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OBJECTIVE: Safer and more effective therapies are needed for the treatment of systemic lupus erythematosus (SLE). B lymphocytes have been shown to play fundamental pathogenic roles in SLE, and therefore, elimination of B cells with the use of rituximab may represent a new therapy for SLE. METHODS: A phase I/II dose-escalation trial of rituximab added to ongoing therapy in SLE was conducted. Rituximab was administered as a single infusion of 100 mg/m2 (low dose), a single infusion of 375 mg/m2 (intermediate dose), or as 4 infusions (1 week apart) of 375 mg/m2 (high dose). CD19+ lymphocytes were measured to determine the effectiveness of B cell depletion. The Systemic Lupus Activity Measure (SLAM) score was used as the primary outcome for clinical efficacy. RESULTS: Rituximab was well tolerated in this patient population, with most experiencing no significant adverse effects. Only 3 serious adverse events, which were thought to be unrelated to rituximab administration, were noted. A majority of patients (11 of 17) had profound B cell depletion (to <5 CD19+ B cells/microl). In these patients, the SLAM score was significantly improved at 2 and 3 months compared with baseline (P = 0.0016 and P = 0.0022, respectively, by paired t-test). This improvement persisted for 12 months, despite the absence of a significant change in anti-double-stranded DNA antibody and complement levels. Six patients developed human antichimeric antibodies (HACAs) at a level > or =100 ng/ml. These HACA titers were associated with African American ancestry, higher baseline SLAM scores, reduced B cell depletion, and lower levels of rituximab at 2 months after initial infusion. CONCLUSION: Rituximab therapy appears to be safe for the treatment of SLE and holds significant therapeutic promise, at least for the majority of patients experiencing profound B cell depletion. Based on these results, controlled trials of rituximab appear to be warranted. [ABSTRACT FROM AUTHOR]

Rheumatoid arthritis (RA) is a complex immune-mediated inflammatory disorder in which patients suffer from inflammatory-erosive arthritis. Recent advances on histopathology heterogeneity of RA synovial tissue revealed three distinct phenotypes based on cellular composition (pauci-immune, diffuse and lymphoid), suggesting that distinct etiologies warrant specific targeted therapy which motivates a need for cost effective phenotyping tools in preclinical and clinical settings. To this end, we developed an automated multi-scale computational pathotyping (AMSCP) pipeline for both human and mouse synovial tissue with two distinct components that can be leveraged together or independently: (1) segmentation of different tissue types to characterize tissue-level changes, and (2) cell type classification within each tissue compartment that assesses change across disease states. Here, we demonstrate the efficacy, efficiency, and robustness of the AMSCP pipeline as well as the ability to discover novel phenotypes. Taken together, we find AMSCP to be a valuable cost-effective method for both pre-clinical and clinical research., (© 2024. The Author(s).)

Rheumatoid arthritis (RA) is an autoimmune disease involving antigen-specific T and B cells. Here, we perform single-cell RNA and repertoire sequencing on paired synovial tissue and blood samples from 12 seropositive RA patients. We identify clonally expanded CD4 + T cells, including CCL5+ cells and T peripheral helper (Tph) cells, which show a prominent transcriptomic signature of recent activation and effector function. CD8 + T cells show higher oligoclonality than CD4 + T cells, with the largest synovial clones enriched in GZMK+ cells. CD8 + T cells with possibly virus-reactive TCRs are distributed across transcriptomic clusters. In the B cell compartment, NR4A1+ activated B cells, and plasma cells are enriched in the synovium and demonstrate substantial clonal expansion. We identify synovial plasma cells that share BCRs with synovial ABC, memory, and activated B cells. Receptor-ligand analysis predicted IFNG and TNFRSF members as mediators of synovial Tph-B cell interactions. Together, these results reveal clonal relationships between functionally distinct lymphocyte populations that infiltrate the synovium of patients with RA., (© 2024. The Author(s).)

Synovial tissue inflammation is a hallmark of rheumatoid arthritis (RA). Recent work has identified prominent pathogenic cell states in inflamed RA synovial tissue, such as T peripheral helper cells; however, the epigenetic regulation of these states has yet to be defined. Here, we examine genome-wide open chromatin at single-cell resolution in 30 synovial tissue samples, including 12 samples with transcriptional data in multimodal experiments. We identify 24 chromatin classes and predict their associated transcription factors, including a CD8 + GZMK+ class associated with EOMES and a lining fibroblast class associated with AP-1. By integrating with an RA tissue transcriptional atlas, we propose that these chromatin classes represent 'superstates' corresponding to multiple transcriptional cell states. Finally, we demonstrate the utility of this RA tissue chromatin atlas through the associations between disease phenotypes and chromatin class abundance, as well as the nomination of classes mediating the effects of putatively causal RA genetic variants., (© 2024. The Author(s).)

Lupus nephritis (LN) is a pathologically heterogenous autoimmune disease linked to end-stage kidney disease and mortality. Better therapeutic strategies are needed as only 30%-40% of patients completely respond to treatment. Noninvasive biomarkers of intrarenal inflammation may guide more precise approaches. Because urine collects the byproducts of kidney inflammation, we studied the urine proteomic profiles of 225 patients with LN (573 samples) in the longitudinal Accelerating Medicines Partnership in RA/SLE cohort. Urinary biomarkers of monocyte/neutrophil degranulation (i.e., PR3, S100A8, azurocidin, catalase, cathepsins, MMP8), macrophage activation (i.e., CD163, CD206, galectin-1), wound healing/matrix degradation (i.e., nidogen-1, decorin), and IL-16 characterized the aggressive proliferative LN classes and significantly correlated with histological activity. A decline of these biomarkers after 3 months of treatment predicted the 1-year response more robustly than proteinuria, the standard of care (AUC: CD206 0.91, EGFR 0.9, CD163 0.89, proteinuria 0.8). Candidate biomarkers were validated and provide potentially treatable targets. We propose these biomarkers of intrarenal immunological activity as noninvasive tools to diagnose LN and guide treatment and as surrogate endpoints for clinical trials. These findings provide insights into the processes involved in LN activity. This data set is a public resource to generate and test hypotheses and validate biomarkers.

