Production Of Pathogenic Antibodies Research Articles

Dihydroartemisinin inhibits follicular helper T and B cells: implications for systemic lupus erythematosus treatment.

Systemic lupus erythematosus (SLE) is a common autoimmune disease, and its pathogenesis mainly involves the aberrant activation of B cells through follicular helper T (Tfh) cells to produce pathogenic antibodies, which requires more effective and safe treatment methods. Dihydroartemisinin (DHA) is the main active ingredient of artemisinin and has immunosuppressive effects. In this study, in vitro experiments confirmed that DHA inhibited Tfh cell induction and weakened its auxiliary function in B cell differentiation; furthermore, DHA directly inhibited B cell activation, differentiation, and antibody production. Furthermore, a mouse model of SLE was established, and we confirmed that DHA significantly reduced the symptoms of SLE and lupus nephritis, and decreased serum immunoglobulin (Ig)G, IgM, IgA, and anti-dsDNA levels. Moreover, DHA reduced the frequencies of total Tfh cells, activated Tfh cells, and B cell lymphoma 6, and interleukin (IL)-21 levels in Tfh cells from the spleen and lymph nodes, as well as the levels of B cells, germinal center B cells, and plasma cells in the spleen, lymph nodes, and kidneys. Additionally, DHA inhibited Tfh cells by blocking IL-2-inducible T cell kinase (ITK) signaling and its downstream nuclear factor (NF)-κB, nuclear factor of activated T cell, and activating protein-1 pathways, and directly inhibited B cells by blocking Bruton's tyrosine kinase (BTK) signaling and the downstream NF-κB and Myc pathways. Overall, our results demonstrated that DHA inhibited Tfh cells by blocking ITK signaling and also directly inhibited B cells by blocking BTK signaling. Therefore, reducing the production of pathogenic antibodies might effectively treat SLE.

SREBP signaling is important for pathogenic B cell responses in allergy.

Abstract Severe IgE antibody-mediated allergic conditions impacts millions worldwide, significantly influencing both the survival and quality of life of those affected. Our previous research indicated the critical role of sterol regulatory element-binding protein (SREBP) signaling in mediating lipid homeostasis during B cell activation and the formation of germinal centers following immunization and viral infection. However, it remained unclear whether this pathway is also involved in the pathogenic B cell responses triggered by chronic allergen sensitization. Our latest findings show that ablation of SREBP signaling in B cells doesn't affect overall IgE levels in the serum. However, during chronic sensitization to house dust mites (HDM), SREBP signaling was essential for allergen-triggered germinal center responses and pathogenic antibody production. These results suggest the potential of targeting the B cell SREBP pathway as a treatment approach for allergies.

Inhibition of Bromodomain and Extraterminal Domain (BET) Proteins Alleviates Chronic Graft- Versus-Host-Disease Via Modulating the CXCR5/CXCL13 Axis

