Recent studies suggest that CD20-mediated B cell depletion may be effective in reducing CNS inflammation in MS8, 9
. In this report, we investigated the immunological consequences of anti-CD20 B cell depletion in EAE induced by MOG protein and MOG p35–55. In MOG-protein-induced EAE, but not in EAE induced by MOG p35–55, activated MOG-reactive B cells participated as APC and promoted differentiation of naïve MOG-specific T cells into proinflammatory Th1 and Th17 cells in vitro. Anti-CD20-mediated B cell depletion ameliorated EAE induced by MOG-protein and suppressed development of Th1 and Th17 cells in vivo. Anti-CD20 treatment initiated after MOG-specific antibodies were generated, led to subsequent reduction in titers. Investigations in rheumatoid arthritis28, 29
and systemic lupus erythematosus30
indicated that administration of anti-CD20 similarly dampened humoral responses although plasma cells, which do not express CD20, were not eliminated13
. While reduction of myelin-specific antibodies may potentiate the therapeutic effect of B cell depletion in a subgroup of MS patients with CNS antibody deposition3,31
, it should be recognized that the benefit of anti-CD20 B cell depletion observed in a six-month placebo-controlled trial in relapsing-remitting MS was not associated with a reduction in antibodies8
. Further, in EAE antibodies elicited by immunization with mouse MOG protein, although self-reactive, are not considered pathogenic19
. Thus, the clinical benefit of anti-CD20 treatment observed in this EAE model more likely reflects a reduction in pro-inflammatory cellular function of MOG-specific B cells.
In both EAE models, anti-CD20 treatment depleted B cells within the CNS of mice with established EAE. In MOG protein-induced EAE, B cells became activated, and a greater number of B cells infiltrated the CNS (). The capability to deplete B cells within the CNS is of particular therapeutic relevance in light of the discovery of ectopic B cell follicles32
within the meninges in some individuals that developed secondary progressive MS, and that formation of these lymphoid follicle-like structures may be associated with elevated risk for irreversible disability33
. The observation that B cells were efficiently depleted within the meninges, suggests that anti-CD20 could be also an attractive candidate for treatment of a subset of patients with secondary progressive MS.
Exacerbation of MOG peptide-induced EAE by CD20 treatment highlights the complexity of B cell function in CNS autoimmunity. Immunization with MOG p35–55 did not promote B cell activation. In contrast to anti-CD20 depletion in MOG protein-induced EAE, which was associated with clinical benefit and reduction in proinflammatory Th1 and Th17 cells within the CNS, CD20-mediated depletion resulted in clinical worsening of MOG p35–55-induced EAE and increased numbers of CNS infiltrating Th1 and Th17 cells. Besides serving as the source for antibody-secreting plasma cells and as APC for T cell activation, some B cell subsets may have an important role in immune regulation of CNS autoimmune disease22, 27, 34
. Evidence suggests that antigen-naive B cells exert anti-inflammatory properties27, 35
, which may inhibit maturation and pro-inflammatory differentiation of other APC in vivo36
. In this regard, it has been observed that dendritic cells isolated from B cell-deficient mice, produce higher levels of IL-12 and promote pro-inflammatory T cell differentiation37
. In conjunction with our observation that after anti-CD20 B cell depletion remaining myeloid APC secreted more pro-inflammatory TNF and less anti-inflammatory IL-10, these findings collectively indicate B cells can regulate other APC and suggest that this B cell characteristic may be abrogated by nonselective anti-CD20-mediated B cell depletion.
Naïve B cells may play an important role in development and maintenance of Treg in vivo22, 38
. Deficiencies in the Treg compartment have been identified in several autoimmune conditions, including MS39, 40
and one of the goals in MS therapy is to correct this imbalance41, 42
. While some studies suggest that anti-CD20 depletion may be associated with a modest increase of Treg 14, 43
, we observed a reduction in numbers of CD25+
Treg in anti-CD20 treatment of EAE induced by either rMOG or MOG peptide. This finding is further supported by our investigations using B cell-deficient μMT44
mice. Similar to unimmunized anti-CD20 B cell-depleted mice, we demonstrated that B cell-deficient μMT or JHT mice contained lower frequencies of CD25+
Treg (), again indicating that B cells participate in Treg homeostasis. There were no obvious qualitative differences in Treg in wild-type and anti-CD20 B cell-depleted mice. In this regard, we did not detect intracellular IL-10 protein production in CD4+
Treg in either isotype-treated, or B cell-depleted mice.