Rheumatoid arthritis is a prototypical autoimmune disease that causes joint inflammation and destruction 1 . There is currently no cure for rheumatoid arthritis, and the effectiveness of treatments varies across patients, suggesting an undefined pathogenic diversity 1,2 . Here, to deconstruct the cell states and pathways that characterize this pathogenic heterogeneity, we profiled the full spectrum of cells in inflamed synovium from patients with rheumatoid arthritis. We used multi-modal single-cell RNA-sequencing and surface protein data coupled with histology of synovial tissue from 79 donors to build single-cell atlas of rheumatoid arthritis synovial tissue that includes more than 314,000 cells. We stratified tissues into six groups, referred to as cell-type abundance phenotypes (CTAPs), each characterized by selectively enriched cell states. These CTAPs demonstrate the diversity of synovial inflammation in rheumatoid arthritis, ranging from samples enriched for T and B cells to those largely lacking lymphocytes. Disease-relevant cell states, cytokines, risk genes, histology and serology metrics are associated with particular CTAPs. CTAPs are dynamic and can predict treatment response, highlighting the clinical utility of classifying rheumatoid arthritis synovial phenotypes. This comprehensive atlas and molecular, tissue-based stratification of rheumatoid arthritis synovial tissue reveal new insights into rheumatoid arthritis pathology and heterogeneity that could inform novel targeted treatments., (© 2023. The Author(s).)

Introduction: Defective lymphatic drainage and translocation of B-cells in inflamed (Bin) joint-draining lymph node sinuses are pathogenic phenomena in patients with severe rheumatoid arthritis (RA). However, the molecular mechanisms underlying this lymphatic dysfunction remain poorly understood. Herein, we utilized multi-omic spatial and single-cell transcriptomics to evaluate altered cellular composition (including lymphatic endothelial cells, macrophages, B-cells, and T-cells) in the joint-draining lymph node sinuses and their associated phenotypic changes and cell-cell interactions during RA development using the tumor necrosis factor transgenic (TNF-Tg) mouse model., Methods: Popliteal lymph nodes (PLNs) from wild-type (n=10) and TNF-Tg male mice with "Early" (5 to 6-months of age; n=6) and "Advanced" (>8-months of age; n=12) arthritis were harvested and processed for spatial transcriptomics. Single-cell RNA sequencing (scRNAseq) was performed in PLNs from the TNF-Tg cohorts (n=6 PLNs pooled/cohort). PLN histopathology and ELISPOT along with ankle histology and micro-CT were evaluated. Histopathology of human lymph nodes and synovia was performed for clinical correlation., Results: Advanced PLN sinuses exhibited an increased Ighg2b/Ighm expression ratio (Early 0.5 ± 0.1 vs Advanced 1.4 ± 0.5 counts/counts; p<0.001 ) that significantly correlated with reduced talus bone volumes in the afferent ankle (R 2 = 0.54, p<0.001 ). Integration of single-cell and spatial transcriptomics revealed the increased IgG2b + plasma cells localized in MARCO + peri-follicular medullary sinuses. A concomitant decreased Fth1 expression (Early 2.5 ± 0.74 vs Advanced 1.0 ± 0.50 counts, p<0.001 ) within Advanced PLN sinuses was associated with accumulation of iron-laden Prussian blue positive macrophages in lymph nodes and synovium of Advanced TNF-Tg mice, and further validated in RA clinical samples. T-cells were increased 8-fold in Advanced PLNs, and bioinformatic pathway assessment identified the interaction between ALCAM + macrophages and CD6 + T-cells as a plausible co-stimulatory mechanism to promote IgG2b class-switching., Discussion: Collectively, these data support a model of flare in chronic TNF-induced arthritis in which loss of lymphatic flow through affected joint-draining lymph nodes facilitates the interaction between effluxing macrophages and T-cells via ALCAM-CD6 co-stimulation, initiating IgG2b class-switching and plasma cell differentiation of the expanded Bin population. Future work is warranted to investigate immunoglobulin clonality and potential autoimmune consequences, as well as the efficacy of anti-CD6 therapy to prevent these pathogenic events., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Kenney, Rangel-Moreno, Peng, Chen, Bruno, Embong, Pritchett, Fox, Becerril-Villanueva, Gamboa-Domínguez, Quataert, Muthukrishnan, Wood, Korman, Anolik, Xing, Ritchlin, Schwarz and Wu.)

