Production Of Pathogenic Autoantibodies Research Articles

AChR Antibody-Positive Myasthenia Gravis

Herein, we describe the mechanisms, diagnostic procedures, and treatment options for acetylcholine receptor (AChR) antibody-positive myasthenia gravis (MG). The upstream pathomechanism of this condition involves AChR-sensitized T cell-dependent B cell proliferation and the subsequent production of pathogenic autoantibodies. Downstream molecules include AChR antibodies that activate complement pathways, resulting in the destruction of motor endplates. We further introduce newly-developed molecular targeted drugs for the treatment of MG that aims to secure patients' health-related quality of life.

Paeonol interferes with lupus nephritis by regulating M1/M2 polarization of macrophages

Systemic lupus erythematosus (SLE) is a complex autoimmune disease of unknown etiology. It is marked by the production of pathogenic autoantibodies and the deposition of immune complexes. Lupus nephritis (LN) is a prevalent and challenging clinical complications of SLE. Cortex Moutan contains paeonol as its main effective component. In this study, using the animal model of SLE induced by R848, it was found that paeonol could alleviate the lupus-like symptoms of lupus mouse model induced by R848 activating TLR7, reduce the mortality and ameliorate the renal damage of mice. In order to explore the mechanism of paeonol on lupus nephritis, we studied the effect of paeonol on the polarization of Raw264.7 macrophages in vitro. The experimental results show that paeonol can inhibit the polarization of macrophages to M1 and promote their polarization to M2, which may be related to the inhibition of MAPK and NF-κB signaling pathways. Our research provides a new insight into paeonol in the treatment of lupus nephritis, which is of great importance for the treatment of systemic lupus erythematosus and its complications.

B-cells in pulmonary arterial hypertension: friend, foe or bystander?

There is an unmet need for new therapeutic strategies that target alternative pathways to improve the prognosis of patients with pulmonary arterial hypertension (PAH). As immunity has been involved in the development and progression of vascular lesions in PAH, we review the potential contribution of B-cells in its pathogenesis and evaluate the relevance of B-cell-targeted therapies. Circulating B-cell homeostasis is altered in PAH patients, with total B-cell lymphopenia, abnormal subset distribution (expansion of naïve and antibody-secreting cells, reduction of memory B-cells) and chronic activation. B-cells are recruited to the lungs through local chemokine secretion, and activated by several mechanisms: 1) interaction with lung vascular autoantigens through cognate B-cell receptors; 2) costimulatory signals provided by T follicular helper cells (interleukin (IL)-21), type 2 T helper cells and mast cells (IL-4, IL-6 and IL-13); and 3) increased survival signals provided by B-cell activating factor pathways. This activity results in the formation of germinal centres within perivascular tertiary lymphoid organs and in the local production of pathogenic autoantibodies that target the pulmonary vasculature and vascular stabilisation factors (including angiotensin-II/endothelin-1 receptors and bone morphogenetic protein receptors). B-cells also mediate their effects through enhanced production of pro-inflammatory cytokines, reduced anti-inflammatory properties by regulatory B-cells, immunoglobulin (Ig)G-induced complement activation, and IgE-induced mast cell activation. Precision-medicine approaches targeting B-cell immunity are a promising direction for select PAH conditions, as suggested by the efficacy of anti-CD20 therapy in experimental models and a trial of rituximab in systemic sclerosis-associated PAH.

Double-Negative T (DNT) Cells in Patients with Systemic Lupus Erythematosus.

