Interferon Signature Research Articles

Case report: Exploring efficacy of tofacitinib in modulating interferon response in five case of anti-MDA5+ dermatomyositis with interstitial lung disease

A case report highlights the challenges faced in managing a 66-year-old Chinese woman diagnosed with anti-MDA5 antibody-positive dermatomyositis (MDA5-DM) complicated by rapidly progressive interstitial lung disease (RP-ILD). Despite aggressive therapeutic interventions, her condition rapidly deteriorated, emphasizing the severity and devastating nature of this subtype of DM. A salient feature of her clinical presentation was the marked elevation of interferon (IFN)-γ and IFN-α levels, underscoring the pivotal role that IFNs play in driving the pathogenesis and progression of MDA5-DM-related RP-ILD. In an attempt to stem the relentless progression, tofacitinib, a Janus kinase (JAK) inhibitor, was applied into her treatment regimen. This therapeutic intervention led to a transient decrease in IFN-related cytokines, offering a glimpse of hope that JAK inhibition could modulate the exaggerated IFN response implicated in the disease. Other four similar cases underscore the critical importance of early and aggressive intervention in MDA5-DM patients, as well as the potential therapeutic avenues involving IFN blockers by JAK inhibitors. Urgent and well-designed clinical trials are imperative to unravel the intricate interplay between RP-ILD and the IFN signature in MDA5-DM, and to evaluate novel therapeutic targets that promise long-term efficacy and safety.

Vitiligo-update on pathogenesis, diagnostics and therapy

Vitiligo is acommon skin disease leading to depigmentation that is associated with progressive destruction of melanocytes. The main subtype, nonsegmental vitiligo (NSV), is considered achronic autoimmune disease leading to activation of melanocyte-specific CD8+ lymphocytes and development of acutaneous immune memory. Vitiligo can result in significant impairment of quality of life and is associated with anumber of comorbidities. Despite the existence of national guidelines and international treatment recommendations, there is astrong need for improved health care for patients with vitiligo. The options in the treatment algorithm for vitiligo are similar and consisted until recently of only topical and systemic corticosteroids, topical calcineurin inhibitors, phototherapy, and surgical interventions. New insights into the pathobiological role of the interferon signature in vitiligo have most recently contributed to the approval of the first vitiligo-specific therapy with the topical Janus kinase1/2 inhibitor ruxolitinib for patients with NSV.

Chronic CRYPTOCHROME deficiency enhances cell-intrinsic antiviral defences.

The within-host environment changes over circadian time and influences the replication and severity of viruses. Genetic knockout of the circadian transcription factors CRYPTOCHROME 1 and CRYPTOCHROME 2 (CRY1-/-/CRY2-/-; CKO) leads to altered protein homeostasis and chronic activation of the integrated stress response (ISR). The adaptive ISR signalling pathways help restore cellular homeostasis by downregulating protein synthesis in response to endoplasmic reticulum overloading or viral infections. By quantitative mass spectrometry analysis, we reveal that many viral recognition proteins and type I interferon (IFN) effectors are significantly upregulated in lung fibroblast cells from CKO mice compared with wild-type (WT) mice. This basal 'antiviral state' restricts the growth of influenza A virus and is governed by the interaction between proteotoxic stress response pathways and constitutive type I IFN signalling. CKO proteome composition and type I IFN signature were partially phenocopied upon sustained depletion of CRYPTOCHROME (CRY) proteins using a small-molecule CRY degrader, with modest differential gene expression consistent with differences seen between CKO and WT cells. Our results highlight the crosstalk between circadian rhythms, cell-intrinsic antiviral defences and protein homeostasis, providing a tractable molecular model to investigate the interface of these key contributors to human health and disease.This article is part of the Theo Murphy meeting issue 'Circadian rhythms in infection and immunity'.

