Clinical Immunology

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.

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.

IgA nephropathy (IgAN) is a common primary glomerulonephritis characterized by glomerular immune-complex deposits with (co)dominant IgA. These deposits are enriched for IgA1 glycoforms with some O-glycans deficient in galactose (Gd-IgA1). Circulating Gd-IgA1 is bound by IgG autoantibodies to form immune complexes, some of which deposit in glomeruli. Genomic and immunologic studies indicate involvement of pro-inflammatory signaling pathways in the production of Gd-IgA1 in IgAN. Genomic studies identified multiple genetic loci associated with IgAN and suggested a convergence on the NF-{kappa}B pathway, including RELA, the gene encoding the NF-{kappa}B subunit p65. However, the mechanisms by which NF-{kappa}B pathways may affect O-glycosylation in IgA1-producing cells are unknown. Using EBV-immortalized B cells derived from peripheral-blood mononuclear cells of IgAN patients and healthy controls that have constitutively activated NF-{kappa}B, we report that inhibition of NF-{kappa}B/p65 by a selective IKK{beta} inhibitor TPCA-1 reduced phosphorylation of NF-{kappa}B/p65 at S536 and decreased production of IgA1 and, conversely, increased Gd-IgA1 production. This was likely related to reduced expression of C1GALT1 gene that encodes the enzyme responsible for galactosylation of IgA1 O-glycans. Flow-cytometry imaging revealed changes in nuclear translocation and co-localization of the NF-{kappa}B/p65 with co-transcriptional factor SP1, a transcriptional activator of C1GALT1, suggesting that NF-{kappa}B pathway affects IgA1 O-glycosylation via SP1 transcriptional control of C1GALT1 expression. Furthermore, prolonged IKK{beta} inhibition altered B cell subpopulations, enhancing generation of cells with a plasmablast-like phenotype, characterized by high SSC MFI and CD138 expression. Together, these findings provide functional evidence for involvement of NF-{kappa}B/p65 and its transcriptional partners in IgA1 O-glycosylation. HighlightsO_LIIKK{beta} inhibition reduced C1GALT1 expression and thereby increased galactose-deficient IgA1 (Gd-IgA1) production in immortalized human B cells. C_LIO_LISP1+ subpopulations, a transcriptional activator of C1GALT1, declined after sustained NF-{kappa}B inhibition. C_LIO_LINF-{kappa}B inhibition shifted a subpopulation of B cells into a plasmablast-like phenotype. C_LIO_LIThis study links NF-{kappa}B signaling with the GWAS-identified RELA susceptibility locus and IgA1 O-glycosylation. C_LI

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterised by sustained type I interferon signalling and widespread immune dysregulation. Low-density neutrophils (LDNs) are expanded in SLE and display pro-inflammatory and tissue-damaging properties. However, their metabolic phenotype remains poorly defined. Here, we performed a comprehensive metabolic characterisation of circulating LDNs and normal-density neutrophils (NDNs) from patients with SLE and matched healthy individuals (HC). Neutrophil subsets were isolated from peripheral blood of SLE patients and HC donors using a two-step protocol of negative selection and Percoll density centrifugation. Immunophenotyping phenotype was carried out by flow cytometry to assess phenotypic expression of common neutrophil markers CD15, CD16, CD10, CD66b, CD62L, MPO, and IL-1{beta}. Bioenergetic profiling of LDNs and NDNs was performed in situ using the Seahorse MitoStress test to measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Metabolic flexibility and phenotypic alterations were assessed in LDNs and NDNs following inhibiting mitochondrial metabolism with oligomycin and glycolysis with 2DG. We found that SLE LDNs exhibit an immature phenotype compared with autologous and healthy NDNs, as determined transcriptionally by C/EBP{varepsilon} and by surface protein expression levels of CD10. Both LDNs and NDNs from SLEDAI[&ge;]4 patients demonstrated significantly elevated ECAR relative to HC neutrophils. Further, SLE LDNs displayed enhanced metabolic flexibility, with the capacity to switch towards a glycolytic phenotype under metabolic stress conditions. Inhibition of glycolysis altered the inflammatory and maturation-associated phenotype of both SLE neutrophil subsets, indicating a direct link between cellular metabolism and pathogenic neutrophil function. Collectively, these findings identify fundamental metabolic alterations in SLE neutrophil subsets and support neutrophil immunometabolism as a potential therapeutic target in SLE.

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.

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.

