Systemic lupus erythematosus (SLE) is an autoimmune disease with heterogeneous clinical manifestations characterized by the generation of pathogenic autoantibodies directed against chromatin and a variety of other nuclear antigens. Although the pathogenesis of SLE is not yet fully understood, a growing body of experimental evidence indicates that B lymphocytes play a central role. In SLE, B cells may disturb immune homeostasis by multiple mechanisms in addition to the production of pathogenic autoantibodies, including autoantigen presentation, cytokine production, and modulation of the T cell repertoire and T cell memory (1–4).
Overall, the implication is that loss of B cell tolerance is likely critical to SLE disease pathogenesis. The precise interplay between genetic defects and environmental influences that must underlie this loss of tolerance and subsequent disease progression remains to be fully elucidated. Investigation of the genetics of lupus in mice and humans suggests the importance of defects in apoptosis, immune complex clearance, and lymphoid signaling (5–7). Defects in lymphoid signaling may include defects that lower the activation threshold of B cells and lead to B cell hyperactivity and immune dysregulation. In human SLE the evidence for B cell hyperactivity is multifold and includes the presence of increased numbers of spontaneous immunoglobulin-secreting peripheral B cells, increased calcium flux upon signaling through the B cell receptor, and expression of high levels of costimulatory molecules CD80, CD86, and CD40 ligand on B cells (8, 9).
Additionally, recent evidence suggests a role in SLE for high serum levels of B lymphocyte stimulator (BLyS), a member of the tumor necrosis factor family of cytokines that promotes B cell maturation and survival and plasma cell differentiation (10, 11). BLyS, along with other cytokines, intrinsic B cell defects, and the abnormal influence of other immune cells, may contribute to the defects in peripheral blood B cell subpopulations that have been observed in SLE. These include a naive B cell lymphopenia, circulating germinal center (GC) founder B cells, and expansion of circulating plasmablasts (12–14).
Given the critical role of B cells in the pathogenesis of SLE, we hypothesized that the targeted elimination of B cells has the potential to induce long-lasting remissions and reestablish B cell tolerance. Rituximab is a chimeric mouse–human monoclonal antibody against the B cell–specific antigen CD20, which depletes B lymphocytes in vivo from the pre-B stage in the bone marrow, when CD20 is first expressed, to the mature B cell stage. Because CD20 is not expressed on early bone marrow B cell precursors and plasma cells, these cells are not susceptible to depletion with rituximab. Rituximab represents an effective treatment of B cell lymphomas and has emerged as a promising potential treatment of several autoimmune diseases in which B cells may play an important role, including lupus (15–19). We recently reported results of a phase I/II dose-escalation trial in which rituximab-induced B cell depletion significantly improved SLE clinical disease activity (18, 20).
As a first examination of the immunologic effects of rituximab in SLE, we evaluated B cell subsets and autoantibodies in patients at baseline, in the setting of selective depletion by anti-CD20 monoclonal antibodies (mAb), and during the B cell recovery phase. This is the first mechanistic study of the effects of B cell depletion on B cell abnormalities in human SLE. Our results indicate that targeted B cell depletion effectively normalizes many of the significant disturbances in peripheral B lymphocyte homeostasis that are characteristic of active SLE.
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- PATIENTS AND METHODS
The results of our study demonstrate that CD20-targeted B cell depletion in the treatment of SLE effectively normalizes the significant disturbances in peripheral B lymphocyte homeostasis that are characteristic of active disease, including naive lymphopenia, expansion of a population of IgD/CD27 double-negative cells, the presence of plasma cell precursors, and expansion of autoreactive memory B cell populations (14). Consistent with this normalization of B cell homeostasis, we recently reported that the subset of patients with effective B cell depletion had a significant clinical improvement as measured by the SLAM (18), despite the fact that the majority of patients had persistently elevated serum autoantibody levels. These results provide support for the notion, initially advanced in murine lupus, that B lymphocytes play a central role in human SLE independent of autoantibody production, and highlight the benefit of further exploring B cell depletion as a treatment of active SLE.