Although B cells account for a significant proportion of the lymphocytic infiltrate in discoid lupus erythematosus (DLE), their contribution to pathogenesis is unknown. In this study, we compare the immune landscape of 17 subjects with DLE with that of 21 subjects with subacute cutaneous lupus erythematosus using transcriptomic and histologic analyses of lesional skin. A few of the subjects (3 of 17 subjects with DLE, and 5 of 21 subjects with subacute cutaneous lupus erythematosus) had concomitant systemic lupus erythematosus. Using a modified Autoimmune Profiling Panel (NanoString Technologies, Seatle, WA), we show that B-cell‒specific genes, including canonical pan‒B cell markers CD19 (P = 0.0060), MS4A1 (CD20) (P = 0.0047), and CD79a (P = 0.0201), are among the most upregulated genes in DLE. Numerous other genes encoding B-cell‒associated proteins, including Igs, BAFF receptors, and FCRL family members, are similarly enriched. Relative cell type scoring reveals that among various inflammatory cell types, only B cells are more prevalent in DLE. Digital whole-image slide analysis of immunohistochemistry for B cells (CD20) and T cells (CD3) supports our gene expression findings of a disproportionately greater B-cell infiltrate in DLE lesions. Overall, this study identifies a B-cell‒predominant signature unique to DLE and highlights the importance of studying the role of cutaneous B cells in DLE pathogenesis., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: To investigate the hypothesis that selective inhibitors of nuclear export (SINE compounds), recently approved for treatment of refractory plasma cell (PC) malignancy, may have potential in the treatment of lupus., Methods: Female NZB/NZW mice were treated with the SINE compound KPT-350 or vehicle control. Tissue specimens were harvested and analyzed by flow cytometry, using standard markers. Nephritis was monitored by determining the proteinuria score and by histologic analysis of kidney specimens. Serum anti-double-stranded DNA (anti-dsDNA) levels were measured by enzyme-linked immunosorbent assay, and total numbers of IgG-secreting and dsDNA-specific antibody-secreting cells were assessed by enzyme-linked immunospot assay., Results: KPT-350 abrogated murine lupus nephritis at both early and late stages of the disease and rapidly impaired generation of autoreactive PCs in germinal centers (GCs). SINE compounds inhibited the production of NF-κB-driven homeostatic chemokines by stromal cells, altering splenic B and T cell strategic positioning and significantly reducing follicular helper T cell, GC B cell, and autoreactive PC counts. KPT-350 also decreased levels of cytokines and chemokines involved in PC survival and recruitment in the kidney of lupus-prone mice. Exportin 1, the target of SINE compounds, was detected in GCs of human tonsils, splenic B cells of lupus patients, and multiple B cell subsets in the kidneys of patients with lupus nephritis., Conclusion: Collectively, our results provide support for the therapeutic potential of SINE compounds, via their targeting of several molecular and cellular pathways critical in lupus pathogenesis, including autoantibody production by plasma cells., (© 2022 American College of Rheumatology.)

Ectopic lymphoid structures (ELS) can develop in rheumatoid arthritis (RA) synovial tissue, but the precise pathways of B cell activation and selection are not well understood. Here, we identify a synovial B cell population characterized by co-expression of a family of orphan nuclear receptors (NR4A1-3), which is highly enriched in RA synovial tissue. A transcriptomic profile of NR4A synovial B cells significantly overlaps with germinal center light zone B cells and an accrual of somatic hypermutation that correlates with loss of naive B cell state. NR4A B cells co-express lymphotoxins α and β and IL-6, supporting functions in ELS promotion. Expanded and shared clones between synovial NR4A B cells and plasma cells and the rapid upregulation with BCR stimulation point to in situ differentiation. Together, we identify a dynamic progression of B cell activation in RA synovial ELS, with NR4A transcription factors having an important role in local adaptive immune responses., Competing Interests: Declaration of interests S.R. is a paid consultant for Gilead, Pfizer, Rheos, and J&J, and is a founder of Mestag. D.E.O. is an inventor of two non-licensed patents; US 63/031,861 entitled “markers and Cellular Antecedents of Rheumatoid Arthritis Flares” and US 63/050,155 entitled “method and System for RNA Isolation from Self-Collected and Small Volume Samples.” S.G. receives research support from Novartis and is a consultant for UCB., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Objective: B cells can become activated in germinal center (GC) reactions in secondary lymphoid tissue and in ectopic GCs in rheumatoid arthritis (RA) synovium that may be tumor necrosis factor (TNF) and lymphotoxin (LT) dependent. This study was undertaken to characterize the peripheral B cell compartment longitudinally during anti-TNF therapy in RA., Methods: Participants were randomized in a 2:1 ratio to receive standard dosing regimens of etanercept (n = 43) or adalimumab (n = 20) for 24 weeks. Eligible participants met the American College of Rheumatology 1987 criteria for RA, had clinically active disease (Disease Activity Score in 28 joints >4.4), and were receiving stable doses of methotrexate. The primary mechanistic end point was the change in switched memory B cell fraction from baseline to week 12 in each treatment group., Results: B cell subsets remained surprisingly stable over the course of the study regardless of treatment group, with no significant change in memory B cells. Blockade of TNF and LT with etanercept compared to blockade of TNF alone with adalimumab did not translate into significant differences in clinical response. The frequencies of multiple activated B cell populations, including CD21- double-negative memory and activated naive B cells, were higher in RA nonresponders at all time points, and CD95+ activated B cell frequencies were increased in patients receiving anti-TNF treatment in the nonresponder group. In contrast, frequencies of transitional B cells-a putative regulatory subset-were lower in the nonresponders., Conclusion: Overall, our results support the notion that peripheral blood B cell subsets are remarkably stable in RA and not differentially impacted by dual blockade of TNF and LT with etanercept or single blockade of TNF with adalimumab. Activated B cells do associate with a less robust response., (© 2021, American College of Rheumatology.)

Background: We have previously shown that SLE BMSC have decreased proliferation, increased ROS, increased DNA damage and repair (DDR), a senescence associated secretory phenotype, and increased senescence-associated β-galactosidase. We have also shown SLE BMSC produce increased amounts of interferon beta (IFNβ), have increased mRNA for several genes induced by IFNβ, and have a pro-inflammatory feedback loop mediated by a MAVS. To better understand the phenotype of SLE BMSC we conducted mRNA sequencing., Methods: Patients fulfilling SLE classification criteria and age and sex matched healthy controls were recruited under an Institutional Review Board approved protocol. Bone marrow aspirates and peripheral blood samples were obtained. BMSC were isolated and grown in tissue culture. Early passage BMSC were harvested and mRNA samples were sent for RNAseq. Serum samples were assayed for IFNβ by ELISA., Results: On the basis of top differentially expressed genes between SLE and healthy controls, SLE patients with high levels of serum IFNβ clustered together while SLE patients with low levels of IFNβ clustered with healthy controls. Those genes differentially expressed in SLE patients generally belonged to known IFN pathways, and showed a strong overlap with the set of genes differentially expressed in IFNβ high subjects, per se. Moreover, gene expression changes induced by treating healthy BMSC with exogenous IFNβ were remarkably similar to gene expression differences in SLE IFNβ high vs low BMSC., Conclusions: BMSCs from SLE patients are heterogeneous. A subgroup of SLE BMSC is distinguished from other SLE BMSC and from controls by increased levels of mRNAs induced by type I interferons. This subgroup of SLE patients had increased levels of IFNβ in vivo., Competing Interests: Declaration of Competing Interest The authors declared that there is no conflict of interest., (Copyright © 2019. Published by Elsevier Ltd.)

