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. Author manuscript; available in PMC: 2016 Jun 1.
Published in final edited form as: J Immunol. 2015 Apr 20;194(11):5077–5084. doi: 10.4049/jimmunol.1402236

B cell antigen presentation is sufficient to drive neuro-inflammation in an animal model of multiple sclerosis1

Chelsea R Parker Harp *, Angela S Archambault *, Julia Sim †, Stephen T Ferris ‡, Robert J Mikesell *, Pandelakis A Koni §, Michiko Shimoda , Christopher Linington ‖, John H Russell †, Gregory F Wu *,‡,#
PMCID: PMC4433779  NIHMSID: NIHMS676021  PMID: 25895531

Abstract

B cells are increasingly regarded as integral to the pathogenesis of multiple sclerosis (MS) in part due to the success of B cell depletion therapy. Multiple B cell-dependent mechanisms contributing to inflammatory demyelination of the central nervous system (CNS) have been explored using experimental autoimmune encephalomyelitis (EAE), a CD4 T cell-dependent animal model for multiple sclerosis (MS). While B cell antigen presentation has been suggested to regulate CNS inflammation during EAE, direct evidence that B cells can independently support antigen-specific autoimmune responses by CD4 T cells in EAE is lacking. Using a newly developed murine model of in vivo conditional expression of MHCII, we previously reported that encephalitogenic CD4 T cells are incapable of inducing EAE when B cells are the sole antigen presenting cell. Herein we find that B cells cooperate with dendritic cells to enhance EAE severity resulting from myelin oligodendrocyte glycoprotein (MOG) immunization. Further, increasing the precursor frequency of MOG-specific B cells, but not addition of soluble MOG-specific antibody, is sufficient to drive EAE in mice expressing MHCII by B cells alone. These data support a model in which expansion of antigen-specific B cells during CNS autoimmunity amplifies cognate interactions between B and CD4 T cells and have the capacity to independently drive neuro-inflammation at later stages of disease.

Introduction

Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system (CNS) affecting close to 2.3 million people worldwide that is a leading cause of disability in young adults (1, 2). Moderately efficacious immune-modulating therapies for MS have been developed, in part with the aid of the CD4 T cell-dependent animal model experimental autoimmune encephalomyelitis (EAE). By generating and presenting target auto-antigens, antigen presenting cells (APCs) play an essential role in coordinating the behavior of CD4 T cells and inflammatory destruction of myelin during EAE (3, 4). Combined expression of MHCII, co-stimulatory molecules and cytokines by APCs regulates CD4 T cell functional traits in both peripheral and CNS compartments and ultimately directs the inflammatory cascade of events resulting in myelin and nerve damage (4, 5). The identity and characteristics of APCs involved in initiating and propagating inflammation within the CNS has been under intense scrutiny (3, 5). While dendritic cells (DCs) have been suggested to serve all required APC roles in EAE and MS, they are not sufficient to generate maximal disease in recombinant myelin oligodendrocyte glycoprotein (rMOG)-immunization models of EAE or for the development of spontaneous optic neuritis (6). Thus, additional APCs must participate in the generation and propagation myelin-reactive CD4 T cells in autoimmune neuro-inflammation. Extensive studies have been performed examining the contribution of other APCs such as monocytes, macrophages and microglia in EAE and suggest that they work in concert with DCs to promote disease (3).

Several studies have identified contributions by another professional APC - B cells - in the pathogenesis of CNS inflammatory demyelination, offsetting the earlier viewpoint that B cells are not required for EAE that was suggested by work in mice genetically deficient in B cells (7). For example, MOG-specific immunoglobulin (Ig) increases disease severity of EAE (8-10) and greater numbers of MOG-specific B cells combined with T cells recognizing cognate antigen results in spontaneous inflammatory demyelination in the CNS (11, 12). Further, B cell depletion after the onset of EAE can ameliorate inflammation and clinical disease (13, 14). Moreover, subsets of B cells identified by their production of IL-10, IL-6 or IL-35 have been shown to modulate the severity of EAE (15-17). Alternatively, B cells have suppressive traits during EAE, as depletion of B cells before peptide immunization can exacerbate disease (13). In sum, B cells are clearly implicated in the pathogenesis of EAE, via multiple mechanisms including cytokine and Ig production, as well as regulation of CD4 T cell function.

The importance of B cells in MS is underscored by the demonstration that B cell depletion therapy can be highly efficacious for certain patients (18). However, the mechanisms by which removal of B cells from MS patients results in clinical benefit remain unclear. While plasma cells and Ig are typical features of the MS plaque (2, 19) and localized intrathecal production of Ig is detected in most patients with MS (20), the efficacy of B cell depletion in MS appears to be independent of any effects on plasma cells or Ig (21-23). Furthermore, follow-up studies on MS patients undergoing B cell depletion revealed alterations in proliferation and pro-inflammatory cytokine production by CD4 T-cells (21). These studies raise questions regarding the degree to which B cell antigen presentation, rather than Ig production, drives neuro-inflammation during MS. B cells have been recognized to function as APCs in neuro-inflammation, particularly after induction of EAE via immunization with rMOG (14). Subsequent work has suggested that B cell antigen presentation is required to initiate disease induced by recombinant human MOG immunization in a B cell-dependent form of EAE (24). However, whether B cells are capable of independently driving CD4 T cell autoreactivity to myelin targets during EAE has not been determined.

Hence, we sought to determine the sufficiency of B cells for APC function during EAE. We began our studies by generating a murine system for the conditional expression of MHCII to restrict expression of MHCII to B cells. We originally observed that B cell antigen presentation is not sufficient for the initiation or propagation of EAE (25). However, due to the clinical evidence for B cell antigen presentation during EAE and MS, we chose to further explore the role for antigen-specific B cells. Herein, we found that maximal disease in protein-induced active EAE models is dependent upon B cell antigen presentation. Further, narrowing the repertoire of B cell specificity to MOG results in enhanced antigen processing and presentation, facilitating EAE development. This is the first demonstration in vivo that B cells can serve as the primary APC and coordinate CD4 T cell autoreactivity to myelin antigens during EAE.

Materials and Methods

Mice

C57BL/6 (B6) mice were purchased from The Jackson Laboratory. BMHCII mice were generated as previously reported (25). DCMHCII mice (26) were kindly provided by Dr. Terri Laufer (University of Pennsylvania). IgHMOG mice (10) were kindly provided by Dr. Hartmut Wekerle (Max Planck Institute of Neurobiology). IAβf/f mice were previously reported (27) and were bred to CD19Cre mice obtained commercially from The Jackson Laboratory. Thymic grafting was performed as described using thymii from B6 mice (6). Animals were housed in a specific pathogen-free barrier facility at Washington University School of Medicine. All breeding and experimental protocols were performed in accordance with protocols reviewed and approved by the Washington University in St. Louis Animal Studies Committee.