B cell deficiency is associated with reduced frequency of FoxP3+ regulatory T cells
Anti-CD20 therapy has been examined in other EAE settings45, 46
. B cell depletion prevented exacerbations in a murine model of spontaneous relapsing-remitting EAE in which Tg T cells and B cells both recognize MOG46
. A recent publication by Matsushita and colleagues45
also demonstrated exacerbation of MOG peptide-induced EAE when B cell-depleting treatment began prior to disease induction. Elegantly, the authors attributed worsening of disease to the absence of an IL-10-producing (B10) regulatory B cell subset. When anti-CD20 treatment started 14 days after immunization, severity of MOG p35–55-induced EAE was ameliorated, leading the authors to conclude that although protective at the time of disease induction, at a later stage, B cells or B cell subsets may promote disease progression. The apparent divergence in outcome of B cell depletion in reversal of MOG peptide-induced disease in our study could reflect differences in experimental procedures, such as dose of MOG p35–55 used for EAE induction, or the nature of the anti-CD20 antibody used47
. One striking difference, however, is that they detected a peptide-specific antibody response upon immunization with their MOG p35–55 preparation, which could have reflected the four-fold higher dose of p35–55 used for EAE induction in their study. While those antibodies did not likely contribute in a pathogenic manner, their appearance may be indicative of B cell activation and maturation following immunization with MOG peptide, which was not observed in this report. Also, in our investigation, B cell depletion in hCD20 Tg mice was achieved using a mouse anti-hCD20 monoclonal antibody. More recently, we tested a mouse anti-mouse (m) CD20 monoclonal antibody for prevention of EAE induced by MOG protein or MOG peptide in non-Tg mice. Consistent with our findings using mouse anti-hCD20, anti-mCD20 treatment suppressed development of proinflammatory T cells and clinical EAE induced by MOG protein, while it promoted development of proinflammatory T cells and exacerbated clinical EAE induced by MOG p35–55. Most importantly, our demonstration that B cells regulate secretion of pro-inflammatory cytokines by monocytes is in agreement with the observation by Matsushita, et al.
that certain B cell subsets have regulatory function, while others support pathogenesis of CNS autoimmune disease. Unlike the results of Matsushita and colleagues, our data indicate that the immunological and clinical outcome of B cell depletion is determined by the activation status and antigen-specificity of B cells, rather than the time of treatment initiation.
While the paradoxical clinical outcomes of CD20-mediated B cell depletion in EAE induced by MOG p35–55 and MOG protein correlated with increased and decreased frequencies of proinflammatory T cells, respectively, it should be recognized that reduction in Treg and augmentation of proinflammatory cytokine expression by remaining APC were common features of CD20 B cell depletion in both models. B cells may undertake additional cellular immune functions, which could have been eliminated by anti-CD20 treatment. It was observed that B cells are capable of capturing protein via their antigen-specific BCR and delivering it to lymph node follicular dendritic cells, more professional APC26
. Through this mechanism of antigen transport, B cells can contribute indirectly to proinflammatory T cell polarization. We have demonstrated that activated MOG-specific B cells, but not naive B cells, serve directly as APC and polarize proinflammatory T cells. Therefore, we favor the possibility that there is a balance, and that the benefit from eliminating MOG protein-activated B cells reflects inhibition of their proinflammatory cellular function, while exacerbation of p35–55-induced EAE relates to depletion of unactivated (naive) B cells that participate in regulation. As was previously observed for myeloid APC, which can be divided into proinflammatory “type I” or anti-inflammatory “type II” classes48, 49
, B cells may exhibit proinflammatory “Be1” or anti-inflammatory “Be2” T cell-polarizing phenotypes50
. In the absence of antigen-activated Be1 cells, CD20 B cell depletion may exacerbate autoimmune disease in some settings51
. Recently, we created Tg mice that contain B cells that express membrane MOG-specific BCR, but cannot secrete antibodies (Molnarfi, N. et al., unpublished). These BCR Tg mice will permit us to distinguish between certain cellular functions of Ag-specific B cells and the role of antibodies in pathogenesis of MOG-induced EAE.
In this report, we studied two distinct EAE models. One cannot conclude that EAE induced by either MOG protein or MOG peptide more closely reflects MS. Each model has its virtues and may emphasize different aspects of pathogenesis52
. APC must process MOG protein through the endocytic pathway for MHC class II-restricted presentation of its encephalitogenic determinant to CD4+
T cells, while MOG p35–55 can be loaded onto MHC II molecules directly6
. We have demonstrated that activated MOG-primed B cells are capable of efficiently presenting MOG protein and promoting differentiation of pathogenic MOG-specific T cells. Immunization with MOG protein elicits a stronger antibody response than does priming to MOG peptide. Our results highlight key differences in cellular and humoral B cell responses to MOG protein and MOG peptide, which could be important when choosing an EAE model for preclinical testing of other novel B cell-targeting agents for MS.
In summary, this study supports the use of anti-CD20-mediated depletion of activated B cells in treatment of CNS autoimmune disease and establishes inhibition of B cell-dependent activation of pathogenic Ag-specific T cells as one immunological mechanism that may contribute to its clinical benefit in MS. In addition, the observations in this report may be relevant to B cell depletion therapy in NMO, which is associated with pathogenic AQP4-specific IgG1, a T cell-dependent antibody subclass53, 54
. Our study cautions that non-selective elimination of B cells may prevent unactivated or regulatory B cells from exerting their beneficial anti-inflammatory influence on other immune cells. Selective depletion of antigen-activated B cells may be a valuable strategy to further improve efficacy of B cell targeted therapies in MS and other inflammatory CNS demyelinating diseases.