Common variable immunodeficiency (CVID) comprises a group of related disorders defined by defects in B cell function and antibody production. Concurrent autoimmune features are common, but the underlying pathogenic mechanisms of autoimmunity in CVID are poorly understood. Overlap in some clinical and laboratory features suggests a shared pathogenesis, at least in part, with systemic lupus erythematosus (SLE). One important part of SLE pathogenesis is loss of B cell tolerance, an aspect that warrants further study in CVID. The study of inherently autoreactive 9G4 + B cells has led to a greater understanding of B cell tolerance defects in lupus. Study of these B cells in CVID has yielded conflicting results, largely due to differences in methodological approaches. In this study, we take a comprehensive look at 9G4 + B cells throughout B cell development in CVID patients and compare patients both with and without autoimmune features. Using flow cytometry to examine B cell subpopulations in detail, we show that only those CVID patients with autoimmune features demonstrate significant expansion of 9G4 + B cells, both in naïve and multiple memory populations. Examination of two autoreactive B cell subsets recently characterized in SLE, the activated naïve (aNAV) and double negative 2 (DN2) B cells, reveals an expanded 9G4 + DN2 population to be common among CVID patients. These results reveal that both multiple central and peripheral B cell tolerance defects are related to autoimmunity in CVID. Furthermore, these data suggest that the autoreactive DN2 B cell population, which has not previously been examined in CVID, may play an important role in the development of autoimmunity in patients with CVID., (Copyright © 2019 Richardson, Slack, Dhillon, Marcus, Barnard, Palanichamy, Sanz, Looney and Anolik.)

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by pathologic T cell-B cell interactions and autoantibody production. Defining the T cell populations that drive B cell responses in SLE may enable design of therapies that specifically target pathologic cell subsets. Here, we evaluated the phenotypes of CD4+ T cells in the circulation of 52 SLE patients drawn from multiple cohorts and identified a highly expanded PD-1hiCXCR5-CD4+ T cell population. Cytometric, transcriptomic, and functional assays demonstrated that PD-1hiCXCR5-CD4+ T cells from SLE patients are T peripheral helper (Tph) cells, a CXCR5- T cell population that stimulates B cell responses via IL-21. The frequency of Tph cells, but not T follicular helper (Tfh) cells, correlated with both clinical disease activity and the frequency of CD11c+ B cells in SLE patients. PD-1hiCD4+ T cells were found within lupus nephritis kidneys and correlated with B cell numbers in the kidney. Both IL-21 neutralization and CRISPR-mediated deletion of MAF abrogated the ability of Tph cells to induce memory B cell differentiation into plasmablasts in vitro. These findings identify Tph cells as a highly expanded T cell population in SLE and suggest a key role for Tph cells in stimulating pathologic B cell responses.

The function of B cells in osteoblast (OB) dysfunction in rheumatoid arthritis (RA) has not been well-studied. Here we show that B cells are enriched in the subchondral and endosteal bone marrow (BM) areas adjacent to osteocalcin + OBs in two murine RA models: collagen-induced arthritis and the TNF-transgenic mice. Subchondral BM B cells in RA mice express high levels of OB inhibitors, CCL3 and TNF, and inhibit OB differentiation by activating ERK and NF-κB signaling pathways. The inhibitory effect of RA B cells on OB differentiation is blocked by CCL3 and TNF neutralization, and deletion of CCL3 and TNF in RA B cells completely rescues OB function in vivo, while B cell depletion attenuates bone erosion and OB inhibition in RA mice. Lastly, B cells from RA patients express CCL3 and TNF and inhibit OB differentiation, with these effects ameliorated by CCL3 and TNF neutralization. Thus, B cells inhibit bone formation in RA by producing multiple OB inhibitors.