EAE

Active and passive EAE were induced according to prior reports (25, 28). Briefly, active EAE was induced in mice by subcutaneous injection of 200 μg MOG35-55 emulsified in complete Freund's adjuvant (CFA; Sigma) containing 500 μg H37RA (Difco, Detroit, MI) and intraperitoneal (i.p.) injection with 200 ng Pertussis Toxin (Enzo Life Sciences) in 0.2 ml PBS on day 0. Additionally, mice received 200ng Pertussis Toxin i.p. 48 hours later. Alternatively, mice were injected with 150 μg rMOG protein (residues 1-125 of human MOG protein (6)) in 500 μg CFA. Passive EAE was established by transfer of 1×107 MOG-specific, Thy1.1+ encephalitogenic cells as reported (25, 29). Mice were observed daily and clinical score were assessed with a five point scoring system, as follows: 0 = no disease; 1 = limp tail; 2 = mild hind limb paresis; 3 = severe hind limb paresis; 4 = complete hind limb plegia or quadriplegia; 5 = moribund or dead.

Flow cytometry

Prior to perfusion, spleens were harvested from experimental mice. Single cell suspensions from spleens were treated with ACK erythrocyte lysis buffer. Mice were perfused with 25 mL of ice-cold PBS and brains and spinal cords were collected from perfused mice and dounce homogenized to obtain single cell suspensions. CNS cells were purified by centrifugation for 30 min in a 30% Percoll (GE Healthcare) solution as previously reported (25). Cells were incubated with the anti-Fc receptor antibody 2.4G2 prior to the addition of antibodies. The following antibodies were purchased from BD Biosciences: CD45-FITC, CD8α-APC-H7, CD19-APC-H7, CD19-BV510, B220-PE-TxRed, B220–PE-CF594, CD11b-AlexaFluor-700, MHC-v450. The following antibodies were purchased from eBioscience: MHCII-Pacific Blue, CD11c-PECy7. The following antibodies were purchased from BioLegend (San Diego, CA): CD138-PE, MHCII (I-A/I-E)-Pacific Blue, Thy1.1-PerCP, CD4-APC. Cells were acquired on a Gallios flow cytometer (Beckman Coulter) and analyzed with FloJo software (TreeStar) with doublets being excluded.

Histology

Mice were sacrificed and perfused with 25mL cold PBS followed by 20mL 4% paraformaldehyde (Sigma-Aldrich). Brains and spinal cords were removed and fixed in 4% paraformaldehyde overnight or longer. Tissues were paraffin-embedded and sectioned at 5μm by the Developmental Biology Histology & Microscopy Core at Washington University in St. Louis. Representative sections were stained for myelination with solochrome cyanine as previously reported (30) and Hematoxylin and Eosin for inflammation. Slides were examined by light microscopy using a Nikon 90i motorized upright digital microscope with camera and Metamorph software (Molecular Devices).

ELISA

Animals were sacrificed by cardiac puncture and blood samples were allowed to clot at RT. Serum was obtained by centrifugation of clotted blood at 15,000×g for 30 min and frozen at -20C until use. Total serum MOG-specific IgG was quantified by plating serial serum dilutions on 96 well plates pre-coated with 10 ug/ml rMOG in coating buffer (Sodium bicarbonate). Plates were blocked with 1% BSA in PBS and incubated with sera overnight at 4C. After washing, MOG-specific IgG retained by the plate-bound rMOG was detected with alkaline phosphatase-conjugated goat-anti mouse IgG F(ab')2 (Jackson Immunoresearch) and SigmaFast p-Nitrophenyl phosphate tablets with SigmaFast Tris Buffer tablets in dH2O (Sigma). Absorbance was measured at 450 nm with a μQuant plate reader and KC Junior software (Biotek) was used for data analysis.

Antibody Transfer

8.18C5 producing hybridoma cells were grown in CELLine Flasks (BD Biosciences). Supernatant was collected and recombinant antibodies were purified with a Hi-Trap Protein G Column HP (Amersham; GE). Antibodies were eluted with 0.1 M glycine into microcentrifuge tubes that contained 1 M Tris and dialyzed against PBS. Antibody concentrations were determined by absorbance at 280 nm. 200ug 8.18C5 was injected i.v. on days 7 and 9 post-transfer of encephalitogenic T cells.

Antigen Presentation Assay

The generation of the MOG-specific T cell hybridoma 204.62 was performed according to standard protocol (31). Briefly, B6 mice were immunized via footpad injection of 10 nM of MOG35-55 emulsified in CFA (Difco, Detroit, MI). Seven days following immunization, the draining popliteal lymph nodes were dispersed and fused according to standard protocols (31). Hybridomas were used in antigen presentation assays according to prior reports (25). Spleens were processed for B cell enrichment by AutoMACS (Miltenyi, Auburn, CA). Single-cell suspensions were plated, washed, and combined with antigen. A total of 5×105 APCs were cultured with 5×104 hybridoma cells with antigen overnight at 37C. Proliferation of CTLL-2 was measured after addition of supernatant by [3H]thymidine incorporation as described (31). EC50 was calculated by constructing a dose-response curve for each population of APCs, using the concentration of rMOG providing half-maximal IL-2 response by the hybridoma 204.62.

Statistics

Mann-Whitney U testing was used for comparisons of EAE severity. Student's t-tests were employed for comparisons between leukocytes and Ig values.

Results

B cell antigen presentation is required for maximal disease severity in EAE

We previously showed that antigen presentation by DCs alone is capable of mediating full disease in active EAE induced by immunization with MOG35-55 but not recombinant MOG protein (rMOG) or for the development of spontaneous optic neuritis (6). Based on these results, along with the results of B cell depletion studies in MS and EAE, we hypothesized that B cell antigen presentation cooperates with DC antigen presentation to maximize the initial response to rMOG protein during active EAE induction. To explore the possible cooperation between DC and B cell antigen presentation during the initiation of EAE, we bred mice in which MHCII expression is restricted to DCs (DCMHCII (26)) to those in which MHCII expression is restricted to B cells (BMHCII (25)), generating mice in which DCs and B cells are the only MHCII-bearing cells (DCMHCIIxBMHCII). After thymic grafting from B6 neonates, mice were immunized with rMOG according to standard protocols (6). While BMHCII and DCMHCII mice were highly resistant to rMOG-induced EAE as reported, the combination of DC and B cell MHCII expression significantly elevated the susceptibility to EAE (Fig.1A), both in terms of incidence and severity (Table I). These results suggest that B cell antigen presentation is required for enhancing the encephalitogenic response by CD4 T cells during EAE.

Fig. 1.

