MS and healthy controls. RNA-seq and ATAC-seq analyses revealed extensive MS-dependent gene expression and chromatin accessibility differences in EBV-transformed, but not in primary B cells. These changes are largely accounted for by the expression levels of EBNA2, an EBV-encoded transcriptional regulator previously implicated in MS. ChIP-seq analysis revealed that EBNA2 binding with its interacting human partners RBPJ, EBF1, and PU.1 is highly enriched at MS genetic risk loci, with extensive EBNA2 allelic binding and increased enrichment at MS genetic risk loci in MS-derived cells. Our findings demonstrate that enhanced EBNA2 activity in MS alters human gene expression, chromatin accessibility, and transcription factor binding in an MS-dependent manner. Collectively, this study provides new insights into the molecular mechanisms through which EBV, particularly EBNA2, interacts with host genetic risk to contribute to MS pathogenesis.

Autophagy has been implicated in several lung diseases, either protecting tissues or driving pathology. Hypersensitivity pneumonitis (HP) is a complex inflammatory lung disease, and autophagy is heavily involved in regulating inflammation. The role of autophagy in HP remains unclear. The aim of our study was to understand the role of autophagy in HP pathogenesis. GFP-LC3 transgenic mice were exposed intranasally to Saccharopolyspora rectivirgula (SR) to induce HP and follow autophagy activation in the lung. Then, we take advantage of our Atg4b-deficient mouse model to assess how autophagy disruption impacts lung inflammation in response to SR antigen challenge. Increased autophagy activation was observed in epithelial and inflammatory cells after SR antigen exposure in GFP-LC3 transgenic lungs. GFP-LC3 puncta colocalized with ATG4B and ATG5 in epithelial and inflammatory cells after antigen exposure. Autophagy impairment limits the inflammatory response after SR antigen exposure in the lungs from the Atg4b-deficient mice when compared to WT mice. To evaluate whether lipopolysaccharide (LPS) exacerbates the inflammatory response in the Atg4b-deficient, a SR+LPS combined treatment was developed and we discovered that LPS aggravates the SR-induced HP in WT but not in Atg4b-deficient mice. Reduced HP severity in Atg4b-deficient mice was associated with decreased expression of NFkB, CCL1, CCL25, CXCL1, TNFR1, IL-13, and IL-17A, diminished CD4+ T cell recruitment and expansion, reduced M2-like macrophages, and decreased granuloma and iBALT development. Our findings highlight autophagy as a critical driver in HP pathogenesis and as a potetial target for novel theraphy development.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and long COVID are complex chronic conditions that often follow infectious triggers with overlapping clinical features but poorly defined pathophysiological relationships. This study aimed to identify disease-specific immune signatures through multiparameter immunophenotyping of monocytes, dendritic cells, and T-cell subsets. A total of 207 participants were included (ME/CFS: n = 103; long COVID: n = 63; healthy controls: n = 41). Peripheral blood mononuclear cells were analyzed using multiparameter flow cytometry. Statistical analyses included non-parametric testing, age-adjusted ANCOVA, correlation network analysis, and principal component analysis (PCA). Long COVID was characterized by increased M2-like monocyte polarization, elevated CD80 expression across monocyte subsets, expansion of dendritic cells, and reduced expression of activation markers, indicating persistent immune activation with features of immune exhaustion. In contrast, ME/CFS exhibited reduced costimulatory molecule expression, impaired CCR7-mediated immune cell trafficking, and less coordinated activation patterns, consistent with a state of immune suppression. Correlation network analysis revealed more extensive and integrated immune interactions in long COVID, while PCA identified distinct immunophenotypic components and enabled moderate discrimination between the two conditions. These findings demonstrate that ME/CFS and long COVID are characterized by distinct immune profiles, supporting the concept of divergent immunopathological mechanisms. The identified signatures may contribute to biomarker development and guide targeted therapeutic approaches.

Despite important advances in understanding the etiopathology of multiple sclerosis, factors determining disease progression remain partially understood and often difficult to predict. Specific diagnostic and prognostic biomarkers are needed to optimize the risk-benefit ratio of treatment for each patient. The aim of our study was to identify a cerebrospinal fluid proteomic signature associated with diagnosis and short- to mid-term prognosis across the multiple sclerosis continuum. Our multicentric cohort study analyzed CSF samples from 120 patients using a proteomics data-independent acquisition strategy. Differentially expressed proteins were identified across diagnostic groups: 62 patients with multiple sclerosis, 15 patients with clinically isolated syndrome, and 43 healthy controls. We also compared the CSF of patients with no evidence of disease activity with those with disease activity at 2 and 5 years of follow-up. A diagnostic and prognostic classification model was built using iterative cross-validated logistic regression models on shared differentially expressed proteins across these two comparisons. A total of 1,257 proteins were quantified, and 162 differentially expressed proteins were identified across comparisons. We identified a set of ten proteins associated with the diagnosis and prognosis of multiple sclerosis, including previously identified potential biomarkers (CH3L2, IGHG1, IGKC, LAMP2, ADA2), proteins known to be involved in the pathophysiology of multiple sclerosis (A0A8J8YUT9, AT2A2, CO3A1) and two yet unreported proteins (DSC2 and MMRN2). Multivariate models based on these proteins achieved good accuracy for the diagnosis of MS compared with CIS (area under the receiver operating characteristics curve [AUROC] up to 80% using 3 proteins) and prognosis (NEDA vs. EDA; AUROC up to 96% at 2 and 5 years; using 5 proteins). These results, which will require further investigation to validate the new biomarkers, open new perspectives on multiple sclerosis pathophysiology and therapeutic targets.

Type I interferons (IFN-Is) play a critical role in innate immunity, modulating the host response. While dysregulated IFN-I signaling has been implicated in autoimmune and infectious disorders, its role in inflammatory bowel disease (IBD) remains unclear. In this study, we extensively assessed the function of IFN-I signaling in human IBD and murine models of colitis. Expression of IFN-I signature genes was elevated in patients with active ulcerative colitis as well as multiple murine models of colitis. Single cell RNA sequencing revealed that upregulated IFN-I signature genes were enriched in myeloid cells, which exhibited increased expression of IFN receptors during mucosal inflammation. Mice carrying gain-of-function alleles of Ifnar1, a subunit of IFN-I receptor, showed heightened IFN-I signaling and altered colonic immune homeostasis at baseline, and were more susceptible to experimental colitis. In contrast, postnatal inhibition of IFNAR1, using either an inducible transgenic mouse model or an anti-IFNAR1 blocking antibody, protected against experimental colitis. Taken together, our findings reveal a previously under-recognized pathogenic role of IFN-I in IBD and provide a rationale for therapeutic intervention targeting this pathway.