Journal of Autoimmunity

ObjectivesIn dermatomyositis patients with anti-Mi2 autoantibodies, autoantibodies can enter muscle cells, leading to the aberrant expression of genes normally repressed by the Mi2/nucleosome remodeling and deacetylation (NuRD) complex. However, the mechanism by which autoantibodies interfere with Mi2/NuRD function remains unclear. This study aimed to identify additional autoantibodies in anti-Mi2-positive patients as well as the specific epitopes recognized by anti-Mi2 and any novel autoantibodies. MethodsPhage ImmunoPrecipitation Sequencing (PhIP-Seq) was used to screen serum samples from anti-Mi2-positive myositis patients for autoantibodies. Enzyme-linked immunosorbent assays (ELISA) and luciferase immunoprecipitation system (LIPS) immunoassays were used to detect autoantibodies in serum samples from myositis patients and healthy controls. ResultsPhIP-Seq identified autoantibodies recognizing the autoimmune regulator (AIRE) in sera from anti-Mi2 autoantibody-positive patients. Both anti-AIRE and anti-Mi2 autoantibodies predominantly recognized a homologous region of the plant homeodomain zinc finger type I (PHD1), which is critical for AIRE and Mi2/NuRD function. ELISA and LIPS testing showed that anti-Mi2 autoantibody-positive patients were positive for anti-AIRE autoantibodies, while AIRE reactivity was largely absent in healthy comparators, anti-Mi2 autoantibody-negative-myositis, and other autoimmune diseases. Affinity-purified anti-Mi2 autoantibodies recognized both Mi2 and AIRE by ELISA, whereas anti-Mi2-depleted immunoglobulin fractions did not recognize either protein. ConclusionsAutoantibodies recognizing Mi2 also recognize AIRE at a homologous PHD1 finger. This region is required by the Mi2/NuRD complex to anchor the nucleosome and consequently repress gene expression. Our findings suggest that anti-Mi2 autoantibodies disrupt NuRD complex function by binding to the PHD1 domain. Further studies are needed to determine if anti-Mi2 autoantibodies bind other PHD1-containing proteins and their functional implications.

BackgroundSjogrens Disease (SjD) is an autoimmune disease characterized by lymphocytic infiltration of salivary and lacrimal glands (LG). The LG produces the protein-rich aqueous component of tears, and SjD-associated autoimmune dacryoadenitis (AD) may thus alter tear autoantibody composition. MethodsThe presence of tertiary lymphoid structures (TLS) in LG from two murine models of SjD-associated AD, male NOD and male NOR mice, were evaluated using immunofluorescence. IgG and IgA reactivity in serum and tears from these models were probed in three studies against a panel of 80-120 autoantigens using autoantibody microarrays relative to serum and tears from healthy male BALB/c mice. Data were analyzed by R package Limma. ResultsAnalysis of immunofluorescence in LG sections from both SjD models showed TLS. Only one autoantibody was significantly elevated in tears and serum in both SjD models across all studies. Three autoantibodies were significantly elevated in serum but not in tears in both SjD models across all studies. Conversely, six IgG and thirteen IgA autoantibodies (6 sharing the same autoantigen) were significantly elevated in tears but not serum in both SjD models. ConclusionNOD and NOR mice with SjD-associated AD have distinct autoantibody profiles in tears and serum. Tear IgA isotype autoantibodies showed a greater diversity than tear IgG autoantibodies. TLS observed in LG are a likely source of the tear autoantibodies.

