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.

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 doseescalation 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. Rituximab is a chimeric mouse/human monoclonal antibody directed against the B cell-specific antigen CD20, which depletes B lymphocytes in vivo from the pre-B cell stage in bone marrow, when CD20 is first expressed, to the mature B cell stage. Due to its efficacy in the depletion of both normal and malignant B cells, rituximab represents an effective treatment for B cell lymphomas and has emerged as a promising treatment for multiple autoimmune diseases, including systemic lupus erythematosus (SLE), as we and other investigators have previously described (1-4).However, the long-term immunologic effects of rituximab, the mechanism(s) whereby B cell depletion Dr.

Objective We postulated that proteasome inhibition (PI) may be useful in the treatment of SLE by targeting plasmacytoid dendritic cells (pDCs) and plasma cells (PCs), both critical to disease pathogenesis. Methods Lupus prone mice were treated with the non-selective PIs carfilzomib and bortezomib, the LMP7-selective immunoproteasome inhibitor ONX 0914, or vehicle control. Tissues were harvested and analyzed by flow cytometry using standard markers. Nephritis was monitored by proteinuria and kidney harvest. Serum anti-dsDNA levels were measured by ELISA and total IgG and dsDNA antibody secreting cells (ASC) by ELIspot. Human PBMCs or mouse bone marrow cells were incubated with TLR agonists and PIs and interferon α measured by ELISA and flow cytometry. Results Early treatment of lupus prone mice with the dual targeting PIs 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 total IgG ASCs, an effect that became more pronounced with prolonged treatment, and was reflected in decreasing serum autoantibodies. Remarkably, proteasome inhibition efficiently suppressed production of interferon α by toll-like receptor activated pDCs in vitro and in vivo, an effect mediated by both an inhibition of pDC survival and function. Conclusions Inhibition of the immunoproteasome is equally efficacious to dual targeting agents in preventing lupus disease progression by targeting two critical pathways in disease pathogenesis, type I interferon activation and autoantibody production by plasma cells.

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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.

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.

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 ؋ 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 (ϳ10 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.Systemic lupus erythematosus (SLE) is a complex autoimmune disease involving multiple organ systems. The immunologic events triggering the onset of clinical manifestations have not been fully defined, but a central role of B cells in the pathogenesis of this disease has been established by studies performed in multiple laboratories, both in mice and in humans (1-3). Given the strong evidence supporting an abnormal B cell compartment in SLE, B cell depletion (BCD) therapy is being investigated as a potential treatment strategy. Several open-label studies, including our own, have demonstrated that BCD provides significant clinical benefit in SLE (4,5). Our studies have shown that clinical improvement after BCD therapy precedes the decline in levels of conventional serum autoantibodies, strongly supporting the notion of antibody-independent pathogenic roles of B cells.

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.