Double-negative T (DNT) cells are a rare and unconventional T-lymphocyte subpopulation lacking both CD4 and CD8 markers. Their immunopathological roles and clinical relevance have yet to be elucidated. Beyond autoimmune lymphoproliferative syndrome (ALPS), these cells may also play a role in rheumatic disorders, including systemic lupus erythematosus (SLE); indeed, these two diseases share several autoimmune manifestations (including nephritis). Moreover, one of the main experimental murine models used to investigate lupus, namely the MRL/lpr mouse, is characterized by an expansion of DNT cells, which can support the production of pathogenic autoantibodies and/or modulate the immune response in this context. However, lupus murine models are not completely consistent with their human SLE counterpart, of course. In this mini review, we summarize and analyze the most relevant clinical studies investigating the DNT cell population in SLE patients. Overall, based on the present literature review and analysis, DNT cell homeostasis seems to be altered in patients with SLE. Indeed, most of the available clinical studies (which include both adults and children) reported an increased DNT cell percentage in SLE patients, especially during the active phases, even though no clear correlation with disease activity and/or inflammatory parameters has been clearly established. Well-designed, standardized, and longitudinal clinical studies focused on DNT cell population are needed, in order to further elucidate the actual contribution of these cells in SLE pathogenesis and their interactions with other immune cells (also implicated and/or altered in SLE, such as basophils), and clarify whether their expansion and/or immunophenotypic aspects may have any immunopathological relevance (and, then, represent potential disease markers and, in perspective, even therapeutic targets) or are just an unspecific epiphenomenon of autoimmunity.

Understanding CD4+ T cells in autoimmune bullous diseases.

Autoimmune bullous diseases (AIBDs) are a group of life-threatening blistering diseases caused by autoantibodies that target proteins in the skin and mucosa. Autoantibodies are the most important mediator in the pathogenesis of AIBDs, and various immune mechanisms contribute to the production of these pathogenic autoantibodies. Recently, significant progress has been made in understanding how CD4+ T cells drive autoantibody production in these diseases. Here, we review the critical role of CD4+ T cells in the production of pathogenic autoantibodies for the initiation and perpetuation of humoral response in AIBDs. To gain an in-depth understanding of CD4+ T-cell pathogenicity, antigen specificity, and mechanisms of immune tolerance, this review covers comprehensive mouse and human studies of pemphigus and bullous pemphigoid. Further exploration of pathogenic CD4+ T cells will potentially provide immune targets for improved treatment of AIBDs.

T peripheral helper cells in autoimmune diseases: What do we know?

The interactions between T cells and B cells are essential for antibody responses and the development of autoimmune diseases. Recently, a distinct subset of T cells capable of helping B cells was established in synovial fluid, and they were termed peripheral helper T (Tph) cells. PD-1hiCXCR5-CD4+ Tph cells express high levels of CXCL13, which drives the formation of lymphoid aggregates and tertiary lymphoid structures, ultimately facilitating the local production of pathogenic autoantibodies. Tph and T follicular helper cells share some key features but can be distinguished by their surface markers, transcriptional regulation, and migration capability. We summarize recent findings on Tph cells in this review and provide a perspective on their potential roles in a range of autoimmune diseases. More clinical and in-depth mechanistic investigations of Tph cells may help to improve the understanding of pathogenesis and further provide novel therapeutic targets in autoimmune diseases.

Ophiopogonin D attenuates the progression of murine systemic lupus erythematosus by reducing B cell numbers.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by immune abnormalities leading to multi-organ damage. The activation of autoreactive B cell differentiation will lead to the production of pathogenic autoantibodies, contributing to the development of SLE. However, the effects of Ophiopogonin D (OP-D) on B cell activation and autoantibody production as well as renal injury in the pathogenesis of SLE remain unclear. MRL/lpr mice, one of the most commonly used animal models of SLE, were intragastrically administered with 5 mg/kg/d OP-D at 17 weeks of age for 3 weeks. The survival rates of mice in each group were monitored for 6 weeks until 23 weeks of age. Proteinuria and serum creatinine levels were measured. Serum levels of immunoglobulin (Ig)G, IgM, and anti-dsDNA autoantibodies were detected by enzyme-linked immunosorbent assay. Numbers of CD19+ B cells in the blood, spleen and bone marrow and numbers of splenic germinal center (GC) B cells were calculated by using flow cytometry. OP-D treatment prolonged survival in MRL/lpr mice. OP-D treatment reduced proteinuria and serum creatinine levels as well as mitigated renal pathological alternation in MRL/lpr mice. Furthermore, serum levels of IgG, IgM, and anti-dsDNA autoantibodies were reduced by OP-D treatment. OP-D lessened not only CD19+ B cells in the spleen and bone marrow but also plasma cells that secreted anti-dsDNA autoantibodies, IgG and IgM in the spleen and bone marrow. OP-D ameliorated the progression of SLE by inhibiting the secretion of autoantibodies though reducing B cell numbers.

