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Autoimmunity, August 2012; 45(5): 400–414 q Informa UK, Ltd. ISSN 0891-6934 print/1607-842X online DOI: 10.3109/08916934.2012.665529

Antibody-independent B cell effector functions in relapsing remitting Multiple Sclerosis: Clues to increased inflammatory and reduced regulatory B cell capacity SARA J. IRELAND, MONICA BLAZEK, CHRISTOPHER T. HARP, BENJAMIN GREENBERG, ELLIOT M. FROHMAN, LAURIE S. DAVIS, & NANCY L. MONSON University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA (Submitted 16 January 2012; accepted 7 February 2012)

Abstract The pathogenic role for B cells in the context of relapsing remitting multiple sclerosis (MS) is incompletely defined. Although classically considered a T cell-mediated disease, B cell-depleting therapies showed efficacy in treating the clinical symptoms of RRMS without decreasing plasma cells or total immunoglobulin (Ig) levels. Here, we discuss the potential implications of antibody-independent B cell effector functions that could contribute to autoimmunity with particular focus on antigen presentation, cytokine secretion, and stimulation of T cell subsets. We highlight differences between memory and naı¨ve B cells from MS patients such as our recent findings of hyper-proliferation from MS memory B cells in response to CD40 engagement. We discuss the implications of IL6 overproduction in contrast to limited IL10 production by B cells from MS patients and comment on the impact of these functions on yet unexplored aspects of B cells in autoimmune disease. Finally, we contextualize B cell effector functions with respect to current immunomodulatory therapies for MS and show that glatiramer acetate (GA) does not directly modulate B cell proliferation or cytokine secretion.

Keywords: B cell cytokines, B cell proliferation, B cell APC function, B cell co-stimulation molecules, B-T interaction

Introduction Relapsing remitting multiple sclerosis (RRMS) is an inflammatory, demyelinating autoimmune syndrome of the central nervous system. While T cells are the critical immune component required to induce disease pathology, more recent evidence highlights B cells as central components of the disease. MS patients treated with B cell-depleting monoclonal antibody therapy show significant improvement in the clinical symptoms of MS, with a reduction in total and new gadolinium enhancing lesions, and relapse incidence [1 –4]. Studies in the mouse model of MS, experimental autoimmune encephalomyelitis (EAE) also demonstrate a role for B cells [5 –8]. How B cells potentiate immune responses and why B celldepletion therapy is effective in a T cell-mediated disease remains unclear. While antibodies are thought to play a role in MS disease pathology,

current B cell-depletion therapies do not target immunoglobulin (Ig) secreting plasma cells and total Ig levels remain unchanged during the time when clinical symptoms were diminished [1 – 4]. Therefore, B cells likely influence MS disease pathology through antibody-independent effector functions. These include antigen presentation, cytokine secretion, and co-stimulation that could impact T cell function (Figure 1). Here we review the capacity for B cell effector functions to modulate immune responses in the context of MS and provide our recent data that highlight intrinsic differences between B cells from MS patients and Healthy Donors (HDs). Memory B cells Like T cells, B cells can be divided into naı¨ve and memory populations [9]. Human memory B cells are readily identifiable as CD19 þ CD27 þ [10]. Mem-

Correspondence: Dr. Nancy L. Monson, Department of Neurology and Neurotherapeutics, UT Southwestern Medical Center, Building J, 3rd floor, Room 132B, Dallas Texas 75390, USA. E-mail: [email protected]

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Figure 1. B cells possess a variety of antibody-independent effector functions that have the capacity to influence T cell responses. B cells can serve as antigen presenting cells (APC) to CD4 þ T cells. B cell APC function is particularly effective when B cells recognize their cognate antigen with the B cell receptor (BCR). Expression of co-stimulatory and inhibitory molecules on the plasma membrane of B cells can stimulate or dampen T cell activation. Cytokines produced by B cells are known to stimulate different T cell subsets, including regulatory (Treg) and inflammatory (TH1 and TH17) cells. In the context of a B-T cell APC couple, B cell cytokines could impact T cell activation and even polarization. However, the direct contributions of these B cell effector functions are currently incompletely defined in response to pathogens and in the context of autoimmunity.

ory B cells have previously encountered cognate antigen and undergone activation, most typically to Tdependent antigens that require T cell help to achieve fulminant activation and maturation. In contrast to their naı¨ve counterparts, memory B cells secrete more pro-inflammatory cytokines and bear somatically hypermutated B cell receptors (BCR)[12,13]. Human memory B cells are more capable of supporting antigen-independent autologous T cell proliferation than naı¨ve B cells [14]. Supernatants from memory B cells, but not naı¨ve B cells, stimulated through CD40 enhanced T cell IFNg secretion to polyclonal stimuli [15]. In fact, the percentage of peripheral blood memory B cells also increases during MS relapse [16]. Furthermore, activated and memory B cells are overrepresented in the CSF of MS patients [17] and have greater capacity to migrate towards elevated chemoattractant factor CXCL13 in CSF from MS patients [17,18]. Thus, memory B cells that are reactive to self-antigens are likely to exert effector functions that promote inflammatory disease pro-

cesses in MS patients through reciprocal B-T cell activation [19] and possibly by priming naı¨ve T cells. B cell Proliferation Although antigen-specific recall and polyclonal proliferative responses by T cells from MS patients have been widely studied [20 – 24], a limited number of investigations have reported on B cell proliferation in MS cohorts. It has been demonstrated that chronically activated B cells are found in the meninges of MS patients where ectopic germinal centers reside [25,26]. Given that CD40/40L interactions are critical for germinal center development and chronic activation of B cells, it is reasonable to suggest that B cells from MS patients may have a hyper-proliferative response to CD40 engagement. To test this, we cultured memory and naı¨ve B cells from HDs and MS patients with high or low dose CD40L for 5 days and quantified B cell proliferation by CFSE dilution (Figure 2). Memory and naı¨ve B cells from HDs proliferated similarly to high (57.3%

402 S. J. Ireland et al.

Figure 2. B cell proliferation in response to high or low dose CD40 engagement. All B cell subsets from HD and MS patients proliferated significantly more on a high dose of CD40L compared to low CD40L. Memory and naive B cells from HD proliferated similarly to both doses of CD40L. In contrast, memory B cells from MS proliferated significantly more than naı¨ve B cells when provoked with low or high doses of CD40L. No differences were observed between HD and MS patients. Student’s unpaired t-test was used to compare proliferation, significance was defined as a p-value at or below 0.05.

vs 59.2%, p ¼ 0.611) and low dose CD40L (9.9% vs 8.5%, p ¼ 0.84). In contrast, memory B cells from MS patients proliferated 1.3 fold more than naı¨ve B cells in response to high dose CD40L (64.9% vs 47.7%, p ¼ 0.003) and 3.4-fold more than naı¨ve B cells at low dose CD40L (34.9% vs 10.3%, p ¼ 0.019). This data suggests that there are unique differences between CD40 stimulated memory and naı¨ve B cells from MS patients as memory B cells appeared to be hyper-responsive to CD40 stimulation. Given increased expression of CD40L on unstimulated peripheral blood T cells from MS patients [27] and mitogen-stimulated T cells from MS patients [28,29], it is likely that the hyper-responsiveness of memory B cells from MS patients to CD40 engagement that we observed in vitro might even be more profound in vivo. A key challenge then becomes halting B cell mediated pathogenesis and restoring regulation of B cell function in MS patients. B cell antigen presentation Studies with murine B cells demonstrated that antigen specific B cells are potent antigen presenting cells (APC)[30 –33]. In fact, B cells are critical APCs in some mouse models of autoimmune diseases [34,35]. Human B cells are also capable of APC function, as they uptake large soluble and particulate antigens

[36 – 38] similar to the murine B cell’s preference for protein antigen [39]. B cells primarily capture cognate antigen through their BCR, which induces BCR mediated cross-linking and internalization [40,41]. Of note, under certain circumstances, B cells can uptake antigen through pinocytosis [42]. Antigens taken up by B cells are targeted to the endocytic compartment where they are processed and can be subsequently presented to CD4 þ T cells in the context of MHC Class II [43]. Initial investigations of human B cells as APC were carried out utilizing Epstein-bar virus (EBV) transformed B cells. Which can process and present particulate exogenous antigen to T cell clones [37]. This methodology proved that B cells can support antigen-specific T cell proliferation to tetanus toxoid (TT) [36]. Others showed that EBV-transformed B cells had the most effective APC function when previously activated T cells were used as responders [44]. In addition, EBV þ B cells from melanoma patients expanded in the same fashion were effectively used as APC to generate melanoma-specific, MHC II restricted CD4 þ T cell clones that proliferated and secreted IFNg in response to melanoma antigen [45]. A potential confounding factor in these early human EBV B cell APC assays is that EBV transformation can induce IL10 production in B cells [46], which may

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Figure 3. CD4 þ T cell proliferation provoked by B cell subsets in the presence of antigen. Total peripheral blood T cells were cultured with autologous memory or naive B cells and antigen for 5 days; proliferation was quantified by CFSE dilution (background proliferation with no antigen was subtracted). A. In response to foreign antigen (tetanus toxoid or mumps virus, plotted together), HD and MS B cells induce similar proliferation by CD4 þ T cells. B. Memory and naive B cells from MS patients induce significantly higher CD4 þ T cell proliferation in cultures containing self antigen (myelin oligodendrocyte glycoprotein 1–125 (rMOG) or myelin basic protein (MBP), plotted together) compared to HDs. There were also a significantly greater number of responders to neuro-antigen in the MS cohorts Statistical analysis on the percentage of proliferation was performed using a student’s unpaired t-test. The number of responders by proliferating was defined as two standard deviations above the mean proliferation in cultures with no antigen and compared using x2 analysis. P-values less than 0.05 were considered significant.