Autoimmune diseases result from immune responses against self-antigens but exhibit marked phenotypic diversity shaped by genetic and environmental factors. Genome-wide association studies (GWAS) have identified susceptibility loci that inform polygenic scores (PGS) for risk prediction. This study integrates phenotypic and genetic data from the UK Biobank(UKB) to characterize disease overlap, genetic heterogeneity, and shared biological mechanisms across autoimmune conditions. Comorbidity patterns were further assessed using patient records from UKB and the TriNetX(TNX). Phenotypic data from 502,371 UKB participants were used to evaluate diagnostic overlap, with a subset of 104,544 individuals analyzed for PGS distributions. Significant variants were identified using genome-wide thresholds, allele frequency, and predicted impact, and shared genes were subsequently mapped to pathways using Hallmark gene sets. Comorbidity across rare and common autoimmune diseases was assessed in the UKB and TNX using ICD-10 codes, focusing on White individuals (71,069,654 in TNX; 502,371 in UKB). Odds ratios for 15 diseases were estimated, and cross-cohort comparisons evaluated reproducibility and cohort-specific differences. PGS analyses revealed both shared and distinct genetic architectures, indicating partial genetic overlap and supporting poly-autoimmunity. Integration of common, rare and impactful variants identified both known and novel gene associations, while pathway analysis highlighted systemic and tissue-specific immune dysregulation. Cross-dataset comparisons confirmed consistent comorbidity patterns but underscored the impact of dataset-specific factors, emphasizing the need for standardized approaches in autoimmune disease research.

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.

Coeliac Disease (CeD) is a chronic gastrointestinal inflammatory disease initiated by dietary gluten in genetically predisposed individuals. While the inflammatory processes which drive tissue destruction in the coeliac duodenum have been extensively characterised, an increased oxidative stress (OS) response has also been suggested to contribute to CeD pathogenesis. However, the precise mechanisms which regulate OS in the coeliac mucosa and whether they impact inflammation remain ill defined. The master anti-oxidant transcriptional regulator Nuclear factor erythroid 2-related factor 2 (Nrf2), and its inhibitor, Kelch like ECH-associated protein 1 (Keap1) have been implicated in chronic gastrointestinal inflammatory diseases, such as ulcerative colitis but have been largely unexplored in the context of CeD. To investigate redox balance in the CeD duodenum, we utilised single cell transcriptomics to assess overall OS and cytoprotective Nrf2 activation across cell subsets in duodenal biopsies from CeD patients. OS induced gene expression was broadly increased across multiple cell subsets in the CeD mucosa. Simultaneously, specific markers of Nrf2 activation were decreased in cell subtypes central to pathogenesis of CeD, including activated CD4+ T cells and intraepithelial T lymphocytes, indicating a distinct redox imbalance in these cells. Furthermore, pharmacological activation of Nrf2 significantly decreased gliadin induced IFNG expression in CeD duodenal biopsies. Taken together, our findings demonstrate that redox imbalance represents a therapeutic opportunity for the modulation of proinflammatory responses that drive the pathogenesis of CeD.

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

Systemic lupus erythematosus (SLE) is associated with infection susceptibility and altered innate immune function. Monocyte metabolism is linked to appropriate cytokine release and bacterial containment. We investigated cytokine production and metabolic programming in the monocyte population from SLE patients and healthy controls following lipopolysaccharide (LPS) stimulation. SLE monocytes displayed increased IL-10, TNF, and IL-8 production, with impaired IL-1{beta} induction. Metabolic profiling revealed altered substrate use, with increased glucose dependence and reduced fatty acid and amino acid oxidation after LPS stimulation. SLE patients exhibited reduced numbers of classical monocytes, expansion of intermediate monocytes, and dysregulated subset-specific metabolic reprogramming in response to LPS. This descriptive study provides a cornerstone for (i) understanding infection susceptibility in SLE, (ii) subset-resolved immunometabolic profiling as a tool in autoimmunity, and (iii) developing future metabolic-targeted therapeutic strategies HighlightsO_LIDescriptive mapping shows SLE monocytes are proinflammatory with glucose dependence after LPS C_LIO_LIClassical and intermediate SLE subsets show divergent baseline metabolic preferences versus healthy C_LIO_LISLE subsets display aberrant LPS responses, i.e.. increased glucose and reduced fatty acid oxidation C_LIO_LIThis study provides a cornerstone for subset-resolved immunometabolism in infection susceptibility. C_LI

