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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. B cells contribute to rheumatoid arthritis pathogenesis and bone erosion, but the underlying mechanisms are still unclear. Here the authors show, using mouse models and patient tissues, that B cells directly inhibit osteoblast differentiation by producing CCL3 and TNF, thereby providing a potentially new direction for arthritis therapy.

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.

While durable antibody responses from long-lived plasma cell (LLPC) populations are important for protection against pathogens, LLPC may be harmful if they produce antibodies against self-proteins or self-nuclear antigens as occurs in autoimmune diseases such as systemic lupus erythematosus (SLE). Thus, the elimination of autoreactive LLPC may improve the treatment of antibody-driven autoimmune diseases. However, LLPC remain a challenging therapeutic target. Here, we compare the matched bone marrow (BM) and peripheral blood (PBL) plasma cell (PC) compartments of SLE and healthy donors (HD). We show a similar distribution of CD138- and CD138+ PC, including putative LLPC (CD19- CD138+ CD38+), between SLE and HD BM. For both SLE and HD, CD138+ PC are at a higher frequency in BM than PBL. Expression of Ki-67 associates with the PBL compartment where it is found on all PC subsets regardless of CD19 or CD138 expression. Transcriptomic analysis identifies an interferon (IFN) gene signature in transitional B cells in the SLE BM, but surprisingly also in the BM PC derived from SLE. BM PC and B cells phosphorylate STAT1 in response to type I IFN stimulation in vitro , but with decreased fold change compared to those from the PBL. While BM PC bind type I IFN receptor-blocking antibody anifrolumab, it is to a lesser degree than circulating B cells. Anti-nuclear autoantibodies (ANA) are found in the BM supernatant and PBL serum of SLE patients. Both SLE and HD BM-derived PC have increased survival compared to their PBL counterparts when treated with verdinexor. In summary, these findings show evidence of IFN activation in BM PC from SLE.

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.

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.

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. 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. Advanced PLN sinuses exhibited an increased expression ratio (Early 0.5 ± 0.1 vs Advanced 1.4 ± 0.5 counts/counts; ) that significantly correlated with reduced talus bone volumes in the afferent ankle (R 0.54, ). Integration of single-cell and spatial transcriptomics revealed the increased IgG2b plasma cells localized in MARCO peri-follicular medullary sinuses. A concomitant decreased expression (Early 2.5 ± 0.74 vs Advanced 1.0 ± 0.50 counts, ) 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. 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.

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.

Restricted V(D)J recombination during fetal development was postulated to limit antibody repertoire breadth and prevent autoimmunity. However, newborn serum contains abundant autoantibodies, suggesting that B cell tolerance during gestation is not yet fully established. To investigate this apparent paradox, we evaluated the reactivities of more than 450 antibodies cloned from single B cells from human fetal liver, bone marrow, and spleen. We found that incomplete B cell tolerance in early human fetal life favored the accumulation of polyreactive B cells that bound both apoptotic cells and commensal bacteria from healthy adults. Thus, the restricted fetal preimmune repertoire contains potentially beneficial self-reactive innate-like B cell specificities that may facilitate the removal of apoptotic cells during development and shape gut microbiota assembly after birth.

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.

B cells are critical players in the orchestration of properly regulated immune responses, providing protection against infectious agents without inflicting autoinflammatory damage. A balanced B cell compartment is also essential to create protective immunity in response to vaccines. This difficult compromise is achieved through the finely regulated participation of multiple B cell populations with different antibody-dependent and independent functions. Both types of functions allow B cells to powerfully modulate other components of the innate and adaptive immune system. For the most part, however, the necessary division of labor among different B cell populations is poorly understood. B cell dysfunction has been implicated in multiple autoimmune conditions. The physiological importance and complexity of B cell functions has been brought to the fore in recent years by the success of rituximab-based B cell depletion therapy (BCDT) in multiple autoimmune diseases including rheumatoid arthritis (RA) and multiple sclerosis (MS) which are conventionally viewed as T-cell mediated conditions. Given the widespread utilization of BCDT in malignant and autoimmune diseases and the key role of B cells in both protective immunity and pathogenic autoimmunity, a better understanding of B cell functions is of the essence and a focus of the research in our division. We are investigating these issues through a variety of approaches, including the study of the phenotype and function of human B cell populations in health, their perturbation in autoimmune disease states, the effects of targeted biologic therapies, and the study of relevant murine models.