have inhibited T cell proliferation and pro-inflammatory cytokine production in these previous studies. The next approach used to study human B cell APC function was to activate total human B cells in vitro with CD40L and IL4 (CD40-B). This is a means to increase expression of co-stimulatory molecules, factors known to be important in the generation of T cell responses [47 – 52] and chemotactic molecules on B cells important for secondary lymphoid organ homing [53]. Murine antigen-specific B cells activated in a similar manner rival the APC capacity of dendritic cells (DC) [54]. This is likely related to the observation that antigen-experienced B cells expressed increased levels of co-stimulatory molecules (CD80, CD86 and CD40) and MHC Class II molecules [11,55]. Initial studies demonstrated that peripheral healthy donor B cells expanded using the CD40-B system were effective APC in the generation of antigen-specific CD4 þ T cell lines [30,45]. In addition, CD40-B cells from HDs or cancer patients expanded viral-specific memory, autoantigen/tumor and neoantigen specific CD8 þ T cell responses [42]. Human peripheral blood mononuclear cells (PBMC)-stimulated with CD40L and IL4 to activate B cells were highly effective in generating neuro-antigen specific autologous CD4 þ and CD8 þ T cell clonal proliferation and IFNg secretion [47]. In our laboratory, we previously demonstrated that peripheral CD40-B cells from MS patients or HDs stimulated autologous CD4 þ T cell proliferation in the presence of foreign or neuroantigens [56]. Taken together, these studies indicate that in vitro activated CD40-B cells act as APC for

CD4 þ and CD8 þ T cells in antigen recall responses and in primary responses to foreign or autoantigen, including neuro-antigens. More recently, our laboratory investigated APC capacity of peripheral blood memory or naı¨ve B cells from MS patients and HDs that were not stimulated prior to culturing with autologous T cells [57]. We found that memory and naı¨ve B cells from HDs and MS patients are equally able to induce autologous CD4 þ T cell proliferation in response to foreign antigen (TT or mumps, Figure 3A). In contrast, proliferative responses of CD4 þ T cells to the neuroantigens myelin oligodendrocyte glycoprotein (MOG) or myelin basic protein (MBP) were virtually nonexistent in HDs, with only one responder from the naı¨ve B cell compartment (defined as two standard deviations above the mean proliferative response in cultures with no antigen). Strikingly, nearly one-third of all MS patients had memory and/or naı¨ve B cells that induced proliferation of autologous CD4 þ T cells in response to neuro-antigens (Figure 3B). From these results, we conclude that B cells from a subgroup of MS patients harbor the ability to stimulate T cell proliferation to neuro-antigens, which are thought to be key in the perpetuation of MS disease pathogenesis. Peripheral B cells from MS patients are also able to induce autologous CD4 þ T cell IFNg production in response to neuro-antigens [57], suggesting that B cells have a direct impact on Th1 responses. Others have shown that T cells from MS patients were unique in their requirement for B cells to achieve maximal proliferative and IFNg responses to polyclonal

404 S. J. Ireland et al. activators (aCD3)[58]. B cell-derived cytokines (LTa, TNFa and IL12) are critical for optimal T cell proliferation and differentiation into IFNg producing cells [15,58]. However, the requirement for these cytokines in antigen specific B-T interactions is not known. It is also unknown how B cells operating as APCs impact particular CD4 þ T cell subsets, although it is clear that B cells possess all the required elements for proficient antigen presentation. The potential impact of cytokines produced by B cells B cells are capable of producing a wide variety of proinflammatory and regulatory cytokines and growth factors [59 –61] that have long been appreciated for their potential to influence B and T cells. Yet, how and the extent to which B cell-derived cytokines impact immune responses and autoimmunity remains incompletely defined. It is clear that B cells directly stimulate IFNg secretion and proliferation of TH1 CD4 þ T cell in an antigen-dependent fashion [57] and/or through provision of cytokines [62]. Demonstrations of altered cytokine production in B cells from MS patients provoke questions of the contribution of B cell cytokines to autoimmune disease and immune responses to pathogens [59]. Here, we focus on cytokines produced by B cells that have the potential to impact Th17 responses and B cell regulatory function. TH17 responses Little is known about how B cells might impact T cell populations, including TH1, TH17, T central or effector memory or even naı¨ve T cells (Figure 1). TH17 cells from HDs produced inflammatory cytokines including IL17, IL21, and IL22 in response to infectious agents. However, TH17 cells are increasingly thought to play an important pathogenic role in autoimmune disorders including MS, Rheumatoid Arthritis (RA), and Inflammatory Bowel Disease (IBD). Few studies have addressed the connection between B cells and TH17 responses. There has been no formal demonstration of the direct impact of B cells on CD4 þ TH17 cells in humans. However, several recent pieces of data strongly, and surprisingly, implicate B cells as potent modulators of TH17 responses in HDs and patients with autoimmune disorders. For example, in HDs, the frequency of memory B cells in the peripheral blood positively correlates with ex vivo IL17 production by CD4 þ T cells [63]. In contrast, patients with the primary immunodeficiency, X-linked agammaglobulinemia (XLA), who have few B cells, have a reduced frequency of peripheral IL17and IL22-producing T cells when stimulated ex vivo

with the potent cytokine inducers, PMA and ionomycin [63]. In patients with Common Variable Immunodeficiency Disorder (CVID), who have a dramatic reduction in B cell maturation (memory and plasma cells), the frequency of CD4 þ IL17 producing T cells is negatively correlated with the number of immature B cells and B cell HLA-DR (MHC II) expression [63]. These correlative data indicate a potential link between B cell memory APC capacity and the generation or maintenance of Th17 cells. Evidence from infectious disease models more conclusively point to a role for B cells in bolstering TH17 responses. Healthy PBMCs depleted of B cells and incubated with C. albicians, a known stimulator of TH17 responses, showed dramatic reductions in IL17 (, 50%) and IL22 (, 20-30%) production, yet IL17 and IL22 production by PBMCs from XLA patients were unaffected by B cell depletion [64]. Human TH17 responses to C. albicans in vitro require memory T cells and APC [65]. This indicates possible B cell involvement with antigen-specific memory TH17 responses; conversely, it does not preclude the notion that B cells indirectly modulate TH17 cells by influencing other immune components, such as macrophages that are important for TH17 responsiveness to C. albicans. Separate evidence from Mycobacterium tuberculosis (MTB) infected individuals gives further credence to the link between B cells and TH17 cells [66]. Studies of autoimmune disorders revealed that B cell depletion also decreased TH17 responses in mice [8] and in humans [58,67,68]. RA patients treated with B cell-depleting therapy show decreased IL17 and IL22 production, as well as decreased expression of RORgT (TH17 transcription factor) but not of IFNg or TNFa in the synovial tissue [68]. One case study showed similar reductions of TH17 cytokines in the peripheral blood of RA patients [67]. In MS patients, therapeutic B cell-depletion reduced total CD4 þ and CD8 þ T cell proliferation and the proliferating fraction of CD4 þ T cells that produced IFNg or IL17 upon ex vivo polyclonal stimulation [58]. In the same study, TNFa and LTa from supernatants of autologous CD40/BCR activated B cells could partially restore T cell proliferation and IFNg production in B cell-depleted T cell cultures from MS patients [58]. Although these studies provide evidence that the presence of B cells is required for fulminate IL17 responses, the absence of B cells is distinct from direct B cell effects on TH17 cells. There are several ways in which B cell effector functions could directly impact TH17 cells. First, B cells might act in concert with non-T immune cells and contribute to a yet unknown, but important aspect of TH17 responses. Second, B cells could present antigen, secrete cytokines and provide costimulation to incite memory Th17 cell responses.

B cell effector functions in MS Finally, B cells could play a role in polarizing naı¨ve T cells to a TH17 phenotype. In the context of human autoimmune disease, it is not known whether B cells reactivate memory T cells or, perhaps, initiate activation and expansion of clonally ignorant naı¨ve T cells. In humans, TGFb, in combination with IL6 [69 – 71], IL1b [72] and/or IL23 [73] is thought to be critical for TH17 polarization. Human B cells from HDs can produce both TGFb [60] and IL6 [61,74]. In fact, IL4 alone induces IL6 production from human CD19 þ B cells that is enhanced in a dose dependent fashion by the addition of BCR crosslinking agents [75]. HD memory B cells, and to a lesser extent naı¨ve B cells, produce IL23 upon stimulation through the BCR alone or in combination with CD40 ligation [15]. Human B cells from HDs also produce robust amounts of IL1b upon BCR stimulation [59]. B cells can produce TNFa and LTa, which, in addition to IL23 [76], are important factors for potentiating TH17 responses. Although it is true that HD B cells generally make lesser amounts of these polarizing cytokines than their myeloid APC counterparts, the expression of such cytokines by B cells has not been thoroughly investigated in the context of autoimmune disease. IL6 is a pleiotropic cytokine produced by B cells, other APCs, and a variety of non-immune cells. IL6 impacts the survival, proliferation and activity of immune cells and acute inflammatory responses. IL6 is important for B cell maturation and impacts CD4 þ T cells [77]. Moreover, IL6 can cross the blood brain barrier [78]. There is some evidence of increased IL6 levels in the periphery and cerebrospinal fluid of MS patients [79,80]; however, these findings are not consistent in all studies [81]. In contrast, IL6 is known to correlate with increased disease activity in RA[82] and IL6 blockade appears to be of therapeutic benefit in RA patients [83]. These reports, along with the potential impact on B and T cell responses