ContextAbdominal symptoms are poorly documented during primary Sjogrens syndrome (pSS). ObjectivesTo describe abdominal symptoms among pSS patients and to assess their association with characteristics of the disease. MethodsOne hundred and fifty patients followed at Hospital and University Center of Limoges were prospectively included and were evaluated using a composite global symptom score (GSS) describing abdominal symptoms and their severity. Data concerning the clinical and biological characteristics of the pSS and abdominal disorders were also collected. ResultsNinety-five per cent of pSS patients suffered from abdominal symptoms with a median GSS of 7.5{+/-}5.5 points out of 30. More than half of the patients experienced abdominal tension (68%), upper abdominal pain (54%), abdominal discomfort (58%) and/or constipation (54%). Regarding the pSS activity ESSDAI score items, general and central nervous system involvement was associated with a high GSS. Regarding the patients symptoms ESSPRI score, there was a positive correlation with the GSS (p<0.01). Multivariate analysis showed a statistical association between a high GSS and seronegative status for SSA, gastroparesis and ESSPRI score (p<0.01 for each one). ConclusionThis study revealed that more than 90% of pSS patients suffered from abdominal symptoms. There is currently no therapeutic recommendation because of the lack of specific study and comprehension of the physiopathological mechanisms involved.

ObjectiveSystemic lupus erythematosus (SLE) is a chronic autoimmune disease causing multi-organ damage. The most specific autoantibody response in SLE, present in 20-30% of patients, targets the Sm-protein and has been shown to recognize a linear Sm-derived B cell epitope containing a post-translational modification (PTM) of arginine termed symmetrical dimethyl arginine (sDMA). As autoantibodies to other PTM-modified proteins are often promiscuous, we aimed to determine the specificity and cross-reactivity of anti-Sm antibodies. MethodsSpecificity and promiscuity/cross-reactivity of anti-Sm IgG were measured by ELISA in SLE patients and healthy donors using peptides containing either sDMA or unmodified arginine. Inhibition and cross-reactivity were determined using competitive ELISA and affinity purification. Recognition of endogenous EBNA1 by anti-Sm IgG was performed by Western blot using lysates of EBV-bearing lymphoblastoid cell lines. ResultsThe sDMA residue is recognized by anti-Sm+ SLE patients regardless of the peptide amino acid sequence, with a modest impact of amino acids flanking sDMA on recognition. Most notably, IgGs targeting sDMA comprise the overall majority ([~]90%) of the anti-Sm antibody repertoire and are highly cross-reactive between SmD3108-122 and several sDMA-containing viral-derived epitopes, including full-length EBNA1. ConclusionOur data implicate that the majority of anti-Sm IgGs target the sDMA residue irrespective of its Sm-context, thus representing a prototypic anti-PTM response. These antibodies are highly promiscuous, recognizing several sDMA-modified targets, including naturally occurring viral sDMA-expressing epitopes. These findings suggest a new mechanism by which molecular mimicry of sDMA-modified viral proteins could contribute to a breach of tolerance in anti-Sm+ SLE patients. KEY MESSAGESO_ST_ABSWhat is already known on this topicC_ST_ABSO_LISymmetrical dimethylarginine (sDMA) residues are present on RGG/RG repeat regions within the SmD1, SmD3, and SmB/B subunits of the Sm protein complex in vivo. C_LIO_LIThe introduction of sDMA is essential for recognition of minimal antigenic epitopes spanning amino acids #95-119 on the SmD1 and #108-122 on the SmD3 subunits by a specific subset of anti-Sm autoantibodies. C_LI What this study addsO_LIWe show that autoantibodies targeting sDMA residues are common in anti-Sm+ SLE patients and represent the majority of the anti-Sm autoantibody repertoire. C_LIO_LIWe demonstrate that anti-sDMA autoantibodies are highly cross-reactive and can also bind to sDMA residues on other targets, including several virus-derived epitopes, thus representing a prototypic anti-modified protein antibody (AMPA) response directed against post-translationally modified proteins through symmetrical dimethylation of arginine. C_LI How this study might affect research, practice, or policyO_LIThe high cross-reactivity of Sm antibodies to various sDMA-containing epitopes, combined with a dominant antigenic specificity, provides mechanistic insight in the potential link between viral infection and tolerance breach in anti-Sm+ SLE patients. C_LI

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.