Introduction: Chronic graft-versus-host disease (cGVHD) is a severe complication of allogeneic hematopoietic cell transplant (allo-HCT) often leading to transplant-mediated mortality. Studies have shown that T follicular helper cells (Tfh) upregulate CXCR5 and migrate into the lymphoid organ follicles in response to CXCL13 promoting germinal center (GC) B cell proliferation and differentiation of plasma cells resulting in pathogenic antibody production and profibrotic pathways associated with cGVHD. In contrast, T follicular regulatory cells (Tfr) modulate the magnitude of the GC response; thus, the balance of Tfr/Tfh could be crucial to mitigating cGVHD.Bromodomain and extra-terminal domain (BET) proteins are epigenetic readers influencing transcription of a multitude of inflammatory genes and our lab has previously reported pharmacological inhibition of BET, through OPN-51107, improves survivability and limits disease progression in murine models of acute GVHD that led to opening the first-in-human clinical trial of OPN-51107 in steroid refractory acute GVHD. We demonstrated BET proteins to be important in modulating acute GVHD pathogenesis, however, mechanisms of BET regulation within cGVHD are largely unknown. Here, using OPN-51107 we aim to further investigate mechanisms of cGVHD pathogenesis and identify successful BET inhibition-based therapeutic approaches. Methods: We used a Bronchiolitis Obliterans model (B6->B10.br) of cGVHD. Mice were administered 120mg/kg cyclophosphamide (D-3 and D-2) and 8Gy irradiation (D-1) as a conditioning regimen followed by B6 T-cell depleted bone marrow cells with and without CD45.1 B6 splenocytes. Mice that did not receive allogeneic splenocytes served as controls and did not develop GVHD. Recipients of allogeneic splenocytes were treated with a BET inhibitor - OPN-51107 at 10mg/kg 3x/weekly, or vehicle, from D28-54(+ 4) via oral gavage. Resting lung elastance and resistance was measured using the Flexivent system. At the time of lung function analysis, lung and liver tissue was harvested and stained via Hesson's Trichrome to identify and quantify collagen deposition. We performed immunofluorescence to quantify alveolar alloantibody deposition. Spleen and lung cells were stimulated with PMA/I and analyzed via spectral flow cytometry. An inflammatory murine cytokine array (#ARY006, R&D Systems) was used to analyze circulating plasma cytokine levels. Results: BET inhibition via OPN-51107 treatment decreased lung resistance (vehicle vs OPN-51107 =1.16Rrs vs 0.83Rrs p<0.05) and elastance (vehicle vs. OPN-51107=62Ers vs. 45Ers p<0.05). OPN-51107 treatment also significantly reduced alveolar antibody (Ig) deposition per area compared to vehicle (vehicle vs. OPN-51107= 4.91% vs. 3.3% p=0.01) and collagen deposition (vehicle vs. OPN-51107=19.0% vs. 12.9% p<0.05) and liver (vehicle vs. OPN-51107=19.3% vs. 2.35% p<0.0001). Congruently, BET inhibition decreased B220-CD19- CD138+mature plasma cells (vehicle vs. OPN-51107=15% vs 9.1% p<0.05) in the lung. Serum cytokine array analysis revealed a significant decrease in circulating CXCL13, relevant to Tfh migration as well as a decrease in other proinflammatory cytokines - CXCL9, MCP-1, IL3, RANTES and G-CSF important for macrophage activation and pathogenicity in OPN-51107 compared to vehicle treated mice. BET inhibition reduced splenic Tfh while increasing Tfr, thereby increasing Tfr:Tfh ratio (vehicle vs. OPN-51107= 5% vs. 9% p<0.05). Furthermore, total splenic CD19+ B-cells (vehicle vs. OPN51107= 70% vs. 33% p<0.001) as well as CXCR5 expression on B-cells were decreased in OPN-51107 treated mice (MFI vehicle vs. OPN-51107= 4744 vs. 571.5 p<0.0001). Conclusion: OPN-51107 improved lung function which correlated with significantly decreased lung fibrosis and antibody deposition in a BO model of cGVHD. BET inhibition reduces cGVHD immune pathology through decreased inflammatory cytokine production, promotion of GC Tfr cell population and modulates B-cell proliferation and/or trafficking. Significance: Using OPN-51107 in a BO model of cGVHD we demonstrate that BET inhibition is a feasible therapeutic strategy to reduce disease severity and improve lung function.

Increased serum IL-2, IL-4, IL-5 and IL-12p70 levels in AChR subtype generalized myasthenia gravis

BackgroundMyasthenia gravis (MG) is an autoimmune disorder affecting neuromuscular junctions. Cytokines play important roles in facilitating the immune response and augmenting the pathogenic antibody production. The current study aims to sensitively characterize the serum levels of cytokines with very low concentration in generalized MG (gMG).MethodsUsing ultrasensitive single-molecule arrays (SIMOA), we measured serum IL-2, IL-4, IL-5 and IL-12p70 in 228 participants including 152 immunotherapy-naïve anti-acetylcholine receptor (AChR) subtype gMG from Huashan MG registry and 76 age-matched healthy controls. Subgroup analysis was then performed by stratifying patients according to the onset ages, MGFA classification, disease duration at baseline.ResultsSerum IL-2, IL-4, IL-5 and IL-12p70 levels were significantly elevated in gMG compared to controls (0.179 pg/mL versus 0.011 pg/mL, P < 0.0001; 0.029 pg/mL versus 0.018 pg/mL, P = 0.0259; 0.215 pg/mL versus 0.143 pg/mL, P = 0.0007; 0.132 pg/mL versus 0.118 pg/mL, P = 0.0401). Subgroup analysis revealed that IL-2 levels were slightly elevated in gMG with MGFA II compared to MGFA III/IV (0.195 pg/mL versus 0.160 pg/mL, P = 0.022), as well as elevated levels of IL-2 (0.220 pg/mL versus 0.159 pg/mL, P = 0.0002) and IL-5 (0.251 pg/mL versus 0.181 pg/mL, P = 0.004) in late-onset gMG compared with the early-onset gMG. gMG patients with a long duration had a significant increased serum IL-12p70 than those with a short duration (0.163 pg/mL versus 0.120 pg/mL, P = 0.011).ConclusionSerum IL-2, IL-4, IL-5 and IL-12p70 levels were increased in AChR subtype gMG using ultrasensitive measurement. Serum cytokines with very low concentrations may provide as potential biomarkers in stratifying gMG patients in future prospective cohort studies.