Fig. 1

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B cell antigen presentation is required for maximal disease severity in EAE. (A) Mean clinical scores ± SEM for WT (square; n=13) and BMHCII (open triangles; n=3) mice, along with thymic-engrafted DCMHCIIxBMHCII (grey filled diamonds; n=10) and DCMHCII (open diamonds; n=8) mice immunized with 150 μg human rMOG. (B) Mean clinical scores ± SEM of WT (square; n=3), BMHCIIxIgHMOG (circle; n=5), and BMHCII (triangle; n=3) mice that received encephalitogenic CD4 T cells. Graph represents one of three independent experiments with at least three mice per experimental group. By Mann-Whitney U test, p < 0.05 (DCMHCIIxBMHCII vs. WT). (C) Median and interquartile range of day of disease onset for WT (square; n=47) and BMHCIIxIgHMOG (circles; n=37) mice that received encephalitogenic CD4 T cells. Graph represents combined data from five independent experiments with three to ten mice per experimental group. By Mann-Whitney two-tailed test, p < 0.0001 (WT vs BMHCIIxIgHMOG).

Table I. Summary of clinical features in active and passive EAE.

Mouse group Model Incidence Day of Onset (median (range)) Maximum Disease* (median (range))
WT**(n=13) Active hMOG 92.3% 12 (9-17) 4 (2-4)
DCMHCII(n=8) Active hMOG 0% - -
DCMHCIIxBMHCII(n=10) Active hMOG 70% 15 (12-20)a 4 (1-4)
BMHCIIxIgHMOG(n=5) Active hMOG 0% - -
WT(n=41) Passive 98% 7 (5-11) 4 (1.5-5)
WT+ 8.18c5(n=11) Passive 100% 9 (7-10)b 5 (2.5-5)c
BMHCIIxIgHMOG(n=38) Passive 97% 14 (7-28)c 2 (0.5-4)c
BMHCII +8.18c5(n=12) Passive 0% - -
IAβf/f(n=9) Passive 100% 8 (6-8) 3 (0.5-3.5)
IAβf/fxCD19Cre(n=26) Passive 100% 7 (7-10) 3 (1-4)
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*

Only mice available at day 30 post-immunization were used to calculate maximum disease scores

**

WT includes B6 mice only

a

p < 0.05 by Mann-Whitney U compared to WT

b

p < 0.005 by Mann-Whitney U compared to WT

c

p < 0.0005 by Mann-Whitney U compared to WT

Based on our findings in DCMHCIIxBMHCII mice, we hypothesized that B cell antigen presentation is necessary for EAE development. Thus, we examined the requirement for B cell antigen presentation during EAE utilizing a genetic model of conditional MHCII deletion (27). We crossed IAβf/f mice (27) with CD19Cre mice to eliminate MHCII expression by B cells (IAβf/fxCD19Cre mice). Using rMOG protein as immunogen, Zamvil and colleagues reported that IAβf/fxCD19Cre mice are resistant to EAE (24). We sought to determine the influence of B cell MHCII expression during secondary phases of EAE. EAE was induced in C57BL/6 (B6) and IAβf/fxCD19Cre mice by transfer of encephalitogenic CD4 T cells. We found that IAβf/fxCD19Cre mice exhibited similar EAE severity compared with WT controls (Table I). The degree of MHCII elimination in IAβf/fxCD19Cre mice has been reported to be very high but with a variable percentage of MHCII+ cells still remaining (27). Thus, we performed flow cytometric assessment of MHCII expression in IAβf/fxCD19Cre mice during EAE, which revealed a sizeable residual expression of MHCII by B cells in blood and spleen (Supplemental Fig. 1). These findings demonstrate incomplete elimination of B cell MHCII expression in IAβf/fxCD19Cre mice with EAE, leaving open the possibility that the severity of disease in mice entirely lacking MHCII expression by B cells may be less. Nonetheless, our results demonstrate a requirement for B cell expression of MHCII during the initiation of EAE but call into question the role for B cell antigen presentation during the propagation of responses by encephalitogenic CD4 T cells during EAE.

Increasing the frequency of MOG-specific B cells permits antigen presentation by B cells to suffice for APC function in passive EAE

These results, along with the reports of several others (21, 24), suggest that B cell antigen presentation is critical to the development of CD4 T cell-mediated inflammatory CNS demyelination. However, B cell MHCII expression alone is not sufficient to support active or passive EAE (25). Thus, we sought to determine the basis of resistance to EAE in BMHCII mice, in which B cells exclusively express MHCII (25). Because cognate B and T cell interactions have been implicated during pathogenic CD4 T cell-dependent responses, including autoimmune CNS demyelination (14, 21), we reasoned that elevating the efficiency with which APCs acquire and present antigen to CD4 T cells would facilitate disease. To test this hypothesis, we increased the precursor frequency of MOG-specific B cells by crossing BMHCII mice to IgHMOG mice that express a receptor highly specific for MOG (BMHCIIxIgHMOG mice). Active immunization of BMHCIIxIgHMOG mice with rMOG did not elicit signs of EAE (Table I). Following receipt of MOG-specific encephalitogenic CD4 T cells WT mice develop clinical evidence of EAE beginning on average at day seven (Fig. 1B; Table I). In contrast, BMHCII mice were entirely resistant to EAE after transfer of encephalitogenic CD4 T cells as previously reported (25). However, BMHCIIxIgHMOG mice with greater B cell specificity for MOG did develop typical signs of EAE, beginning on average 17 days following transfer of donor T cells (Fig. 1B; Table I). Of note, a statistically significant and reproducible delay in onset was observed (Fig. 1C), and a mild, but statistically significant, reduction in disease severity was also consistently seen compared to WT mice with passive EAE (Table I).

B cell antigen presentation mediates inflammatory demyelination with reduced CNS accumulation of MHCII

The primary site of inflammatory damage in this murine system of EAE in mice on the B6 background is the spinal cord (32). Hence, we examined spinal cords from WT, BMHCII and BMHCIIxIgHMOG mice for pathologic changes. Histologic assessment revealed inflammatory infiltrates and foci of demyelination in WT spinal cords after passive EAE induction. Similarly, regions of inflammatory demyelination were observed in spinal cord samples from BMHCIIxIgHMOG mice that developed passive EAE (Fig. 2). The lack of clinical disease and the absence of myelin loss or inflammatory infiltrates within the spinal cord in BMHCII mice were consistent with previous findings(25) (Fig. 2). No differences in anatomic regions of immune cells infiltration or myelin loss were observed between WT and BMHCIIxIgHMOG mice.

Fig. 2.

Fig. 2

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Histopathological examination of spinal cord sections from formalin-fixed, paraffin-embedded tissue. Hematoxylin & eosin (H&E) staining of WT (top), BMHCII (middle) and BMHCIIxIgHMOG (bottom) mice at peak of disease (Left panels) after the induction of passive EAE. Solochrome cyanine staining for myelin of the indicated groups at peak of disease (Right Panels). Scale bar in each image = 100um. Images are illustrative of staining from at least six mice per group collected from at least three different experiments.