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prompted us to investigate the potential for B cells to produce IL6 in MS patients. We stimulated the naı¨ve and memory B cell subsets from HDs and MS patients with conditions known to induce cytokine production (see methods), sampled supernatants at either day 3 or 5 of culture and assayed for cytokine levels by ELISA. We were able to detect IL6 in all culture conditions tested for both naı¨ve and memory B cells from MS patients and HDs (Table I). IL6 production by naı¨ve and memory B cells from MS patients was more robust than IL6 production by HDs, and even rivaled that of mitogen-stimulated myeloid APC [84]. As shown in Table I, naı¨ve B cells from MS patients stimulated with high dose CD40L plus IL4 produced the largest quantity of IL6 (1263 pg/mL) that was significantly more than HD naı¨ve B cells ( p ¼ 0.0019). Similarly, naı¨ve B cells from MS patients produced more IL6 than HD in response to CD40L plus IL2 and CpG ( p ¼ 0.0031). Memory B cells from MS patients stimulated with high dose CD40L plus IL-4 produced only 43 pg/mL of IL6, but when BCR stimulation was added, IL6 production was increased 18.2-fold (789.5 pg/mL) and was significantly greater than that of HD memory B cells in the same condition ( p ¼ 0.0007). Stimulation of memory B cells from MS patients with CD40L, IL2 and CpG induced significantly more IL6 than from memory B cells of HDs ( p ¼ 0.016). These data provide evidence that B cells from MS patients produce more IL6 than their HD counterparts to a variety of stimuli. The underlying mechanism of this exaggerated IL6 production is a topic of active investigation in our laboratory. This is potentially linked to both T cell responses in MS and hyperproliferation of B cells. Further studies are necessary to elucidate whether IL6 produced by B cells (or some other aspect of B cell function) impacts TH17 polarization, T cell or B cell function. Of note, anti-IL6 receptor (a-IL6r) therapy is approved for treating RA and Castleman’s disease (a rare B cell malignancy). Anti-IL6r therapy has been

Table I. Memory and naı¨ve B cell cytokine production after 3 or 5 days of culture with polyclonal stimuli from HD and MS patients. IL6 production Day 3 (mean, in pg/mL)

IL10 production Day 5 (mean, in pg/mL)

CD40L CD40L IL4 CD40L IL4 BCR CD40L IL2 CpG CD40L CD40L IL4 CD40L IL4 BCR CD40L IL2 CpG HD naı¨ve HD memory MS naı¨ve MS memory

75 62 44 183

165 57 1263b 43

157 53 568 789c

59 64 484b 256a

113 22 18d 1

94 96 10 1

134 89 27d 1

156a 101 25 11

Supernatants were assayed for IL6 and IL10 by ELISA, results are presented as the mean for each group in pg/mL. Naı¨ve B cells from HC tended to produce more IL10 than MS patients and significantly more in cultures with CD40L, IL2 and CpG. Memory B cells from MS patients and HDs secrete similar amounts of IL-10; however, the number of HD memory B cells that responded by producing IL10 was significantly greater than MS patients in cultures with CD40L and CD40L plus IL4 and BCR cross-link. Student’s unpaired t-test was used to compare HD to MS production of cytokines within memory or naı¨ve subsets for each stimulation, the number of responders (defined as two standard deviations above the mean of MS B cells with CD40L only) was compared using chi-squared test.; a # 0.05 t-test.; b # 0.01 t-test.; c # 0.001 t-test.; d Chi-squared #0.05.

406 S. J. Ireland et al. tried in a variety of autoimmune and B cell malignant disorders, but has not been tested in MS [85] though multiple reports in mouse models of MS support a role for IL6 in disease pathology [86 –92]. It is tempting to speculate that a-IL6r therapeutics would show efficacy in MS. However, anti-IL23/12p40 [93] and TNF-a [94,95] therapies, designed to block TH17 expansion in vivo, do not have predictable outcomes in MS patients. B cell-derived IL-10 and regulatory B cells Interleukin 10 is another pleiotropic cytokine capable of acting on both B and T cells, which is of great interest to regulatory B cell effector functions [96]. Recent investigations primarily focused on IL10 as a regulatory and anti-inflammatory cytokine, although more historical observations defined the important role of IL10 as a survival and maturation factor with the ability to potentiate autoimmune diseases involving B cells [97,98]. Human B cells secrete IL10 to a variety of stimuli including CD40L, BCR cross-link [46], and CpG alone [74] or in combination with CD40L [99]. IL10 acts in an autocrine or paracrine fashion on B cells to increase antibody production [55]. In fact, both IL10 and IL6 are important for supporting antibody secretion [46]. Exposure of B cells to IL10 tends to lead to B cell survival and differentiation into plasma cells [100]. The most well-known role for IL10 is that of an anti-inflammatory molecule and IL10 is secreted by regulatory B and T cells, but also by macrophages [101] and by some subsets of dendritic cells [102,103]. IL10 dampens immune responses in several ways. First, IL10 limits antigen presentation through down-modulation of MHC Class II and coreceptors (CD80 and CD86) expression on APC [96,97,104,105]. IL10 can also directly suppress CD4 þ T cell proliferation and cytokine secretion induced by CD28 stimulation in healthy control peripheral blood cells and T cell lines [104], as well as increase T and B cell sensitivity to apoptosis [106]. Thus, it became of interest to determine the impact of B cell derived IL10 on inflammation associated with MS. Initial reports that serum titers of IL10 in MS patients were elevated [107] further intensified the pursuit. However, the first indication of a prominent role of IL10 production by B cells in controlling disease was established in mouse models of MS [108– 111]. More recently it has been shown that IL10 producing B cells are critical for the induction of FOXP3 þ T regulatory cells and for limiting TH17 and TH1 T cell responses in a mouse model of arthritis [112]. B cell production of IL10 plays a role in controlling disease severity in mouse models of MS [108 –111]. Such data is highly suggestive of a central role for IL10 producing B cells in the regulation of MS.

However, the impact of IL10 production by B cells in MS patients is in its advent of being understood. Previous studies in our laboratory and others showed a distinct deficit in IL10 production by B cells from MS patients [17,56,58,113] and suggest that the lack of IL10 producing B cells allows for progression of the inflammation associated with the disease. However, it is known that helminth infection can overcome the deficit in peripheral B cell production of IL10 in MS patients and restore it to that of HD [114]. There are also multiple reports of putative regulatory B cells (Breg) in MS patients. Peripheral B cells from MS patients stimulated with CpG produce less IL10 than HDs, and the frequency of CD27 þ memory B cells from MS patients that secrete IL10 is significantly less than HDs or MS patients in remission [115] indicating the importance of frequency and fluctuations in regulatory or inflammatory B cell populations. A separate study found that the frequency of peripheral B cells (CD27þCD24hi), thought to harbor Breg, was increased in MS patients compared to HD when stimulated with CD40L plus CpG or lipopolysaccharide; however several patients were receiving immunomodulatory therapy at the time of blood draw [116]. A third study found that the number of IL10 producing B cells from total peripheral blood B cells from MS patients is similar to HD in unstimulated cultures, yet was decreased in MS patients stimulated with CpG alone [113]. The differences in assay conditions, cohorts and methods for quantifying B cell IL10 production led us to investigate whether B cells from our treatment naı¨ve RRMS patient cohort were competent IL10 producers. We also reasoned that naı¨ve and memory B cells may have different capacities to produce IL10, considering the historical understanding that the impact of IL10 on activated B cells is different than the impact of IL10 on naı¨ve B cells [100]. Thus, we cultured naı¨ve or memory B cells from HD and MS patients with polyclonal stimuli and asked whether B cells from MS patients and HDs were equally capable of producing IL10. Our results indicate that naı¨ve B cells from HDs stimulated with CD40L alone produced significant amounts of IL10 (average ¼ 113.3 pg/mL), which was enhanced with the addition of IL4 þ BCR (134.4 pg/mL) or IL2 þ CpG (156.7 pg/mL)(Table I). However, none of these conditions induced robust IL10 production by either naı¨ve or memory B cells from treatment naı¨ve MS patients (Table I). Thus, although both memory and naı¨ve B cells from MS patients are capable of producing IL10, the production is minimal compared to that produced by naı¨ve and memory B cells from HDs. In separate studies, memory B cells secreted robust amounts of IL10 [61,117]. Further investigation is needed to understand the role of IL10 producing B cells in MS

B cell effector functions in MS patients with careful attention to culturing conditions, types of B cells in the cultures, clinical status of the patients in the cohorts, and surface marker combinations used to identify IL10 producing B cells. Ratio of IL6 and IL10 expression The deficit in IL10 production by B cells from MS patients combined with excessive production of IL6 by B cells from MS patients has the potential to impact the balance of regulatory and inflammatory signals in this disease. Of particular interest is that B cells from patients with Behcet’s disease produce increased IL6, with limited IL10 production in response to B cell activating factor (BAFF) stimulation[116], just as we have observed in our MS cohort. Moreover, reports have shown that increases in the ratio of IL6 to IL10 were prognostic of poor outcomes in systemic inflammatory response syndrome [118] and infant sepsis [119]. In addition, the overabundance of IL-6 in RA patients is known to block inhibitory effects of IL10 on CD4 þ T cells [120]. This led us to compare the IL6/IL10 ratio produced by B cells in HDs and MS patients. The IL6/IL10 production ratios by memory and naı¨ve B cells from HDs and MS patients are presented in Figure 4. These data demonstrate that memory and naı¨ve B cells from MS patients are more likely to secrete IL6 in response to a broad spectrum of stimuli, while memory and naı¨ve B cells from HDs are more likely to secrete IL10 in response to a broad spectrum of stimuli. These data highlight the intrinsic dysregulation of proliferation and cytokine production by B cells from MS patients. Our observations and other studies have

