IL-5-deficient mice are susceptible to experimental autoimmune encephalomyelitis
Catherine Weir1,
Claude C. A. Bernard2 and
B. Thomas Bäckström1
1 Malaghan Institute of Medical Research, Department of Pathology and Molecular Medicine, Wellington School of Medicine, PO Box 7060, Wellington South, New Zealand 2 Neuroimmunology Laboratory, Department of Biochemistry, La Trobe University, Victoria, 3086, Australia
Correspondence to: T. Bäckström; E-mail: tbackstrom{at}malaghan.org.nz
Transmitting editor: D. Tarlinton
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Abstract
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Experimental autoimmune encephalomyelitis (EAE) is an animal model commonly used to investigate mechanisms involved in the activation of self-reactive T cells. Whereas auto-reactive Th1 cells are believed to be involved in the generation of EAE, Th2 cells can induce EAE in immunocompromised hosts. Since the Th2 cytokine IL-5 can influence the nature and severity of disease, we investigated the role of IL-5 in the EAE model. Wild-type C57BL/6J and IL-5/ mice were immunized with myelin oligodendrocyte glycoprotein (MOG)3555 peptide and the development of EAE observed. Our results show that IL-5/ mice developed EAE with a similar day of onset and comparable severity to wild-type mice. Primed T cells isolated from IL-5/ mice proliferated equally to wild-type cells in response to antigen challenge with MOG3555. Antigen-specific T cells from IL-5/ mice produced IFN-
and tumor necrosis factor-
, but no IL-4 or IL-10, indicating that a predominant Th1 environment was induced following immunization. No differences in the types of cells infiltrating into the central nervous system were observed between IL-5/ and wild-type mice. Our results suggest that IL-5 is not directly involved in the initiation or effector phase of MOG3555-induced EAE in immunocompetent C57BL/6J mice.
Keywords: autoimmunity, experimental autoimmune encephalomyelitis, IL-5, multiple sclerosis, Th1, Th2
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Introduction
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Experimental autoimmune encephalomyelitis (EAE) is accepted as the principal animal model of the human disease multiple sclerosis (MS). EAE can be induced in experimental animals by immunization with homogenized central nervous system (CNS) tissue, purified myelin or specific myelin antigens together with complete Freunds adjuvant (CFA) and pertussis toxin (1). Immunization generally produces an inflammatory response that activates macrophages and dendritic cells (DC). This process leads to up-regulation of MHC, co-stimulatory molecules and presentation of myelin antigens to self-reactive CD4+ T cells (2). Recent research has shown that DC expressing high levels of MHC class II and B7 molecules can efficiently present myelin oligodendrocyte glycoprotein (MOG) peptides to naive T cells and induce EAE, suggesting that DC play a critical role in the initiation of this autoimmune disease (35). Presentation of MOG peptides initiates sufficient T cell activation that results in proliferation and cytokine production of self-reactive CD4+ T cells. These CD4+ T cells then migrate to the CNS where they recognize the myelin antigen. Finally, these events lead to the secretion of inflammatory cytokines responsible for the immunopathology found in EAE.
CD4+ T cells can be divided into two subsets based on the array of cytokines they produce upon antigenic stimulation (6). Cells of the Th1 phenotype are primarily involved in cell-mediated immunity, and produce the inflammatory cytokines IL-2, IFN-
, tumor necrosis factor (TNF)-
and lymphotoxin. Th2 cells are involved in the humoral immune response, and produce the cytokines IL-4, IL-5, IL-6, IL-10 and IL-13 (7,8). After induction of an immune response with either a foreign or self-antigen, it has been found that the response is dominated by one particular Th subset over another. For example, the generation of an inflammatory Th1 response is essential for the clearance of intracellular pathogens. In addition, encephalitogenic T cells of the Th1 phenotype are common to most models of EAE. Transfer of CD4+ Th1 cells specific for the myelin antigens myelin basic protein (MBP), proteolipid protein or MOG induce EAE (1). The cytokines produced by the Th1 cells activate microglial cells and other cells present in the CNS, which have been demonstrated in conjunction with astrocytes to phagocytose myelin (9). In addition, macrophages and some polymorphonuclear cells, B cells and CD8+ T cells may also enter the CNS with the T cells, which in the presence of the inflammatory cytokines attack the myelin surrounding the nerve cell (10,11). In contrast to Th1, cells of the Th2 phenotype are responsible for the clearance of helminth infections and have also been implicated in the pathogenesis of systemic autoimmune diseases via the production of autoantibodies (12). In the EAE model, non-encephalitogenic Th2 cells recognizing the same peptide can inhibit the proliferation of encephalitogenic T cells via the production of IL-10 (13). Conversely, auto-reactive Th2 cells can, under special circumstances, induce rather than protect against EAE (14,15). It is, therefore, important to investigate the role of both Th1- and Th2-associated cytokines on EAE.