One surprising finding in our study was the remarkable variability in B cell depletion achieved. Multiple factors likely underlie this variability, including differences in rituximab pharmacokinetics and Fc receptor polymorphisms that directly impact effector mechanisms of B cell killing mediated by the anti-CD20 monoclonal antibody (20, 34). Moreover, this was a dose-escalation study, with only 4 subjects receiving high-dose rituximab; thus, the frequency of depletion failure in patients receiving full-dose therapy remains to be determined. However, our results suggest that disease activity and B cell activation may also be associated with impaired B cell depletion. Thus, SLE patients with circulating CD38high B cells (IgD+ GC founder cells and IgD− plasma cell precursors) were more likely to have incomplete B cell depletion. There is precedent for this finding in that B cells from autoimmune mice appear to be more resistant to depletion with monoclonal antibodies than are their nonautoimmune counterparts (30). Thus, lupus B cells might be globally resistant to depletion because of intrinsic abnormalities or anomalous costimulatory signals (delivered by cytokines or through cognate interactions) that could favor B cell survival over apoptosis (35). We also recently reported that B cell activation is associated with down-modulation of surface CD20 expression via movement into lipid rafts and endocytosis, providing a potential mechanistic explanation for resistance to B cell depletion (36).
Alternatively, discrete B cell subsets that are preferentially expanded in active SLE, such as CD38high GC founder cells, might be less susceptible to rituximab, thereby accounting for the ineffective depletion observed in some patients. In turn, incomplete depletion of GC founder cells could reflect either intrinsic resistance of these cells and/or ongoing exuberant GC reactions that may be central to the pathogenesis of SLE, as we recently suggested (37). Therefore, our own work indicates that the expansion of autoreactive VH4.34 IgG memory cells in SLE represents defective censoring of these cells in the GC in a disease-specific manner not shared by other autoimmune conditions, such as rheumatoid arthritis (ref. 37, and Anolik J, et al: unpublished observations). An association between incomplete B cell depletion and overactive GC reactions in SLE is also suggested by the fact that residual switched memory B cells (the presumed product of GC reactions) were still detectable in the peripheral blood of SLE patients who had effective B cell depletion. Whether B cells in the peripheral lymphoid tissue of SLE patients are resistant to full depletion with rituximab needs to be confirmed—a feasible goal for future clinical trials through tonsil biopsy of select patients.
Regardless of the mechanisms responsible for incomplete B cell depletion in our cohort, the detection of even small numbers of residual peripheral blood memory and plasma cells after rituximab therapy raises important questions about the degree of B cell depletion necessary for clinical and/or serologic response or cure. The correlation between clinical response and effective B cell depletion (arbitrarily defined as depletion of B cells to <1% of the total peripheral blood lymphocytes [absolute cell count <5 cells/μl]) suggests that complete B cell depletion is not necessary for clinical response. However, it is possible that more complete depletion or repeated depletion with multiple courses of rituximab will be necessary for a lasting clinical response and full restoration of B cell tolerance in the majority of patients. Thus, complete depletion of pathogenic autoreactive B cells for a prolonged period of time may be necessary (although perhaps not sufficient) to normalize abnormalities in other immune compartments that may be critical to disease pathogenesis, including T cells and dendritic cells (4, 38–41). In turn, this normalization in other cell compartments may be critical to preventing the reemergence of an autoreactive B cell repertoire upon immune reconstitution (42–45).
Ideally, B cell depletion provides the immune system with a second chance for proper regulation of emerging autoreactive B lymphocytes and establishment of a normal B cell repertoire (i.e., restoration of tolerance). However, the precise nature and appropriate measures of B cell tolerance in human SLE remain elusive, although critically important to define. Thus, as highlighted by the variability in serum autoantibody response in our study, measurement of autoantibodies represents only an indirect marker that could also reflect the persistence of plasma cells with a heterogeneous lifespan. We suggest that determining the fate of autoreactive VH4.34 B cells represents an additional and powerful biomarker of tolerance in general and of GC censoring in particular. Therefore, normalization of autoreactive VH4.34 memory B cells in select patients treated with rituximab may reflect restoration of a B cell tolerance checkpoint at the level of the GC, although proof of this hypothesis will require examination of peripheral lymphoid tissue. Moreover, how reproducible this finding is in different SLE patients and the immunologic basis of response variability are important areas for future study.