BACKGROUNDGiant cell arteritis (GCA) is a large/medium-vessel granulomatous vasculitis, and the PD-1/PD-L1 coinhibitory pathway seems to be implicated in its pathogenesis. CD4 T cells expressing high PD-1 levels, CD4+CXCR5-PD-1hi peripheral helper (Tph) and CD4+CXCR5+PD-1hi follicular helper T cells (Tfh), are key mediators of autoimmunity. Their frequencies are elevated in the peripheral blood of subjects with several autoimmune conditions but have not been investigated in GCA. Our objective was to study the frequency of circulating Tph (cTph) and Tfh (cTfh) in patients with newly diagnosed GCA (nGCA). METHODSProspective, non-interventional study on consecutive patients referred to our ultrasound GCA fast-track clinic over a period of 24 months. Peripheral blood was drawn immediately upon initial diagnosis. For each patient, an age and gender-matched healthy control (HC) was included. PBMCs isolated by Ficoll-Hypaque were examined by cytometry. Patients were subsequently treated with standard therapy according to the updated 2018 EULAR recommendations. RESULTS65 nGCA patients were included. As compared with HC, nGCA patients presented at baseline with an increased frequency of cTph and cTfh cells. Among the 46 patients who could be followed up for 12 months, 19 experienced a relapse. The baseline frequency of cTph and cTfh cells had been significantly lower in patients who relapsed as compared with those who did not. A cTph cell frequency <0.56 predicted relapse with a sensitivity of 90% and specificity of 93%. CONCLUSIONnGCA patients demonstrate increased baseline cTph and cTfh cell frequencies. Lower baseline proportions of cTph and cTfh cells associate with relapse.

BackgroundLupus nephritis (LN) is a severe manifestation of systemic lupus erythematosus (SLE) characterized by immune-mediated renal damage. While intrarenal CD8+ T cell infiltration has been linked to disease activity in patients, their pathogenic contribution remains unclear, partly due to the lack of mechanistic insight from murine models. MethodsWe investigated renal CD8+ T cell infiltration in female B6.NZMSle1/Sle2/Sle3 lupus-prone mice across different ages, comparing them to C57BL/6 controls. Histopathology was assessed using human-derived NIH Activity and Chronicity Indices, complemented by fibrosis quantification, immunohistochemistry, and digital image analysis. Kidney T cell subsets were evaluated by flow cytometry, and transcriptomic profiling was performed using microarrays. ResultsLupus-prone mice developed progressive kidney injury resembling human LN, with increasing NIH activity scores, collagen deposition, however with interindividual heterogeneity. CD8+ T cell infiltrates were significantly elevated in lupus kidneys as early as 3 months, rising with age and correlating with histological activity. CD8+ T cells localized to periglomerular and peritubular regions but did not predominate over CD4+ T cells, as confirmed by flow cytometry. Transcriptomic analyses revealed age-dependent upregulation of interferon (IFN)-stimulated genes, B cell-associated transcripts, and extracellular matrix remodeling pathways, while T cell- related signatures were more variable. ConclusionsB6.NZMSle1/Sle2/Sle3 mice recapitulate several histopathological and molecular features of human LN, including progressive fibrosis and intrarenal CD8+ T cell infiltration that correlate with disease severity. However, the absence of CD8+ predominance suggests limitations of this model for dissecting CD8+ T cell-specific contributions to LN pathogenesis. Yet, these findings underscore the need to identify renal antigens driving CD8+ T cell responses in human LN.