Adaptive and innate immune pathogenesis of bullous pemphigoid: A review.

Bullous pemphigoid (BP) is an autoimmune blistering disease that primarily affects elderly individuals. The presentation of BP is heterogeneous, typically manifesting as microscopic subepidermal separation with a mixed inflammatory infiltrate. The mechanism of pemphigoid development is unclear. B cells play a major role in pathogenic autoantibody production, and T cells, type II inflammatory cytokines, eosinophils, mast cells, neutrophils, and keratinocytes are also implicated in the pathogenesis of BP. Here, we review the roles of and crosstalk between innate and adaptive immune cells in BP.

Altered Circulating Follicular T Helper Cell Subsets and Follicular T Regulatory Cells Are Indicators of a Derailed B Cell Response in Lupus, Which Could Be Modified by Targeting IL-21R.

Systemic lupus erythematosus (SLE) is characterized by the breakdown of self-tolerance, the production of high-affinity pathogenic autoantibodies and derailed B cell responses, which indicates the importance of central players, such as follicular T helper (TFH) subsets and follicular T regulatory (TFR) cells, in the pathomechanism of the disease. In this study, we aimed to analyze the distribution of the circulating counterparts of these cells and their association with disease characteristics and B cell disproportions in SLE. We found that the increased percentage of activated circulating TFH (cTFH) and cTFR cells was more pronounced in cutaneous lupus; however, among cTFH subsets, the frequency of cTFH17 cells was decreased in patients with lupus nephritis. Furthermore, the decreased proportion of cTFH17 cells was associated with low complement C4 levels and high disease activity scores. We also investigated whether the blocking of the IL-21 receptor (IL-21R) with an anti-IL-21R monoclonal antibody inhibits the B cell response, since IL-21 primarily produced by TFH cells potentially promotes humoral immunity. We observed that anti-IL-21R inhibited plasmablast generation and immunoglobulin production. Our study demonstrated that, besides cTFR/cTFH imbalance, cTFH17 cells play a crucial role in SLE pathogenesis, and modulating cTFH-B cell interaction through the IL-21/IL-21R pathway may be a promising therapeutic strategy to suppress the pathological B cell response.

Abnormal lower expression of GPR183 in peripheral blood T and B cell subsets of systemic lupus erythematosus patients

G protein-coupled receptor 183 (GPR183) has been indicated to mediate the migration and localisation of immune cells in T cell-dependent antibody responses. Systemic lupus erythematosus (SLE) is a canonical autoimmune disease involving B cell-mediated tolerance destruction and excessive pathogenic autoantibody production, in which multiple GPCRs play a role. To date, there has been no systematic study regarding the expression of GPR183 in lymphocyte subsets of SLE patients. In this research, firstly, we observed the expression trends of GRP183 in various T and B cell subsets in human tonsil tissues. These lymphocyte subsets include CD4+, CD8+, naïve T, effector T, Tfh, activated Tfh, Th1, Th2, Th17, Treg, CD19+CD27-, CD19+CD27+, naïve B, germinal centre B, memory B, and plasma cells. Further, compared with healthy controls (HCs), GPR183 expression levels in above peripheral blood lymphocyte subsets of patients with SLE were reduced overall. The differential expression of GPR183 expression between inactive and active SLE patients indicates that GPR183 expression may be concerned with the disease activity of SLE. This was further confirmed through the strong negative correlation with SLEDAI score and positive correlation with serum complement protein C3, C4 and C1q levels. Further receiver operating characteristic (ROC) curve analysis revealed that GPR183 expression in circulating CD27-IgD+ B cells may be beneficial in distinguishing between inactive and active SLE patients. In addition, type I interferon stimulation could down-regulate the expression of GPR183 in peripheral blood T and B cell subsets. Aberrant expression of GPR183 may provide some novel insights into disease activity prediction and underlying pathogenesis of SLE.