Systemic Lupus Erythematosus (SLE) is characterized by B cells lacking IgD and CD27 (double negative; DN). We show that DN cell expansions reflected a subset of CXCR5 - CD11c + cells (DN2) representing pre-plasma cells (PC). DN2 cells predominated in African-American patients with active disease and nephritis, anti-Smith and anti-RNA autoantibodies. They expressed a T-bet transcriptional network; increased Toll-like receptor-7 (TLR7); lacked the negative TLR regulator TRAF5; and were hyper-responsive to TLR7. DN2 cells shared with activated naive cells (aNAV), phenotypic and functional features, and similar transcriptomes. Their PC differentiation and autoantibody production was driven by TLR7 in an interleukin-21 (IL-21)-mediated fashion. An in vivo developmental link between aNAV, DN2 cells, and PC was demonstrated by clonal sharing. This study defines a distinct differentiation fate of autoreactive naive B cells into PC precursors with hyper-responsiveness to innate stimuli, as well as establishes prominence of extra-follicular B cell activation in SLE, and identifies therapeutic targets., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Detailed molecular analyses of cells from rheumatoid arthritis (RA) synovium hold promise in identifying cellular phenotypes that drive tissue pathology and joint damage. The Accelerating Medicines Partnership RA/SLE Network aims to deconstruct autoimmune pathology by examining cells within target tissues through multiple high-dimensional assays. Robust standardized protocols need to be developed before cellular phenotypes at a single cell level can be effectively compared across patient samples., Methods: Multiple clinical sites collected cryopreserved synovial tissue fragments from arthroplasty and synovial biopsy in a 10% DMSO solution. Mechanical and enzymatic dissociation parameters were optimized for viable cell extraction and surface protein preservation for cell sorting and mass cytometry, as well as for reproducibility in RNA sequencing (RNA-seq). Cryopreserved synovial samples were collectively analyzed at a central processing site by a custom-designed and validated 35-marker mass cytometry panel. In parallel, each sample was flow sorted into fibroblast, T-cell, B-cell, and macrophage suspensions for bulk population RNA-seq and plate-based single-cell CEL-Seq2 RNA-seq., Results: Upon dissociation, cryopreserved synovial tissue fragments yielded a high frequency of viable cells, comparable to samples undergoing immediate processing. Optimization of synovial tissue dissociation across six clinical collection sites with ~ 30 arthroplasty and ~ 20 biopsy samples yielded a consensus digestion protocol using 100 μg/ml of Liberase™ TL enzyme preparation. This protocol yielded immune and stromal cell lineages with preserved surface markers and minimized variability across replicate RNA-seq transcriptomes. Mass cytometry analysis of cells from cryopreserved synovium distinguished diverse fibroblast phenotypes, distinct populations of memory B cells and antibody-secreting cells, and multiple CD4 + and CD8 + T-cell activation states. Bulk RNA-seq of sorted cell populations demonstrated robust separation of synovial lymphocytes, fibroblasts, and macrophages. Single-cell RNA-seq produced transcriptomes of over 1000 genes/cell, including transcripts encoding characteristic lineage markers identified., Conclusions: We have established a robust protocol to acquire viable cells from cryopreserved synovial tissue with intact transcriptomes and cell surface phenotypes. A centralized pipeline to generate multiple high-dimensional analyses of synovial tissue samples collected across a collaborative network was developed. Integrated analysis of such datasets from large patient cohorts may help define molecular heterogeneity within RA pathology and identify new therapeutic targets and biomarkers.

B cells play a central role in systemic lupus erythematosus (SLE) pathophysiology but dysregulated pathways leading to a break in B cell tolerance remain unclear. Since Toll-like receptor 9 (TLR9) favors the elimination of autoreactive B cells in the periphery, we assessed TLR9 function in SLE by analyzing the responses of B cells and plasmacytoid dendritic cells (pDCs) isolated from healthy donors and patients after stimulation with CpG, a TLR9 agonist. We found that SLE B cells from patients without hydroxychloroquine treatment displayed defective in vitro TLR9 responses, as illustrated by the impaired upregulation of B cell activation molecules and the diminished production of various cytokines including antiinflammatory IL-10. In agreement with CD19 controlling TLR9 responses in B cells, decreased expression of the CD19/CD21 complex on SLE B cells was detected as early as the transitional B cell stage. In contrast, TLR7 function was preserved in SLE B cells, whereas pDCs from SLE patients properly responded to TLR9 stimulation, thereby revealing that impaired TLR9 function in SLE was restricted to B cells. We conclude that abnormal CD19 expression and TLR9 tolerogenic function in SLE B cells may contribute to the break of B cell tolerance in these patients.

Objective: Despite considerable advances in the understanding of systemic lupus erythematosus (SLE), there is still an urgent need for new and more targeted treatment approaches. We previously demonstrated that small-molecule blockade of G protein βγ subunit (Gβγ) signaling inhibits acute inflammation through inhibition of chemokine receptor signal transduction. We undertook this study to determine whether inhibition of Gβγ signaling ameliorates disease in a mouse model of SLE., Methods: Lupus-prone (NZB × NZW)F1 female mice were prophylactically or therapeutically treated with the small-molecule Gβγ inhibitor gallein. Tissue samples were analyzed by flow cytometry and immunohistochemistry. The development and extent of nephritis were assessed by monitoring proteinuria and by immunohistochemical analysis. Serum immunoglobulin levels were measured by enzyme-linked immunosorbent assay, and total IgG and anti-double-stranded DNA (anti-dsDNA) antibody-secreting cells were measured by enzyme-linked immunospot assay., Results: Gallein inhibited accumulation of T cells and germinal center (GC) B cells in the spleen. Both prophylactic and therapeutic treatment reduced GC size, decreased antibody-secreting cell production in the spleen, and markedly decreased accumulation of autoreactive anti-dsDNA antibody-secreting cells in kidneys. Gallein also reduced immune complex deposition in kidneys. Finally, gallein treatment dramatically inhibited kidney inflammation, prevented glomerular damage, and decreased proteinuria. Mechanistically, gallein inhibited immune cell migration and signaling in response to chemokines in vitro, which suggests that its mechanisms of action in vivo are inhibition of migration of immune cells to sites of inflammation and inhibition of immune cell maturation., Conclusion: Overall, these data demonstrate the potential use of gallein or novel inhibitors of Gβγ signaling in SLE treatment., (© 2016, American College of Rheumatology.)

Objective: Rheumatoid arthritis (RA) is a systemic autoimmune disease that often leads to joint damage. The mechanisms of bone damage in RA are complex, involving activation of bone-resorbing osteoclasts (OCs) by synoviocytes and Th17 cells. This study was undertaken to investigate whether B cells play a direct role in osteoclastogenesis through the production of RANKL, the essential cytokine for OC development., Methods: RANKL production by total B cells or sorted B cell subpopulations in the peripheral blood and synovial tissue from healthy donors or anti-cyclic citrullinated peptide-positive patients with RA was examined by flow cytometry, real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and immunohistochemical analysis. To define direct effects on osteoclastogenesis, B cells were cocultured with CD14+ monocytes, and OCs were enumerated by tartrate-resistant acid phosphatase staining., Results: Healthy donor peripheral blood B cells were capable of expressing RANKL upon stimulation, with switched memory B cells (CD27+IgD-) having the highest propensity for RANKL production. Notably, switched memory B cells in the peripheral blood from RA patients expressed significantly more RANKL compared to healthy controls. In RA synovial fluid and tissue, memory B cells were enriched and spontaneously expressed RANKL, with some of these cells visualized adjacent to RANK+ OC precursors. Critically, B cells supported OC differentiation in vitro in a RANKL-dependent manner, and the number of OCs was higher in cultures with RA B cells than in those derived from healthy controls., Conclusion: These findings reveal the critical importance of B cells in bone homeostasis and their likely contribution to joint destruction in RA., (© 2016, American College of Rheumatology.)