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identified a defect in IL10 production by B cells from MS patients. Whether this decreased ability to secrete IL10 impacts the regulatory function of B cells in MS patients remains unknown. In addition, the increased expression of IL6 in B cells from MS patients we identified here could impact not only B and T cell function, but also contribute to tissue inflammation. In the context of APC capacity, increased IL6 expression might influence T cell polarization or function. Finally, the altered ratios of cytokines secreted by B cells to polyclonal stimuli indicate an intrinsic predisposition to or initiation of an inflammatory program that is unique to B cells from MS patients. Future studies are needed to address the mechanism of this skewed cytokine production and how immunomodulatory therapies might restore balanced cytokine secretion in B cells. Impact of immunomodulatory therapies on B cells: Type 1 interferons Both IFNb-1b and IFNb-1a are FDA approved therapeutics for treating RRMS. While their effects on T cell responses and other myeloid cells are the subject of numerous investigations [16,121], there are few reports on the impact of IFN therapies on B cells, particularly with respect to IFNb-1a. Recently the impact of IFNb-1b on B cells was shown in vitro where activated B cells exposed to IFNb-1b had decreased CD40 and CD80 costimulatory molecule expression [76,122], and decreased IL23 and IL1b secretion but increased IL10, IL12 and IL27 production [76]. Supernatants from IFNb-1b exposed B cell cultures inhibited IL-17

Figure 4. B cells from MS patients secrete altered ratios of IL6 to IL10. Comparison of the IL6 on day 3 and IL10 on day 5 production from individual cultures in response to polyclonal stimuli. A. Naive B cells from MS patients exhibit exaggerated IL6 production that is disproportionate to IL10 production compared to HD in response to CD40L high plus IL4. B. Memory B cells from MS patients also have an IL6-skewed cytokine profile in response to CD40L high plus IL4 and BCR cross-link. Mean IL6: IL10 ratios are provided below the panels. Students unpaired t-test used to identify statistical differences between ratios defined as a p-value at or below 0.05.

408 S. J. Ireland et al. Table II. Direct exposure of B cells to GA does not alter proliferation. CD40L high

CD40L low

CD40L CD40L IL4 CD40L IL4 BCR CD40L IL2 CpG CD40L CD40L IL4 CD40L IL4 BCR CD40L IL2 CpG Naı¨ve HD Memory HD Naı¨ve MS Memory MS

0.95 0.27 0.72 0.72

0.25 0.45 0.42 0.63

0.26 0.76 0.74 0.80

0.97 0.17 0.81 0.87

0.87 0.58 0.99 0.91

0.98 0.80 0.68 0.19

0.74 0.50 0.55 0.32

0.63 0.41 0.87 0.41

Values in the table represent p-values comparing B cell proliferation under the stimulation combination in the absence of GA to B cell proliferation under the same stimulation combination in the presence of GA.

secretion by PBMC [76]. IFNb-1b also reduced the ability of B cells to induce proliferation and IFNg secretion by T cells [76,123]. The frequency of CD27 þ peripheral memory B cells is diminished with IFNb therapy, as well as expression of CD86 [16,17]. In the same study, CCR5 expression was also decreased on total CD19 þ and naı¨ve B cells, but not memory B cells [16]. Although IFNb-1a receptor is expressed on B cells and they respond to the cytokine, few studies have addressed the effect of this IFN treatment on B cells. It is known that IFNb-1a does induce inhibitory proteins in B cells [124]. Collectively, these observations demonstrate that the existing IFN therapies can impact effector functions of B cells that include co-stimulation, cytokine production and APC activity.

Impact of immunomodulatory therapies on B cells: Glatiramer acetate Glatiramer acetate (GA) is a random polymer composed of amino acids in a similar ratio to a myelin basic protein (MBP), a candidate auto-antigen in MS, and is an FDA approved immunomodulatory therapy for the treatment of MS. Antibodies isolated from the CNS of some MS patients bind MBP [125 –128] and B cells from peripheral blood can bind MBP or GA directly [56,57]. Although GA can bind directly to HLA molecules [129], it is possible that MBP-specific B cells bind GA via the BCR and are processed to HLA in a specific manner. In fact, our laboratory found that FITC labeled MBP or GA can directly

bind purified B cells. While GA was considered to modulate T cells by binding directly to MHC II and interfering with self-antigen presentation, some recent evidence showed that myeloid APC treated in vitro with GA had increased IL10 expression and decreased IL-12p70 [130] and TNF-a [131]. These effects of GA have not been assayed in human B cells. Most interestingly, B cells from mice treated in vivo with GA produced more IL10 than controls, with a concomitant decrease in the co-stimulatory molecules, CD80 and CD86 [109,110]. Adoptive transfer of in vivo GA exposed B cells confers resistance to the mouse model of MS, shown by a reduction in IFNg and IL17 responses [110]. In the same study, B cells were required for the mechanism of GA activity in vivo. These studies prompted us to ask whether direct exposure of human B cells to GA in vitro alters their cytokine production profile or proliferative capacity. To test this hypothesis, we incubated memory or naı¨ve B cells from HDs and MS patients with polyclonal stimuli in the presence or absence of GA. We found that the presence of GA in B cell cultures did not alter proliferation (Table II) or cytokine secretion by B cells (Table III). These data show in vitro treatment of purified B cells with GA does not directly impact the B cell functions tested here. Future studies are necessary to determine whether human B cell activity is altered by therapeutic treatment with GA similarly to what was observed in mice. Furthermore, it is possible that in both mice and humans, altered B cell activity is not resultant of a direct interaction between B cells and GA.

Table III. Direct exposure of B cells to GA does not alter IL6 or IL10 production. IL6 Day 3

IL10 Day 5

CD40L CD40L IL4 CD40L IL4 BCR CD40L IL2 CpG CD40L CD40L IL4 CD40L IL4 BCR CD40L IL2 CpG Naı¨ve HD Memory HD Naı¨ve MS Memory MS

0.46 0.65 0.65 0.89

0.34 0.24 0.86 0.37

0.88 0.48 0.41 0.54

0.28 0.63 0.73 0.74

0.80 0.31 0.81 0.31

0.22 0.87 0.81 0.42

0.73 0.47 0.94 0.36

0.94 0.86 0.97 0.66

Values in the table represent p-values comparing IL6 or IL10 production by B cells under the stimulation combination in the absence of GA to IL6 or IL10 production by B cells under the same stimulation combination in the presence of GA.

B cell effector functions in MS Conclusions B cells have the capacity to act as regulatory and inflammatory effector cells in the context of relapsing remitting multiple sclerosis. Memory B cells in particular are fully equipped to participate in immune responses in an antibody secretion independent manner. Here, we discussed the potential role of B cell APC capacity, cytokine secretion and memory B cells on MS with a focus on how these functions impact T cells. B cells subsets from MS patients have significantly greater propensity to induce autologous CD4 þ T cell responses to self antigens than HDs, while maintaining a similar response to foreign antigen. We also showed that B cell subsets from MS patients exhibit increased IL6 production that is not coordinately regulated with IL10 production as it is in memory and naı¨ve B cells from HDs. Our studies suggest that how currently approved immunomodulatory therapies could impact B cell effector functions, but demonstrated that one therapeutic, GA, does not directly act on B cells to induce regulatory function via secretion of IL10 or dampen proliferative capacity. Taken together, there are a multitude of B cell effector functions that have the potential to explain the efficacy of B cell-depleting therapies. These B cell functions are just beginning to be understood and they are worthy of future investigations to increase our knowledge of the role of B cells in autoimmune responses, particularly in the context of MS. Methods Human samples: Relapsing remitting MS (RRMS) patients were recruited at the University of Texas Southwestern Medical Center according to Institutional Review Board approved criteria. Cells were obtained by leukapheresis. Patients were not on corticosteroid treatment within 60 days prior to leukapheresis and had never received immunomodulatory therapy. All patients had at least one relapse in the 2 years prior, but not within 60 days of leukapheresis. Patient characteristics are provided elsewhere [57]. PBMC from HDs and MS patients were isolated by ficoll paque (GE Healthcare) density gradient centrifugation and cryopreserved at -1208C on the same day in 50% pooled human serum (Gemini Bioproducts), 10% DMSO (Fisher) and 40% RPMI 1640 (Cellgro). B cell purification and isolation: Total B cells were isolated from PBMC by magnetic activated cell separation (MACS) using a-CD19 microbeads (Miltenyi) according to the manufacturers instructions. Purity was typically above 95%. Total CD19 þ B cells were stained with CD19-PECy5, CD27-PE and IgD-FITC (BD Bioscience) and sorted into naı¨ve (CD19 þ IgD þ CD27-) and memory

