Jennifer Barnard

Germinal center exclusion of autoreactive B cells is defective in human systemic lupus erythematosus.

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.

Novel Human Transitional B Cell Populations Revealed by B Cell Depletion Therapy

Transitional cells represent a crucial step in the differentiation and selection of the mature B cell compartment. Human transitional B cells have previously been variably identified based on the high level of expression of CD10, CD24, and CD38 relative to mature B cell populations and are expanded in the peripheral blood following rituximab-induced B cell-depletion at reconstitution. In this study, we take advantage of the gradual acquisition of the ABCB1 transporter during B cell maturation to delineate refined subsets of transitional B cells, including a late transitional B cell subset with a phenotype intermediate between T2 and mature naive. This late transitional subset appears temporally following the T1 and T2 populations in the peripheral compartment after rituximab-induced B cell reconstitution (and is thus termed T3) and is more abundant in normal peripheral blood than T1 and T2 cells. The identity of this subset as a developmental intermediate between early transitional and mature naive B cells was further supported by its ability to differentiate to naive during in vitro culture. Later transitional B cells, including T2 and T3, are found at comparatively increased frequencies in cord blood and spleen but were relatively rare in bone marrow. Additional studies demonstrate that transitional B cells mature across a developmental continuum with gradual up-regulation of mature markers, concomitant loss of immature markers, and increased responsiveness to BCR cross-linking in terms of proliferation, calcium flux, and survival. The characterization of multiple transitional B cell subpopulations provides important insights into human B cell development.

Beneficial effect of novel proteasome inhibitors in murine lupus via dual inhibition of type I interferon and autoantibody-secreting cells

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.

Cutting Edge: Anti-Tumor Necrosis Factor Therapy in Rheumatoid Arthritis Inhibits Memory B Lymphocytes via Effects on Lymphoid Germinal Centers and Follicular Dendritic Cell Networks

Rheumatoid arthritis (RA) is mediated by a proinflammatory cytokine network with TNF at its apex. Accordingly, drugs that block TNF have demonstrated significant efficacy in the treatment of RA. A great deal of experimental evidence also strongly implicates B cells in the pathogenesis of RA. Yet, it remains unclear whether these two important players and the therapies that target them are mechanistically linked. In this study we demonstrate that RA patients on anti-TNF (etanercept) display a paucity of follicular dendritic cell networks and germinal center (GC) structures accompanied by a reduction in CD38+ GC B cells and peripheral blood memory B cell lymphopenia compared with healthy controls and RA patients on methotrexate. This study provides initial evidence in humans to support the notion that anti-TNF treatment disrupts GC reactions at least in part via effects on follicular dendritic cells.

Prolonged effects of short‐term anti‐CD20 B cell depletion therapy in murine systemic lupus erythematosus

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.

Neutrophil-Mediated IFN Activation in the Bone Marrow Alters B Cell Development in Human and Murine Systemic Lupus Erythematosus

Inappropriate activation of type I IFN plays a key role in the pathogenesis of autoimmune disease, including systemic lupus erythematosus (SLE). In this study, we report the presence of IFN activation in SLE bone marrow (BM), as measured by an IFN gene signature, increased IFN regulated chemokines, and direct production of IFN by BM-resident cells, associated with profound changes in B cell development. The majority of SLE patients had an IFN signature in the BM that was more pronounced than the paired peripheral blood and correlated with both higher autoantibodies and disease activity. Pronounced alterations in B cell development were noted in SLE in the presence of an IFN signature with a reduction in the fraction of pro/pre-B cells, suggesting an inhibition in early B cell development and an expansion of B cells at the transitional stage. These B cell changes strongly correlated with an increase in BAFF and APRIL expression in the IFN-high BM. Furthermore, we found that BM neutrophils in SLE were prime producers of IFN-α and B cell factors. In NZM lupus-prone mice, similar changes in B cell development were observed and mediated by IFN, given abrogation in NZM mice lacking type-I IFNR. BM neutrophils were abundant, responsive to, and producers of IFN, in close proximity to B cells. These results indicate that the BM is an important but previously unrecognized target organ in SLE with neutrophil-mediated IFN activation and alterations in B cell ontogeny and selection.

Expansion of Activated Peripheral Blood Memory B Cells in Rheumatoid Arthritis, Impact of B Cell Depletion Therapy, and Biomarkers of Response

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.

Long-Term B Cell Depletion in Murine Lupus Eliminates Autoantibody-Secreting Cells and Is Associated with Alterations in the Kidney Plasma Cell Niche

Autoantibodies to dsDNA, produced by autoreactive plasma cells (PCs), are a hallmark of systemic lupus erythematosus and play a key role in disease pathogenesis. Recent data suggest that autoreactive PCs accumulate not only in lymphoid tissues, but also in the inflamed kidney in lupus nephritis. We hypothesized that the variable efficacy of anti-CD20 (rituximab)–mediated B cell depletion in systemic lupus erythematosus may be related to the absence of an effect on autoreactive PCs in the kidney. In this article, we report that an enrichment of autoreactive dsDNA Ab-secreting cells (ASCs) in the kidney of lupus-prone mice (up to 40% of the ASCs) coincided with a progressive increase in splenic germinal centers and PCs, and an increase in renal expression for PC survival factors (BAFF, a proliferation-inducing ligand, and IL-6) and PC attracting chemokines (CXCL12). Short-term treatment with anti-CD20 (4 wk) neither decreased anti-dsDNA nor IgG ASCs in different anatomical locations. However, long-term treatment (12 wk) significantly reduced both IgG- and dsDNA-specific ASCs. In addition, long-term treatment substantially decreased splenic germinal center and PC generation, and unexpectedly reduced the expression for PC survival factors in the kidney. These results suggest that prolonged B cell depletion may alter the PC survival niche in the kidney, regulating the accumulation and maintenance of autoreactive PCs.