Although B cell depletion therapy (BCDT) is effective in a subset of rheumatoid arthritis (RA) patients, both mechanisms and biomarkers of response are poorly defined. Here we characterized abnormalities in B cell populations in RA and the impact of BCDT in order to elucidate B cell roles in the disease and response biomarkers. In active RA patients both CD27+IgD- switched memory (SM) and CD27-IgD- double negative memory (DN) peripheral blood B cells contained significantly higher fractions of CD95+ and CD21- activated cells compared to healthy controls. After BCD the predominant B cell populations were memory, and residual memory B cells displayed a high fraction of CD21- and CD95+ compared to pre-depletion indicating some resistance of these activated populations to anti-CD20. The residual memory populations also expressed more Ki-67 compared to pre-treatment, suggesting homeostatic proliferation in the B cell depleted state. Biomarkers of clinical response included lower CD95+ activated memory B cells at depletion time points and a higher ratio of transitional B cells to memory at reconstitution. B cell function in terms of cytokine secretion was dependent on B cell subset and changed with BCD. Thus, SM B cells produced pro-inflammatory (TNF) over regulatory (IL10) cytokines as compared to naïve/transitional. Notably, B cell TNF production decreased after BCDT and reconstitution compared to untreated RA. Our results support the hypothesis that the clinical and immunological outcome of BCDT depends on the relative balance of protective and pathogenic B cell subsets established after B cell depletion and repopulation.

NOTCH-dependent signaling pathways are critical for normal bone remodeling; however, it is unclear if dysfunctional NOTCH activation contributes to inflammation-mediated bone loss, as observed in rheumatoid arthritis (RA) patients. We performed RNA sequencing and pathway analyses in mesenchymal stem cells (MSCs) isolated from transgenic TNF-expressing mice, a model of RA, to identify pathways responsible for decreased osteoblast differentiation. 53 pathways were dysregulated in MSCs from RA mice, among which expression of genes encoding NOTCH pathway members and members of the noncanonical NF-κB pathway were markedly elevated. Administration of NOTCH inhibitors to RA mice prevented bone loss and osteoblast inhibition, and CFU-fibroblasts from RA mice treated with NOTCH inhibitors formed more new bone in recipient mice with tibial defects. Overexpression of the noncanonical NF-κB subunit p52 and RELB in a murine pluripotent stem cell line increased NOTCH intracellular domain-dependent (NICD-dependent) activation of an RBPjκ reporter and levels of the transcription factor HES1. TNF promoted p52/RELB binding to NICD, which enhanced binding at the RBPjκ site within the Hes1 promoter. Furthermore, MSC-enriched cells from RA patients exhibited elevated levels of HES1, p52, and RELB. Together, these data indicate that persistent NOTCH activation in MSCs contributes to decreased osteoblast differentiation associated with RA and suggest that NOTCH inhibitors could prevent inflammation-mediated bone loss.

Objective: To investigate the hypothesis that proteasome inhibition may have potential in the treatment of SLE, by targeting plasmacytoid dendritic cells (PDCs) and plasma cells, both of which are critical in disease pathogenesis., Methods: Lupus-prone mice were treated with the nonselective proteasome inhibitors carfilzomib and bortezomib, the immunoproteasome inhibitor ONX 0914, or vehicle control. Tissue was harvested and analyzed by flow cytometry using standard markers. Nephritis was monitored by evaluation for proteinuria and by histologic analysis of kidneys. Serum anti-double-stranded DNA (anti-dsDNA) levels were measured by enzyme-linked immunosorbent assay (ELISA), and total IgG and dsDNA antibody-secreting cells (ASCs) by enzyme-linked immunospot assay. Human peripheral blood mononuclear cells or mouse bone marrow cells were incubated with Toll-like receptor (TLR) agonists and proteasome inhibitors, and interferon-α (IFNα) levels were measured by ELISA and flow cytometry., Results: Early treatment of lupus-prone mice with the dual-targeting proteasome inhibitors carfilzomib or bortezomib or the immunoproteasome-specific inhibitor ONX 0914 prevented disease progression, and treatment of mice with established disease dramatically abrogated nephritis. Treatment had profound effects on plasma cells, with greater reductions in autoreactive than in total IgG ASCs, an effect that became more pronounced with prolonged treatment and was reflected in decreasing serum autoantibody levels. Notably, proteasome inhibition efficiently suppressed production of IFNα by TLR-activated PDCs in vitro and in vivo, an effect mediated by inhibition of both PDC survival and PDC function., Conclusion: Inhibition of the immunoproteasome is equally efficacious as dual targeting agents in preventing lupus disease progression by targeting 2 critical pathways in disease pathogenesis, type I IFN activation and autoantibody production by plasma cells., (Copyright © 2012 by the American College of Rheumatology.)

Systemic lupus erythematosus (SLE) is a complex disease characterized by numerous autoantibodies and clinical involvement in multiple organ systems. The immunological events triggering the onset and progression of clinical manifestations are also complex and multi-step, including breach of tolerance in the adaptive immune system, amplification of autoimmunity through innate and adaptive immune system dysregulation, and end-organ damage. Studies of murine genetic manipulations and human risk variants have provided important clues to the cellular and molecular pathogenesis of SLE, operating at multiple of these steps. The breakdown of B-cell tolerance is probably a defining and early event in the disease process and may occur by multiple pathways, including alterations in factors that affect B-cell activation thresholds, B-cell longevity, and apoptotic cell processing. Examples of amplification of autoimmunity on the adaptive immune system side include disturbances in B-cell/T-cell collaboration. B cells can also amplify innate immune cell activation via antibody-dependent and antibody-independent mechanisms. Indeed, one of the key amplification loops in SLE is the activation of plasmacytoid dendritic cells via autoantibodies and RNA-containing and DNA-containing immune complexes, which act as Toll-like receptor ligands, stimulating the secretion of large quantities of IFNα. A more recent link between the innate and adaptive immune system in SLE includes the neutrophil, which can be primed by interferon and autoantibodies to release neutrophil extracellular traps as an additional source of immunogenic DNA, histones, and neutrophil proteins. The innate immune system activation then feeds back, driving autoreactive B-cell and T-cell survival and maturation. This self-perpetuating disease cycle creates the opportunity for targeted treatment inventions at multiple steps.