At both peak of disease and during chronic stages of passively-induced EAE, flow cytometric analysis of spinal cord tissue revealed a typical inflammatory composition of activated microglia with infiltrating macrophages and lymphocytes in spinal cords from both WT and BMHCIIxIgHMOG mice (Fig. 3A&B). When mice reached peak of disease, the fractions of CD45intCD11bint microglia and CD11b-CD45hi lymphocytes were elevated in BMHCIIxIgHMOG compared to WT mice. However, the absolute numbers of these cells were comparable between groups (Fig. 3C); the discrepancy between percent and absolute number of cells in part likely reflects the difference in overall composition of mononuclear cells between groups, considering the lack of endogenous CD4+ T cells in BMHCIIxIgHMOG mice. At late stages of passive EAE, no differences in frequency or absolute number of microglia or lymphocytes were found between WT and BMHCIIxIgHMOG mice (Fig. 3D). Notably, both the fraction and absolute number of MHCII-bearing cells within the spinal cord were drastically reduced in BMHCIIxIgHMOG mice compared with WT mice during the chronic phases of disease (Fig. 3C&D). Because of the requirement for antigen presentation within the CNS (4, 33), we hypothesized that an accumulation of B cells within the spinal cord during EAE in BMHCIIxIgHMOG mice would be evident. However, similar to WT mice, B cells were observed in low proportion and number at both early and late timepoints in BMHCIIxIgHMOG mice (Fig. 3C&D), consistent with prior description of minimal B cell CNS infiltration during standard models of EAE in C57BL/6 mice (34, 35). These results demonstrate that increased numbers of B cells specific for cognate antigen can function alone to propagate autoreactive CD4 T cell inflammatory responses in the CNS, resulting in typical clinico-pathologic features of EAE.

Fig. 3.

Fig. 3

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Cellular composition of spinal cord mononuclear infiltrates is similar for WT and BMHCIIxIgHMOG mice with passive EAE. (A) Flow cytometric analysis of microglia (CD45intCD11bint), activated microglia and infiltrating macrophages (CD11bhiCD45hi) and lymphocytes (CD11b-CD45hi) at peak of disease. Plots are representative of ten mice for each group from five independent experiments. (B) Flow cytometric analysis of frequencies of CD45intCD11bint, CD11bhiCD45hi, and CD11b-CD45hi mononuclear cells at chronic stages of disease. Plots are representative of five mice for each group from two independent experiments. (C) Frequencies (left) and absolute numbers (right) of CNS mononuclear cells at peak of disease. Graphs are representative of 10 mice for each group from five independent experiments. (D) Frequencies (left) and absolute numbers (right) of inflammatory CNS cells at chronic stages of disease. Graphs are representative of five mice for each group from two independent experiments. * p < 0.001; ** p < 0.05; *** p < 0.01 by two-tailed Student's t-test.

MOG-specific immunoglobulin facilitates EAE independently of serum Ig levels

Approximately 30% of B cells from IgHMOG mice express BCR specific for MOG (10). While soluble Ig can facilitate CNS demyelination (36-38), elevating the BCR specificity for MOG is also likely to enhance recognition of target antigen along with increasing the efficiency of antigen processing and presentation by B cells (39, 40). We sought to distinguish the role of soluble MOG-specific Ig in B cell-mediated EAE. Thus, we quantified serum Ig recognizing MOG protein and its conformational epitopes in WT, BMHCII, and BMHCIIxIgHMOG mice. As expected, low levels of serum MOG-specific IgG were detected in WT mice with passive EAE (Fig. 4A). These low levels of anti-MOG IgG were similar to the levels found in WT or BMHCII mice without disease (Fig. 4A). In contrast, serum from naïve BMHCIIxIgHMOG mice contained approximately twice the amount of MOG-specific IgG in comparison to WT and BMHCII mice (p < 0.0001; Fig. 4A). The level of MOG-specific IgG increased after the development of passive EAE in BMHCIIxIgHMOG mice, but this difference was not statistically significant (p = 0.07; Fig. 4A). Serial dilutions were performed to quantify the range of IgG in each group (Fig. 4B). These results suggest that the presence of serum MOG-specific Ig may act to facilitate EAE in BMHCIIxIgHMOG mice. Transfer of the MOG-specific monoclonal antibody 8.18C5 into animals exhibiting clinical signs of EAE is known to increase disease severity and speed the course of disease (9, 38) or initiate disease after immunization in B cell deficient mice (36). Thus, we examined whether soluble Ig specific for MOG can trigger disease induction in BMHCII mice. Transfer of 8.18C5 seven days after induction of EAE resulted in significantly elevated levels of serum MOG-specific IgG (p = 0.0006; Fig. 4A). As expected, transfer of 8.18C5 resulted in enhanced disease severity in WT mice (Fig. 5), demonstrating the efficacy of 8.18C5 in the positive control arm of the experiment. In contrast, administration of 8.18C5 to BMHCII mice did not alter resistance to the development of EAE (Fig. 5). Identical EAE resistance was observed when serum from protein-immunized WT mice with EAE was administered to BMHCII mice (data not shown).

Fig. 4.

Fig. 4

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Soluble MOG-specific IgG is not sufficient to facilitate passive EAE in BMHCII mice. Sera were obtained from naive WT (n=3), WT with EAE (n=12), naïve BMHCIIxIgHMOG (n=5), BMHCIIxIgHMOG with EAE (n=16), BMHCII recipients of encephalitogenic CD4 T cells (n=13) mice and BMHCII recipients of encephalitogenic CD4 T cells plus 8.18C5 (n=2) mice beyond peak of disease from three independent experiments. Serum was assayed by ELISA using MOG protein. (A) MOG-specific IgG serum titers at 1/1000 dilution expressed as mean OD at 405nm ± SEM determined by duplicate samples using ELISA. (B) rMOG-specific IgG titers from WT (square), naïve BMHCIIxIgHMOG (open circle), BMHCIIxIgHMOG with EAE (shaded circle), BMHCII (triangle) and BMHCII recipients of 8.18C5 (X) mice expressed as mean OD at 405nm ± SEM from duplicate serial dilutions as indicated on the x-axis.

Fig. 5.

Fig. 5

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Mean clinical scores of WT (n=12) and BMHCII (n=12) mice, half of which were treated with MOG-specific antibody 8.18C5 and half treated with PBS seven and nine days after induction of passive EAE. p < 0.0005 by Mann-Whitney U test comparing WT receiving 8.18C5 vs. WT.

MOG-specific BCR enhances antigen-specific responses by CD4 T cells

As soluble MOG-specific IgG was not sufficient to induce disease in our model system, we hypothesized that BMHCIIxIgHMOG mice are susceptible to disease as a result of enhanced detection, acquisition, and presentation of self-antigens by B cells due to the higher frequency of MOG-specific B cells. To test this, we co-cultured a highly sensitive MOG-specific T cell hybridoma with rMOG and B cells from WT, BMHCII, or BMHCIIxIgHMOG spleens. Whole WT spleens were capable of stimulating MOG-specific CD4 T cells. In comparison, MOG-specific CD4 T cell responses were slightly reduced with populations of B cells enriched from WT spleens (Fig. 6A). A substantial elevation in T cell responses elicited from BMHCIIxIgHMOG mice splenic B cells compared with WT B cells was observed, even more so when compared with BMHCII B cells (Fig. 6A). Specifically, half-maximal stimulation was achieved at 381 nM of rMOG using B cells enriched from WT spleens in comparison to 70.81 nM rMOG using B cells from BMHCIIxIgHMOG mice. Of note, processing and presentation of rMOG by DCs in DCMHCII mice is not deficient (Fig. 6B and C). These results demonstrate the exquisite capacity for B cells from BMHCIIxIgHMOG mice to capture and present antigen to CD4 T cells which reflects passive EAE induction when B cells are the only functional APC.