P0103 Systems biology analysis of cytokine signalling in IBD highlights the importance of TYK2-dependent pathways

Abstract Background Ulcerative colitis (UC) and Crohn’s disease (CD), collectively inflammatory bowel disease (IBD), are characterized by chronic gastrointestinal tract inflammation. Recently, there has been interest in the use of selective oral tyrosine kinase 2 (TYK2) inhibitors for IBD treatment, which inhibit signalling downstream of multiple pro-inflammatory cytokines and may have an improved safety profile relative to non-specific Janus kinase (JAK) inhibition. Here, we use a transcriptomic approach to assess the involvement of TYK2-dependent signalling pathways in IBD, and how these are impacted by currently available treatments. Methods TYK2-dependent gene signatures were characterized using cytokine stimulation studies and co-expression analysis. Involvement of signature modules in IBD was assessed using disease model data generated from 4059 biopsy samples from 2993 unique patients in 15 UC and 38 CD clinical studies, including assessments of golimumab, infliximab, ustekinumab, and vedolizumab. Pathway differential expression analysis was performed using Gene Set Enrichment Analysis (GSEA) and single-sample GSEA methods.1,2 Results Seven TYK2-dependent gene signature modules were defined, including two for type I interferon (IFN), three for IL-23, and two for IL-12. Type I IFN signatures were strongly enriched in both UC and CD, while enrichment of IL-23 was stronger for UC than for CD, and enrichment of IL-12 was stronger for CD than for UC. In each case, gene signature module enrichment was associated with greater disease severity. Pathway enrichment analysis of data from individual clinical studies demonstrated that specific type I IFN and IL-12 modules were significantly reduced, but not completely ameliorated, following treatment with golimumab, infliximab, ustekinumab, or vedolizumab. Further assessment of response to therapy in these studies showed that enrichment of type I IFN, IL-23, and IL-12 signature modules was associated with non-response to golimumab, infliximab, and vedolizumab. Conclusion Enrichment of TYK2-dependent gene signatures, including type I IFN, IL-23, and IL-12, is associated with both UC and CD. These TYK2-dependent enrichments are not fully mitigated through currently available treatments, and may be associated with non-response to treatment. It is important that all three signalling pathways are considered in the development of future treatment options.

P0212 Non-canonical STING signaling in intestinal epithelial cells guides DNA damage-driven stem cell function and protects from intestinal carcinogenesis

Abstract Background RNaseH2 plays a critical role in removing mis-bounded RNA from the DNA double strand helix1. Absence of RNaseH2 results in spontaneous double-stranded DNA breaks and cytosolic release of dsDNA2. We have previously shown that epithelial deficiency of RNaseH2b (H2bΔIEC) leads to a p53-dependent stem cell suppression and additional deficiency of p53 results in spontaneous intestinal carcinogenesis3. Due to the central role of cGAS/STING in sensing dsDNA, we aim to investigate the role of epithelial STING in a mouse model of chronic epithelial DNA damage and tumor prevention in this study. Methods DNA damage was assessed using comet assay in ModeK cell line with cGAS and STING knock out. Protein kinase activity were assessed by Pamgene Kinase Assay. Intestinal organoids were generated from H2bΔIEC and H2bΔIEC/Tmem173-/-mice. Colony forming assay and EdU staining were performed in all genotypes to assess the impact of STING on epithelial stem cell function and proliferation were assessed by CellTiter Glo assay. RNA sequencing was performed in intestinal organoids. Spontaneous carcinogenesis was assessed in 1-year aged mice. Results Loss of RNaseH2b results in activation of cGAS/STING pathway and subsequent upregulation of IFN-I signature. Deficiency of cGAS/STING lead to impaired IFN-I independent DNA damage repair which were accessed by WB and comet assay. During the DNA damage response, STING but not cGAS mediates the P53 pathway and the P21-CDK axis. Organoid EdU stanning displayed restored proliferation in H2bΔIEC/Tmem173-/- compared to H2bΔIEC. RNA sequencing from H2bΔIEC/Tmem173-/-organoid further confirmed decreased G2/M DNA damage checkpoint and P53 signature compared to H2bΔIEC. In vivo, we show that H2bΔIEC/Tmem173-/- mice have downregulated DNA damage repair and developed spontaneous small bowel adenocarcinoma after 53 weeks. Tumor derived from H2bΔIEC/Tmem173-/- mice show sensitivity to CDK inhibitor consistent with in vitro validation. Conclusion We provide evidence that non-canonical role of epithelial STING mediates DNA damage response and guides P21-CDK1 axis in preventing tumor genesis. Our research provided evidence understand the role of STING in DNA damage response and intestine carcinogenesis.