Introduction: As a group, rheumatoid arthritis (RA) patients exhibit increased risk of infection, and those treated with anti-tumor necrosis factor (TNF) therapy are at further risk. This increased susceptibility may result from a compromised humoral immune response. Therefore, we asked if short-term effector (d5-d10) and memory (1 month or later) B cell responses to antigen were compromised in RA patients treated with anti-TNF therapy., Methods: Peripheral blood samples were obtained from RA patients, including a subset treated with anti-TNF, and from healthy controls to examine influenza-specific responses following seasonal influenza vaccination. Serum antibody was measured by hemagglutination inhibition assay. The frequency of influenza vaccine-specific antibody secreting cells and memory B cells was measured by EliSpot. Plasmablast (CD19+IgD-CD27hiCD38hi) induction was measured by flow cytometry., Results: Compared with healthy controls, RA patients treated with anti-TNF exhibited significantly decreased influenza-specific serum antibody and memory B cell responses throughout multiple years of the study. The short-term influenza-specific effector B cell response was also significantly decreased in RA patients treated with anti-TNF as compared with healthy controls, and correlated with decreased influenza-specific memory B cells and serum antibody present at one month following vaccination., Conclusions: RA patients treated with anti-TNF exhibit a compromised immune response to influenza vaccine, consisting of impaired effector and consequently memory B cell and antibody responses. The results suggest that the increased incidence and severity of infection observed in this patient population could be a consequence of diminished antigen-responsiveness. Therefore, this patient population would likely benefit from repeat vaccination and from vaccines with enhanced immunogenicity.

Objective: Although B cells are implicated in the pathogenesis of systemic lupus erythematosus, the role of B cell depletion (BCD) as a treatment is controversial, given the variable benefit in human disease. This study was undertaken to test the effects of BCD therapy in a murine lupus model to better understand the mechanisms, heterogeneity, and effects on disease outcomes., Methods: (NZB x NZW)F(1) female mice with varying degrees of disease severity were treated with an anti-mouse CD20 (anti-mCD20) antibody (IgG2a), BR3-Fc fusion protein (for BAFF blockade), or control anti-human CD20 monoclonal antibody (approximately 10 mg/kg each). Tissue samples were harvested and analyzed by flow cytometry. The development and extent of nephritis were assessed by monitoring proteinuria (using a urine dipstick) and by immunohistochemical analysis of the kidneys. Serum immunoglobulin levels were measured by enzyme-linked immunosorbent assay., Results: After a single injection of anti-mCD20, BCD was more efficient in the peripheral blood, lymph nodes, and spleen compared with the bone marrow and peritoneum of normal mice as well as younger mice with lupus. Since depletion of the marginal zone and peritoneal B cells was incomplete and variable, particularly in older mice with established nephritis, a strategy of sequential weekly dosing was subsequently used, which improved the extent of depletion. BAFF blockade further enhanced depletion in the spleen and lymph nodes. Early BCD therapy delayed disease onset, whereas BCD therapy in mice with advanced disease reduced the progression of nephritis. These effects were long-lasting, even after B cell reconstitution occurred, and were associated with a reduction in T cell activation but no significant change in autoantibody production., Conclusion: The lasting benefit of a short course of BCD therapy in lupus-prone mice with an intact immune system and established disease highlights the validity of this treatment approach.

Recently, two large randomized controlled trials of distinct biologic therapies in systemic lupus erythematosus, B-cell depletion with rituximab and co-stimulatory blockade with CTLA4Ig (abatacept), failed to meet primary endpoints. Given the great need for new treatments in lupus, these results were met with disappointment and have left the rheumatology and immunology community searching for an explanation. Are these experimental agents ineffective in lupus or are there trial design issues or other considerations? In this commentary, we discuss our perspective on these results within the context of current understanding of the pathophysiology of lupus and the mechanism of action of biologic therapies.

Objective: Recent data suggest that the reconstituting peripheral B cell compartment after B cell depletion therapy may be functionally immature, with a preponderance of transitional B cells and a paucity of memory B cells. This study was undertaken to determine the magnitude, duration, and cause of these defects in rituximab-treated systemic lupus erythematosus (SLE) patients., Methods: Fifteen patients with SLE previously treated with rituximab as part of a phase I/II dose-escalation study were evaluated during a long-term followup (mean followup period 41 months). B cells from peripheral blood and tonsils were assessed using multicolor flow cytometry, and their developmental pathway was classified based on the expression of defined surface markers., Results: Reconstitution of peripheral blood CD27+ memory B cells was delayed for several years after B cell depletion therapy in a subset of patients with prolonged clinical responses and autoantibody normalization. This delay correlated with the degree of expansion of B cells of a transitional phenotype during the B cell reconstitution phase (P = 0.005) and the absence of baseline autoantibodies directed against extractable nuclear antigens (RNP, Sm, Ro antigen, La antigen). Despite the paucity of peripheral blood memory cells and the prolonged expansion of functionally immature transitional B cells, tonsil biopsy tissues revealed active germinal center (GC) reactions, but with decreased Fc receptor homolog 4-positive memory B cells., Conclusion: These results suggest heterogeneity in the B cell depletion and reconstitution process that impacts clinical and immunologic outcomes in SLE. The presence of GC reactions, but with altered memory B cell subpopulations in tonsils, suggests that peripheral blood memory cell reconstitution lags behind a slow secondary lymphoid tissue recovery, with important implications for immunologic competence and tolerance.