Staphylococcus aureus peptidoglycan (PGN) induces pathogenic autoantibody production via autoreactive B cell receptor clonal selection, implications in systemic lupus erythematosus

ObjectivesThere is an intricate interplay between the microbiome and the immune response impacting development of normal immunity and autoimmunity. However, we do not fully understand how the microbiome affects production of natural-like and pathogenic autoantibodies. Peptidoglycan (PGN) is a component of the bacterial cell wall which is highly antigenic. PGNs from different bacteria can differ in their immune regulatory activities. MethodsC57BL/6 and MRL/lpr mice were intraperitoneally injected with saline or PGN from Staphylococcus aureus or Bacillus subtilis. Spleen anti-double-stranded DNA (dsDNA) IgG + B cells were sorted for B-cell receptor sequencing. Serum autoantibody levels and kidney damage were analyzed. Further, the association between plasma S. aureus translocation and systemic lupus erythematosus (SLE) pathogenesis was assessed in women. ResultsAdministration of B. subtilis PGN induced natural-like anti-dsDNA autoantibodies (e.g., IgM, short lived IgG response, and no tissue damage), whereas S. aureus PGN induced pathogenic anti-dsDNA autoantibodies (e.g., prolonged IgG production, low IgM, autoantibody-mediated kidney damage) in C57BL/6 and/or MRL/lpr mice. However, serum total IgG did not differ. S. aureus PGN induced antibodies with reduced clonality and greater hypermutation of IGHV3-74 in splenic anti-dsDNA IgG + B cells from C57BL/6 mice. Further, S. aureus PGN promoted IgG class switch recombination via toll-like receptor 2. Plasma S. aureus DNA levels were increased in women with SLE versus control women and correlated with levels of lupus-related autoantibodies and renal involvement. ConclusionsS. aureus PGN induces pathogenic autoantibody production, whereas B. subtilis PGN drives production of natural nonpathogenic autoantibodies.

A systematic review and meta-analysis of HLA class II associations in patients with IgG4 autoimmunity

Autoimmune diseases caused by pathogenic IgG4 subclass autoantibodies (IgG4-AID) include diseases like MuSK myasthenia gravis, pemphigus vulgaris or thrombotic thrombocytopenic purpura. Their etiology is still unknown. Polymorphisms in the human leukocyte antigen (HLA) gene locus, particularly in HLA-DRB1, are known genetic susceptibility factors for autoimmune diseases. We hypothesized a similar role for HLA polymorphisms in IgG4-AID and conducted a systematic review and meta-analysis with case–control studies on IgG4-AID based on MOOSE/ HuGENet guidelines. Genotype (G) and allele (A) frequencies of HLA-DQB1*05 (G: OR 3.8; 95% CI 2.44–5.9; p < 0.00001; A: OR 2.54; 95% CI 1.82–3.55; p < 0.00001) and HLA-DRB1*14 (G: OR 4.31; 95% CI 2.82–6.59; p < 0.00001; A: OR 4.78; 95% CI 3.52–6.49; p < 0.00001) and the HLA-DRB1*14-DQB1*05 haplotype (OR 6.3; 95% CI 3.28–12.09; p < 0.00001/OR 4.98; 95% CI 3.8–6.53; p < 0.00001) were increased while HLA-DRB1*13 (G: OR 0.48; 95% CI 0.34–0.68; p < 0.0001; A: OR 0.46; 95% CI 0.34–0.62; p < 0.00001) was decreased in IgG4-AID patients. In conclusion, the HLA-DQB1*05, HLA-DRB1*14 alleles and the HLA-DQB1*05-DRB1*14 haplotype could be genetic risk factors that predispose for the production of pathogenic IgG4 autoantibodies and the HLA-DRB1*13 allele may protect from IgG4 autoimmunity.