A comprehensive understanding of the genetic predisposition associated with the initiation and development of Sjogrens syndrome (SjS) is imperative. This would not only enrich our knowledge of the pathogenesis underlying this autoimmune disease but also address the long-standing clinical challenges of more timely diagnosis and effective treatment to retain organ function and improve prognosis. In this study, we used whole exome sequencing analysis of 50 patients with SjS to investigate the predisposing variants, genes, and their associated biological functions. Hundreds of predisposing genes were identified, and numerous biological processes and pathways were highlighted; suggesting a heterogeneity of genetic predisposition to SjS. Female patients carrying a greater number of enriched variants tended to have higher levels of serum IgG and corresponding systemic involvement, demonstrating the pivotal role of genetic predisposition in the pathogenesis of SjS. Biological function analysis indicated that a subset of SjS and neuropathies may share a similar genetic predisposition. Our results showed that extracellular matrix-receptor interactions, macrophage-associated biological functions, and motor proteins may play important roles in the pathogenesis of SjS, and macrophage-associated biological functions may be associated with early onset SjS in female patients. Furthermore, the identification of highly enriched variants in the patient cohort provides the possibility of advancing the diagnosis of SjS. In conclusion, our study provides an extensive framework for analysis of the genetic predisposition to SjS which can facilitate further focused and in-depth investigation of the pathogenetic mechanisms of specific genes, biological processes, and pathways; thereby contributing to the pathophysiology, diagnosis, and therapeutics of SjS.

Lupus nephritis (LN) occurs in up to 50% of patients with systemic lupus erythematosus (SLE), and is a major contributor to mortality and morbidity. LN presents as a highly heterogeneous disease both in histopathology and response to therapy. The molecular and cellular processes leading to renal damage and to the heterogeneity of the disease are not well understood. To elucidate the processes underpinning the heterogeneity of LN, we applied singlecell RNA-sequencing (scRNA-seq) to renal biopsies from LN patients. Skin biopsies were evaluated as a source of biomarkers for monitoring kidney disease. Type-I interferon (IFN) response signatures were identified in tubular cells and keratinocytes, differentiating LN patients from healthy controls. Non-responders associated with higher IFN signatures in both tissue compartments. Moreover, non-response was also associated with a fibrotic signature in the tubular cells. Receptor-ligand interaction analysis indicated that the fibrotic process is likely mediated by FGF receptors with the initiating signal originating from infiltrating leukocytes. Differential expression analysis of tubular cells between proliferative and membranous LN pointed to several fibrosis-relevant pathways, which may offer insight into their histological differences. In summary, scRNA-seq was applied to LN to deconstruct its heterogeneity and provide novel targets for personalized approaches to therapy.

ObjectivesPlant homeodomain (PHD) fingers are present in many chromatin-binding proteins. We recently discovered that anti-Mi2 autoantibodies recognize PHD fingers in Mi2 and AIRE. The purpose of this study was to characterize anti-Mi2 autoantibody recognition of PHD fingers in SP140L and TIF1{gamma} as well as to explore recognition of TIF1{gamma} by both anti-TIF1{gamma} and anti-Mi2 autoantibodies. MethodsLuciferase immunoprecipitation system (LIPS) assays were performed to detect autoantibodies against full-length and protein fragments of SP140L and TIF1{gamma} in serum samples from myositis patients, disease controls, and healthy controls. ResultsAnti-Mi2 autoantibodies recognized SP140L. When a 49 amino acid fragment of the PHD finger of SP140L was used as the target, the specificity for selectively detecting anti-Mi2 autoantibodies increased. Additionally, anti-Mi2 autoantibodies weakly bound TIF1{gamma} compared to anti-TIF1{gamma} autoantibodies. Excluding the TIF1{gamma} PHD finger from the TIF1{gamma} target autoantigen eliminated cross-reactivity with anti-Mi2 autoantibodies, confirming that anti-Mi2 autoantibodies specifically target the PHD finger of TIF1{gamma}. Switching two amino acids in the TIF1{gamma} PHD finger to resemble those in AIRE markedly enhanced anti-Mi2 autoantibody immunoreactivity. Anti-TIF1{gamma} autoantibodies primarily recognized the N-terminal fragment outside of the PHD finger, indicating this region contains the immunodominant epitopes. ConclusionsAnti-Mi2 autoantibodies recognize the PHD fingers of SP140L and TIF1{gamma}. TIF1{gamma} is recognized by two different myositis-specific autoantibodies: anti-Mi2 autoantibodies bind the C-terminal PHD domain and anti-TIF1{gamma} autoantibodies predominantly bind the N-terminal region. Removing the PHD finger from the anti-TIF1{gamma} target autoantigen can improve the specificity of anti-TIF1{gamma} autoantibody assays by reducing cross-reactivity with anti-Mi2 autoantibodies.