Not only is EAE characterized by infiltration of inflammatory antigen-specific T cells into the CNS, but also by increased bloodbrain barrier permeability, astrocytic hypertrophy and demyelination (16). During clinical attacks of EAE there is an increased expression of IL-1, IL-2, IFN-
, TNF-
, perforin and IL-6 in the CNS, whilst during remission there is a decline in the expression of these cytokines and increased expression of transforming growth factor-ß and IL-10 (17,18). These results indicate that disease induction may be a product of Th1 cells, and recovery from EAE results from a shift to Th2 cells and their cytokines. Along these lines, during EAE, the presence of IL-4 in the target organ is sufficient to down-regulate auto-reactive Th1 cells (17,18). Treatment with rIL-4 suppresses the induction of EAE and mice unable to produce IL-4 show increased clinical signs of EAE (19,20). This suggests that the ability of Th2 responses to modify the induction of Th1 CD4+ T cells in vivo is dependent on IL-4. Another Th2 cytokine with a diverse role is IL-5, which has been demonstrated to be an important chemotactic factor for the recruitment of eosinophils to target tissue such as the lung during airway-induced eosinophilia (21,22). In the EAE model, the absence of IFN-
results in large numbers of polymorphonuclear cells entering the CNS, associated with the development of a rapidly progressing lethal disease (23,24). Increased numbers of polymorphonuclear cells entering the CNS are also found after adoptive transfer of MBP-specific Th2 lines, expressing high levels of IL-5 (25). Similar to IFN-
/ mice, the IL-5-expressing MBP-specific Th2 lines induce a mortal non-classical form of EAE. Together, these results indicate that Th2 cells and cytokines possess the potential to be involved in autoimmune diseases. However, little is known about the role of IL-5 during the course of EAE in immunocompetent hosts.
To address the role of IL-5 on the clinical course of EAE, mice with a disrupted IL-5 gene were immunized with the MOG3555 peptide and EAE studied. Our results indicate that IL-5 is not directly involved in the generation of MOG3555-specific IFN-
-producing T cells or their migration to the CNS. Therefore, either IL-5 does not play an essential role on the development of MOG3555 peptide-induced EAE, or other cytokines are able to compensate for the lack of IL-5.
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Methods
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Mice
In all experiments sex-matched C57BL/6J mice (H-2b), originally purchased from the Jackson Laboratory (Bar Harbor, ME) and IL-5/ mice (C57BL/6-IL5TM1Kopf), obtained from M. Kopf (Basel Institute of Immunology, Basel, Switzerland), were used at 68 weeks of age. The mice were bred by the Biomedical Research Unit, Wellington School of Medicine. Experimental protocols were approved by the Wellington School of Medicine Animal Ethics Committee and performed according to the guidelines.
MOG peptide
The MOG3555 peptide used corresponds to amino acid residues 3555 of the mouse sequence (MEVGWYRSPF SRVVHLYRNGK). The peptide was synthesized by Mimotopes (Clayton, Australia) and the purity was >97%.