ObjectivesLupus nephritis (LN) is a severe complication of systemic lupus erythematosus (SLE), leading to progressive renal fibrosis and functional decline. Understanding the interplay between immune cells and stromal cells is needed to develop effective therapeutic strategies. Here, we investigated the landscape of macrophage-fibroblast interactions in human LN and validated these findings in mouse models. MethodsWe characterized distinct fibroblast subsets and their interactions with renal macrophages using single-cell RNA sequencing (scRNAseq) of 156 human LN biopsies and 30 healthy controls from the AMP-SLE cohort, and spatial transcriptomics of biopsies from 6 LN patients. In vitro co-culture studies using mouse models were performed to further define functional consequences of these interactions. ResultsWe identified two myofibroblast subsets: a pro-inflammatory subset (Myofib1) enriched in the tubulointerstitium, and a fibrotic/remodeling subset (Myofib2) in glomeruli, both correlating with the histologic chronicity index. Spatial transcriptomics revealed different colocalization patterns, with Myofib1 interacting with activated resident macrophage (RM) subsets and Myofib2 with glomerular infiltrating disease-associated macrophages. In vitro co-culture studies demonstrated that nephritic RMs promote a pro-inflammatory, remodeling fibroblast phenotype that impairs wound healing and drives a Myofib1-like gene program, whereas disease-associated macrophages generated profibrotic fibroblasts with dysregulated reparative capacity. Cell-cell communication analyses identified key ligand-receptor interactions mediating this crosstalk, including Spp1/integrins, Sema4/PlexinB, and NAMPT/INSR. ConclusionsOur data reveal a spatially and functionally heterogeneous landscape of macrophage-fibroblast crosstalk in LN. These findings advance our understanding of renal fibrogenesis in LN, highlighting specific fibro-inflammatory circuits that may represent therapeutic targets to prevent chronic renal damage.

ObjectiveSystemic sclerosis (SSc) predominantly affects females but exhibits greater disease severity in males, suggesting sex differences underlying SSc pathogenesis. We sought to define sex-associated alterations in the peripheral immune landscape of patients with SSc. MethodsWe performed high-dimensional immune profiling of PBMCs from 37 healthy donors (68% female) and 37 patients with SSc (11 limited, 26 diffuse; 68% female) using 30-color spectral flow cytometry, quantifying 56 immune cell subsets per donor. We conducted sex-stratified comparisons and correlation analysis, and used principal component analysis followed by linear discriminant analysis to derive a sex-discriminant immune cellular module. ResultsDiffuse cutaneous SSc (dcSSc) was associated with a distinct immune landscape characterized by increased monocyte and decreased natural killer-like and B cell frequencies, suggesting a myeloid-skewed peripheral immunophenotype. Males exhibited greater enrichment of innate immune subsets, including monocyte and dendritic cell subsets, while females exhibited greater enrichment of adaptive immune subsets. Among T cells, dcSSc was associated with coordinated remodeling across CD4+ and CD8+ subsets, including expansion of stem cell memory T cells (Tscm), and increased regulatory T cells, Th17 skewing, and decreased effector-memory CD8+ subsets. Females exhibited greater proportions of naive- and Tscm, and males exhibited higher proportions of effector-memory subsets. Integrating these data, we identified a sex-discriminant immune module comprised of 20 cell types that distinguishes males and females with dcSSc. ConclusionsSSc is associated with sex-specific differences in the peripheral immune landscape. A sex-associated immune program, further amplified in disease, may contribute to the paradox of female-biased susceptibility and male-biased severity in SSc.

Long-lived plasma cells (LLPC) sustain humoral immunity but also contribute to the persistence of pathogenic autoantibodies in autoimmune diseases. New therapies targeting LLPC are therefore desirable. Recent studies have shown increased expression of Slc12a2, encoding the Na+ -K+ -Cl- cotransporter (NKCC1), in LLPC. This study investigated whether NKCC1 activity was required for plasma cell survival, persistence or secretion of antibodies. Across in vitro and in vivo settings, mouse plasma cell survival was undiminished by treatment with the NKCC1 inhibitor bumetanide. Acute in vivo bumetanide treatment did not diminish plasma cell numbers, nor show any demonstrable impact on the survival of phenotypically mature I-A/I-EloSLAMF6lo plasma cells. With genetic plasma cell timestamping, even the survival of persistent LLPC was unaffected by bumetanide. Plasma cell secretory capacity, assessed by measuring IgM and IgG2b secretion in culture over three days, was also unaltered by bumetanide. Overall, these results show that pharmacological inhibition of NKCC1 is not sufficient to impair plasma cell survival, persistence or antibody secretion. Despite elevated Slc12a2 mRNA expression in LLPC, NKCC1 alone does not represent a critical plasma cell survival pathway, highlighting the resilience of plasma cells and the challenges associated with therapeutically targeting LLPC.