New Treatment Strategies for IgA Nephropathy: Targeting Plasma Cells as the Main Source of Pathogenic Antibodies.

Immunoglobulin A nephropathy (IgAN) is a rare autoimmune disorder and the leading cause of biopsy-reported glomerulonephritis (GN) worldwide. Disease progression is driven by the formation and deposition of immune complexes composed of galactose-deficient IgA1 (Gd-IgA1) and Gd-IgA1 autoantibodies (anti-Gd-IgA1 antibodies) in the glomeruli, where they trigger complement-mediated inflammation that can result in loss of kidney function and end-stage kidney disease (ESKD). With the risk of progression and limited treatment options, there is an unmet need for therapies that address the formation of pathogenic Gd-IgA1 antibody and anti-Gd-IgA1 antibody-containing immune complexes. New therapeutic approaches target immunological aspects of IgAN, including complement-mediated inflammation and pathogenic antibody production by inhibiting activation or promoting depletion of B cells and CD38-positive plasma cells. This article will review therapies, both approved and in development, that support the depletion of Gd-IgA1-producing cells in IgAN and have the potential to modify the course of this disease. Ultimately, we propose here a novel therapeutic approach by depleting CD38-positive plasma cells, as the source of the autoimmunity, to treat patients with IgAN.

HDAC6 knockout alleviates pristane-induced lupus

Abstract Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by excessive inflammation and production of pathogenic antibodies. Histone deacetylase 6 (HDAC6) is a class IIb histone deacetylase. It has been reported that selective HDAC6 inhibition decreases inflammation in lupus mice models. C57BL/6 mice develop SLE-like symptoms following pristane injection. In this study, sex and age-matched wild type and HDAC6−/− mice on the C57BL/6 background were administered 0.5 ml pristane or PBS intraperitoneally (i.p.) at 8–12 weeks-of-age and were euthanized 10 days later. At sacrifice, body weight and spleen weight were measured, sera were collected, splenocytes and peritoneal cells were harvested for flow cytometry. We found pristane administration increased the spleen weight with no difference between WT mice and HDAC6−/− mice. Flow cytometry results showed that there was no difference in T cell or B cell populations in the spleen. Pristane administration promoted the population of CD11b+ Ly6Chigh inflammatory monocytes and CD11b+Ly6G+ neutrophils. Peritoneal recruitment of these inflammatory monocytes and neutrophils in HDAC6−/− mice was significantly decreased compared to the WT mice. Pristane administration also induced the interferon (IFN) signature genes, like Mx1, Oas1a, Irf7, Irf9, Cxcl10, Isg15. qRT-PCR revealed that these IFN signature genes were decreased in HDAC6−/− mice compared to the WT mice. In summary, our results show that HDAC6 knockout inhibits the recruitment of inflammatory monocytes and neutrophils in the peritoneum in early inflammation response to pristane. HDAC6 deletion also inhibited the expression of IFN signature genes with pristine stimulation.

Clinical significance of anti-SSA/Ro antibody in Neuromyelitis optica spectrum disorders