Early assessment of IL8 and PD1+ Treg predicts response and guides treatment monitoring in cemiplimab-treated cutaneous squamous cell carcinoma

PurposeAnti-programmed cell death 1 (PD1) is the first-choice treatment in patients with advanced cutaneous squamous cell carcinoma (cSCC), when curative options are unavailable. However, reliable biomarkers for patient selection are...

The Inhibitory Effects of Alpha 1 Antitrypsin on Endosomal TLR Signaling Pathways.

Endosomal toll-like receptors (TLRs) TLR7, TLR8, and TLR9 play an important role in systemic lupus erythematosus (SLE) pathogenesis. The proteolytic processing of these receptors in the endolysosome is required for signaling in response to DNA and single-stranded RNA, respectively. Targeting this proteolytic processing may represent a novel strategy to inhibit TLR-mediated pathogenesis. Human alpha 1 antitrypsin (hAAT) is a protease inhibitor with anti-inflammatory and immunoregulatory properties. However, the effect of hAAT on endosomal TLRs remains elusive. In this study, we first tested the effect of hAAT on TLR9 signaling in dendritic cells (DCs). We showed that hAAT inhibited TLR9-mediated DC activation and cytokine production. Human AAT also lowered the expressions of interferon signature genes. Western blot analysis showed that hAAT reduced the expression of the active form (cleaved) of TLR9 in DCs, indicating a novel mechanism of hAAT function in the immune system. We next tested the effect of hAAT on TLR7/8 signaling. Similar to the effect on TLR9 signaling, hAAT also inhibited R848 (TLR7 and 8 agonist)-induced DC activation and functions and lowered the expressions of interferon signature genes. Our in vivo studies using hAAT transgenic mice also showed that hAAT attenuated R848-induced pathogenesis. Specifically, hAAT completely blocked the R848 induction of germinal center T cells (GC T), B cells (GC B), and plasma cells (GC PCs), as well as T follicular T helper cells (TFH), which are all critical in lupus development. These data demonstrated that hAAT inhibited TLR7/8 and TLR9 signaling pathways, which are critical for lupus development. These findings not only advanced the current knowledge of hAAT biology, but also implied an insight into the clinical application of hAAT.

Distinct PathophysiologicPathways Support Stratification of Sjögren's Disease Based on Symptoms, Clinical, and Routine Biological Data.

Recently, three distinct phenotypes of patients with Sjögren disease (SjD) have been described based on cluster analysis: B cell active with low symptoms (BALS), high systemic activity (HSA), and low systemic activity with high symptoms (LSAHS). We aimed to assess whether these clusters were associated with distinct biomarkers and the prognostic value of interferon (IFN) signature. The Assessment of Systemic Signs and Evolution in Sjögren's Syndrome cohort is a 20-year prospective cohort of patients with SjD. The following biomarkers were compared: IFN-α2, IFN-γ, CXCL10, CXCL13, BAFF, interleukin (IL)-7, fms-like tyrosine kinase 3 ligand, CCL19, and tumor necrosis factor receptor 2 (TNF-RII). IFN signature was assessed using transcriptomic analysis. We then compared systemic and symptomatic evolution, and the risk of new immunosuppressant prescription and of lymphoma, according to the IFN signature across the three clusters. A total of 395 patients (94% female, median age 53 [interquartile range 43-63] years) were included. Higher levels of CXCL13, IL-7, and TNF-RII were found in the BALS and HSA clusters compared with the LSAHS cluster. A high IFN signature was mainly found in the BALS cluster (57%, vs 48% and 38% in the HSA and LSAHS clusters, respectively). This IFN signature was mainly driven by type I IFN, with higher levels of IFN-α2. In the BALS cluster, a high IFN signature was associated with a higher risk of new immunosuppressant treatment (hazard ratio 9.38; 95% confidence interval 1.22-72.16). All lymphoma occurred in patients with high IFN signature. The three SjD clusters displayed distinct expressions of IFN signature and markers of T and B cell activation, confirming distinct pathophysiologic mechanisms. High IFN signature could predict systemic evolution in the BALS cluster.