Rituximab - Progress but Still Not a Final Resolution for Pemphigus Patients: Clinical Report From a Single Center Study.

Pemphigus is a rare autoimmune disease characterized by the production of pathogenic autoantibodies against desmosomal adhesion proteins, desmoglein 1 and 3. The pathophysiological process leads to the development of blisters and erosions on mucosal and/or skin surfaces as the main clinical manifestation of the disease. Rituximab emerged as the first-line therapeutic option for pemphigus due to its ability to induce remission by depleting peripheral B lymphocytes. Our aim was to assess the efficacy of rituximab in the treatment of patients in Croatia. A single-center, retrospective study was conducted on 19 patients treated with rituximab following a rheumatoid arthritis dosing protocol between October 2015 and March 2021, with a mean follow-up of 24.1 months. After the first rituximab cycle, two patients achieved complete remission off therapy (10.5%), and six patients achieved complete remission on minimal therapy (31.6%). Partial remission was observed among ten patients (52.6%). Eight patients (44.4%) relapsed after the first rituximab cycle. The mean relapse time was 21 months. Seven patients received two rituximab cycles, and three patients received three cycles. Overall, 13 out of 19 patients experienced complete remission at some point during the study, while there were no non-responders after the rituximab treatment. No statistically significant associations were observed between age, sex, type of disease involvement and clinical remission, either on or off therapy. A steady decrease in anti-desmoglein 1 and anti-desmoglein 3 levels was measured among all patients following rituximab treatment. One patient experienced a treatment-related adverse event of infectious etiology (cellulitis). One patient died following the first rituximab cycle, with the cause of death likely not to be associated with the treatment. Rituximab is an effective disease-modifying agent in the treatment of pemphigus with the main benefit of reducing corticosteroid exposure and steroid-related side effects among pemphigus patients. However, a feature of rituximab therapy is high relapse rates and the need for repeated treatment cycles to achieve complete remission. Developing an optimal protocol for rituximab treatment and finding suitable markers for predicting relapse will improve the management of pemphigus patients.

Preclinical stage abundance and nuclear antigen reactivity of faecal Immunoglobulin A vary among males and females of lupus-prone mouse models.

Systemic lupus erythematosus (SLE) is characterized by the production of pathogenic autoantibodies with nuclear antigen (nAg) specificity. Using (SWRxNZB)F1 (SNF1) mice, we showed higher levels of Immunoglobulin A (IgA) production in the intestine and the nAg reactivity of faecal IgA under lupus susceptibility. Here, we determined whether the faecal IgA abundance and nAg reactivity are higher in, different among, various lupus-prone preclinical models (MRL/lpr, NZBxNZW-F1, SNF1, NZM2410 and NZM2328). We also determined whether the faecal IgA nAg reactivity at preseropositive ages correlates with the eventual serum autoantibody levels in males and females of these mouse models. We show that age-dependent increase in the abundance and nAg reactivity of faecal IgA can vary among different lupus-prone mouse models. Importantly, faecal IgA in these mice show significant levels of nAg reactivity, starting as early as at juvenile age. Furthermore, the pre-seropositive stage nAg reactivity of faecal IgA in most lupus-prone strains correlates well with that of eventual, seropositive stage systemic autoantibody levels. Gender differences in serum autoantibody levels were preceded by similar differences in the faecal IgA abundance and nAg reactivity. These observations suggest that faecal IgA features, nAg reactivity particularly, could serve as a biomarker for early prediction of the eventual systemic autoimmunity in lupus-prone subjects.