Background: Neuromyelitis optica spectrum disorder (NMOSD) is an autoimmune inflammatory disease of the central nervous system (CNS), also described as CNS autoimmune astrocytopathy, due to the production of pathogenic antibodies against aquaporin-4 (AQP4) expressed on the foot of astrocytes. NMOSD coexists with autoimmune diseases and related autoantibodies [anti-Sjogren's syndrome A (anti-SSA)/Ro antibody, anti-Sjogren's syndrome B (anti-SSB)/La antibody, anti-nuclear (anti-ANA) antibodies, anti-double-stranded DNA (anti-dsDNA) antibody, anti-thyroglobulin antibody, and anti-thyroid peroxidase antibody].Objectives: No precise conclusion has been drawn on the role of the anti-SSA/Ro antibody in NMOSD. Therefore, the aim of this work was to evaluate whether the anti-SSA/Ro antibody has an impact on the clinical manifestation or prognosis of NMOSD.Methods: Data were retrospectively collected from 102 patients with NMOSD diagnosed by experienced neurologists. The study population was divided into two groups based on the serum anti-SSA/Ro antibody status: NMOSD with or without anti-SSA/Ro antibody. The clinical, neuroimaging and laboratory parameters were compared between the two groups, including the neurological symptoms, MRI results, frequency of systemic autoantibodies, Expanded Disability Status Scale (EDSS), and NMOSD relapse rate. The EDSS and relapse were applied as measures of the NMOSD patient prognostic value. Cox regression analysis was used to evaluate the prognostic impact of anti-SSA/Ro antibody on NMOSD.Results: Among the 102 NMOSD patients, striking differences were observed in the positive rate of AQP4-IgG (89.2% vs. 72.3%, p = 0.046) between those patients with and without the anti-SSA/Ro antibody. In addition, NMOSD patients with anti-SSA/Ro antibody showed the presence of more frequent anti-ANA antibodies (p = 0.002), anti-SSB/La antibody (p < 0.001), anti-dsDNA antibody (p < 0.002), Sjogren's syndrome (SS, p < 0.001) and systemic lupus erythematosus (SLE, p = 0.045). Univariate and multivariate Cox regression analysis were performed to confirm that the anti-SSA/Ro antibody affected the EDSS score and the relapse of NMOSD patients. The analysis of the survival curve revealed that the EDSS score in the NMOSD patients positive for the anti-SSA/Ro antibody reached 4.0 (p = 0.035) and relapsed (p = 0.039) earlier than in the negative group.Conclusion: The anti-SSA/Ro antibody could be associated with disease activity and severe disability in NMOSD.

TCDD Inhibition of IgG1 Production in Experimental Autoimmune Encephalomyelitis (EAE) and In Vitro.

The environmental contaminant 2,3,7,8-tetrachlorodibenzo-para-dioxin (TCDD) is a ligand for the aryl hydrocarbon receptor (AhR). TCDD is well-characterized to produce immunotoxicity, including suppression of antibody production. Previously we showed that TCDD inhibited myelin oligodendrocyte glycoprotein (MOG) peptide-specific IgG and attenuated disease in experimental autoimmune encephalomyelitis (EAE) model in mice. Thus, the purpose of this study was to characterize the effects of TCDD on IgG subclasses in EAE and in vitro and assess effects in B cells derived from various tissues. TCDD modestly suppressed intracellular IgG expression in splenocytes (SPLC), but not bone marrow (BM) or lymph node (LN) cells. To further understand TCDD’s effects on IgG, we utilized LPS and LPS + IL-4 in vitro to stimulate IgG3 and IgG1 production, respectively. TCDD preferentially suppressed IgG1+ cell surface expression, especially in SPLC. However, TCDD was able to suppress IgG1 and IgG3 secretion from SPLC and B cells, but not BM cells. Lastly, we revisited the EAE model and determined that TCDD suppressed MOG-specific IgG1 production. Together these data show that the IgG1 subclass of IgG is a sensitive target of suppression by TCDD. Part of the pathophysiology of EAE involves production of pathogenic antibodies that can recruit cytolytic cells to destroy MOG-expressing cells that comprise myelin, so inhibition of IgG1 likely contributes to TCDD’s EAE disease attenuation.

Plasma exchange for severe immune-related adverse events from checkpoint inhibitors: an early window of opportunity?

Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of several advanced malignancies leading to durable remission in a subset of patients. Their rapidly expanding use has led to an increased frequency of immune-related adverse events (irAEs). The pathogenesis of irAEs is poorly understood but may involve aberrant activation of T cells leading to inflammatory cytokine release or production of pathogenic antibodies leading to organ damage. Severe irAEs can be extremely debilitating and, in some cases, life threatening. IrAEs may not always be corticosteroid responsive or may require excessively high, often toxic, corticosteroid doses. Therapeutic plasma exchange (PLEX) is a treatment modality that has shown promising results for the management of certain severe irAEs, including irAEs that are not mentioned in current treatment guidelines. PLEX may attenuate ongoing irAEs and prevent delayed irAEs by accelerating clearance of the ICI, or by acutely removing pathogenic antibodies, cytokines, and chemokines. Here, we summarize examples from the literature in which PLEX was successfully used for the treatment of irAEs. We posit that timing may be a critical factor and that earlier utilization of PLEX for life-threatening irAEs may result in more favorable outcomes. In individuals at high risk for irAEs, the availability of PLEX as a potential therapeutic mitigation strategy may encourage life-saving ICI use or rechallenge. Future research will be critical to better define which indications are most amenable to PLEX, particularly to establish the optimal place in the sequence of irAE therapies and to assess the ramifications of ICI removal on cancer outcomes.