SOX2-induced IL1α-mediated immune suppression drives epithelial dysplasia malignant transformation.

Squamous cell carcinomas (SCC) are often preceded by potentially malignant precursor lesions, most of which remain benign. The terminal exhaustion phenotypes of effector T-cells and the accumulation of myeloid-derived suppressor cells (MDSC) have been thoroughly characterized in established SCC. However, it is unclear what precancerous lesions harbor a bona fide high risk for malignant transformation and how precancerous epithelial dysplasia drives the immune system to the point of no return. Here we show that expression of SRY-box transcription factor 2 (SOX2) in precancerous lesions imparts an irreversible risk that recruits suppressive myeloid cells by promoting the release of CCL2. We developed a unique genetically engineered mouse model (GEMM) to recapitulate the malignant transformation of epithelial dysplasia to SCC in the oral mucosa with high histologic and phenotypic fidelity. Using a combination of longitudinal human specimens and the Sox2-GEMM, we found that the myeloid cells in precancerous epithelial dysplasia exhibit a distinctive dichotomous profile featuring high levels of IL-1α-SLC2A1 and low levels of type-I interferon (IFN-I) signatures, which occurs before SCC emerges histologically. Brief priming of myeloid cells with IL-1α desensitizes them to IFN-I agonists and makes myeloid-derived suppressor cells (MDSC) even more suppressive of T-cell activation. Mechanistically, IL-1 activation represses the expression of DHHC3/7 enzymes, which are responsible for the palmitoylation of stimulator of interferon genes (STING). Early blockade of IL1 signaling using pharmacologic and genetic approaches similarly reduces MDSC and SLC2A1 high myeloid cells, suppresses epithelial dysplasia transformation, and extends survival. This work establishes a previously unrecognized SOX2-CCL2-IL1 pathway that leads to irreversible immune escape when precancerous epithelial lesions transform.

Topical Mupirocin Treatment Reduces Interferon and Myeloid Signatures in Cutaneous Lupus Erythematous Lesions Through Targeting of Staphyloccal Species.

Cutaneous lupus erythematosus (CLE) is an inflammatory skin manifestation of systemic lupus erythematosus. Type I interferons (IFNs) promote inflammatory responses and are elevated in CLE lesions. We recently reported that CLE lesions are frequently colonized with Staphylococcus aureus. Here, we follow up via a proof-of-concept study to investigate whether type I IFN and inflammatory gene signatures in CLE lesions can be modulated with mupirocin, a topical antibiotic treatment against S aureus-mediated skin infections. Participants with active CLE lesions (n = 12) were recruited and randomized into a week of topical treatment with either 2% mupirocin or petroleum jelly vehicle. Paired samples were collected before and after seven days of treatment to assess microbial lesional skin responses. Microbial samples from nares and lesional skin were used to determine baseline and posttreatment Staphylococcus abundance and microbial community profiles by 16S ribosomal RNA gene sequencing. Inflammatory responses were evaluated by bulk RNA sequencing of lesional skin biopsies. We identified 173 differentially expressed genes in CLE lesions after topical mupirocin treatment. Decreased lesional Staphylococcus burden correlated with decreased IFN pathway signaling and inflammatory gene expression and barrier dysfunction. Interestingly, mupirocin treatment lowered skin monocyte levels, and this mupirocin-associated depletion of monocytes correlated with decreased inflammatory gene expression. Mupirocin treatment decreased lesional Staphylococcus, and this correlated with decreased IFN signaling and inflammatory gene expression. This study suggests a topical antibiotic could be employed to decrease lupus skin inflammation and type I IFN responses by reducing Staphylococcus colonization.