RNASE2 Mediates Age-Associated B Cell Expansion Through Monocyte Derived IL-10 in Patients With Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is characterized by the production of pathogenic autoantibodies. Ribonuclease A family member 2 (RNase2) is known to have antiviral activity and immunomodulatory function. Although RNASE2 level has been reported to be elevated in SLE patients based on mRNA microarray detection, its pathologic mechanism remains unclear. Here, we confirmed that RNASE2 was highly expressed in PBMCs from SLE patients and associated with the proportion of CD11c+T-bet+ B cells, a class of autoreactive B cells also known as age-associated B cells (ABCs). We showed that reduction of RNASE2 expression by small interfering RNA led to the decrease of ABCs in vitro, accompanied by total IgG and IL-10 reduction. In addition, we demonstrated that both RNASE2 and IL-10 in peripheral blood of lupus patients were mainly derived from monocytes. RNASE2 silencing in monocytes down-regulated IL-10 production and consequently reduced ABCs numbers in monocyte-B cell co-cultures, which could be restored by the addition of recombinant IL-10. Based on above findings, we concluded that RNASE2 might induce the production of ABCs via IL-10 secreted from monocytes, thus contributing to the pathogenesis of SLE.

Elevated CD19+Siglec-10+ B cell levels are correlated with systemic lupus erythematosus disease activity

BackgroundSystemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by B cell dysregulation and the breakdown of self-tolerance, leading to pathogenic autoantibody production. Human Siglec-10 is a member of the sialic acid-binding immunoglobulin-type lectin (Siglec) family and a B cell surface coreceptor that inhibits B cell receptor-induced signalling. However, to date, no report has investigated CD19+Siglec-10+ B cells in SLE patients. Thus, this study aimed to measure the population of CD19+Siglec-10+ B cells in patients with SLE and its correlation with disease activity. MethodsFlow cytometry was employed to measure the population of CD19+Siglec-10+ B cells in peripheral blood mononuclear cells (PBMCs) of both SLE patients and healthy controls (HCs). The correlation of the proportion of CD19+Siglec-10+ B cells with the values of SLE disease activity was analysed. PBMCs from HCs were challenged with serum from active SLE, inactive SLE, or HCs, and the proportion of CD19+Siglec-10+ B cells was then assessed. The effect of dexamethasone (DEX) or hydroxychloroquine (HCQ) treatment on the proportion of CD19+Siglec-10+ B cells in PBMCs from SLE patients was also determined. ResultsThe proportion of CD19+Siglec-10+ B cells in SLE patients was significantly elevated (P < 0.05), correlated positively with the SLEDAI score (r = 0.304; P = 0.018) and negatively with complement component 3 (C3) (r = −0.283; P = 0.04). In vitro assays indicated that sera from active SLE patients could significantly enhance the proportion of CD19+Siglec-10+ B cells (P < 0.05), while HCQ treatment significantly attenuated their proportions (P < 0.01). ConclusionsThe elevation of CD19+Siglec-10+ B cells and their correlation with disease activity may suggest a role for Siglec-10 in the pathogenesis and progression of SLE and provide a serum biomarker for SLE activity.

Pemphigus-The Crux of Clinics, Research, and Treatment during the COVID-19 Pandemic.

Pemphigus is a rare autoimmune disease characterised by the production of pathogenic autoantibodies in response to different desmosome proteins. The pathophysiological process leads to the development of blisters and erosions on mucosal and/or skin surfaces. The classical clinical variants of pemphigus are pemphigus vulgaris and pemphigus foliaceus. A diagnostic delay is very common in pemphigus, especially among patients with mucosal involvement. However, in recent years we have witnessed considerably fewer patients with extensive mucocutaneous manifestations, since patients with oral lesions are referred to dermatologists to start the treatment much sooner than they had been previously. Among non-classical variants of pemphigus, unusual cases with discrepancies between autoantibody profiles and clinics challenge the “desmoglein compensation theory”. The identification of several other autoantigens that perform a role in the pathogenesis of different variants of pemphigus will progress immunodermatology towards an approach that will determine personalized pemphigus subtypes for each patient. Comorbidities among patients are primarily associated with the prolonged use of corticosteroids and other immunosuppressive agents. The SARS-CoV-2 pandemic raised concerns regarding the immunosuppressive effects of treatment and the risk of a more complicated COVID-19 infection, as well as on the ability to develop an adequate vaccine response.

MHC haplotype and B cell autoimmunity: Correlation with pathogenic IgG autoantibody subclasses and Fc glycosylation patterns.