The immunomodulatory effect of koumine on B cells under dependent and independent responses by T cells

Dysregulated activation of polyclonal B cells and production of pathogenic antibodies are involved in the development of rheumatoid arthritis (RA). Therefore, targeted B cell therapy is effective against RA. Gelsemium elegans (Gardn. & Champ.) Benth., a toxic plant widely distributed in Southeast Asia, has been used for treating rheumatoid pain, neuropathic pain, spasticity, skin ulcers, and cancers for many years in traditional Chinese medicine. Koumine, an alkaloid monomer from Gelsemium elegans Benth., exerts therapeutic effects against RA. However, whether koumine affects B cells remains unknown. In this study, the effect of koumine on B cells under T cell-independent (TI) and T cell-dependent (TD) immune responses is investigated in vitro and in vivo. Mouse primary B cells were obtained by immunomagnetic bead sorting, and immunomodulatory effects of koumine on the activation, proliferation, and differentiation of B cells were determined in TI and TD models induced by lipopolysaccharide (LPS) and anti-CD40 antibodies in vitro, respectively. The humoral immune responses of TI and TD were established using NP-AECM-FICOLL and NP-CGG in C57BL/6J mice, respectively. We found that koumine inhibited B cell differentiation in the TI model and inhibited B cell activation and proliferation in the TD model in vitro. Koumine also inhibited antibody secretion in TI immune response, TD initial immune response, and in TD secondary immune response. Our results reveal that koumine has a direct and indirect immune regulatory effect on B cells, showing that it can directly inhibit the differentiation and secretion of autoantibodies after abnormal activation of B cells, and indirectly inhibit the activation and proliferation of TD B cells to reduce the secretion of antibodies. It may be an important mechanism for its anti-RA effect in mice, providing a rationale and laboratory data support for the application of koumine in anti-human RA therapy.

To build a plasma cell.

To build a plasma cell.

Meant to B: B cells as a therapeutic target in systemic lupus erythematosus.

B cells have a prominent role in the pathogenesis of systemic lupus erythematosus (SLE). They are mediators of inflammation through the production of pathogenic antibodies that augment inflammation and cause direct tissue and cell damage. Multiple therapeutic agents targeting B cells have been successfully used in mouse models of SLE; however, these preclinical studies have led to approval of only one new agent to treat patients with SLE: belimumab, a monoclonal antibody targeting B cell-activating factor (BAFF). Integrating the experience acquired from previous clinical trials with the knowledge generated by new studies about mechanisms of B cell contributions to SLE in specific groups of patients is critical to the development of new treatment strategies that will help to improve outcomes in patients with SLE. In particular, a sharper focus on B cell differentiation to plasma cells is warranted.

Spontaneous antibody production caused by regulatory T cell deficiency occurs through a germinal center-independent pathway

Foxp3+ regulatory T cells (Tregs) are essential for the prevention of autoantibody and allergen-specific IgE production. Treg deficiency causes an elevation of the serum levels of these pathogenic antibodies, accompanied by spontaneous germinal center (GC) formation. However, it remains to be determined whether excessive and pathogenic antibody production induced by Treg deficiency requires a GC response. Here, we demonstrate that spontaneous antibody production observed in Foxp3 conditional-knockout mice did not need GC formation. Foxp3 and Bcl6 conditional-double knockout mice exhibited spontaneous elevations of IgG1, IgG2c, and IgE levels even though they showed impaired production of IgG1 and IgE specific for the immunized antigen. Furthermore, the IgG1 and IgE antibodies specific for auto- and food-antigens were produced independently of GCs. These data suggested that a GC response was unnecessary for pathogenic antibody production caused by Treg deficiency.