Background and objectivesMyasthenia gravis associated with antibodies against muscle-specific kinase (MuSK-MG) is a well-characterized IgG4-autoimmune disease, however, the mechanisms driving IgG4 predominance remain poorly understood. This study investigated whether promoter DNA methylation of cytokine genes involved in IgG4 class switching is associated with this immune response. MethodsPeripheral blood mononuclear cells were isolated from MuSK-MG patients (n=36), acetylcholine receptor myasthenia gravis (AChR-MG) patients as disease controls (n=7), and sex-matched healthy controls (n=12). Promoter DNA methylation of IL4, IL10, and IL13 was assessed by methylation-sensitive high-resolution melting and relative cytokine mRNA expression by qPCR. Associations with clinical variables, and antibody levels were subsequently evaluated. ResultsMuSK-MG patients showed lower median IL13 promoter methylation compared with healthy controls (p = 0.004). Median IL4 promoter methylation was also reduced in MuSK-MG compared with healthy controls (p < 0.001) and AChR-MG disease controls (p < 0.001), whereas no differences were observed for IL10 promoter methylation. Relative mRNA expression of IL4 (p = 0.0005), IL10 (p = 0.0462), and IL13 (p = 0.0002) was increased in MuSK-MG compared with AChR-MG. Compared with healthy controls, only IL4 expression remained significantly increased (p < 0.0001). Promoter methylation was inversely correlated with relative mRNA expression for IL4 (p < 0.0001), while IL13 showed a similar but non-significant trend (p = 0.054), no association was observed for IL10. Multivariable analysis demonstrated that treatment at sampling was independently associated with lower IL10 and IL13 promoter methylation, whereas no associations were observed with age, sex, disease phase, or disease duration. Promoter methylation did not correlate with total serum IgG4 or anti-MuSK IgG4 levels. DiscussionMuSK-MG is associated with selective hypomethylation of IL4 and IL13 promoters accompanied by increased cytokine gene expression, while IL10 promoter methylation remains unchanged. The association between treatment and IL10 and IL13 promoter methylation suggests that immunosuppressive therapy may influence epigenetic regulation in MuSK-MG. Together, these findings support a role for epigenetic dysregulation of Th2-associated cytokines in the immunological environment associated with IgG4 subclass switch. To our knowledge, this is the first study investigating IL4, IL10, and IL13 promoter DNA methylation in MuSK-MG.

Objective: Tissue-specific biomarkers that are reliable and associated with clinical manifestations of idiopathic inflammatory myopathy (IIM) are lacking. The blood circulation in individuals serves as a central conduit, allowing communication between tissues and facilitating clearance and recycling of tissue-derived proteins. Thus we applied a dual antibody-based proximity extension assay to profile the plasma proteome in subtypes of patients with IIM. Methods: Plasma samples from 201 patients diagnosed with IIM at Karolinska University Hospital were analyzed using Olink. Clinical manifestations, disease activity measurements, and laboratory results were collected. First, we evaluated differential protein abundance across IIM subtypes. Then we calculated tissue scores for each disease subtype using tissue gene expression databases. Finally, interferon (IFN) and complement pathway scores were calculated. Results: Plasma from patients with dermatomyositis was enriched for IFN signaling and skin associated proteins, anti-synthetase syndrome (ASyS) for lung associated proteins, immune-mediated necrotizing myopathy (IMNM) for muscle associated proteins, and inclusion body myositis for T cell response proteins. Patients with IMNM showed the highest tissue score for skeletal muscles, and ASyS for lung tissue score. Skeletal muscle scores correlated strongly with muscle disease activity visual analog scale scores, creatinine kinase levels, and muscle weakness score. Patients with interstitial lung disease along with and those with anti-Jo1 autoantibodies showed differential enrichment of surfactant proteins and IFN pathway activation. Conclusions: Circulatory proteome is a promising tool to capture distinct tissue-specific responses that correlate with disease activity and tissue injury in patients with IIM.