Neutrophils Slow Disease Progression in Murine Lupus via Modulation of Autoreactive Germinal Centers

Neutrophils are well characterized as mediators of peripheral tissue damage in lupus, but it remains unclear whether they influence loss of self-tolerance in the adaptive immune compartment. Lupus neutrophils produce elevated levels of factors known to fuel autoantibody production, including IL-6 and B cell survival factors, but also reactive oxygen intermediates, which can suppress lymphocyte proliferation. To assess whether neutrophils directly influence the progression of autoreactivity in secondary lymphoid organs (SLOs), we characterized the localization and cell–cell contacts of splenic neutrophils at several stages in the progression of disease in the NZB/W murine model of lupus. Neutrophils accumulate in SLO over the course of lupus progression, preferentially localizing near T lymphocytes early in disease and B cells with advanced disease. RNA sequencing reveals that the splenic neutrophil transcriptional program changes significantly over the course of disease, with neutrophil expression of anti-inflammatory mediators peaking during early-stage and midstage disease, and evidence of neutrophil activation with advanced disease. To assess whether neutrophils exert predominantly protective or deleterious effects on loss of B cell self-tolerance in vivo, we depleted neutrophils at different stages of disease. Neutrophil depletion early in lupus resulted in a striking acceleration in the onset of renal disease, SLO germinal center formation, and autoreactive plasma cell production. In contrast, neutrophil depletion with more advanced disease did not alter systemic lupus erythematosus progression. These results demonstrate a surprising temporal and context-dependent role for neutrophils in restraining autoreactive B cell activation in lupus.

THU0263 Kpt-350, A Selective Inhibitor of Nuclear Export (SINE) Compound, Effectively Reduces Interferon-Alpha Activation and Autoreactive Plasma Cells in Murine Lupus

Background There is great interest in developing new treatment approaches for systemic lupus erythematosus (SLE), but the biologic therapies under investigation have yielded disappointing results. Recently the nuclear export protein XPO1 (Exportin 1/CRM1) has surfaced as an attractive target for the treatment of inflammatory disorders like SLE. Selective Inhibitor of Nuclear Export (SINE) compounds are potent, orally-available, and well-tolerated XPO1 (Exportin 1/CRM1) agonists. In vitro and in vivo, SINE compounds exert apoptotic and anti-inflammatory effects by mediating nuclear retention of important XPO1 cargos like the NFκB pathway regulatory protein, IκB (figure). Objectives This study evaluated the efficacy of the SINE compound KPT-350 on disease progression, interferon-α (IFN-α) production by plasmacytoid dendritic cells (pDCs) and autoantibody production by plasma cells (PCs) in a murine model of SLE. Methods Cohorts of nephritic NZB/W F1 lupus-prone female mice, with established disease (elevated anti-dsDNA antibody titer and proteinuria) were treated with low or high dose KPT-350 (5 or 7.5 mg/kg, respectively) or a vehicle control three times per week for 8 weeks (n=8/group). Proteinuria was monitored and kidney histology assessed. Spleen, bone marrow (BM) and kidney cells were harvested and analyzed by flow cytometry. Antibody secreting cells (ASCs) and germinal centers (GCs) were enumerated and measured by ELIspot and immunofluorescent staining. Serum samples and RNA were collected for Luminex assay and qPCR. Effects on pDC production of IFN-α were assessed using in vitro cultures of BM cells stimulated with CpG 2216 10ng/ml for 5 hours. Results Both high and low doses of KPT-350 prevented nephritis progression with a statistically significant reduction in urine protein levels in treated groups (p<0.05 beginning at 6 weeks). Moreover, nephritis was improved histologically (GN score: 3.3±1.2 vehicle vs. 1.0±-0 SINE; interstitial nephritis: 2±-1.1 vehicle vs. 0±-0 SINE; lipoid infiltrate score: 1.3±-0.6 vehicle vs. 0±-0 SINE). In the spleen, levels of GC B cells (CD19+PNA+CD95+) and IgG and anti-DNA ASCs were dramatically reduced. ASCs were also reduced in the BM and became undetectable in the kidney. Importantly, serum anti-DNA antibodies decreased with treatment but total IgG levels remained intact. KPT-350 inhibited the production of IFN-α by pDCs in a strong, dose-dependent fashion (% of vehicle: 1 uM 88%, 10 uM 66%, 100 um 39%, and 1000 uM 0%). Additionally, IFN-α regulated chemokines in the serum of treated mice were decreased (MCP-1: 250±-90 pg/mL vehicle vs. 54±-26 pg/mL SINE, p=0.049), as were the renal mRNA levels of three PC survival factors: BAFF (38% of vehicle, p<0.05), APRIL (19% of vehicle, p<0.05) and IL6 (40% of vehicle). Conclusions SINE compounds influence the generation, survival and function of PCs and other immune cells through a multifactorial mechanism involving pDCs, GCs and ASCs. It is likely that a common process, namely inhibition of the canonical NFκB pathway, underlies KPT-350s effect. Collectively, our findings suggest that, by simultaneously modulating IFN-α activation and NFκB signaling, SINE compounds might slow disease progression in SLE. Disclosure of Interest J. Rangel-Moreno: None declared, W. Wang: None declared, T. Owen: None declared, J. Barnard: None declared, B. Klebanov Employee of: Karyopharm Therapeutics, Inc, Y. Landesman Employee of: Karyopharm Therapeutics, Inc, S. Tamir Employee of: Karyopharm Therapeutics, Inc, S. Shacham Shareholder of: Karyopharm Therapeutics, Inc, Employee of: Karyopharm Therapeutics, Inc, J. Anolik: None declared