Induction of EAE
Female C57BL/6J and IL-5/ mice were injected with 50 µg MOG3555 peptide in CFA (Difco, Detroit, MI) containing 400 µg of Mycobacterium tuberculosis. The 0.2 ml emulsion was injected s.c. in both flanks. Mice received 500 ng of pertussis toxin (List Biological, Campbell, CA) in 0.5 ml PBS i.p. immediately after and 2 days following immunization. The mice were observed daily for clinical signs and scores were assigned based on the following scale: 1, flaccid tail; 2, hind limb weakness or abnormal gait; 3, complete hind limb paralysis; 4, complete hind limb paralysis and forelimb paralysis; 5, complete paralysis.
Proliferative T cell responses
Lymph nodes were harvested from mice 14 days after immunization and single-cell suspensions prepared. Lymphocytes, 5 x 105 cells/well in 200 µl, were incubated in 96-well flat-bottom plates with the indicated amounts of antigen in IMDM supplemented with 5% FCS, 2 mM glutamine, 100 U/ml penicillin G, 100 µg/ml streptomycin sulfate and 5 x 105 M 2-mercaptoethanol (all from Gibco/BRL Life Technologies, Auckland, New Zealand). Cells were cultured in the presence of various concentrations of peptide or concanavalin A. Cultures were incubated for 72 h, 0.5 µCi of [3H]thymidine added to each well and plates harvested after an additional 16 h of culture.
Cytokine ELISA and ELISPOT
Lymph node cells were prepared and stimulated with peptide as for the proliferation assay. Supernatants were removed after 48 h and ELISAs performed as follows. Nunc 96-well immunoplates were coated overnight at 4°C with 12 µg/ml of the capture antibody. The plates were washed and incubated with 1% BSA in PBS. Culture supernatants and standards were added to the plates, and incubated at room temperature for 2 h. The plates were then washed and incubated with biotinylated anti-cytokine antibody at room temperature for 2 h. After washing, plates were incubated with streptavidin-conjugated horseradish peroxidase (Amersham Pharmacia, Uppsala, Sweden) and developed using the substrate TMB (Sigma, St Louis, MO). The following antibody pairs were used: IFN-
, AN18 and XMG-D6biotin; IL-4, 11B11 and BVD6-24G2biotin (PharMingen, San Diego, CA). For detection of cytokine-secreting cells by ELISPOT, lymph node cells or lymphocytes isolated from the CNS were stimulated for 48 h with various concentrations of MOG3555 or concanavalin A in a 96-well ELISA plate coated with the capture antibody for each specific cytokine. Spots were developed by alkaline phosphatase-conjugated streptavidin and 5'BCIP as substrate (Sigma).
Histology
Wild-type and IL-5/ mice were immunized with 50 µg MOG3555, as described above. Brains and spinal cords were removed 30 days later, and fixed in 10% buffered formalin (Sigma). Paraffin-embedded sections (6 µm thick) were stained with either hematoxylin & eosin or Luxol fast blue to assess cellular infiltration and demyelination respectively.
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Results
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IL-5 has no effect on the development of EAE
The aim of this study was to determine if the absence of IL-5 has any effect on the development of EAE. To investigate this, mice that were deficient in IL-5 were immunized with the MOG3555 peptide. In addition, control wild-type mice were also immunized in the same manner along with mice deficient in IL-4. Mice lacking IL-4 are more susceptible to EAE, an observation supported by other researchers data (19,20) and by us (this manuscript). Typically following immunization with the MOG peptide, wild-type and IL-5/ mice developed clinical symptoms at day 10, and then the disease became more severe with maximum clinical scores of 2.1 and 2.3 respectively (Fig. 1 and Table 1). The disease was indistinguishable with no statistical differences in the day of onset and disease severity between both groups (Table 1). The results suggest that IL-5 has no significant effect on the development of EAE. The IL-4/ mice showed clinical symptoms at a similar time point. However, these mice developed EAE with a greater clinical severity, as expected from previous work (19,20).

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Fig. 1. IL-5 has no effect on the development of EAE. C57BL/6J wild-type (n = 1 3), IL-4 (n = 8) and IL-5 (n = 16) gene-deficient mice were immunized with 50 µg MOG3555 in CFA s.c. followed by 500 ng pertussis toxin i.p. The mice were observed daily for clinical signs of EAE. Results are combined data from four separate experiments and plotted as mean clinical scores ± SEM.