FC21 Sexual dimorphism in keratinocyte response to IL-36 cytokines

Abstract Pustular psoriasis is a devastating subtype of psoriasis associated with significant morbidity and mortality. Pustular psoriasis is the only form of psoriasis that shows prominent sex bias, with the majority of cases found in women. Pustular psoriasis, and to a lesser extent plaque psoriasis, is characterized by the prominent involvement of the IL-36 family of cytokines, which consists of three pro-inflammatory cytokines: IL-36A, IL-36B and IL-36G, and the IL-36 receptor antagonist (IL-36RA/IL36RN). All three IL-36 members and the IL-36 receptor antagonist are increased (5- to 10-fold) in psoriatic skin. Single-cell and spatial-seq data demonstrated that IL-36 responses are primarily localized to the supraspinous compartment of the epidermis and strongly correlate with and act downstream of both IL-17A and TNF responses. CRISPR/Cas9 targeted knocking out of each IL-36 family member in keratinocytes demonstrates that both IL36G and IL36R KOs, but not IL36A KO, suppress both IL-17A and TNF responses (P < 0.001). Notably, the suppressive role of IL36G KO occurred in the absence of neutrophil proteases, which have been considered primary activators of the IL-36 axis in the skin. Bulk RNA-seq data from primary keratinocytes (n = 47) showed marked sex bias in IL-36G response, with female keratinocytes having a more robust pro-inflammatory response to IL-36G compared with male keratinocytes (P < 0.001). Furthermore, female keratinocytes showed higher expression of the type I IFN, IFNK (P < 0.001; FC-3.84), and IFN signature genes, such as MX1 (P < 0.001; FC-4.78), indicating an association between IFN-κ and the IL-36 axis in female keratinocytes. These data provide novel insights into IL-36 biology, demonstrate its role in amplifying IL-17 and TNF responses in the epidermis, and suggest that the sex bias of IL-36 response may contribute to the marked female bias of pustular forms of psoriasis.

Type I interferon-stimulated genes predict clinical response to belimumab in systemic lupus erythematosus.

The type I interferon (IFN-I) response is crucial in systemic lupus erythematosus (SLE). The mRNA level of interferon-stimulated genes (ISGs) is widely used for evaluating the activity of IFN in SLE. However, the character of ISGs in belimumab-treated SLE patients has not be reported. In this study, we enrolled 53 SLE patients undergoing belimumab treatment and assessed their clinical responses at 3, 6, and 12 months. The expression levels of 25 ISGs in Peripheral blood mononuclear cells (PBMCs) were quantified at baseline and at 3 months using quantitative real-time PCR. Using Least absolute shrinkage and selection operator (LASSO)-logistic regression, five genes (CXCL10, EPSTI1, HECR6, IFI27, IFIH1) were identified to predict belimumab efficacy. The IFN signature score, a multivariate logistic regression model based on the change rates of these genes, positively predicted the SLE responder index (SRI) at 12 months, with an area under curve of 0.940 in receiver operating characteristic and favorable outcomes in decision curve analysis. Patients with an IFN signature score ≥0 had higher SRI response rates, better clinical markers (including SLE disease activity index 2000 scores, anti-dsDNA, IgG levels, daily doses of prednisone, and higher complement C3 and C4 levels), and faster B cell decline than those with scores <0. In conclusion, after 3 months of belimumab treatment, the expression levels of IFN-I-inducible genes varied, and the IFN signature score reliably forecasted the SRI response at 6 and 12 months.

Evaluation of IRF7 mRNA and its Association with Promoter Methylation in Kashmiri (North-Indian) Patients with Systemic Sclerosis: A Case-Control Study.

The interferon regulatory factor 7 (IRF7), a member of the IRF family of transcription factors, plays a major role in the regulation of numerous aspects of an immune response and has increasingly been surveyed to determine the aetiology and pathogenesis of systemic sclerosis (SSc). Objective: This study aimed to investigate the transcriptional levels of IRF7 mRNA in peripheral blood mononuclear cells (PBMCs) and the impact of promoter methylation on IRF7 mRNA expression in SSc patients compared to healthy controls. PBMCs were obtained from confirmed 40 naïve SSc cases and 20 healthy controls for IRF-7 expression and methylation analysis. mRNA expression was performed using the quantitative real-time polymerase chain reaction (SYBR green method) concerning the housekeeping gene. A promoter methylation profile study was carried out by bisulfite treatment of DNA, followed by methylation-specific polymerase chain reaction (MS-PCR) in SSc cases against controls. The relative expression analysis revealed that the selected IRF7 gene was upregulated in the patient group compared to healthy controls (p=0.003). In addition, mRNA expression of IRF7 was significantly increased in the limited cutaneous group compared to the diffuse cutaneous group. Moreover, SSc cases had hypomethylated IRF7 promoters compared to controls, and the significant impact of IRF7 promoter methylation on mRNA expression was observed (p=0.001). IRF7 overexpression in PBMCs from SSc patients may be caused by IRF7 promoter demethylation, and this aberrant expression of IRF7 in SSc might provide a link between the prominent IFN signature and the development of SSc.

