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 and Multiple Sclerosis, often conventionally viewed as T-cell mediated conditions. Given the exploration of BCDT in various autoimmune diseases and the key role of B cells in both protective immunity and pathogenic autoimmunity, it is important to better understand B cell functions in disease. This is particularly highlighted by the recent failure of two large clinical trials of BCDT in human SLE, raising doubts about the therapeutic efficacy of this approach. However, we find that B cell depletion has robust effects in a murine lupus model, resulting in both significant delays in disease onset when used in a prevention regimen and improvement in nephritis when used for therapeutic treatment of advanced disease. This indicates that B cells are critical for both the initiation and maintenance of autoimmunity in this spontaneous SLE model, and even short-term B cell depletion may have lasting benefit.
In light of data regarding the variability in effects of BCD in human SLE (5
), it is notable that lupus prone mice with more active nephritis display some resistance to B cell depletion with anti-CD20. Although this phenomenon has been demonstrated previously (16
), our data extend these findings, particularly given that the prior study relied predominantly on human CD20 transgenic MRL-lpr mice that express lower levels of human CD20 as compared with endogenous CD20, utilization of unusually large quantities of anti-CD20 antibody (1–10 mg; 10–100-fold higher than here or the comparable dosing in humans) of a less efficient isotype (IgG1), and observed very inefficient B cell depletion (<50%). In our hands, resistance of B cells to anti-CD20 appears to be multi-factorial and at least in part related to more rapid drug clearance, suggesting that it could be overcome by more frequent or higher dosing. The mechanistic basis for more rapid clearance of drug may include accelerated clearance through the reticuloendothelial system and/or formation of an antibody response against the anti-mCD20. However, we were unable to detect the latter (data not shown). It is also likely that there are B cell intrinsic factors that contribute to the resistance to depletion in autoimmune mice. Although it remains controversial how anti-CD20 mediates B cell depletion, with evidence for complement-dependent cytotoxicity, antibody dependent cellular cytotoxicity, and induction of apoptosis, Fc receptor dependent mechanisms via the latter two pathways may predominate in vivo (15
). Thus it is plausible that an imbalance of B cell survival/apoptotic pathways, a defect shared among genetically distinct mouse models (27
), plays a significant role in depletion resistance, a hypothesis we are currently exploring. Notably, we find that combined treatment with anti-CD20 and BAFF blockade improves the extent of depletion suggesting that elevated levels of BAFF contribute to enhanced B cell survival. This has been suggested in normal mice (15
) but not previously demonstrated in lupus, a disease where such mechanisms may be particularly relevant given the excess BAFF found in both mice (24
) and man (28
The availability of anti-mCD20 provides the opportunity to monitor tissue depletion of B cells in a systematic fashion that is not possible in human studies. It is interesting that there is a tissue and subset specific hierarchy of sensitivity of B cells to depletion, with peripheral blood B cells most sensitive, tissue B cells (spleen and LN) less so, and peritoneal and BM B cells most resistant- the latter likely explained by the low CD20 expression by the majority of BM B cells. The greater difficulty depleting tissue B cells is consistent with prior publications (15
) and emerging though more limited data in humans (30
). We find that in the spleen residual B cells are predominantly of a MZ and memory phenotype, with plasma cells also resistant due to lack of CD20 expression. The resistance of MZ B cells to depletion has been reported in human CD20 transgenic mice (15
) as well as more recently in autoimmune models using some anti-mCD20 (21
) but not others (29
). This suggests that antibody specific, microenvironment, and mouse strain effects are likely to be important in resistance of B cells to anti-CD20 depletion. The increased resistance of the MZ to depletion in lupus mice may be due in part to excess BAFF and the dependence of MZ B cell survival on this cytokine given that MZ depletion is significantly improved with combination BAFF blockade. We also find residual memory B cells in the spleen although, in contrast to prior studies (15
), GC B cells appear to be quite sensitive to depletion. This suggests strain specific differences in the sensitivity of certain B cell subsets to depletion. Alternatively, the factors maintaining spontaneous GCs in lupus prone mice may be different from GCs in Peyer’s patches or splenic GCs arising after immunization (15
). Of note, the disruption of spontaneous GCs and ectopic GCs may be an important mechanism of action of BCD in the treatment of lupus. The presence of residual memory B cells is notable given our prior findings in human studies of relative resistance of memory B cells to depletion in a subset of SLE patients (32
). In accord with this data, it has recently been demonstrated that the human spleen is a reservoir for long-lived memory B cells that are resistant to B cell depletion (31
). This may be beneficial for maintenance of protective immunity after BCDT but limit the deletion of autoreactive memory. It is interesting that a number of recent reports in mouse models of BCD find inhibition of both primary and secondary immune responses, suggesting that memory B cells are deleted (33
). However, this may depend on the timing of anti-CD20 treatment relative to the establishment of a memory response, as well as the host microenvironment. It is also possible that autoreactive memory B cells are protected in inflammatory niches, either within target tissue, spleen, or peritoneum.