Fig. 6.

Fig. 6

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(A) WT splenocytes (black square) or MACS-enriched B cells from WT (open square), BMHCIIxIgHMOG (circle) or BMHCII (triangle) spleens were cultured with the MOG-specific CD4 T cell hybridoma, 204.62 and serial dilutions of rMOG in triplicate. Proliferation of CTLL-2 cells incubated with supernatants was measured by [3H]thymidine incorporation. Graph is representative of two independent experiments. (B) Splenocytes from WT (black square), BMHCII (triangle), BMHCIIxIgHMOG (circle), DCMHCII (open diamond), or DCMHCIIxBMHCII (grey filled diamonds) spleens were cultured with the MOG-specific CD4 T cell hybridoma, 204.62 and serial dilutions of rMOG in triplicate. Proliferation of CTLL-2 cells incubated with supernatants was measured by [3H]thymidine incorporation. Graph is representative of three independent experiments. (C) Splenocytes from WT (black square), BMHCII (triangle), BMHCIIxIgHMOG (circle), DCMHCII (open diamond), or DCMHCIIxBMHCII (grey filled diamonds) spleens were cultured with the MOG-specific CD4 T cell hybridoma, 204.62 and serial dilutions of MOG35-55 peptide in triplicate. Proliferation of CTLL-2 cells incubated with supernatants was measured by [3H]thymidine incorporation. Graph is representative of three independent experiments.

Discussion

Based on our in vivo system of conditional MHCII expression, we originally observed that B cell antigen presentation alone was insufficient to initiate or propagate EAE (25). In our present study we find that B cells are capable of independently performing APC functions during EAE, but only when enough B cells recognize cognate antigen. This is the first demonstration that B cells can serve all antigen presentation functions during passive EAE. The importance of B cell antigen presentation during the initiation of autoimmune CNS demyelination is also highlighted by our finding that B cells cooperate with DCs to greatly augment disease severity in an active immunization EAE model. These results reveal the capacity of B cells to directly regulate cognate CD4 T cell responses during autoimmunity and highlight the selective ability for B cell antigen presentation to propagate CD4 T cell auto-reactivity targeting the CNS.

Molnarfi et al (24) recently developed a system for studying B cell antigen presentation that is complementary to the model used in the present study. Using a T cell- and B cell-dependent active immunization model of EAE (7), they demonstrated the necessity of B cell expression of MHCII, rather than antibody production, for initiating a pathogenic T cell response during neuro-inflammation. In contrast, our work demonstrates the functional cooperation between DC and B cell antigen presentation during the initiation of active EAE, as well as the sufficiency of B cell antigen presentation for disease progression in passive EAE. Certainly other APCs are capable of complementing DCs in the activation of autoreactive CD4 T cell responses. Macrophage and monocyte APCs have been implicated in the initiation of EAE (41), and microglia have long been suspected of facilitating CD4 T cell autoreactivity in EAE (42). Nonetheless, our results implicate a role for B cell antigen presentation in EAE and suggest potential value in targeting cognate B cell-T cell interactions in MS.

The requirement for MOG-specific B cell receptors (BCRs) to facilitate EAE in our system suggests that MOG-specific Ig may be critical for disease induction. However, we were unable to elicit disease in BMHCII mice by addition of either soluble Ig specific for MOG or serum from immunized WT mice. These findings are consistent with the hypothesis that membrane bound BCR specific for MOG is critical to the propagation of CD4 T cell autoreactivity, likely for efficient capture of target antigen. Indeed, recently published work by Zamvil and colleagues demonstrates a critical role for B cell APC function independent of Ig secretion (24). Immunization with rhMOG is a well-characterized B cell-dependent model of active EAE (7, 36, 43). However, there are critical differences in amino acid sequences of the main encephalitogenic peptide (MOG35-55) processed from human and rodent MOG protein that could affect the immune response through altered antigen detection, processing, and presentation. Using rodent rMOG allowed us to study a relevant autoimmune reaction in BMHCIIxIgHMOG mice, as the 8.18c5 heavy chain of IgHMOG mice was developed specifically in response to rodent MOG.

To confirm the Ig-independent nature of our EAE model, we treated BMHCII mice that had received encephalitogenic T cells with either 8.18c5 or serum from rMOG immunized WT mice. While previous research indicates that serum from WT mice is not pathogenic, we used this to control for the possible immune-activating effects of serum components like cytokines and chemokines. Additionally, our ex vivo antigen presentation results support the hypothesis that MOG-specific BCR enhances the ability for B cells to present cognate antigen, with B cells from BMHCIIxIgHMOG mice eliciting approximately five-fold greater stimulation of MOG-specific CD4 T cells compared with WT B cells. These data do not detract from the pathogenic role of soluble MOG-specific Ig in vivo, but rather demonstrate that a distinct pathway dependent upon membrane-bound MOG-specific Ig is critical at later stages during EAE as a mechanism to capture and process target antigen.

We believe that our work may have implications regarding the pathogenesis of MS. The presence of intrathecally generated oligoclonal Ig has been used as a diagnostic feature of MS (2). While the targets of these antibodies remains unclear(44, 45), the presence of oligoclonal Ig in the CSF space in most patients with MS raises the important question about our model of where B cell cognate interactions with T cells occurs during disease. Clonal expansion and Ig class switching and secretion are direct results of B cell antigen presentation to cognate T cells. Indeed, Lambracht-Washington, et al reported that clonally expanded B cells from the CSF of MS patients produce Ig with reactivities to myelin basic protein and some polyreactivity to GFAP and CNPase (46). In our model, up to 30% of B cells recognize a CNS-specific antigen, a situation that perhaps reflects the prevalence or expansion of B cells in MS patients. Whether or not B cells acquire and present antigen in the CNS compartment is a critical question, one that is amenable to address using our unique system.