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.

Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal Infections (PANDAS) is characterized by prepubertal abrupt onset of obsessive-compulsive disorder (OCD). The sine qua non is group A streptococcus (GAS) infection, which is hypothesized to elicit an IgG-class anti-GAS antibody response that cross-reacts with antigens in the basal ganglia. However, the association between GAS antibody (GAS-IgG) levels and PANDAS has been inconsistent, and qualitative differences in GAS-IgG profiles have not been carefully evaluated in well-phenotyped cohorts. Moreover, independent studies have yet to converge on anti-neural autoantibodies that are specific to PANDAS. Here, we used phage display immunoprecipitation sequencing (PhIP-Seq) to perform ultra-deep anti-pathogen antibody repertoire profiling of serum from definitive pediatric PANDAS patients (N = 34) collected as part of a prior double-blind, placebo-controlled clinical trial of intravenous immunoglobulin (IVIg). PANDAS cases were compared to pediatric controls without a history of neuropsychiatric illness (N = 31). To assess for objective evidence of neuroglial injury, serum neurofilament light (NfL) and glial fibrillary acidic protein (GFAP) levels were compared to healthy pediatric controls. Within PANDAS, NfL and GFAP levels were compared between pre- and post-treatment sera. To evaluate for central autoantibodies, a subset of baseline cerebrospinal fluid (CSF) samples (N = 25) was profiled by full-length human protein microarray. Though GAS reactivity by PhIP-Seq was well correlated with clinical anti-DNaseB and anti-streptolysin O titers, there were no quantitative or qualitative differences in GAS-IgG profiles between PANDAS and controls. Furthermore, NfL and GFAP levels did not differ between cases and controls. Within PANDAS, changes in NfL or GFAP levels at six weeks did not differ between placebo and IVIg groups. However, CSF autoantibody profiling by protein microarray revealed infrequent but notable candidate autoantibodies. In one patient, we identified autoantibodies against Argonaute family proteins (AGO-IgG), a marker of autoimmune sensory neuropathy. Longitudinal measurement of AGO-IgG in sera revealed that titers were unchanged after placebo, but decreased after IVIg, coinciding with symptomatic improvement, including a decrease in that patients CY-BOCS score. Overall, these results do not support an etiologic role for GAS-IgG in PANDAS. However, some individuals diagnosed with PANDAS may harbor anti-neural autoantibodies.

Germinal matrix-intraventricular hemorrhage (GM-IVH) is one of the most frequent and severe neurological complications in preterm infants (PT). It triggers an inflammatory response accompanied by neuronal and glial injury and may progress to post-hemorrhagic ventricular dilatation (PHVD), thereby increasing long term disability and cognitive deficits. Nevertheless, the characteristics and evolution of the associated pathology is poorly understood. To assess neuroimmune response and neuropathology induced by GM-IVH, we quantified cytokines, glial activation and neurodegeneration makers in cerebrospinal fluid collected from 12 patients with grades III/IV GM-IVH and PHVD and 5 controls neonates from the onset of pathology up to 2 months of age. Additionally, to evaluate long-term deficits and behavioral outcomes, we used standard behavioral test including Bayley Scales of Infant and Toddler Development, Third Edition (Bayley-III) at 2 years of age. Interestingly, we found that while pathology markers such as ubiquitin carboxy-terminal hydrolase L1 (UCHL1), alpha II spectrin breakdown product 145 (SBDP145) and myelin basic protein (MBP) are elevated in PT, their level decline over time. Furthermore, cytokine profiling identified two divergent temporal trajectories (i.e., diminishing or sustained) that correspond with either neuronal or astrocytic markers. Specifically, diminishing cytokines including IL-6, IL-8, and IP-10 decreased with age and were correlated with neuronal markers such as SBDP145, UCH-L1, and MBP. In contrast, sustained cytokines such as IFN-{gamma}, IL-7, IL-13, and MCP-1 remained elevated or unchanged throughout the study period and were positively correlated with astrocyte reactivity marker GFAP. Notably, sustained cytokines were consistent with worse motor function and behavioral outcome. Together, longitudinal CSF analysis in PT with severe GM-IVH and PHVD identifies a cytokine profile that declines and correlates with neuronal and glial injury markers, and another that remains sustained and correlates with gliosis and adverse neurodevelopmental outcomes. These findings highlight potential CSF biomarkers associated with disease progression and long-term neurological impairment, providing a foundation for future evaluation of candidate therapeutic interventions.

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.

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.