Importantly, we find that B cell depletion prevents the progression of lupus nephritis in both early and late disease despite the variability in extent of depletion. Similar to human SLE (34
), there is a lack of a significant effect of BCD on serum autoantibodies. Our results significantly extend prior observations with the demonstration that autoreactive antibody secreting cells in spleen and bone marrow are not impacted by B cell depletion, at least at early time points. We are currently exploring whether B cell depletion longer term alters the pro-inflammatory milieu that contributes to the maintenance of long-lived autoreactive plasma cells. Different mechanisms of action have been invoked to explain the benefit of BCD in SLE and other autoimmune diseases including the elimination of autoantibodies, decreased T cell activation, the expansion of Treg cells, the disruption of ectopic lymphoid tissue, and the elimination of effector B and other cells from target organs (35
). The lasting benefit seen here of a short course of BCDT in NZB/NZWF1 mice even after B cell reconstitution suggests either profound effects of BCD on other cell populations and/or the emergence of a B regulatory cell population. Regarding the former possibility, we do find a significant impact of B cell depletion on the T cell compartment with decreases in activated and memory T cells. Notably, BCD has been shown to inhibit antigen-specific CD4 T cell expansion in both collagen-induced arthritis and autoimmune diabetes mouse models (39
). Whether this is mediated via direct B cell APC functions or indirectly through B cell cytokine secretion or other B cell functions remains an important area of future study.
It has been known for many years that B cells can produce cytokines with immunosuppressive, polarizing, inflammatory, and tissue-organizing properties, yet the potential biologic relevance of cytokine-producing B cells was largely unappreciated. However, recent findings have renewed interest in this area and have raised the intriguing possibility that cytokine-producing B cells actively modulate both humoral and cellular immune responses (40
). From an autoimmunity standpoint, B cells may either stimulate or inhibit pathogenic responses. A pathogenic role for B cells in SLE is strongly supported by mouse models that are genetically B cell deficient (41
), although these models have some limitations including the presence of immunologic and lymphoid organ structural defects engendered by the genetic absence of B cells and the inability to study effects on established disease. Our data firmly establish the pathogenic role of B cells in lupus disease development and progression, adding to results in human CD20 transgenic lupus prone mice (16
) and B cell depletion with other approaches (42
). On the other hand, evidence is accumulating for regulatory B cells (11
) capable of preventing or suppressing autoimmunity in different mouse models (13
). This protective role may be mediated by inducing T cell anergy during antigen presentation or inducing Treg expansion or activity (11
). These activities are mediated, at least in part, by the B cell production of IL-10 or TGFβ and may control a variety of auto-inflammatory diseases including: inflammatory arthritis, inflammatory bowel disease, autoimmune diabetes, experimental autoimmune encephalitis, and lupus (12
Given the hierarchy of sensitivity of distinct B cell subsets to anti-mCD20, it is important to consider the actual nature and mechanisms of action of Breg cells and the impact of BCD. Mouse Breg activity has been variously assigned to cells with a transitional (in particular, T2-MZ Precursors – T2/MZP), MZ, or B1 phenotype, and lupus resistance has been associated with expansion of MZ cells (13
). Our observation that expansion of transitional B cells correlates with long-term remission in SLE patients treated with BCDT is also consistent with a regulatory nature for certain B cell subsets in humans (32
). Thus, it is interesting that the B cell subsets most resistant to anti-mCD20 include the MZ and B1 cells, leading us to speculate that BCD shifts the balance of protective versus pathogenic B cell functions. Our results are also the first to carefully delineate the kinetics and phenotype of B cell reconstitution in autoimmune mice and impact on clinical and immunologic outcomes. The predominance of an immature transitional phenotype is in keeping with human B cell depletion therapy (50
) and, in the context of persistent disease suppression, highlights the potential regulatory function of these cells. Further delineation of the cytokine secreting ability of these discrete B cell subsets in the context of the autoimmune process and B cell depletion is necessary.
In conclusion, our results indicate that B cells are critical for both the initiation and maintenance of autoimmunity in SLE. B cell depletion is associated with a reduction in T cell memory and activation but a lack of significant effect on autoantibodies and autoreactive B cell memory. The lasting benefit of a short course of BCDT in lupus prone mice suggests a favorable shift in the balance of protective versus pathogenic B cell functions and supports the validity of this approach in the treatment of lupus.