Monitoring anti-B cell immunotherapies in autoimmune diseases: Go with the flow. A Position Paper of the Italian Society for Clinical Cell Analysis (ISCCA)

During the past decades autoimmune diseases have been usually treated with immunosuppressive drugs mostly active on T-Cell mediated responses. Only in recent years, with our extended knowledge of the pathogenic mechanisms of autoreactive disorders and the tremendous development of new therapeutic monoclonal antibodies, anti-B-Cell therapies have emerged as a new option for treating autoimmune diseases. The rationale for this changeover from T-Cell to B-Cell targeted therapies resides in the recently accumulated evidence of the role of B-Cells in the pathogenesis of autoimmune diseases and in the generation of tissue damage. Targeting memory and effector BCells may then disrupt the production of pathogenic antibodies, counteract the role of B-Cells in sustaining antigen presentation to T-Cells and block the synthesis of B-Cell activation cytokines. The anti-CD20 monoclonal antibody Rituximab was first introduced more than 20 years ago for the treatment of CD20+ chronic B-lymphoproliferative disorders, and was then successfully experimented in the treatment of an ever-increasing spectrum of autoimmune diseases. Newer anti-CD20 monoclonal antibodies have been introduced more recently, which vary in their biological effects. The need for laboratory indicators that may help the rational usage and follow-up of anti-CD20 treatments has now emerged, due to the high variability of individual response, to the markedly different outcomes in the various diseases and to the controversial role of pathogenic autoantibodies as indicators of disease activity. Flow cytometric (FCM) analyses to identify and enumerate the B-cell functional subsets in the peripheral blood have been developed in recent years. They can be used to assess the degree and the persistence of memory B-Cell depletion, the quality and the timing of B-Cell reconstitution, along with the highly sensitive FCM counting technique needed for the detection of extremely low cell levels. The long-term aim of this innovative approach is to provide clinicians with a tool for a safer and more rational usage of anti-CD20 agents.

Single-Cell RNA-Seq Identifies Potentially Pathogenic B Cell Populations That Uniquely Circulate in Patients with Chronic Gvhd

While B cells are known to contribute to the pathogenesis of chronic graft-versus-host disease (cGVHD) in mice, it has been challenging to elucidate intrinsic mechanisms of tolerance loss in patients. To identify distinct and potentially targetable B-cell subsets in cGVHD, we employed single-cell RNA-Seq along with an unsupervised hierarchical clustering analysis, targeting 10,000 single B cells from each of eight patients who were &amp;gt;12 months post-allogeneic hematopoietic stem cell transplantation (HCT) and either had active cGVHD manifestations (n=4) or never developed cGVHD (n=4). Bioinformatics analysis of pooled cell data (using R with Seurat extension package) identified 6 major B cell clusters common to all patients (Figure 1A). "Intra-cluster" gene comparison (using R package DESeq2, false-discovery rate 0.05) revealed numerous differentially expressed genes between patient groups. The greatest number of differentially-expressed genes occurred in a cluster referred to herein as 'Cluster 6' (Figure 1A, in yellow with asterisk). Within Cluster 6, B cells from active cGVHD patients expressed significantly increased ITGAX (CD11c, Padj =0.007), TNFRSF13B (TACI, a receptor for BAFF, Padj =0.003), IGHG1 (IgG1, Padj =9.3e-06) and IGHG3 (IgG3, Padj =1.7e-12), along with 44 additional genes (to be discussed). Thus, Cluster 6 in cGVHD patients may represent a CD11cpos, BAFF-responsive B cell subset primed to undergo isotype switching in response to alloantigen. Flow cytometry analysis on PBMCs from an independent HCT patient cohort (n=10) confirmed that CD11cpos B cells were indeed significantly expanded in cGVHD (P &amp;lt; 0.01, Figure 1B), and revealed these B cells were also TACIpos, CD19high, forward scatter high (FSChigh) blast-like cells (Figure 1C). We found that these CD11cpos B cells had mixed expression of CD21, CD27, IgD and CD24 (Figure 1C). Remarkably, other recent studies on bulk patient B cells have suggested that similar CD11cposCD21negCD19highT-BETpos cells are critical drivers of humoral autoimmunity in diseases including systemic lupus erythematosus (SLE; Scharer et al. 2019; Rubtsova et al. 2017; Rubtsov et al. 2011). This subset now identified by single-cell RNA-Seq is consistent with a population of TACIhigh B cells that produced IgG in response to BAFF treatment ex vivo (Sarantopoulos 2009). Data suggest we have identified functionally distinct and potentially targetable B cell subpopulations. We are employing functional assays to determine whether the additional molecular pathways now elucidated account for our previous work showing greater ex vivo B cell survival rates and hyper-responsiveness to surrogate antigen (Allen et al. 2012, 2014), certain TLR agonists (Suthers et al. 2017), and NOTCH ligand (Poe et al. 2017). In addition to more deeply characterizing B-cell subsets in cGVHD, our single-cell RNA-Seq analyses identified several genes significantly altered across multiple B cell clusters in the cGVHD group, implicating more broad alterations of some genes in this disease. Among these is CKS2, a critical cell cycle regulator, which was significantly increased in cGVHD B cells (Padj 1.0e-10 to 0.018, depending on the cluster evaluated). Increased CKS2 expression was validated by qPCR analysis on B cells from a separate HCT patient cohort with or without cGVHD (P &amp;lt; 0.001, Figure 1D), suggesting that the majority of cGVHD B cells are primed to enter the cell cycle at multiple stages of differentiation when exposed to the proper stimuli. In summary, we used an unbiased approach to identify and further characterize an extrafollicular CD11cposTACIposCD19high B cell population in cGVHD patients that appears to be activated and undergoing active IgG isotype switching. This plasmablast-like B cell population is potentially amenable to therapeutic intervention to prevent pathogenic antibody production. Importantly, we also identify gene alterations across the cGVHD peripheral B cell compartment that potentially underpin promotion of hyperactivated B cells in this disease. Therapeutic strategies to target these pathways will also be discussed. This work was supported by a National Institutes of Health grant, R01HL129061. Disclosures Ho: Omeros Corporation: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Research Funding; Jazz Pharmaceuticals: Consultancy. Horwitz:Abbvie Inc: Membership on an entity's Board of Directors or advisory committees. Rizzieri:Celgene, Gilead, Seattle Genetics, Stemline: Other: Speaker; AbbVie, Agios, AROG, Bayer, Celgene, Gilead, Jazz, Novartis, Pfizer, Sanofi, Seattle Genetics, Stemline, Teva: Other: Advisory Board; AROG, Bayer, Celgene, Celltron, Mustang, Pfizer, Seattle Genetics, Stemline: Consultancy; Stemline: Research Funding.