Women are predisposed to systemic lupus erythematosus (SLE) with a prevalence ratio of up to 9:1 over men. Multiple mouse strains including NZM2328 exhibit strong female dominance in developing spontaneous lupus as in humans with SLE. While lupus-prone mice can develop disease under germ free (GF) condition, the role of gut microbiota in female bias for lupus nephritis is not investigated systematically. Here, using specific pathogen free (SPF) and GF NZM2328 mice, and employing microbiota-depletion and microbial-association strategies, we show that microbiota influences lupus-like disease outcomes differently in males and females. Female NZM2328 mice with intact microbiota presents higher inflammation factor expression, including X-chromosome linked TLRs, in the distal gut and systemic compartments, and higher activation of genes and biological pathways such as neutrophil extracellular trap (NET) formation and complement and coagulation cascade (CCC) pathways, associating with their higher disease susceptibility. Gut microbiota-depletion as well as GF derivation eliminated not only the modest differences in the serum and fecal antibody levels and nAg reactivity, but also the gender bias in the timing of clinical stage disease onset as well as systemic NET and CCC pathway activation. Reciprocally, conventionalization of GF NZM2328 mice at juvenile age restored the female bias in intestinal and systemic autoantibody levels, pro-inflammatory immune pathway activation, and the timing of clinical stage disease onset. Overall, our observations show that, while genetic susceptibility appears to be the cause of lupus-like disease in NZM2328 mice, differential activation of NET and CCC pathways in males and females upon exposure to gut microbes, in combination with host-factors, causes gender bias in disease outcomes. We conclude that microbiota exposure-dependent protection of males and overactivation of NET and CCC pathways in females could be contributing to the female bias in lupus-like disease in NZM2328 mice.

Background and AimsCeliac disease (CeD) is a chronic digestive autoimmune disorder affecting approximately 1% of the worldwide population. It is driven by T cells activated by specific HLA-DQ2 or HLA-DQ8 molecules leading to the destruction of intestinal villi. We aimed to characterize shared CeD-specific T cells in patients on and off a gluten-free diet (GFD) from a large cohort of cases and controls. MethodsWe performed bulk TCR{beta} immune sequencing of the peripheral blood of 1,604 biopsy-confirmed CeD patients (1,339 on GFD, 265 on normal diet) and 1,100 controls. We identified over 300 TCR{beta}s enriched in CeD cases versus controls in an HLA-aware manner, controlling for CeD risk alleles. ResultsCeD-associated TCR{beta}s were found to be more predictive of disease than previously characterized gliadin and glutenin-binding TCRs in a validation cohort. Furthermore, the clonal breadth of these TCR{beta}s was associated with increased intestinal damage. Immune sequencing of the peripheral blood also uncovered repertoire-level differences between CeD patients and controls. CeD patients displayed significantly higher productive clonality compared to age-matched controls as well as expansion of TCR{beta}s specific to cytomegalovirus (CMV) and Epstein-Barr Virus (EBV). ConclusionsThese findings underscore the value of unbiased immune repertoire sequencing to identify novel biomarkers for autoimmune disease and to discover new disease mechanisms which can improve both diagnosis and treatment of disease.