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(CD19 þ CD27 þ ) populations on a FACS Aria (BD Biosciences, custom order system). Cell Culture: Memory and naı¨ve B cell subsets were stained with CFSE as previously described [57]. 1 £ 105 naı¨ve or memory B cells were cultured for 5 days in u-bottom 96-well plates (Corning) in 200 mL minimal media (RMPI 1640, Cellgro; 10% fetal calf serum, Hyclone; 100 mg/mL penicillin 100 U/mL streptomycin and 2 mM L-glutamine, Fisher Scientific). Immortalized Swiss mouse fibroblast cells (NIH-3T3) stably expressing membrane bound human CD40L (CD40LþNIH-3T3) were a kind gift from Gordon Freeman. CD40LþNIH-3T3 cells were grown in minimal media supplemented with 200 mg/mL gentamicin/G418 (Fisher). NIH3T3 cells were sub-lethally irradiated with 10,000 rads from a Cs137 source to prevent proliferation and seeded into 96 well plates at 500 (low CD40L) or 2000 (high CD40L) cells per well. CD40LþNIH3T3 cells were allowed to adhere overnight before the media was removed and B cell cultures added. Further stimulation of B cells was carried out with 10 ng/mL of recombinant human IL-2 (Gibco) or IL-4 (R&D Systems). Goat anti-human IgM/IgG (Jackson Immunoresearch) was added at a final concentration of 0.5 mg/mL and class-B CpG (Invivogen CpG ODN 2006 or Coley Pharmaceuticals CpG ODN 10103, CpG ODN) was used at a final concentration of 5 mg/mL. Quantification of Cytokines: Culture supernatants were collected on day 3 and replaced with fresh media. Supernatants were frozen at -208C for batched analysis. IL10, LTa (BD Pharmingen), IL12p70, TNFa, and IL6 (eBioscience) were quantified by ELISA. The lowest detection level for IL6 and TNFa was 1 pg/mL, 5 pg/mL for LTa and 9.5 pg/mL for IL12p70. Statistical Analysis: the unpaired Student’s t-test was performed to compare cytokine concentration and proliferative responses; a p-value less than 0.05 was considered statistically significant. The number of responders for each cytokine was defined as two standard deviations above the mean cytokine concentration of naı¨ve B cells from MS patients stimulated with CD40L alone. Chi-square analysis was used to determine the statistical significance between the number of responders. Acknowledgements The authors wish to thank the MS patients and healthy donors who have contributed samples to these studies. We also thank the CONQUER repository team and the UTSWMC FLOW CYTOMETRY CORE for their support in obtaining samples and collecting data for these studies. Finally, we thank TEVA Neuroscience for supporting the B-T project for the past 5 years.

410 S. J. Ireland et al. Declaration of Interest: Dr. Nancy Monson is a paid consultant for MedImmune, and serves as a scientific advisor for Genentech. Funding for this project is provided by TEVA Neuroscience. The authors have no other interests to declare. The authors alone are responsible for the content and writing of the paper. References [1] Bar-Or A, Calabresi PA, Arnold D, Markowitz C, Shafer S, Kasper LH, Waubant E, Gazda S, Fox RJ, Panzara M, Sarkar N, Agarwal S, Smith CH. Rituximab in relapsing-remitting multiple sclerosis: a 72-week, open-label, phase I trial. Ann Neurol 2008;63(3):395 –400. [2] Hauser SL, Waubant E, Arnold DL, Vollmer T, Antel J, Fox RJ, Bar-Or A, Panzara M, Sarkar N, Agarwal S, LangerGould A, Smith CHB-cell. depletion with rituximab in relapsing-remitting multiple sclerosis. N Engl J Med 2008; 358(7):676 –688. [3] Marriott JJ, O’Connor PW. Emerging therapies in relapsingremitting multiple sclerosis. Rev Recent Clin Trials 2010; 5(3):179–188. [4] Kappos L, Li D, Calabresi PA, O’Connor P, Bar-Or A, Barkhof F, Yin M, Leppert D, Glanzman R, Tinbergen J, Hauser SL. Ocrelizumab in relapsing-remitting multiple sclerosis: a phase 2, randomised, placebo-controlled, multicentre trial. Lancet 2011; 378(9805):1779–1787. [5] Weber MS, Prod’homme T, Patarroyo JC, Molnarfi N, Karnezis T, Lehmann-Horn K, Danilenko DM, EasthamAnderson J, Slavin AJ, Linington C, Bernard CC, Martin F, Zamvil SSB-cell. activation influences T-cell polarization and outcome of anti-CD20 B-cell depletion in central nervous system autoimmunity. Ann Neurol 2010;68(3):369–383. [6] Lyons JA, Ramsbottom MJ, Cross AH. Critical role of antigen-specific antibody in experimental autoimmune encephalomyelitis induced by recombinant myelin oligodendrocyte glycoprotein. Eur J Immunol 2002;32(7): 1905– 1913. [7] Lyons JA, San M, Happ MP, Cross AH. B cells are critical to induction of experimental allergic encephalomyelitis by protein but not by a short encephalitogenic peptide. Eur J Immunol 1999;29(11):3432–3439. [8] Monson NL, Cravens P, Hussain R, Harp CT, Cummings M, de Pilar Martin M, Ben LH, Do J, Lyons JA, Lovette-Racke A, Cross AH, Racke MK, Stuve O, Shlomchik M, Eagar TN. Rituximab therapy reduces organ-specific T cell responses and ameliorates experimental autoimmune encephalomyelitis. PLoS One 2011;6(2):e17103. [9] Klein U, Rajewsky K, Kuppers R. Human immunoglobulin (Ig)M þ IgD þ peripheral blood B cells expressing the CD27 cell surface antigen carry somatically mutated variable region genes: CD27 as a general marker for somatically mutated (memory) B cells. J Exp Med 1998;188(9): 1679– 1689. [10] Agematsu K, Hokibara S, Nagumo H, Komiyama A. CD27: a memory B-cell marker. Immunol Today 2000;21(5): 204–206. [11] Wagle NM, Cheng P, Kim J, Sproul TW, Kausch KD, Pierce SKB-lymphocyte. signaling receptors and the control of class-II antigen processing. Curr Top Microbiol Immunol 2000;245(2):101–126. [12] Pascual V, Liu YJ, Magalski A, de Bouteiller O, Banchereau J, Capra JD. Analysis of somatic mutation in five B cell subsets of human tonsil. J Exp Med 1994;180(1):329–339. [13] Tangye SG, Avery DT, Deenick EK, Hodgkin PD. Intrinsic differences in the proliferation of naive and memory human

[14]

[15]

[16] [17]

[18]

[19] [20]

[21]

[22]

[23]

[24]

[25]

[26]

[27]

[28]

B cells as a mechanism for enhanced secondary immune responses. J Immunol 2003;170(2):686–694. Good KL, Avery DT, Tangye SG. Resting human memory B cells are intrinsically programmed for enhanced survival and responsiveness to diverse stimuli compared to naive B cells. J Immunol 2009;182(2):890–901. Gagro A, Servis D, Cepika AM, Toellner KM, Grafton G, Taylor DR, Branica S, Gordon J, Type I. cytokine profiles of human naive and memory B lymphocytes: a potential for memory cells to impact polarization. Immunology 2006; 118(1):66–77. Niino M, Hirotani M, Miyazaki Y, Sasaki H. Memory and naive B-cell subsets in patients with multiple sclerosis. Neurosci Lett 2009;464(1):74– 78. Haas J, Bekeredjian-Ding I, Milkova M, Balint B, Schwarz A, Korporal M, Jarius S, Fritz B, Lorenz HM, Wildemann B. B cells undergo unique compartmentalized redistribution in multiple sclerosis. J Autoimmun 2011;37(4):289 –299. Sellebjerg F, Bornsen L, Khademi M, Krakauer M, Olsson T, Frederiksen JL, Sorensen PS. Increased cerebrospinal fluid concentrations of the chemokine CXCL13 in active MS. Neurology 2009;73(23):2003–2010. McHeyzer-Williams LJ, McHeyzer-Williams MG. Antigenspecific memory B cell development. Annu Rev Immunol 2005;23:487–513. Markovic-Plese S, Fukaura H, Zhang J, al-Sabbagh A, Southwood S, Sette A, Kuchroo VK, Hafler DA. T cell recognition of immunodominant and cryptic proteolipid protein epitopes in humans. J Immunol 1995;155(2): 982 –992. Hafler DA, Benjamin DS, Burks J, Weiner HL. Myelin basic protein and proteolipid protein reactivity of brain- and cerebrospinal fluid-derived T cell clones in multiple sclerosis and postinfectious encephalomyelitis. J Immunol 1987; 139(1):68–72. Crawford MP, Yan SX, Ortega SB, Mehta RS, Hewitt RE, Price DA, Stastny P, Douek DC, Koup RA, Racke MK, Karandikar NJ. High prevalence of autoreactive, neuroantigen-specific CD8 þ T cells in multiple sclerosis revealed by novel flow cytometric assay. Blood 2004;103(11): 4222–4231. Karandikar NJ, Crawford MP, Yan X, Ratts RB, Brenchley JM, Ambrozak DR, Lovett-Racke AE, Frohman EM, Stastny P, Douek DC, Koup RA, Racke MK. Glatiramer acetate (Copaxone) therapy induces CD8(þ) T cell responses in patients with multiple sclerosis. J Clin Invest 2002;109(5): 641 –649. Kim HJ, Antel JP, Duquette P, Alleva DG, Conlon PJ, Bar-Or A. Persistence of immune responses to altered and native myelin antigens in patients with multiple sclerosis treated with altered peptide ligand. Clin Immunol 2002;104(2): 105 –114. Krumbholz M, Theil D, Cepok S, Hemmer B, Kivisakk P, Ransohoff RM, Hofbauer M, Farina C, Derfuss T, Hartle C, Newcombe J, Hohlfeld R, Meinl E. Chemokines in multiple sclerosis: CXCL12 and CXCL13 up-regulation is differentially linked to CNS immune cell recruitment. Brain 2006; 1(Pt 1):200–211. Cepok S, von Geldern G, Grummel V, Hochgesand S, Celik H, Hartung H, Hemmer B. Accumulation of class switched IgD-IgM- memory B cells in the cerebrospinal fluid during neuroinflammation. J Neuroimmunol 2006; 180(1 – 2): 33–39. Teleshova N, Bao W, Kivisakk P, Ozenci V, Mustafa M, Link H. Elevated CD40 ligand expressing blood T-cell levels in multiple sclerosis are reversed by interferon-beta treatment. Scand J Immunol 2000;51(3):312– 320. Pelfrey CM, Moldovan IR, Cotleur AC, Zamor N, Rudick RA. Effects of sex hormones on costimulatory molecule