Cervus and cucumis peptides ameliorates bone erosion in experimental arthritis by inhibiting osteoclastogenesis

ObjectiveRheumatoid arthritis is an autoimmune disease characterised by inflammation and bone loss, leading to joint destruction and deformity. The cervus and cucumis polypeptide (CCP) injection, one of the traditional Chinese...

Dysregulation of humoral immunity in Foxp3 conditional-knockout mice

Foxp3+ regulatory T cells (Tregs) are crucial for maintaining tolerance to self-antigens and preventing autoimmune diseases. Loss of Foxp3 expression leads to autoimmunity and disrupts humoral immune responses, including hyperproduction of immunoglobulin E (IgE). Elucidation of how Tregs control antibody production can lead to the development of new therapies for autoimmune and allergic diseases. However, premature death of Foxp3-deficient mice makes it difficult to analyze the roles of Tregs in humoral immunity of adult mice. In this study, we developed Foxp3 conditional-knockout mice (Foxp3floxR26CreERT2) in which the Foxp3 gene was inducibly deleted by tamoxifen administration. After oral administration of tamoxifen, titers of immunoglobulins, particularly IgG2c and IgE, were increased in Foxp3floxR26CreERT2 mice compared with that in controls. Under these conditions, CD4+ T cells from Foxp3floxR26CreERT2 mice had increased expression of several activation markers, including inducible costimulator and CD40 ligand, as well as the cytokines interleukin 4 and interferon gamma. In addition, the proportions of T follicular helper (Tfh) cells and germinal center (GC) B cells were increased in Foxp3floxR26CreERT2 mice compared with those in controls. These results indicated that Tregs controlled excessive or pathogenic antibody production by suppressing Tfh cell differentiation and GC formation. Furthermore, these data suggested that Foxp3floxR26CreERT2 mice could be a useful tool for screening therapeutic agents.

Long-lasting persistence of large B-cell clones in hepatitis C virus-cured patients with complete response of mixed cryoglobulinaemia vasculitis.