Systemic lupus erythematosus (SLE) is an autoimmune disease in which the bodys immune system mistakenly attacks healthy tissue in many body parts, symptoms vary among people and may be mild to severe. Red rash on the face is the most common symptom, others include painful and swollen joints, fever, chest pain, and feeling tired [1]. Both genetics and environmental factors are identified to be involved in SLE [2]. Currently, there is no cure for SLE, and corticosteroids are usually used as one of the treatment methods, but long-term use leads to side effects [3]. Identifying new therapeutic targets is crucial for improving the treatment of Systemic Lupus Erythematosus (SLE). By integrating bulk and single-cell RNA-seq data with spatial transcriptomics of keratinocytes, we found KLHDC7B to be upregulated in UV-treated HaCaT cells, SLE skin keratinocytes, and spatially resolved epidermal samples. These findings suggest KLHDC7B as a potential therapeutic target for SLE.

BackgroundGastrointestinal (GI) involvement in systemic sclerosis (SSc) affects up to 90% of patients and is a major driver of morbidity and mortality. Despite its clinical importance, GI disease in SSc is highly heterogeneous, with upper and lower GI manifestations representing distinct phenotypic extremes whose underlying immunologic basis remains poorly defined. MethodsWe performed unbiased, proteome-wide autoantibody profiling using a human protein microarray comprising >21,000 full-length proteins (>80% of the human proteome). Sera from patients with SSc and isolated upper GI dysmotility (n=23), isolated lower GI dysmotility (n=17), and non-SSc controls (n=20) were analyzed. Enriched autoantibodies were identified using Fishers exact test, and unsupervised clustering was applied to define serology-based patient subsets and relate immune signatures to clinical phenotypes. ResultsDistinct autoantibody profiles differentiated patients with upper versus lower GI disease. Upper GI-predominant SSc was characterized by enrichment of previously unreported autoantibodies, including those targeting TiSSc1/2 (newly identified proteins encoded within the MIRLET7BHG locus), FAM9C, SPATA20, FAM110D, EMILIN1, CARD14, SMN1, KCTD7, and PHYHD1, whereas lower GI disease was associated with antibodies against HAO2, KLHL7, SUFU, APPL1, BNIP2, UCHL3, ZNF385A, LIMD1, MAGEA9, and PPP2R3C. Serology-driven clustering identified four reproducible subgroups with distinct patterns of GI, pulmonary, vascular, and autonomic involvement, defining clinically meaningful disease phenotypes that extend beyond traditional anatomic classification. ConclusionsProteome-scale serological profiling reveals previously unrecognized autoimmune signatures underlying GI heterogeneity in SSc. These findings support a shift from anatomy-based to serology-defined classification of SSc GI disease and provide a foundation for biomarker development, patient stratification, and precision medicine approaches in this population.

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.

ObjectivesSj{square}grens disease (SjD) diagnosis requires either positive anti-SSA antibodies or a labial salivary gland biopsy with a positive focus score (FS). One-third of SjD patients lack anti-SSA antibodies (SSA-), requiring a positive FS for diagnosis. Our objective was to identify novel autoantibodies to diagnose seronegative SjD. MethodsIgG binding to a high density whole human peptidome array was quantified using sera from SSA- SjD cases and matched non-autoimmune controls. We identified the highest bound peptides using empirical Bayesian statistical filters, which we confirmed in an independent cohort comprising SSA- SjD (n=76), sicca controls without autoimmunity (n=75), and autoimmune controls (SjD features but not meeting SjD criteria; n=41). In this external validation, we used non-parametric methods for peptide abundance and controlled false discovery rate in group comparisons. For predictive modeling, we used logistic regression, model selection methods, and cross-validation to identify clinical and peptide variables that predict SSA- SjD and FS positivity. ResultsIgG against a peptide from D-aminoacyl-tRNA deacylase (DTD2) was bound more in SSA- SjD than sicca controls (p=.004) and more than combined controls (sicca and autoimmune controls combined; p=0.003). IgG against peptides from retroelement silencing factor-1 (RESF1) and DTD2, were bound more in FS-positive than FS-negative participants (p=.010; p=0.012). A predictive model incorporating clinical variables showed good discrimination between SjD versus control (AUC 74%) and between FS-positive versus FS-negative (AUC 72%). ConclusionWe present novel autoantibodies in SSA- SjD that have good predictive value for SSA- SjD and FS-positivity. KEY MESSAGESO_LIWhat is already known on this topic - Seronegative (anti-SSA antibody negative [SSA-]) Sjogrens disease (SjD) requires a labial salivary gland biopsy for diagnosis, which is challenging to obtain and interpret. C_LIO_LIWhat this study adds - We identified novel autoantibodies in SSA- SjD that, when combined with readily available clinical variables, provide good predictive ability to discriminate 1) SSA- SjD from control participants and 2) abnormal salivary gland biopsies from normal salivary gland biopsies. C_LIO_LIHow this study might affect research, practice or policy - This study provides novel diagnostic antibodies addressing the critical need for improvement of SSA- SjD diagnostic tools. C_LI