B cell effector functions in MS

[29]

[30] [31]

[32]

[33] [34]

[35]

[36]

[37]

[38]

[39]

[40]

[41]

[42]

[43]

[44]

[45]

expression in multiple sclerosis. J Neuroimmunol 2005; 167(1–2):190– 203. Kosmaczewska A, Bilinska M, Ciszak L, Noga L, Pawlak E, Szteblich A, Podemski R, Frydecka I. Different patterns of activation markers expression and CD4 þ T-cell responses to ex vivo stimulation in patients with clinically quiescent multiple sclerosis (MS). J Neuroimmunol 2007;189(1– 2): 137–146. Lanzavecchia A. Antigen-specific interaction between T and B cells. Nature 1985;314(6011):537– 539. Malynn BA, Romeo DT, Wortis HH, Antigen-specific B. cells efficiently present low doses of antigen for induction of T cell proliferation. J Immunol 1985;135(2):980–988. Sanders VM, Uhr JW, Vitetta ES. Antigen-specific memory and virgin B cells differ in their requirements for conjugation to T cells. Cell Immunol 1987;104(2):419–425. Bottomly K, Janeway CA, Jr. Antigen presentation by B cells. Nature 1989;337(6202):24. Yan J, Harvey BP, Gee RJ, Shlomchik MJ, Mamula MJ. B cells drive early T cell autoimmunity in vivo prior to dendritic cell-mediated autoantigen presentation. J Immunol 2006; 177(7):4481–4487. O’Neill SK, Shlomchik MJ, Glant TT, Cao Y, Doodes PD, Finnegan A. Antigen-specific B cells are required as APCs and autoantibody-producing cells for induction of severe autoimmune arthritis. J Immunol 2005;174(6):3781–3788. Issekutz T, Chu E, Geha RS. Antigen presentation by human B cells: T cell proliferation induced by Epstein Barr virus B lymphoblastoid cells. J Immunol 1982;129(4):1446–1450. Lombardi G, del Gallo F, Vismara D, Piccolella E, de Martino C, Garzelli C, Puglisi C, Colizzi V. Epstein-Barr virus-transformed B cells process and present Mycobacterium tuberculosis particulate antigens to T-cell clones. Cell Immunol 1987;107(2):281–292. Vidard L, Kovacsovics-Bankowski M, Kraeft SK, Chen LB, Benacerraf B, Rock KL. Analysis of MHC class II presentation of particulate antigens of B lymphocytes. J Immunol 1996;156(8):2809–2818. Constant S, Schweitzer N, West J, Ranney P, Bottomly K. B lymphocytes can be competent antigen-presenting cells for priming CD4 þ T cells to protein antigens in vivo. J Immunol 1995;155(8):3734–3741. Batista FD, Neuberger MS. B cells extract and present immobilized antigen: implications for affinity discrimination. EMBO J 2000;19(4):513 –520. Aluvihare VR, Khamlichi AA, Williams GT, Adorini L, Neuberger MS. Acceleration of intracellular targeting of antigen by the B-cell antigen receptor: importance depends on the nature of the antigen-antibody interaction. EMBO J 1997;16(12):3553–3562. von Bergwelt-Baildon M, Schultze JL, Maecker B, Menezes I, Nadler LM. Correspondence re R. Lapointe et al., CD40stimulated B lymphocytes pulsed with tumor antigens are effective antigen-presenting cells that can generate specific T cells. Cancer Res 2003;63:2836– 2843, Cancer Res 2004; 64(11):4055–6; author reply 6 –7. Kleijmeer MJ, Morkowski S, Griffith JM, Rudensky AY, Geuze HJ. Major histocompatibility complex class II compartments in human and mouse B lymphoblasts represent conventional endocytic compartments. J Cell Biol 1997;139(3):639–649. Chu ET, Lareau M, Rosenwasser LJ, Dinarello CA, Geha RS. Antigen presentation by EBV-B cells to resting and activated T cells: role of interleukin 1. J Immunol 1985; 134(3):1676–1681. Lapointe R, Bellemare-Pelletier A, Housseau F, Thibodeau J, Hwu P, CD40-stimulated B. lymphocytes pulsed with tumor antigens are effective antigen-presenting cells that can

[46] [47] [48]

[49]

[50]

[51] [52]

[53]

[54]

[55]

[56]

[57]

[58]

[59] [60]

[61]

411

generate specific T cells. Cancer Res 2003;63(11): 2836–2843. Burdin N, Rousset F, Banchereau J. B-cell-derived IL-10: production and function. Methods 1997;11(1):98– 111. Arbour N, Lapointe R, Saikali P, McCrea E, Regen T, Antel JP. A new clinically relevant approach to expand myelin specific T cells. J Immunol Methods 2006;310(1–2):53–61. Schultze JL, Michalak S, Seamon MJ, Dranoff G, Jung K, Daley J, Delgado JC, Gribben JG, Nadler LM. CD40activated human B cells: an alternative source of highly efficient antigen presenting cells to generate autologous antigen-specific T cells for adoptive immunotherapy. J Clin Invest 1997;100(11):2757– 2765. Baccam M, Woo SY, Vinson C, Bishop GA. CD40-mediated transcriptional regulation of the IL-6 gene in B lymphocytes: involvement of NF-kappa B, AP-1, and C/EBP. J Immunol 2003;170(6):3099–3108. Rousset F, Garcia E, Banchereau J. Cytokine-induced proliferation and immunoglobulin production of human B lymphocytes triggered through their CD40 antigen. J Exp Med 1991;173(3):705–710. Splawski JB, Fu SM, Lipsky PE. Immunoregulatory role of CD40 in human B cell differentiation. J Immunol 1993; 150(4):1276– 1285. Gray D, Dullforce P, Jainandunsing S. Memory B cell development but not germinal center formation is impaired by in vivo blockade of CD40-CD40 ligand interaction. J Exp Med 1994;180(1):141–155. von Bergwelt-Baildon M, Shimabukuro-Vornhagen A, Popov A, Klein-Gonzalez N, Fiore F, Debey S, Draube A, Maecker B, Menezes I, Nadler LM, Schultze JL. CD40-activated B cells express full lymph node homing triad and induce T-cell chemotaxis: potential as cellular adjuvants. Blood 2006; 107(7):2786– 2789. Ahmadi T, Flies A, Efebera Y, Sherr DH. CD40 Ligandactivated, antigen-specific B cells are comparable to mature dendritic cells in presenting protein antigens and major histocompatibility complex class I- and class II-binding peptides. Immunology 2008;124(1):129 –140. Bar-Or A, Oliveira EM, Anderson DE, Krieger JI, Duddy M, O’Connor KC, Hafler DA. Immunological memory: contribution of memory B cells expressing costimulatory molecules in the resting state. J Immunol 2001;167(10):5669–5677. Harp CT, Lovett-Racke AE, Racke MK, Frohman EM, Monson NL. Impact of myelin-specific antigen presenting B cells on T cell activation in multiple sclerosis. Clin Immunol 2008;128(3):382– 391. Harp CT, Ireland S, Davis LS, Remington G, Cassidy B, Cravens PD, Stuve O, Lovett-Racke AE, Eagar TN, Greenberg BM, Racke MK, Cowell LG, Karandikar NJ, Frohman EM, Monson NL. cells from a subset of treatmentnaive relapsing-remitting multiple sclerosis patients elicit CD4(þ ) T-cell proliferation and IFN-gamma production in response to myelin basic protein and myelin oligodendrocyte glycoprotein. Eur J Immunol 2010;40(10):2942–2956. Bar-Or A, Fawaz L, Fan B, Darlington PJ, Rieger A, Ghorayeb C, Calabresi PA, Waubant E, Hauser SL, Zhang J, Smith CH. Abnormal B-cell cytokine responses a trigger of Tcell-mediated disease in MS? Ann Neurol 2010;67(4): 452 –461. Pistoia V. Production of cytokines by human B cells in health and disease. Immunol Today 1997;18(7):343–350. Appel H, Neure L, Kuhne M, Braun J, Rudwaleit M, Sieper J. An elevated level of IL-10- and TGFbeta-secreting T cells, B cells and macrophages in the synovial membrane of patients with reactive arthritis compared to rheumatoid arthritis. Clin Rheumatol 2004;23(5):435–440. Agrawal S, Gupta S. TLR1/2, TLR7, and TLR9 signals directly activate human peripheral blood naive and memory B

412 S. J. Ireland et al.

[62]

[63]

[64]

[65]

[66]

[67]

[68]

[69]

[70]

[71]

[72]

[73]