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IL-5 has no effect on T cell proliferation in response to MOG3555
Since IL-5 had no effect on the onset or development of EAE, we next determined if MOG-specific cells generated in mice deficient for IL-5 respond to MOG peptide in a similar manner to cells generated in wild-type mice. Fourteen days after immunization, lymphocytes were isolated from draining lymph nodes of IL-5/ and wild-type mice. The cells were stimulated with various concentrations of MOG3555 or concanavalin A as a control (Fig. 2). The results show that MOG3555-specific T cells were generated in both groups of mice and there was no difference in proliferation to the control mitogen concanavalin A or the MOG peptide (Fig. 2). This suggests that the absence of IL-5 does not lead to altered T cell proliferation. Taken together, these results show that MOG3555-specific T cells are generated in both wild-type and IL-5/ mice, and cells from both groups respond strongly to MOG peptide re-stimulation.

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Fig. 2. MOG3555-specific T cells are present in both wild-type and IL-5/ mice. C57BL/6J and IL-5/ mice were immunized with 50 µg MOG3555 in CFA s.c. followed by 500 ng pertussis toxin i.p. Lymph node cells were isolated 14 days after immunization and stimulated in vitro with concanavalin A (A) or MOG3555 (B) in triplicate wells. [3H]Thymidine was added for the last 16 h of culture. Results are combined from four separate experiments and plotted as mean c.p.m. ± SEM.
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IL-5/ mice have similar numbers of MOG3555-specific IFN-
-producing cells in the periphery
While the above result showed that MOG3555-specific T cells were present in the lymph nodes of both wild-type and IL-5/ mice, and that these cells can respond to MOG peptide re-stimulation, the possibility existed that the number of MOG-specific T cells in the periphery differed between IL-5/ and wild-type mice. Therefore, peptide-primed lymph node cells were stimulated with MOG3555 or concanavalin A for 48 h, and the number of IFN-
- and IL-4-producing cells determined by ELISPOT. As revealed by the numbers of IFN-
-specific spots following concanavalin A or MOG3555 stimulation, the two groups of mice had similar numbers of cells capable of producing IFN-
(Fig. 3A and B). In addition, no IL-4-producing cells were detected upon concanavalin A or MOG peptide stimulation (Fig. 3C and D). These results suggest that IL-5 has little effect on the number of MOG-specific IFN-
-producing T cells generated.

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Fig. 3. Similar number of MOG3555-specific cells in the periphery of IL-5/ mice. Lymphocytes were isolated from the lymph nodes of C57BL/6J and IL-5/ mice 14 days following immunization. The cells were stimulated for 48 h with concanavalin A (A and C) or MOG3555 (B and D) on anti-IFN- (A and B) or anti-IL-4 (C and D) antibody-coated plates in triplicate wells. The spots from cytokine-producing cells were developed after the culture period with 5'BCIP. Results are plotted as mean spots/106 ± SEM, combined from two representative experiments.
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IL-5/ mice show no difference in Th1/Th2 cytokine profile
To determine if the absence of IL-5 had any effect on the generation of Th1 and Th2 cells, cytokine ELISAs were performed. Concanavalin A stimulation induced a profound Th1 response with IFN-
and TNF-
production, but no IL-4, by both IL-5/ and wild-type cells (Fig. 4A, C and E). More importantly, we found that MOG3555-specific T cells from IL-5/ and wild-type mice produced similar levels of IFN-
and TNF-
(Fig. 4B and D). In addition, no MOG3555-specific IL-4 was detected from the cells of either the wild-type or IL-5/ mice (Fig. 4F). These results demonstrate that the absence of IL-5 does not alter the Th1/Th2 phenotype or level of the cytokines produced. Thus, even in the absence of IL-5, a predominant peptide-specific Th1 response was generated, with no up-regulation of the Th2 cytokines IL-4 (Fig. 4) or IL-10 (data not shown).