We therefore propose a model in which myelin auto-reactivity initiates when B cells cooperate with DCs to prime CD4 T cells, followed by germinal center reactions and B cell clonal expansion that results in B cells assuming a primary role in antigen presentation to propagate CD4 T cell responses. The number of B cells expressing BCRs specific for cognate auto-antigen is thus highly relevant in promoting the likelihood of B and T cell interactions to perpetuate disease. While the upper limit of antigen-specific B cells required for disease in our model has been defined as approximately 30% by previous characterizations of the IgHMOG mouse (10), our attempts to define a threshold for auto-reactive B cells in EAE initially involved administration of rMOG and adjuvant several days prior to the initiation of passive EAE in BMHCII mice. BMHCII mice treated with rMOG and CpG or rMOG with CpG and anti-CD40 antibody were resistant to passive EAE (data not shown). Thus, activating MOG-specific B cells via pre-conditioning with antigen and adjuvant does not overcome the barrier to EAE when B cells function as the sole APC. Nonetheless, a logical extension of our findings is identifying the clinically relevant threshold for B cell antigen specificity. Theoretically, enhanced efficacy of B cell depletion therapy could be achieved by specifically targeting auto-reactive B cells that have expanded to deleterious levels, a concept that is recognized (e.g. (46)) but impeded by the confusion over actual CNS targets of lymphocytes. Perhaps antigen-specific targeting of B cells is more practical in neuromyelitis optica, an inflammatory disease similar to MS in which B and T cell auto-reactivity toward a specific protein target - aquaporin 4 - is defined (47).

What remains to be demonstrated is the requirement for DCs at later stages of EAE and MS. In fact, this is highly controversial given the recent observation that DC depletion may exacerbate EAE (48). However, in contrast to DCs that are short-lived, B cells can mature into long-lived memory phenotype with the capacity to indefinitely extend autoimmune responses. Additionally, depletion of B cells may unleash potent pro-inflammatory monocyte-derived APCs in the setting of MS (49), and the complexity of B cell targeted therapy in MS has recently been highlighted by the recent findings of the clinical trial using the B cell depleting agent Atacicept that led to exacerbation of MS rather than improvement (50).

Our data demonstrate that minimal MHCII expression within the CNS is required during the propagation of EAE. In light of the requirement for the generation of target antigens from CNS tissue (51, 52), these data suggest that myelin antigens derived from the CNS can be generated in sufficient quantity by a relatively minimal number of APCs. The small numbers of B cells observed in the CNS of BMHCIIxIgHMOG mice that develop EAE may be a result of B cells being more facile in capturing soluble protein antigens. Alternatively, protein antigens may be directly targeted from the periphery by B cells after an initial breach of the CNS compartment, particularly by highly activated T cells. This would serve as an early step in the genesis of cognate antigens resulting in the delay in disease observed in BMHCIIxIgHMOG mice compared with WT mice. In specific circumstances, accumulation of B cells in the CNS during MS and EAE does occur in the form of ectopic lymphoid follicles (34, 53). Whether this process requires MHCII has yet to be explored.

Supplementary Material

1

Acknowledgments

We thank Gurumoorthy Krishnamoorthy and Hartmut Wekerle for their generous provision of the IgHMOG mice and advice on experimental approaches. We would like to thank Drs. Anne Cross, Robyn Klein, Raj Apte, Emil Unanue and Laura Piccio for critical review of the manuscript, as well as Soomin Shin and Gretchen McGee for other technical support. We would like to acknowledge the technical assistance of Bryan Bollman for completion of the histochemical experiments.

Footnotes

1

Funded by the National Institutes of Health (NINDS) grants K08NS062138 and 1R01NS083678.

