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/ l). 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-doublestranded DNA antibody and complement levels. Six patients developed human antichimeric antibodies (HACAs) at a level >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.

Objective. To examine immunization responses in patients with rheumatoid arthritis (RA) treated with rituximab and to investigate the effects of rituximabinduced CD20؉ B cell depletion on immune responses to tetanus toxoid (T cell-dependent antigen), pneumococcal polysaccharide (T cell-independent antigen), and keyhole limpet hemocyanin (KLH) (neoantigen) and on delayed-type hypersensitivity (DTH).Methods. In a controlled trial, we enrolled 103 patients with active RA receiving a stable dose of methotrexate (MTX). Tetanus toxoid, pneumococcal polysaccharide, and KLH vaccines as well as a Candida albicans skin test were administered to 1 group of patients receiving rituximab plus MTX (called rituximab-treated patients) for 36 weeks and to 1 group of patients receiving MTX alone for 12 weeks. The primary end point was the proportion of patients with a Ն4-fold rise in antitetanus IgG levels. Antitetanus, antipneumococcal, and anti-KLH serum IgG levels were measured prior to and 4 weeks following vaccine administration. The DTH response to C albicans was measured 2-3 days following placement.Results. Responses to tetanus toxoid vaccine (Ն4-fold rise) were similar in both groups (39.1% of rituximab-treated patients and 42.3% of patients treated with MTX alone). The ability to maintain a positive DTH response to the C albicans skin test was comparable in both groups (77.4% of rituximab-treated patients and 70% of patients treated with MTX alone), showing no effect of rituximab treatment. Rituximab-treated patients had decreased responses to pneumococcal polysaccharide vaccine (57% of patients had a 2-fold rise in titer in response to >1 serotype, compared with 82% of patients treated with MTX alone) and to KLH vaccine (47% of patients had detectable anti-KLH IgG, compared with 93% of patients treated with MTX alone).Conclusion. Recall responses to the T celldependent protein antigen tetanus toxoid as well as DTH responses were preserved in rituximab-treated RA patients 24 weeks after treatment. Responses to neoantigen (KLH) and T cell-independent responses to pneumococcal vaccine were decreased, but many patients were able to mount responses. These data suggest that polysaccharide and primary immunizations should be administered prior to rituximab infusions to maximize responses.ClinicalTrials.gov identifier: NCT00282308.

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.

Rituximab is a promising new therapy for SLE. The variability of responses in patients with SLE may be related to HACA formation. The failure to respond to immunisations is surprising, in view of the apparently low risk of infections. Better biological markers are necessary to follow these patients during treatment.

B cells are critical players in the orchestration of properly regulated immune responses, providing protection against infectious agents without inflicting auto-inflammatory 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.

No abstract