IL-5 has no effect on the infiltration of cells into the CNS
To determine if IL-5 had any effects on the infiltration of cells into the CNS, the spinal cords and brains were removed 30 days following immunization with MOG3555. The hematoxylin & eosin-stained sections revealed that there were no phenotypic or cellular differences of the infiltrating lymphoid cells in the CNS between IL-5/ and wild-type mice (Fig. 5). There were several inflammatory foci consisting of mainly mononuclear cells present in the spinal cord (Fig. 5) and brain stem (data not shown) of both groups of mice. These results indicate that IL-5 does not have a significant role on the immunopathology of MOG3555-induced EAE. However, even though the number of infiltrating cells was similar, preliminary FACS analysis showed a difference in the percentage of CNS-resident B cells (7.4% in IL-5-deficient mice and 27.7% in C57BL/6J mice; data not shown).

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Fig. 5. Comparable CNS infiltration in C57BL/6J and IL-5/ mice. Mice were immunized as described in Fig. 1. The brains and spinal cords from mice with a disease score of 34 were removed 30 days following immunization and hematoxylin & eosin staining performed on paraffin sections. Only spinal cords from C57BL/6J (A) and IL-5/ mice (B) are shown. PBS control immunized mice showed no clinical signs (data not shown). Representative spinal cords sections from one of two experiments with similar results are shown. Original magnifications x200.
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Discussion
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In this study we have examined the effects of IL-5 on the autoimmune disease EAE, using IL-5/ and wild-type control C57BL/6J mice immunized with the encephalitogenic MOG3555 peptide. As the induction phase of EAE is associated with CD4+ cells of the Th1 phenotype and the recovery phase with Th2 cells, we predicted that the Th2 cytokine IL-5 ought to play a significant role during the chronic phase of EAE. These predictions are based on the facts that the absence of cytokine IL-4 (19,20) or IL-10 (20,26,27) leads to increased clinical disease during the course of EAE, suggesting a critical role for these Th2 cytokines in the down-regulation of encephalitogenic Th1 cells. However, we found no significant clinical differences in the mice lacking IL-5 in comparison with the wild-type animals (Fig. 1 and Table 1). In our experiments we used IL-4/ mice as controls. As previously reported, IL-4/ mice develop more severe EAE in comparison to the C57BL/6J and IL-5/ mice (Fig. 1).
IL-5 is a potent chemoattractant and cell differentiation factor for eosinophils and basophils (22). Therefore, the absence of IL-5 during the course of EAE may have led to a reduction in the numbers of cells migrating to the lymph node and responding to the MOG peptide. However, no difference in T cell proliferation following immunization is observed between the IL-5/ and wild-type mice (Fig. 2). This indicates that IL-5 is not important for proliferation of the MOG3555-specific T cells upon encounter with the peptide. Although IL-5 has no effect on the development of proliferative T cell responses, ELISPOT assays were preformed to determine the number of MOG3555-specific T cells producing IFN-
present in the lymph nodes. The result showed that there is no reduction in the numbers of antigen-specific T cells in lymph nodes of IL-5-deficient compared with wild-type mice (Fig. 3). Furthermore, cytokine ELISAs were used to determined if there was a shift in the Th1/Th2 balance. No differences in the cytokine profiles were found. Similar levels of IFN-
and TNF-
are produced in response to MOG3555 by T cells from IL-5/ and wild-type mice (Fig. 4). In addition, no IL-4 or IL-10 was detected, indicating a predominant Th1 environment is generated even in the absence of IL-5 (Fig. 4 and data not shown). Furthermore, IL-5/ mice show no significant pathological consequences since they developed similar clinical disease (Fig. 1 and Table 1) and had equivalent cellular infiltration in the CNS as wild-type animals (Fig. 5).
The interplay between various cytokines during EAE is complex. During the course of disease in wild-type animals, increased IFN-
production correlates with increased disease severity (28). On the other hand, mice deficient in the Th1 cytokine IFN-
acquire an enhanced disease phenotype (23). These mice develop an atypical EAE, characterized by neutrophillic infiltration into the CNS (23,24), in which IL-5 may be involved given its influence on neutrophil migration and maturation. Thus, EAE can develop in the absence of the Th1 cytokine IFN-
, in which case the Th2 cytokine IL-5 may play a role. However, using IFN-
competent mice we find that the absence of IL-5 does not influence the severity of EAE (Fig. 1 and Table 1).