References

  • 1.Compston A, McDonald IR, Noseworthy J, Lassmann H, Miller DH, Smith KJ, Wekerle H, Confavreux C. McAlpine's Multiple Sclerosis. Churchill Livingstone; Philadelphia: 2006. [Google Scholar]
  • 2.Frohman EM, Racke MK, Raine CS. Multiple sclerosis--the plaque and its pathogenesis. N Engl J Med. 2006;354:942–955. doi: 10.1056/NEJMra052130. [DOI] [PubMed] [Google Scholar]
  • 3.Chastain EM, Duncan DS, Rodgers JM, Miller SD. The role of antigen presenting cells in multiple sclerosis. Biochim Biophys Acta. 2011;1812:265–274. doi: 10.1016/j.bbadis.2010.07.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Becher B, Bechmann I, Greter M. Antigen presentation in autoimmunity and CNS inflammation: how T lymphocytes recognize the brain. J Mol Med. 2006;84:532–543. doi: 10.1007/s00109-006-0065-1. [DOI] [PubMed] [Google Scholar]
  • 5.Wu GF, Alvarez E. The immunopathophysiology of multiple sclerosis. Neurol Clin. 2011;29:257–278. doi: 10.1016/j.ncl.2010.12.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Wu GF, Shindler KS, Allenspach EJ, Stephen TL, Thomas HL, Mikesell RJ, Cross AH, Laufer TM. Limited sufficiency of antigen presentation by dendritic cells in models of central nervous system autoimmunity. J Autoimmun. 2011;36:56–64. doi: 10.1016/j.jaut.2010.10.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Oliver AR, Lyon GM, Ruddle NH. Rat and human myelin oligodendrocyte glycoproteins induce experimental autoimmune encephalomyelitis by different mechanisms in C57BL/6 mice. J Immunol. 2003;171:462–468. doi: 10.4049/jimmunol.171.1.462. [DOI] [PubMed] [Google Scholar]
  • 8.Genain CP, Nguyen MH, Letvin NL, Pearl R, Davis RL, Adelman M, Lees MB, Linington C, Hauser SL. Antibody facilitation of multiple sclerosis-like lesions in a nonhuman primate. J Clin Invest. 1995;96:2966–2974. doi: 10.1172/JCI118368. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Pollinger B, Krishnamoorthy G, Berer K, Lassmann H, Bosl MR, Dunn R, Domingues HS, Holz A, Kurschus FC, Wekerle H. Spontaneous relapsing-remitting EAE in the SJL/J mouse: MOG-reactive transgenic T cells recruit endogenous MOG-specific B cells. J Exp Med. 2009;206:1303–1316. doi: 10.1084/jem.20090299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Litzenburger T, Fassler R, Bauer J, Lassmann H, Linington C, Wekerle H, Iglesias A. B lymphocytes producing demyelinating autoantibodies: development and function in gene-targeted transgenic mice. J Exp Med. 1998;188:169–180. doi: 10.1084/jem.188.1.169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Bettelli E, Baeten D, Jager A, Sobel RA, Kuchroo VK. Myelin oligodendrocyte glycoprotein-specific T and B cells cooperate to induce a Devic-like disease in mice. J Clin Invest. 2006;116:2393–2402. doi: 10.1172/JCI28334. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Krishnamoorthy G, Lassmann H, Wekerle H, Holz A. Spontaneous opticospinal encephalomyelitis in a double-transgenic mouse model of autoimmune T cell/B cell cooperation. J Clin Invest. 2006;116:2385–2392. doi: 10.1172/JCI28330. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.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;118:3420–3430. doi: 10.1172/JCI36030. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Weber MS, Prod'homme T, Patarroyo JC, Molnarfi N, Karnezis T, Lehmann-Horn K, Danilenko DM, Eastham-Anderson J, Slavin AJ, Linington C, Bernard CC, Martin F, Zamvil SS. B-cell activation influences T-cell polarization and outcome of anti-CD20 B-cell depletion in central nervous system autoimmunity. Ann Neurol. 2010;68:369–383. doi: 10.1002/ana.22081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Matsushita T, Horikawa M, Iwata Y, Tedder TF. Regulatory B cells (B10 cells) and regulatory T cells have independent roles in controlling experimental autoimmune encephalomyelitis initiation and late-phase immunopathogenesis. J Immunol. 2010;185:2240–2252. doi: 10.4049/jimmunol.1001307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Barr TA, Shen P, Brown S, Lampropoulou V, Roch T, Lawrie S, Fan B, O'Connor RA, Anderton SM, Bar-Or A, Fillatreau S, Gray D. B cell depletion therapy ameliorates autoimmune disease through ablation of IL-6-producing B cells. J Exp Med. 2012;209:1001–1010. doi: 10.1084/jem.20111675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Shen P, Roch T, Lampropoulou V, O'Connor RA, Stervbo U, Hilgenberg E, Ries S, Dang VD, Jaimes Y, Daridon C, Li R, Jouneau L, Boudinot P, Wilantri S, Sakwa I, Miyazaki Y, Leech MD, McPherson RC, Wirtz S, Neurath M, Hoehlig K, Meinl E, Grutzkau A, Grun JR, Horn K, Kuhl AA, Dorner T, Bar-Or A, Kaufmann SH, Anderton SM, Fillatreau S. IL-35-producing B cells are critical regulators of immunity during autoimmune and infectious diseases. Nature. 2014;507:366–370. doi: 10.1038/nature12979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Hauser SL, Waubant E, Arnold DL, Vollmer T, Antel J, Fox RJ, Bar-Or A, Panzara M, Sarkar N, Agarwal S, Langer-Gould A, Smith CH. B-cell depletion with rituximab in relapsing-remitting multiple sclerosis. N Engl J Med. 2008;358:676–688. doi: 10.1056/NEJMoa0706383. [DOI] [PubMed] [Google Scholar]
  • 19.Lucchinetti CF, Bruck W, Lassmann H. Evidence for pathogenic heterogeneity in multiple sclerosis. Ann Neurol. 2004;56:308. doi: 10.1002/ana.20182. [DOI] [PubMed] [Google Scholar]
  • 20.Franciotta D, Columba-Cabezas S, Andreoni L, Ravaglia S, Jarius S, Romagnolo S, Tavazzi E, Bergamaschi R, Zardini E, Aloisi F, Marchioni E. Oligoclonal IgG band patterns in inflammatory demyelinating human and mouse diseases. J Neuroimmunol. 2008;200:125–128. doi: 10.1016/j.jneuroim.2008.06.004. [DOI] [PubMed] [Google Scholar]
  • 21.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 T-cell-mediated disease in MS? Ann Neurol. 2010;67:452–461. doi: 10.1002/ana.21939. [DOI] [PubMed] [Google Scholar]
  • 22.Cross AH, Stark JL, Lauber J, Ramsbottom MJ, Lyons JA. Rituximab reduces B cells and T cells in cerebrospinal fluid of multiple sclerosis patients. J Neuroimmunol. 2006;180:63–70. doi: 10.1016/j.jneuroim.2006.06.029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Piccio L, Naismith RT, Trinkaus K, Klein RS, Parks BJ, Lyons JA, Cross AH. Changes in B- and T-lymphocyte and chemokine levels with rituximab treatment in multiple sclerosis. Arch Neurol. 2010;67:707–714. doi: 10.1001/archneurol.2010.99. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Molnarfi N, Schulze-Topphoff U, Weber MS, Patarroyo JC, Prod'homme T, Varrin-Doyer M, Shetty A, Linington C, Slavin AJ, Hidalgo J, Jenne DE, Wekerle H, Sobel RA, Bernard CC, Shlomchik MJ, Zamvil SS. MHC class II-dependent B cell APC function is required for induction of CNS autoimmunity independent of myelin-specific antibodies. J Exp Med. 2013;210:2921–2937. doi: 10.1084/jem.20130699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Archambault AS, Carrero JA, Barnett LG, McGee NG, Sim J, Wright JO, Raabe T, Chen P, Ding H, Allenspach EJ, Dragatsis I, Laufer TM, Wu GF. Cutting edge: Conditional MHC class II expression reveals a limited role for B cell antigen presentation in primary and secondary CD4 T cell responses. J Immunol. 2013;191:545–550. doi: 10.4049/jimmunol.1201598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Lemos MP, Fan L, Lo D, Laufer TM. CD8alpha+ and CD11b+ dendritic cell-restricted MHC class II controls Th1 CD4+ T cell immunity. J Immunol. 2003;171:5077–5084. doi: 10.4049/jimmunol.171.10.5077. [DOI] [PubMed] [Google Scholar]
  • 27.Shimoda M, Li T, Pihkala JP, Koni PA. Role of MHC class II on memory B cells in post-germinal center B cell homeostasis and memory response. J Immunol. 2006;176:2122–2133. doi: 10.4049/jimmunol.176.4.2122. [DOI] [PubMed] [Google Scholar]