Hepatitis C virus (HCV)-related mixed cryoglobulinaemia vasculitis (MCV) is characterized by the expansion of rheumatoid factor-producing B-cell clones. The aim of this study was to assess whether B-cell clones may persist in these patients after the clearance of the virus with antiviral therapy, and whether their persistence influences clinical outcomes. Forty-five HCV-cured MCV patients were followed up for a median of 18.5 (range 9-38) months after the clearance of HCV. Circulating B-cell clones were detected using flow cytometry either by the skewing of kappa/lambda ratio or by the expression of a VH 1-69-encoded idiotype. The clinical response of vasculitis was 78% complete, 18% partial and 4% null. However, cryoglobulins remained detectable in 42% of patients for more than 12months. Circulating B-cell clones were detected in 18 of 45 patients, and in 17 of them persisted through the follow-up; nine of the latter patients cleared cryoglobulins and had complete response of vasculitis. Several months later, two of these patients had relapse of MCV. B-cell clones persist in MCV patients long after HCV infection has been cleared but halt the production of pathogenic antibody. These 'dormant' cells may be reactivated by events that perturb B-cell homeostasis and can give rise to the relapse of cryoglobulinaemic vasculitis.

LRRK2 is involved in the pathogenesis of system lupus erythematosus through promoting pathogenic antibody production

BackgroundSystemic lupus erythematosus (SLE) is a prototypic autoimmune disease characterized by the presence of pathogenic autoantibodies associated with polyclonal B cell hyperreactivity. Previous study reported that autophagy-related gene Leucine-rich repeat kinase 2 (LRRK2) was likely a susceptible gene for SLE. However, the pathogenic function of LRRK2 in SLE is undefined.MethodsUsing quantitative PCR, we compared the expression levels of LRRK2 in B cells between SLE patients and healthy controls. The expression levels of LRRK2 in in vitro induced CD19hi B cells and naïve B cells were compared as well based on RNA-seq assay. A pristane-induced lupus-like mouse model was used to explore the effects of LRRK2 on the development of SLE. IgG level, B cell subsets in the spleens and bone marrows and pathological features in the kidneys were compared between wildtype (WT) and Lrrk2−/− littermates.ResultsIt was obvious that LRRK2 expression was dramatically up-regulated in primary B cells from SLE patients compared to those from healthy controls, as well as in activated CD19hi B cells. More significantly, LRRK2 expression in B cells was positively correlated with system lupus erythematosus disease activity index (SLEDAI), an indicator for disease severity, and serum IgG levels in SLE patients. Negative correlations were observed between LRRK2 expression and serum C3 or C4 levels, two clinical features associated with SLE-related nephritis. LRRK2 deficiency reduced the death rate of pristane treated mice. Decreased levels of total IgG and autoantibody were detected in the serum with less deposition of immune complexes and attenuated pathological symptoms in the kidneys of Lrrk2−/− mice. Consistent with the reduction in IgG production, the percentages of germinal center B cells and plasma cells decreased significantly as well with LRRK2 deficiency.ConclusionsOur study demonstrates that LRRK2 expression is upregulated in B cells from SLE patients with strong correlations to disease severity. LRRK2 deficiency largely attenuates the pathogenic progress of lupus-like features in pristane-induced mice. This is probably achieved through affecting B cell terminal differentiation and subsequent immunoglobulin production.

A Functionally Different Immune Phenotype in Cattle Is Associated With Higher Mastitis Incidence.

A novel vaccine against bovine viral diarrhea (BVD) induced pathogenic antibody production in 5–10% of BVD-vaccinated cows. Transfer of these antibodies via colostrum caused Bovine neonatal pancytopenia (BNP) in calves, with a lethality rate of 90%. The exact immunological mechanisms behind the onset of BNP are not fully understood to date. To gain further insight into these mechanisms, we analyzed the immune proteome from alloreactive antibody producers (BNP cows) and non-responders. After in vitro stimulation of peripheral blood derived lymphocytes (PBL), we detected distinctly deviant expression levels of several master regulators of immune responses in BNP cells, pointing to a changed immune phenotype with severe dysregulation of immune response in BNP cows. Interestingly, we also found this response pattern in 22% of non-BVD-vaccinated cows, indicating a genetic predisposition of this immune deviant (ID) phenotype in cattle. We additionally analyzed the functional correlation of the ID phenotype with 10 health parameters and 6 diseases in a retrospective study over 38 months. The significantly increased prevalence of mastitis among ID cows emphasizes the clinical relevance of this deviant immune response and its potential impact on the ability to fight infections.