With regard to the Th1-associated cytokine IL-12 (p35/p40), recent data has revealed that IL-12p35-, but not IL-12p40-, deficient mice are susceptible to EAE (29). The cytokine IL-23 consists of a p19 subunit combined with the p40 subunit of IL-12 (30) and most studies investigating the role of IL-12 on EAE have used IL-12p40 gene-deficient mice (26) or IL-12p40-specific mAb (31,32). These data demonstrate a redundancy of the IL-12 system and suggest that EAE can develop in the absence of IL-12 (p35/p40), but not IL-23 (p19/p40) (29). Notably, a Th1-type response to MOG3555 is generated in wild-type and, although weaker, disease-susceptible IL-12p35/ mice. In contrast, disease-resistant IL-12p40/ mice produce a Th2-type response with high levels of IL-4, IL-5 and IL-10 (29). Albeit the fact that IL-12p40/ mice show elevated IL-5 levels, no atypical EAE with neutrophillic infiltration into the CNS develops. Since IL-12p40/ mice also show elevated levels of IL-4 and IL-10, it is likely that these cytokines are responsible for the disease resistance phenotype, masking any effect of IL-5.
Whether B cells play a role in EAE is controversial and confusing. In this regard, Hjelmström et al., Eugster et al. and Wolf et al. have all shown that B cell-deficient mice develop EAE after MOG3555 or MBP Ac111 immunization (3335). In addition, results from Cross and Holmdahls laboratories have shown that B cells play a significant role in EAE, since B cell-deficient mice are resistant to MOG protein-induced, but not MOG peptide-induced EAE (3638). A recent report complicates this issue. Fillatreau et al. have shown that B cell-deficient mice develop a more severe EAE upon immunization with recombinant MOG protein (39). The reason for the discrepancy between the work of Fillatreau et al. and Cross and Holmdahls laboratories is not clear. Fillatreau et al. suggested that their use of homologous mouse MOG protein rather than heterologous human or rat recombinant MOG protein, as used by the other laboratories, could explain the disparity. We have found, using FACS analyses, reduced numbers of CNS-infiltrating B cells of sick IL-5/ mice in comparison to wild-type mice (7.4 and 27.7% respectively), with no difference in the percentage of peripheral B cell. Whether IL-5 plays a more significant role in a B cell-dependent EAE model using recombinant MOG protein is currently under investigation.
Taken together, our results suggest that IL-5 has no significant role in the development of EAE or that an unknown cytokine could compensate for the absence of IL-5. However, IL-5 appears to have immunomodulatory functions in MS, since relapsing-remitting MS patients treated with glatiramer acetate show a reduced relapse frequency (40) with a concomitant increase in IL-5-producing T cells (41). Although the mechanism for the immunomodulatory function of glatiramer acetate is not fully understood, Vieira et al. have shown that glatiramer acetate-primed DC drive the development of anti-inflammatory Th2 cells (42). Therefore, although there is no evidence for a direct function of IL-5 in the immunopathology of MS, a better understanding of its potential therapeutic role in a clinical setting is desired.
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Acknowledgements
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We thank members of the Malaghan Institute of Medical Research for critical reading of the manuscript, and Ann Thornton and Joan Nicol for technical laboratory assistance. This work was supported by grants from the National Multiple Sclerosis Society and the Wellington Medical Research Foundation of New Zealand, the NH & MRC and the Bethlehem Griffiths Research Foundation of Australia, and by generous donations from the J. B. Were and Son Trust and Toward a Cure Australia. B. T. B. is the recipient of the Wellcome Trust Senior Research Fellowship in Medical Science, New Zealand.
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Abbreviations
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CFAcomplete Freunds adjuvant
CNScentral nervous system
DCdendritic cell
EAEexperimental autoimmune encephalomyelitis
MBPmyelin basic protein
MOGmyelin oligodendrocyte glycoprotein
MSmultiple sclerosis
TNFtumor necrosis factor
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