[74]

cell subsets to produce cytokines, chemokines, and hematopoietic growth factors. J Clin Immunol 2011;31(1):89– 98. Wagner M, Poeck H, Jahrsdoerfer B, Rothenfusser S, Prell D, Bohle B, Tuma E, Giese T, Ellwart JW, Endres S, Hartmann G. IL-12p70-dependent Th1 induction by human B cells requires combined activation with CD40 ligand and CpG DNA. J Immunol 2004;172(2):954–963. Barbosa RR, Silva SP, Silva SL, Melo AC, Pedro E, Barbosa MP, Pereira-Santos MC, Victorino RM, Sousa AE. Primary B-cell deficiencies reveal a link between human IL-17producing CD4 T-cell homeostasis and B-cell differentiation. PLoS One 2011;6(8):e22848. van de Veerdonk FL, Lauwerys B, Marijnissen RJ, Timmermans K, Di Padova F, Koenders MI, Gutierrez-Roelens I, Durez P, Netea MG, van der Meer JW, van den Berg WB, Joosten LA. The anti-CD20 antibody rituximab reduces the Th17 cell response. Arthritis Rheum 2011;63(6): 1507–1516. van de Veerdonk FL, Marijnissen RJ, Kullberg BJ, Koenen HJ, Cheng SC, Joosten I, van den Berg WB, Williams DL, van der Meer JW, Joosten LA, Netea MG. The macrophage mannose receptor induces IL-17 in response to Candida albicans. Cell Host Microbe 2009;5(4):329 –340. Zhang M, Wang Z, Graner MW, Yang L, Liao M, Yang Q, Gou J, Zhu Y, Wu C, Liu H, Zhou B, Chen X. B cell infiltration is associated with the increased IL-17 and IL-22 expression in the lungs of patients with tuberculosis. Cell Immunol 2011;270(2):217–223. Yamamoto A, Sato K, Miyoshi F, Shindo Y, Yoshida Y, Yokota K, Nakajima K, Akiba H, Asanuma Y, Akiyama Y, Mimura T. Analysis of cytokine production patterns of peripheral blood mononuclear cells from a rheumatoid arthritis patient successfully treated with rituximab. Mod Rheumatol 2010;20(2):183 –187. van de Veerdonk FLLB, Marijnissen RJ, Timmermans K, Di Padova F, Koenders MI, Gutierrez-Roelens I, Durez P, Netea MG, van der Meer JW, van den Berg WB, Joosten LA. The anti-CD20 antibody rituximab reduces the Th17 cell response. Arthritis Rheum 2011;63(6):1507–1516. Bettelli E, Carrier Y, Gao W, Korn T, Strom TB, Oukka M, Weiner HL, Kuchroo VK. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature 2006;441(7090):235– 238. Benwell RK, Lee DR. Essential and synergistic roles of IL1 and IL6 in human Th17 differentiation directed by TLR ligand-activated dendritic cells. Clin Immunol 2010;134(2): 178–187. Mangan PR, Harrington LE, O’Quinn DB, Helms WS, Bullard DC, Elson CO, Hatton RD, Wahl SM, Schoeb TR, Weaver CT. Transforming growth factor-beta induces development of the T(H)17 lineage. Nature 2006; 441(7090):231–234. Acosta-Rodriguez EV, Napolitani G, Lanzavecchia A, Sallusto F. Interleukins 1beta and 6 but not transforming growth factor-beta are essential for the differentiation of interleukin 17-producing human T helper cells. Nat Immunol 2007;8(9):942 –949. Volpe E, Servant N, Zollinger R, Bogiatzi SI, Hupe P, Barillot E, Soumelis V. A critical function for transforming growth factor-beta, interleukin 23 and proinflammatory cytokines in driving and modulating human T(H)-17 responses. Nat Immunol 2008;9(6):650–657. Cognasse F, Hamzeh-Cognasse H, Lafarge S, Chavarin P, Pozzetto B, Richard Y, Garraud O. Identification of two subpopulations of purified human blood B cells, CD27CD23 þ and CD27high CD80 þ , that strongly express cell surface Toll-like receptor 9 and secrete high levels of interleukin-6. Immunology 2008;125(3):430–437.

[75] Smeland EB, Blomhoff HK, Funderud S, Shalaby MR, Espevik T. Interleukin 4 induces selective production of interleukin 6 from normal human B lymphocytes. J Exp Med 1989;170(4):1463–1468. [76] Ramgolam VS, Sha Y, Marcus KL, Choudhary N, Troiani L, Chopra M, Markovic-Plese S. B cells as a therapeutic target for IFN-beta in relapsing-remitting multiple sclerosis. J Immunol 2011;186(7):4518– 4526. [77] Dienz O, Rincon M. The effects of IL-6 on CD4 T cell responses. Clin Immunol 2009 Jan;130(1):27–33. [78] Banks WA, Kastin AJ, Gutierrez EG. Penetration of interleukin-6 across the murine blood-brain barrier. Neurosci Lett 1994;179(1–2):53–56. [79] Kleine TO, Zwerenz P, Graser C, Zofel P. Approach to discriminate subgroups in multiple sclerosis with cerebrospinal fluid (CSF) basic inflammation indices and TNF-alpha, IL-1beta, IL-6, IL-8. Brain Res Bull 2003;61(3):327–346. [80] Laurenzi MA, Siden A, Persson MA, Norkrans G, Hagberg L, Chiodi F. Cerebrospinal fluid interleukin-6 activity in HIV infection and inflammatory and noninflammatory diseases of the nervous system. Clin Immunol Immunopathol 1990; 57(2):233–241. [81] Maimone D, Gregory S, Arnason BGW, Reder AT. Cytokine levels in the cerebrospinal fluid and serum of patients with multiple sclerosis. J Neuroimmunol 1991;32:67 –74. [82] Milman N, Karsh J, Booth RA. Correlation of a multicytokine panel with clinical disease activity in patients with rheumatoid arthritis. Clin Biochem 2010;43(16 – 17): 1309–1314. [83] Tanaka T, Kishimoto T. Immunotherapeutic implication of IL-6 blockade. Immunotherapy 2012;4(1):87– 105. [84] Palmer CD, Mutch BE, Workman S, McDaid JP, Horwood NJ, Foxwell BM. Bmx tyrosine kinase regulates TLR4induced IL-6 production in human macrophages independently of p38 MAPK and NFkapp}B activity. Blood 2008; 111(4):1781– 1788. [85] Snir A, Kessel A, Haj T, Rosner I, Slobodin G, Toubi E. AntiIL-6 receptor antibody (tocilizumab): a B cell targeting therapy. Clin Exp Rheumatol 2011;29(4):697 –700. [86] Quintana A, Muller M, Frausto RF, Ramos R, Getts DR, Sanz E, Hofer MJ, Krauthausen M, King NJ, Hidalgo J, Campbell IL. Site-specific production of IL-6 in the central nervous system retargets and enhances the inflammatory response in experimental autoimmune encephalomyelitis. J Immunol 2009;183(3):2079– 2088. [87] Gijbels K, Van Damme J, Proost P, Put W, Carton H, Billiau A. Interleukin 6 production in the central nervous system during experimental autoimmune encephalomyelitis. Eur J Immunol 1990;20(1):233 –235. [88] Eugster HP, Frei K, Kopf M, Lassmann H, Fontana A. IL-6deficient mice resist myelin oligodendrocyte glycoproteininduced autoimmune encephalomyelitis. Eur J Immunol 1998;28(7):2178– 2187. [89] Mendel I, Katz A, Kozak N, Ben-Nun A, Revel M. Interleukin-6 functions in autoimmune encephalomyelitis: a study in gene-targeted mice. Eur J Immunol 1998;28(5): 1727– 1737. [90] Samoilova EB, Horton JL, Hilliard B, Liu TS, Chen Y. IL-6deficient mice are resistant to experimental autoimmune encephalomyelitis: roles of IL-6 in the activation and differentiation of autoreactive T cells. J Immunol 1998; 161(12):6480 –6486. [91] Serada S, Fujimoto M, Mihara M, Koike N, Ohsugi Y, Nomura S, Yoshida H, Nishikawa T, Terabe F, Ohkawara T, Takahashi T, Ripley B, Kimura A, Kishimoto T, Naka T. IL-6 blockade inhibits the induction of myelin antigen-specific Th17 cells and Th1 cells in experimental autoimmune encephalomyelitis. Proc Natl Acad Sci U S A 2008;105(26): 9041–9046.