277 The transcriptome of Sweet syndrome provides novel insights into its pathogenesis and reveals a prominent interferon signature

277 The transcriptome of Sweet syndrome provides novel insights into its pathogenesis and reveals a prominent interferon signature

COVID-19 progression and convalescence in common variable immunodeficiency patients show dysregulated adaptive immune responses and persistent type I interferon and inflammasome activation

Common variable immunodeficiency (CVID) is the most prevalent primary immunodeficiency, marked by hypogammaglobulinemia, poor antibody responses, and increased infection susceptibility. The COVID-19 pandemic provided a unique opportunity to study the effects of prolonged viral infections on the immune responses of CVID patients. Here we use single-cell RNA-seq and spectral flow cytometry of peripheral blood samples before, during, and after SARS-CoV-2 infection showing that COVID-19 CVID patients display a persistent type I interferon signature at convalescence across immune compartments. Alterations in adaptive immunity include sustained activation of naïve B cells, increased CD21low B cells, impaired Th1 polarization, CD4+ T central memory exhaustion, and increased CD8+ T cell cytotoxicity. NK cell differentiation is defective, although cytotoxicity remains intact. Monocytes show persistent activation of inflammasome-related genes. These findings suggest the involvement of intact humoral immunity in regulating these processes and might indicate the need for early intervention to manage viral infections in CVID patients.

Inborn Error of WAS Presenting with SARS-CoV-2-Related Multisystem Inflammatory Syndrome in Children.

Multisystem inflammatory syndrome in children (MIS-C) has been reported in patients with inborn errors of immunity (IEI), providing insights into disease pathogenesis. Here, we present the first case of MIS-C in a child affected by Wiskott-Aldrich syndrome (WAS) gene mutation, elucidating underlying predisposing factors and the involved inflammatory pathways. Genetic analysis revealed a frameshift truncating variant in the WAS gene, resulting in WAS protein expression between mild and severe forms, despite a clinical phenotype resembling X-linked thrombocytopenia (XLT). IL-1β secretion by LPS-stimulated peripheral blood mononuclear cells from patient during MIS-C was lower compared to healthy subjects but increased during follow-up. Conversely, the percentage of ASC (apoptosis-associated speck-like protein containing a CARD) specks in the patient's circulating monocytes during the acute phase was higher than in healthy subjects. The type I interferon (IFN) signature during MIS-C was normal, in contrast to the raised IFN signature measured far from the acute event. This case supports the association of IEI with MIS-C, potentially linked to delayed immune responses to SARS-CoV-2. The XLT phenotype underlies a subclinical immunodysregulation involving the NLRP3 inflammasome and the type-I IFN response.

Pathogenesis of Sjögren's disease: one year in review 2024.

The pathogenesis of Sjögren's disease (SjD) is still elusive; however, the disease is widely recognised as a multistep disorder triggered by the interplay of environmental, hormonal and genetic factors. Innate immune system plays a crucial role in the initiation of the inflammatory process, but the amplification and the perpetuation of the autoimmune process require a continual interaction between the innate and adaptive immune systems. Several important contributions elucidating SjD pathogenesis have been recently published due to emerging technologies. This review provides an overview of the recent literature focusing, in the first part, on new insights into genetic and epigenetics studies. In the second part, we will discuss new findings related to salivary epithelial glandular cells and their interaction with other immune cells, type I interferon signature and innate immunity. Finally, as ectopic germinal centres like structures in the salivary glands of patients with SjD have been critically involved in autoreactive B cell activation and have been associated with progression towards B cell lymphomas, we will focus on new insights into their regulation in SjD and novel insights into the transition to lymphoma. Hopefully, a better comprehension of SjD complexity will pave the way to highly targeted therapeutic strategies.