The observed variability in autoantibody response also raises important questions regarding autoreactive plasma cell biology in SLE. Autoreactive B cells could be successfully and fully eliminated by rituximab and not reemerge upon immune reconstitution, yet anti-dsDNA levels (and VH4.34 antibodies) remain elevated because of the presence of long-lived autoreactive plasma cells that continue to produce autoantibody. Alternatively, precursor B cells (memory B cells in particular, based on our results) may be incompletely depleted and provide a reservoir for the continuous production of autoreactive plasma cells. Distinguishing between these 2 possibilities is quite difficult but has important implications for the pathogenesis and treatment of human SLE and the origin of autoimmune memory. Indeed, controversy has surrounded the question of whether autoimmune memory in SLE is attributable to long-lived autoreactive plasma cells and/or continuous stimulation of autoreactive memory B cells, through either self antigen or polyclonal activation (46, 47). Although the latter process likely plays an important role, there is increasing experimental evidence that a fraction of plasmablasts are long-lived, are important in maintaining humoral antibody memory, and can contribute to autoimmune disease. As reported by Slifka et al (48), in mice noncycling bone marrow–derived plasmablasts can be transferred to antigen-naive mice and survive and secrete antibody for more than a year in the absence of detectable memory B cells (48).
In autoimmune NZB/NZW mice, although the continuous production of high numbers of short-lived plasma cells is important to hypergammaglobulinemia and the autoimmune process, a fraction of the autoreactive plasmablasts are noncycling and long-lived (49, 50). Moreover, these long-lived plasma cells reside not only in the bone marrow, as conventionally thought, but also in additional survival niches (e.g., the kidneys and possibly other inflamed tissues) and may be particularly resistant to treatment interventions (47, 50).
In human SLE, the persistence of some autoantibodies even during quiescent disease or following immunosuppression has been taken to reflect the presence of long-lived plasma cells. We had originally surmised that anti-dsDNA is likely produced by short-lived plasma cells, because the levels of these autoantibodies can be dramatically reduced by treatment with high-dose steroids with or without immunosuppressive drugs, whereas the levels of total IgG and other autoantibodies such as anti-Sm are, in contrast, unchanged. The existence of short-lived plasma cell populations in SLE is supported by our data demonstrating a significant decrease in peripheral plasmablast expansion immediately after rituximab treatment in select patients. Although it is possible that the peripheral blood plasmablasts did not die off but rather homed to the bone marrow, other lymphoid tissue, or inflamed tissue, this would be less consistent with the serum autoantibody normalization in these patients. Regardless, the fact that rituximab treatment significantly decreased plasma cell expansion in this cohort highlights the fact that B cell depletion can alter plasma cell homeostasis in SLE.
In contrast, the persistence of elevated anti-dsDNA and VH4.34 autoantibody titers in the majority of our patients despite normalization of autoreactive memory B cell numbers in some suggests that the lifespan of autoreactive plasmablasts, even those conventionally thought of as short-lived, may vary in different patients. In fact, the lifespan of autoreactive plasmablasts may be heterogeneous even in individual patients. This possibility is supported by the kinetics of the anti-dsDNA decrease in serologic responders, with anti-dsDNA titers decreasing immediately after rituximab treatment but not normalizing for well over 12 months. As described for NZB/NZW mice, the survival of autoreactive plasmablasts in human SLE similarly may be dependent on microenvironment. Whether plasmablast survival niches are provided by the bone marrow or other sites (such as inflamed target tissue) and how steroids or other treatment agents might alter the microenvironment are important unresolved questions.
In conclusion, this study is the first to show evidence that in SLE, B cell depletion therapy with rituximab dramatically improves abnormalities in B cell homeostasis, with a decreased proportion of autoreactive memory B cells after treatment. The persistence of serum autoantibodies in most patients is noteworthy and raises important questions regarding the relative contribution of long-lived autoreactive plasma cells versus memory B cells to the autoimmune process in SLE. It seems reasonable that for maximal clinical efficacy and induction of long-term remissions with rituximab, full reestablishment of B cell tolerance with elimination of autoreactive memory and plasma cell populations will be necessary. Whether this goal can be consistently achieved using repeated cycles of rituximab and/or combination therapy is an important area of future investigation.