B cell effector functions in MS [92] Gijbels K, Brocke S, Abrams JS, Steinman L. Administration of neutralizing antibodies to interleukin-6 (IL-6) reduces experimental autoimmune encephalomyelitis and is associated with elevated levels of IL-6 bioactivity in central nervous system and circulation. Mol Med 1995;1(7):795–805. [93] Segal BM, Constantinescu CS, Raychaudhuri A, Kim L, Fidelus-Gort R, Kasper LH. Repeated subcutaneous injections of IL12/23 p40 neutralising antibody, ustekinumab, in patients with relapsing-remitting multiple sclerosis: a phase II, double-blind, placebo-controlled, randomised, doseranging study. Lancet Neurol 2008;7(9):796–804. [94] van Oosten BW, Barkhof F, Truyen L, Boringa JB, Bertelsmann FW, von Blomberg BM, Woody JN, Hartung HP, Polman CH. Increased MRI activity and immune activation in two multiple sclerosis patients treated with the monoclonal anti-tumor necrosis factor antibody cA2. Neurology 1996;47(6):1531–1534. [95] Mohan N, Edwards ET, Cupps TR, Oliverio PJ, Sandberg G, Crayton H, Richert JR, Siegel JN. Demyelination occurring during anti-tumor necrosis factor alpha therapy for inflammatory arthritides. Arthritis Rheum 2001;44(12): 2862–2869. [96] Moore KW, de Waal Malefyt R, Coffman RL, O’Garra A. Interleukin-10 and the interleukin-10 receptor. Annu Rev Immunol 2001;19:683–765. [97] Moore KW, O’Garra A, de Waal Malefyt R, Vieira P, Mosmann TR. Interleukin-10. Annu Rev Immunol 1993;11: 165–190. [98] Beebe AM, Cua DJ, de Waal Malefyt R. The role of interleukin-10 in autoimmune disease: systemic lupus erythematosus (SLE) and multiple sclerosis (MS). Cytokine Growth Factor Rev 2002;13(4–5):403– 412. [99] Hanten JA, Vasilakos JP, Riter CL, Neys L, Lipson KE, Alkan SS, Birmachu W. Comparison of human B cell activation by TLR7 and TLR9 agonists. BMC Immunol 2008;9:39. [100] Itoh K, Hirohata S. The role of IL-10 in human B cell activation, proliferation, and differentiation. J Immunol 1995;154(9):4341–4350. [101] de Waal Malefyt R, Abrams J, Bennett B, Figdor CG, de Vries JE. Interleukin 10(IL-10) inhibits cytokine synthesis by human monocytes: an autoregulatory role of IL-10 produced by monocytes. J Exp Med 1991;174(5):1209–1220. [102] Hart AL, Al-Hassi HO, Rigby RJ, Bell SJ, Emmanuel AV, Knight SC, Kamm MA, Stagg AJ. Characteristics of intestinal dendritic cells in inflammatory bowel diseases. Gastroenterology 2005;129(1):50–65. [103] Ng SC, Plamondon S, Kamm MA, Hart AL, Al-Hassi HO, Guenther T, Stagg AJ, Knight SC. Immunosuppressive effects via human intestinal dendritic cells of probiotic bacteria and steroids in the treatment of acute ulcerative colitis. Inflamm Bowel Dis 2010;16(8):1286–1298. [104] de Waal Malefyt R, Yssel H, de Vries JE. Direct effects of IL10 on subsets of human CD4 þ T cell clones and resting T cells. Specific inhibition of IL-2 production and proliferation. J Immunol 1993;150(11):4754–4765. [105] Koppelman B, Neefjes JJ, de Vries JE, de Waal Malefyt R. Interleukin-10 down-regulates MHC class II alphabeta peptide complexes at the plasma membrane of monocytes by affecting arrival and recycling. Immunity 1997;7(6): 861 –871. [106] Marra LE, Zhang ZX, Joe B, Campbell J, Levy GA, Penninger J, Zhang L. IL-10 induces regulatory T cell apoptosis by up-regulation of the membrane form of TNFalpha. J Immunol 2004;172(2):1028–1035. [107] Rodriguez-Sainz Mdel C, Sanchez-Ramon S, de Andres C, Rodriguez-Mahou M, Munoz-Fernandez MA. Th1/Th2 cytokine balance and nitric oxide in cerebrospinal fluid and serum from patients with multiple sclerosis. Eur Cytokine Netw 2002;13(1):110–114.

413

[108] Begum-Haque S, Christy M, Ochoa-Reparaz J, Nowak EC, Mielcarz D, Haque A, Kasper LH. Augmentation of regulatory B cell activity in experimental allergic encephalomyelitis by glatiramer acetate. J Neuroimmunol 2011; 232(1–2):136–144. [109] Begum-Haque S, Sharma A, Christy M, Lentini T, OchoaReparaz J, Fayed IF, Mielcarz D, Haque A, Kasper LH. Increased expression of B cell-associated regulatory cytokines by glatiramer acetate in mice with experimental autoimmune encephalomyelitis. J Neuroimmunol 2010;219(1– 2):47–53. [110] Kala M, Rhodes SN, Piao WH, Shi FD, Campagnolo DI, Vollmer TL. B cells from glatiramer acetate-treated mice suppress experimental autoimmune encephalomyelitis. Exp Neurol 2010;221(1):136–145. [111] Matsushita T, Yanaba K, Bouaziz JD, Fujimoto M, Tedder TF. Regulatory B cells inhibit EAE initiation in mice while other B cells promote disease progression. J Clin Invest 2008; 118(10):3420–3430. [112] Carter NA, Vasconcellos R, Rosser EC, Tulone C, MunozSuano A, Kamanaka M, Ehrenstein MR, Flavell RA, Mauri C. Mice lacking endogenous IL-10-producing regulatory B cells develop exacerbated disease and present with an increased frequency of Th1/Th17 but a decrease in regulatory T cells. J Immunol 2011;186(10):5569–5579. [113] Hirotani M, Niino M, Fukazawa T, Kikuchi S, Yabe I, Hamada S, Tajima Y, Sasaki H. Decreased IL-10 production mediated by Toll-like receptor 9 in B cells in multiple sclerosis. J Neuroimmunol 2010;221(1–2):95 –100. [114] Correale J, Farez M, Razzitte G. Helminth infections associated with multiple sclerosis induce regulatory B cells. Ann Neurol 2008;64(2):187–199. [115] Knippenberg S, Peelen E, Smolders J, Thewissen M, Menheere P, Cohen Tervaert JW, Hupperts R, Damoiseaux J. Reduction in IL-10 producing B cells (Breg) in multiple sclerosis is accompanied by a reduced naive/memory Breg ratio during a relapse but not in remission. J Neuroimmunol 2011;239(1–2):80 –86. [116] Iwata Y, Matsushita T, Horikawa M, Dilillo DJ, Yanaba K, Venturi GM, Szabolcs PM, Bernstein SH, Magro CM, Williams AD, Hall RP, St Clair EW, Tedder TF. Characterization of a rare IL-10-competent B-cell subset in humans that parallels mouse regulatory B10 cells. Blood 2011; 117(2):530–541. [117] Bancos S, Phipps RP. Memory B cells from older people express normal levels of cyclooxygenase-2 and produce higher levels of IL-6 and IL-10 upon in vitro activation. Cell Immunol 2010;266(1):90–97. [118] Taniguchi T, Koido Y, Aiboshi J, Yamashita T, Suzaki S, Kurokawa A. Change in the ratio of interleukin-6 to interleukin-10 predicts a poor outcome in patients with systemic inflammatory response syndrome. Crit Care Med 1999;27(7):1262–1264. [119] Ng PC, Li K, Wong RP, Chui K, Wong E, Li G, Fok TF. Proinflammatory and anti-inflammatory cytokine responses in preterm infants with systemic infections. Arch Dis Child Fetal Neonatal Ed 2003;88(3):F209–F213. [120] Yamana J, Yamamura M, Okamoto A, Aita T, Iwahashi M, Sunahori K, Makino H. Resistance to IL-10 inhibition of interferon gamma production and expression of suppressor of cytokine signaling 1 in CD4 þ T cells from patients with rheumatoid arthritis. Arthritis Res Ther 2004;6(6): R567–R577. [121] Korporal M, Haas J, Balint B, Fritzsching B, Schwarz A, Moeller S, Fritz B, Suri-Payer E, Wildemann B. Interferon beta-induced restoration of regulatory T-cell function in multiple sclerosis is prompted by an increase in newly generated naive regulatory T cells. Arch Neurol 2008;65(11): 1434–1439.

414 S. J. Ireland et al. [122] Liu Z, Pelfrey CM, Cotleur A, Lee JC, Rudick RA. Immunomodulatory effects of interferon beta-1a in multiple sclerosis. J Neuroimmunol 2001;112(1– 2):153–162. [123] Jiang H, Milo R, Swoveland P, Johnson KP, Panitch H, DhibJalbut S. Interferon beta-1b reduces interferon gammainduced antigen-presenting capacity of human glial and B cells. J Neuroimmunol 1995;61(1):17–25. [124] Christophi GP, Panos M, Hudson CA, Tsikkou C, Mihai C, Mejico LJ, Jubelt B, Massa PT. Interferon-beta treatment in multiple sclerosis attenuates inflammatory gene expression through inducible activity of the phosphatase SHP-1. Clin Immunol 2009;133(1):27–44. [125] Cruz M, Olsson T, Ernerudh J, Hojeberg B, Link H. Immunoblot detection of oligoclonal anti-myelin basic protein IgG antibodies in cerebrospinal fluid in multiple sclerosis. Neurology 1987;37(9):1515 –1519. [126] Warren KG, Catz I. Relative frequency of autoantibodies to myelin basic protein and proteolipid protein in optic neuritis and multiple sclerosis cerebrospinal fluid. J Neurol Sci 1994; 121(1):66–73. [127] Cash E, Weerth S, Voltz R, Kornhuber M. Cells of cerebrospinal fluid of multiple sclerosis patients secrete

[128]

[129]

[130]

[131]

antibodies to myelin basic protein in vitro. Scand J Immunol 1992;35(6):695–701. Gerritse K, Deen C, Fasbender M, Ravid R, Boersma W, Claassen E. The involvement of specific anti myelin basic protein antibody-forming cells in multiple sclerosis immunopathology. J Neuroimmunol 1994;49(1–2):153– 159. Fridkis-Hareli M, Teitelbaum D, Gurevich E, Pecht I, Brautbar C, Kwon OJ, Brenner T, Arnon R, Sela M. Direct binding of myelin basic protein and synthetic copolymer 1 to class II major histocompatibility complex molecules on living antigen-presenting cells– specificity and promiscuity. Proc Natl Acad Sci USA 1994;91(11):4872–4876. Kim HJ, Ifergan I, Antel JP, Seguin R, Duddy M, Lapierre Y, Jalili F, Bar-Or A. Type 2 monocyte and microglia differentiation mediated by glatiramer acetate therapy in patients with multiple sclerosis. J Immunol 2004;172(11): 7144–7153. Weber MS, Starck M, Wagenpfeil S, Meinl E, Hohlfeld R, Farina C. Multiple sclerosis: glatiramer acetate inhibits monocyte reactivity in vitro and in vivo. Brain 2004;6(Pt 6): 1370–1378.