Genome-wide association studies (GWAS) have identified many genes that are associated with the development of certain autoimmune disorders, but the MHC haplotypes still represent the most prevalent genetic risk factor for many autoimmune diseases. The mechanisms by which MHC-associated genetic susceptibility translates into B cell autoimmunity and the development of autoimmune diseases are complex. There is increasing evidence that the MHC haplotype modulates autoreactive B cell responses in multiple ways. Instead of merely inhibiting the production of IgG autoantibodies and mediating complete immunological tolerance, the non-permitting MHC haplotypes seem to facilitate the production of IgG autoantibodies exhibiting Fc glycosylation patterns that are associated with reduced pathogenicity and a protective cytokine profile of T follicular helper (Tfh) cells. Here, we discuss mechanisms linking MHC haplotypes to the production of pathogenic IgG autoantibodies, which could be relevant for the development of improved diagnosis, particularly in the context of individual medicine.

The dynamic epigenetic regulation of the inactive X chromosome in healthy human B cells is dysregulated in lupus patients

Systemic lupus erythematous (SLE) is a female-predominant disease characterized by autoimmune B cells and pathogenic autoantibody production. Individuals with two or more X chromosomes are at increased risk for SLE, suggesting that X-linked genes contribute to the observed sex bias of this disease. To normalize X-linked gene expression between sexes, one X in female cells is randomly selected for transcriptional silencing through X-chromosome inactivation (XCI), resulting in allele-specific enrichment of epigenetic modifications, including histone methylation and the long noncoding RNA XIST/Xist on the inactive X (Xi). As we have previously shown that epigenetic regulation of the Xi in female lymphocytes from mice is unexpectedly dynamic, we used RNA fluorescence in situ hybridization and immunofluorescence to profile epigenetic features of the Xi at the single-cell level in human B cell subsets from pediatric and adult SLE patients and healthy controls. Our data reveal that abnormal XCI maintenance in B cells is a feature of SLE. Using single-cell and bulk-cell RNA sequencing datasets, we found that X-linked immunity genes escape XCI in specific healthy human B cell subsets and that human SLE B cells exhibit aberrant expression of X-linked genes and XIST RNA interactome genes. Our data reveal that mislocalized XIST RNA, coupled with a dramatic reduction in heterochromatic modifications at the Xi in SLE, predispose for aberrant X-linked gene expression from the Xi, thus defining a genetic and epigenetic pathway that affects X-linked gene expression in human SLE B cells and likely contributes to the female bias in SLE.

CD52 Is Elevated on B cells of SLE Patients and Regulates B Cell Function.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by B cell dysregulation and breaks in tolerance that lead to the production of pathogenic autoantibodies. We performed single-cell RNA sequencing of B cells from healthy donors and individuals with SLE which revealed upregulated CD52 expression in SLE patients. We further demonstrate that SLE patients exhibit significantly increased levels of B cell surface CD52 expression and plasma soluble CD52, and levels of soluble CD52 positively correlate with measures of lupus disease activity. Using CD52-deficient JeKo-1 cells, we show that cells lacking surface CD52 expression are hyperresponsive to B cell receptor (BCR) signaling, suggesting an inhibitory role for the surface-bound protein. In healthy donor B cells, antigen-specific BCR-activation initiated CD52 cleavage in a phospholipase C dependent manner, significantly reducing cell surface levels. Experiments with recombinant CD52-Fc showed that soluble CD52 inhibits BCR signaling in a manner partially-dependent on Siglec-10. Moreover, incubation of unstimulated B cells with CD52-Fc resulted in the reduction of surface immunoglobulin and CXCR5. Prolonged incubation of B cells with CD52 resulted in the expansion of IgD+IgMlo anergic B cells. In summary, our findings suggest that CD52 functions as a homeostatic protein on B cells, by inhibiting responses to BCR signaling. Further, our data demonstrate that CD52 is cleaved from the B cell surface upon antigen engagement, and can suppress B cell function in an autocrine and paracrine manner. We propose that increased expression of CD52 by B cells in SLE represents a homeostatic mechanism to suppress B cell hyperactivity.