Explainable deep neural networks for predicting sample phenotypes from single-cell transcriptomics.

Recent advances in single-cell RNA-Sequencing (scRNA-Seq) technologies have revolutionized our ability to gather molecular insights into different phenotypes at the level of individual cells. The analysis of the resulting data poses significant challenges, and proper statistical methods are required to analyze and extract information from scRNA-Seq datasets. Sample classification based on gene expression data has proven effective and valuable for precision medicine applications. However, standard classification schemas are often not suitable for scRNA-Seq due to their unique characteristics, and new algorithms are required to effectively analyze and classify samples at the single-cell level. Furthermore, existing methods for this purpose have limitations in their usability. Those reasons motivated us to develop singleDeep, an end-to-end pipeline that streamlines the analysis of scRNA-Seq data training deep neural networks, enabling robust prediction and characterization of sample phenotypes. We used singleDeep to make predictions on scRNA-Seq datasets from different conditions, including systemic lupus erythematosus, Alzheimer's disease and coronavirus disease 2019. Our results demonstrate strong diagnostic performance, validated both internally and externally. Moreover, singleDeep outperformed traditional machine learning methods and alternative single-cell approaches. In addition to prediction accuracy, singleDeep provides valuable insights into cell types and gene importance estimation for phenotypic characterization. This functionality provided additional and valuable information in our use cases. For instance, we corroborated that some interferon signature genes are consistently relevant for autoimmunity across all immune cell types in lupus. On the other hand, we discovered that genes linked to dementia have relevant roles in specific brain cell populations, such as APOE in astrocytes.

IFN-associated B cell hyperactivity is highly enriched in SLE patients harboring Sm/RNP antibodies.

Systemic Lupus Erythematosus (SLE) is an autoimmune disease characterized by an array of autoantibodies, in particular anti-nuclear antibodies (ANA). The disease is also hallmarked by an expansion of plasmablasts (PB) in peripheral blood. How these relate to autoantibody production is not clear. Here, we aimed to understand B cell alterations in SLE and their relationship to immunoglobulin levels and autoantibody production. We demonstrate that a subgroup of SLE patients is characterized by a high frequency of PB relative to memory B cells (high PB/M). Patients with this phenotype more frequently had high disease activity. Despite low overall frequencies of memory B cells, these patients exhibited an increased activation in the switched CD27+ memory compartment and a strong IFN signature in PB. Repertoire analysis revealed a highly polyclonal expansion and enrichment for IgG1 expressing PB in patients with a high PB/M ratio which was reflected in increased serum IgG levels. Importantly, the hyperactive B cell phenotype was highly enriched in patients harboring Sm/RNP autoantibodies (OR: 9.17 (2.97-26.0)). In summary, we show for the first time a direct relationship between IFN and PB expansion in a subgroup of SLE patients, highly enriched in those harboring Sm/RNP antibodies. These results provide insight into the pathways leading to B cell hyperactivity and autoantibody production which may guide the tailoring of B cell- and IFN-targeted therapies.

Defective germinal center selection results in persistence of self-reactive B cells from the primary to the secondary repertoire in Primary Antiphospholipid Syndrome

Primary antiphospholipid syndrome (PAPS) is a life-threatening clotting disorder mediated by pathogenic autoantibodies. Here we dissect the origin of self-reactive B cells in human PAPS using peripheral blood and bone marrow of patients with triple-positive PAPS via combined single-cell RNA sequencing, B cell receptors (BCR) repertoire profiling, CITEseq analysis and single cell immortalization. We find that antiphospholipid (aPL)-specific B cells are present in the naive compartment, polyreactive, and derived from the natural repertoire. Furthermore, B cells with aPL specificities are not eliminated in patients with PAPS, persist until the memory and long-lived plasma cell stages, likely after defective germinal center selection, while becoming less polyreactive. Lastly, compared with the non-PAPS cells, PAPS B cells exhibit distinct IFN and APRIL signature as well as dysregulated mTORC1 and MYC pathways. Our findings may thus elucidate the survival mechanisms of these autoreactive B cells and suggest potential therapeutic targets for the treatment of PAPS.