  • 28.Racke MK. Experimental autoimmune encephalomyelitis (EAE) Curr Protoc Neurosci. 2001;Chapter 9 doi: 10.1002/0471142301.ns0907s14. Unit9 7. [DOI] [PubMed] [Google Scholar]
  • 29.Archambault AS, Sim J, Gimenez MA, Russell JH. Defining antigen-dependent stages of T cell migration from the blood to the central nervous system parenchyma. Eur J Immunol. 2005;35:1076–1085. doi: 10.1002/eji.200425864. [DOI] [PubMed] [Google Scholar]
  • 30.Shin S, Walz KA, Archambault AS, Sim J, Bollman BP, Koenigsknecht-Talboo J, Cross AH, Holtzman DM, Wu GF. Apolipoprotein E mediation of neuro-inflammation in a murine model of multiple sclerosis. J Neuroimmunol. 2014;271:8–17. doi: 10.1016/j.jneuroim.2014.03.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Lovitch SB, Walters JJ, Gross ML, Unanue ER. APCs present A beta(k)-derived peptides that are autoantigenic to type B T cells. J Immunol. 2003;170:4155–4160. doi: 10.4049/jimmunol.170.8.4155. [DOI] [PubMed] [Google Scholar]
  • 32.Stromnes IM, Goverman JM. Active induction of experimental allergic encephalomyelitis. Nat Protoc. 2006;1:1810–1819. doi: 10.1038/nprot.2006.285. [DOI] [PubMed] [Google Scholar]
  • 33.Bartholomaus I, Kawakami N, Odoardi F, Schlager C, Miljkovic D, Ellwart JW, Klinkert WE, Flugel-Koch C, Issekutz TB, Wekerle H, Flugel A. Effector T cell interactions with meningeal vascular structures in nascent autoimmune CNS lesions. Nature. 2009;462:94–98. doi: 10.1038/nature08478. [DOI] [PubMed] [Google Scholar]
  • 34.Peters A, Pitcher LA, Sullivan JM, Mitsdoerffer M, Acton SE, Franz B, Wucherpfennig K, Turley S, Carroll MC, Sobel RA, Bettelli E, Kuchroo VK. Th17 cells induce ectopic lymphoid follicles in central nervous system tissue inflammation. Immunity. 2011;35:986–996. doi: 10.1016/j.immuni.2011.10.015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.McGeachy MJ, Stephens LA, Anderton SM. Natural recovery and protection from autoimmune encephalomyelitis: contribution of CD4+CD25+ regulatory cells within the central nervous system. J Immunol. 2005;175:3025–3032. doi: 10.4049/jimmunol.175.5.3025. [DOI] [PubMed] [Google Scholar]
  • 36.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;32:1905–1913. doi: 10.1002/1521-4141(200207)32:7<1905::AID-IMMU1905>3.0.CO;2-L. [DOI] [PubMed] [Google Scholar]
  • 37.Weber MS, Hemmer B, Cepok S. The role of antibodies in multiple sclerosis. Biochim Biophys Acta. 2011;1812:239–245. doi: 10.1016/j.bbadis.2010.06.009. [DOI] [PubMed] [Google Scholar]
  • 38.Schluesener HJ, Sobel RA, Linington C, Weiner HL. A monoclonal antibody against a myelin oligodendrocyte glycoprotein induces relapses and demyelination in central nervous system autoimmune disease. J Immunol. 1987;139:4016–4021. [PubMed] [Google Scholar]
  • 39.Lanzavecchia A. Antigen-specific interaction between T and B cells. Nature. 1985;314:537–539. doi: 10.1038/314537a0. [DOI] [PubMed] [Google Scholar]
  • 40.Pape KA, Catron DM, Itano AA, Jenkins MK. The humoral immune response is initiated in lymph nodes by B cells that acquire soluble antigen directly in the follicles. Immunity. 2007;26:491–502. doi: 10.1016/j.immuni.2007.02.011. [DOI] [PubMed] [Google Scholar]
  • 41.Ajami B, Bennett JL, Krieger C, McNagny KM, Rossi FM. Infiltrating monocytes trigger EAE progression, but do not contribute to the resident microglia pool. Nat Neurosci. 2011;14:1142–1149. doi: 10.1038/nn.2887. [DOI] [PubMed] [Google Scholar]
  • 42.Heppner FL, Greter M, Marino D, Falsig J, Raivich G, Hovelmeyer N, Waisman A, Rulicke T, Prinz M, Priller J, Becher B, Aguzzi A. Experimental autoimmune encephalomyelitis repressed by microglial paralysis. Nat Med. 2005;11:146–152. doi: 10.1038/nm1177. [DOI] [PubMed] [Google Scholar]
  • 43.Marta CB, Oliver AR, Sweet RA, Pfeiffer SE, Ruddle NH. Pathogenic myelin oligodendrocyte glycoprotein antibodies recognize glycosylated epitopes and perturb oligodendrocyte physiology. Proc Natl Acad Sci U S A. 2005;102:13992–13997. doi: 10.1073/pnas.0504979102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.O'Connor KC, Appel H, Bregoli L, Call ME, Catz I, Chan JA, Moore NH, Warren KG, Wong SJ, Hafler DA, Wucherpfennig KW. Antibodies from inflamed central nervous system tissue recognize myelin oligodendrocyte glycoprotein. J Immunol. 2005;175:1974–1982. doi: 10.4049/jimmunol.175.3.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Owens GP, Bennett JL, Lassmann H, O'Connor KC, Ritchie AM, Shearer A, Lam C, Yu X, Birlea M, DuPree C, Williamson RA, Hafler DA, Burgoon MP, Gilden D. Antibodies produced by clonally expanded plasma cells in multiple sclerosis cerebrospinal fluid. Ann Neurol. 2009;65:639–649. doi: 10.1002/ana.21641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Lambracht-Washington D, O'Connor KC, Cameron EM, Jowdry A, Ward ES, Frohman E, Racke MK, Monson NL. Antigen specificity of clonally expanded and receptor edited cerebrospinal fluid B cells from patients with relapsing remitting MS. J Neuroimmunol. 2007;186:164–176. doi: 10.1016/j.jneuroim.2007.03.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Varrin-Doyer M, Spencer CM, Schulze-Topphoff U, Nelson PA, Stroud RM, Cree BAC, Zamvil SS. Aquaporin 4-specific T cells in neuromyelitis optica exhibit a Th17 bias and recognize Clostridium ABC transporter. Annals of Neurology. 2012;72:53–64. doi: 10.1002/ana.23651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Yogev N, Frommer F, Lukas D, Kautz-Neu K, Karram K, Ielo D, von Stebut E, Probst HC, van den Broek M, Riethmacher D, Birnberg T, Blank T, Reizis B, Korn T, Wiendl H, Jung S, Prinz M, Kurschus FC, Waisman A. Dendritic cells ameliorate autoimmunity in the CNS by controlling the homeostasis of PD-1 receptor(+) regulatory T cells. Immunity. 2012;37:264–275. doi: 10.1016/j.immuni.2012.05.025. [DOI] [PubMed] [Google Scholar]
  • 49.Lehmann-Horn K, Schleich E, Hertzenberg D, Hapfelmeier A, Kumpfel T, von Bubnoff N, Hohlfeld R, Berthele A, Hemmer B, Weber MS. Anti-CD20 B-cell depletion enhances monocyte reactivity in neuroimmunological disorders. J Neuroinflammation. 2011;8:146. doi: 10.1186/1742-2094-8-146. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Kappos L, Hartung HP, Freedman MS, Boyko A, Radü EW, Mikol DD, Lamarine M, Hyvert Y, Freudensprung U, Plitz T, van Beek J. Atacicept in multiple sclerosis (ATAMS): a randomised, placebo-controlled, double-blind, phase 2 trial. The Lancet Neurology. 2014;13:353–363. doi: 10.1016/S1474-4422(14)70028-6. [DOI] [PubMed] [Google Scholar]
  • 51.Slavin AJ, Soos JM, Stuve O, Patarroyo JC, Weiner HL, Fontana A, Bikoff EK, Zamvil SS. Requirement for endocytic antigen processing and influence of invariant chain and H-2M deficiencies in CNS autoimmunity. J Clin Invest. 2001;108:1133–1139. doi: 10.1172/JCI13360. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Tompkins SM, Padilla J, Dal Canto MC, Ting JP, Van Kaer L, Miller SD. De novo central nervous system processing of myelin antigen is required for the initiation of experimental autoimmune encephalomyelitis. J Immunol. 2002;168:4173–4183. doi: 10.4049/jimmunol.168.8.4173. [DOI] [PubMed] [Google Scholar]
  • 53.Magliozzi R, Columba-Cabezas S, Serafini B, Aloisi F. Intracerebral expression of CXCL13 and BAFF is accompanied by formation of lymphoid follicle-like structures in the meninges of mice with relapsing experimental autoimmune encephalomyelitis. J Neuroimmunol. 2004;148:11–23. doi: 10.1016/j.jneuroim.2003.10.056. [DOI] [PubMed] [Google Scholar]

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