This study presents "aUToAntiBody Comprehensive Database (UT-ABCD)", a comprehensive catalog of autoantibody profiles in 284 human individuals. The subjects include patients diagnosed with Coronavirus disease 2019 (COVID-19; n = 73), systemic sclerosis (SSc; n = 32), systemic lupus erythematosus (SLE; n = 60), anti-neutrophil cytoplasmic antibody-associated vasculitis (AAV; n = 29), atopic dermatitis (AD; n = 26), as well as healthy controls (HC; n = 64). Our investigation employs proteome-wide autoantibody screening (PWAbS) that utilizes 13,352 autoantigens displayed on wet protein arrays (WPAs). Our WPAs display human proteins synthesized in vitro utilizing a wheat germ cell-free system, maintained in a hydrated state. Our findings demonstrated significant elevation in the number of IgG autoantibody positivity in COVID-19, SSc, SLE, AAV, and AD patients compared to HCs. Employing machine learning, we distinguished COVID-19 cases with high accuracy based on autoantibody profiles, notably identifying antibodies against proteins encoded by BCORP1 and KAT2A as highly specific to COVID-19 (specificity: 87% and 97%, respectively). Our research highlights the effectiveness of integrating PWAbS and autoantigenomics in exploring immune responses in COVID-19 and other diseases. It provides a deeper understanding of the autoimmunity landscape in human disorders and introduces a new bioresource for further investigation.

BackgroundSystemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by a loss of self-tolerance, causing inflammation and tissue damage in multiple organs. Animal models have advanced our understanding of SLEs molecular basis, but the FDAs recent elimination of animal testing requirements for drug approval has raised concerns about their validity, prompting a reevaluation of their role in basic research, especially for heterogeneous diseases like SLE. MethodsFour different spontaneous SLE mouse models were studied: MRLlpr/lpr, NZB/W, BXSB.Yaa, and Tlr7.Tg6. Transcriptome sequencing from blood, spleen, and kidney, flow cytometry from the spleen, and cytokines and autoantibody measurement in plasma were performed at four time points. Similar molecular data from human SLE patients was used for the integration. ResultsThe study identified specific molecular pathways driving the phenotype in each mouse model and established optimal time points for future experimental designs. By comparing these pathways to human SLE, the most similar ones and their relationship with disease activity were identified, providing crucial insight into translational relevance. Importantly, disease severity across models was linked to the extent and timing of molecular dysregulations. As expected, MRLlpr/lpr showed the most aggressive phenotype with early immune activation and apoptosis dysregulation, while Tlr7.Tg6 presented late-onset signatures associated with interferon and inflammation. Shared molecular features with human SLE included interferon responses, T and B cell depletion, and neutrophil activation. Integration analysis revealed distinct yet overlapping immune pathways between models and species, with some signatures such as age-associated B cells and double-negative memory T cells being model-specific but potentially relevant to early disease processes. ConclusionsThese findings build a valuable framework for future SLE research, reinforcing the utility of mouse models in studying specific molecular pathways related to human SLE pathogenesis and heterogeneity. The integration of longitudinal mouse data with human transcriptomes highlights the models that best recapitulate key aspects of human disease, offering guidance for the study of specific immunopathological mechanisms or therapeutic targets.