Systemic lupus erythematosus (SLE) is a complex disease characterized by the production of autoantibodies and clinical involvement in multiple organ systems. The immunological events triggering the onset of clinical manifestations have not yet been fully defined, but a central role for B cells in the pathogenesis of this disease has been brought to the fore in the last several years by work performed at multiple laboratories in both mice and humans. B cell defects that have been defined include abnormal expression or function of key signaling molecules, dysregulation of cytokines with key B cell effects, and perturbations in B cell developmental subsets. Many of these defects may contribute to or be reflective of abnormalities in B cell tolerance. Both antibodydependent and antibody-independent mechanisms of B cells are important in SLE. Thus, autoantibodies contribute to autoimmunity by multiple mechanisms, including immunecomplex mediated type III hypersensitivity reactions and type II antibody-dependent cytotoxicity, and by instructing innate immune cells to produce pathogenic cytokines, including IFNalpha, TNF, and IL-1. Autoantibody-independent B cell functions have been postulated to include antigen-presentation, T-cell activation and polarization, and dendritic cell (DC) modulation. Several of these functions are mediated by the ability of B cells to produce immuno-regulatory cytokines, chemokines, and lymphangiogenic growth factors and by their critical contribution to lymphoid tissue development and organization, including the development of ectopic tertiary lymphoid tissue. Given the large body of evidence implicating abnormalities in the B cell compartment in SLE,there has been a recent therapeutic focus on developing interventions that target the B cell compartment by multiple mechanisms.

Breach of B cell tolerance is central to the pathogenesis of systemic lupus erythematosus (SLE). However, how B cell tolerance is subverted in human SLE is poorly understood due to difficulties in identifying relevant autoreactive B cells and in obtaining lymphoid tissue. We have circumvented these limitations by using tonsil biopsies to study autoreactive B cells (9G4 B cells), whose regulation is abnormal in SLE. Here we show that 9G4 B cells are physiologically excluded during the early stages of the GC reaction before acquiring a centroblast phenotype. Furthermore, we provide evidence to indicate that an anergic response to B cell receptor stimulation may be responsible for such behavior. In contrast, in SLE, 9G4 B cells progressed unimpeded through this checkpoint, successfully participated in GC reactions, and expanded within the post-GC IgG memory and plasma cell compartments. The faulty regulation of 9G4 B cells was not shared by RA patients. To our knowledge, this work represents the first comparative analysis of the fate of a specific autoreactive human B cell population. The results identify a defective tolerance checkpoint that appears to be specific for human SLE.

Objective: B lymphocyte depletion has recently emerged as a promising approach to the treatment of systemic lupus erythematosus (SLE). As part of a phase I/II dose-ranging trial of rituximab in the treatment of SLE, we evaluated the fate of discrete B cell subsets in the setting of selective depletion by anti-CD20 monoclonal antibody and during the B cell recovery phase., Methods: B cell depletion and phenotype were examined by flow cytometry of peripheral blood mononuclear cells for CD19, CD20, CD27, IgD, and CD38 expression. Changes in autoreactive B lymphocytes and plasma cells were assessed by determination of serum autoantibody levels (anti-double-stranded DNA and VH4.34) and by direct monitoring of a unique autoreactive B cell population bearing surface antibodies whose heavy chain is encoded by the VH4.34 gene segment., Results: Compared with normal controls, SLE patients displayed several abnormalities in peripheral B cell homeostasis at baseline, including naive lymphopenia, expansion of a CD27-,IgD- (double negative) population, and expansion of circulating plasmablasts. Remarkably, these abnormalities resolved after effective B cell depletion with rituximab and immune reconstitution. The frequency of autoreactive VH4.34 memory B cells also decreased 1 year posttreatment, despite the presence of low levels of residual memory B cells at the point of maximal B cell depletion and persistently elevated serum autoantibody titers in most patients., Conclusion: This study is the first to show evidence that in SLE, specific B cell depletion therapy with rituximab dramatically improves abnormalities in B cell homeostasis and tolerance that are characteristic of this disease. The persistence of elevated autoantibody titers may reflect the presence of low levels of residual autoreactive memory B cells and/or long-lived autoreactive plasma cells.

Most highly estrogen-responsive genes possess multiple estrogen-responsive elements (EREs) that act synergistically to activate expression. Synergism between EREs appears to depend on structural features of the EREs and the promoter. To examine the activation process, we cloned single or multiple tandem copies of the consensus ERE into reporter plasmids. These plasmids contained either a chloramphenicol acetyl transferase reporter gene driven by a minimal promoter or a luciferase reporter gene driven by the Simian virus 40 (SV40) promoter. Using MCF-7 human breast cancer cells, we demonstrate that synergism among EREs depends on the number of EREs, their spacing, and the distance of the EREs from the promoter. The induction capacity of EREs falls off slowly with distance from the promoter. Remarkably, multiple EREs can induce effectively and synergize even when they are located more than 2000 nucleotides from the promoter. For EREs located immediately upstream of the promoter, both the distance separating the EREs and the distance to the promoter have to be optimal for synergy. Altering either distance changes the response from synergistic to additive. For distant EREs, presumed to interact by a looping mechanism at the promoter, the length of DNA between the EREs and the promoter is not critical. Synergy among closely spaced EREs that are far from the promoter only requires an optimal distance separating the ERE centers of symmetry. Interestingly, very widely separated EREs can also synergize, presumably also because of their ability to interact by looping. The estrogen response from single or multiple tandem copies of ERE half-palindromes near the SV40 promoter was also tested. The negligible induction capacity of a single half-site was not significantly increased in multiple sites. The biological role of half-EREs is not apparent in the system employed here.