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The Journal of Immunology, 2006, 177: 7364-7376.
Copyright © 2006 by The American Association of Immunologists, Inc.

Epitope Specificity of Autoreactive T and B Cells Associated with Experimental Autoimmune Encephalomyelitis and Optic Neuritis Induced by Oligodendrocyte-Specific Protein in SJL/J Mice1

Nathali Kaushansky2,*, Ming-Chao Zhong2,*, Nicole Kerlero de Rosbo*, Romana Hoeftberger{dagger}, Hans Lassmann{ddagger} and Avraham Ben-Nun3,*

* Department of Immunology, The Weizmann Institute of Science, Rehovot, Israel; and {dagger} Clinical Institute of Neurology and {ddagger} Center for Brain Research, Department of Neuroimmunology, Medical University of Vienna, Vienna, Austria


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The encephalitogenic potential of oligodendrocyte-specific protein (OSP) in mice, its specific localization in the intralamellar tight junctions in CNS myelin, and the detection of autoreactivity against OSP in multiple sclerosis (MS) strongly suggest the relevance of autoreactivity against OSP in the pathogenesis of MS. In this study, we have characterized the autoimmune T and B cells that are associated with clinicopathological manifestations of OSP-induced MS-like disease in mice by using recombinant soluble mouse OSP (smOSP) and synthetic overlapping peptides spanning smOSP. SJL/J mice immunized with smOSP developed chronic relapsing clinical experimental autoimmune encephalomyelitis accompanied with intense perivascular and parenchymal inflammatory infiltrates, widespread demyelination, axonal loss, and remarkable optic neuritis. The smOSP-primed lymph node cells reacted predominantly against OSP55–80 and to a lesser extent also to OSP22–46 and OSP179–207. Unexpectedly, in vitro selection with smOSP resulted in pathogenic smOSP-specific CD4+ T cells that reacted equally well against OSP55–80, OSP22–46, OSP45–66, and OSP179–207. Fine analysis of the anti-OSP autoimmunity revealed that the disease is primarily associated with CD4+ T cells directed against the major (OSP55–80) and the minor (OSP179–207) encephalitogenic regions that were further delineated, both in vitro and in vivo, to OSP55–66 and OSP194–207, respectively. In contrast, the OSP-induced Abs were predominantly directed against OSP22–46; these Abs were mostly of IgG1 isotype, but high levels of IgG2a and IgG2b and significant levels of IgE were also observed. The reactivity of pathogenic T cells to two encephalitogenic regions, OSP55–80 and OSP179–207, and their diverse TCRVβ gene repertoire may impose difficulties for epitope-directed or TCR-targeting approaches to immune-specific modulation of OSP-related pathogenesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Multiple sclerosis (MS)4 is an autoimmune disease of the central nervous system (CNS) characterized by primary demyelination and axonal damage. The etiology of the disease is unknown. However, ample evidence suggests that, once initiated, autoimmune mechanisms directed against myelin components in the CNS play an important pathogenic role. Because the disease primarily involves damage to the CNS myelin, studies by many laboratories over the last four decades have focused on investigating the following myelin proteins as potential target Ags for the autoimmune attack in MS: myelin basic protein (MBP), proteolipid protein (PLP), myelin oligodendrocyte glycoprotein (MOG), myelin-associated glycoprotein, 2',3'-cyclic nucleotide 3'-phosphodiesterase, myelin-oligodendrocytic basic protein (MOBP), and oligodendrocyte-specific protein (OSP) (reviewed in Refs. 1, 2, 3). Several nonmyelin components, such as glial fibrillary acidic protein, S100β, heat shock proteins, and transaldolase, have also been proposed as potential target Ags in MS (1, 3). Although autoimmune T and/or B cell reactivity against most of these Ags could be detected in the peripheral blood of MS patients, only MBP, PLP, MOG (1, 2, 3), and more recently also MOBP (4, 5, 6), OSP (7, 8, 9, 10), and myelin-associated glycoprotein (11), for which an encephalitogenic potential has been demonstrated upon immunization of laboratory animals, can be considered as bona fide primary target Ags in MS.

OSP is a hydrophobic 207-aa-long protein with four predicted transmembrane domains that has been localized to CNS myelin (12, 13, 14) and shown by immunoelectron microscopy to be a specific component of tight junction strands in myelin sheaths of oligodendrocytes; it is also expressed in Sertoli cells of the testes (15). OSP, which constitutes 7% of the CNS myelin proteins (14), is an integral membrane protein important in the formation and maintenance of the compact lamellar structure of myelin in the CNS (16). Based on sequence similarity and its ability to form tight junctions in fibroblasts, OSP is a member of the claudin family of tight junction proteins (OSP/claudin-11) (15).

The function of OSP as a structural protein essential for the formation of tight junctions and maintenance of the compact lamellar structure of CNS myelin was strongly indicated by studies in OSP knockout mice (17) and more convincingly so by studies in PLP/DM20 and OSP/claudin-11 double-knockout mice that presented with markedly abnormal myelin compaction and smaller axon diameters (18). One of the significant structural differences between CNS myelin and peripheral nervous system myelin is the presence of intralamellar tight junctions in CNS myelin. Therefore, an autoimmune attack against OSP would affect the integrity of the myelin sheaths in the CNS only, suggesting the possibility that OSP may be a highly relevant candidate target autoantigen in the CNS-specific disease MS.

Based on the demonstrated encephalitogenic potential of OSP in H-2s (7, 8) and H-2b mice (8) and the detection of anti-OSP Abs and OSP-reactive T cells in MS (9, 10), OSP can be included in the category of encephalitogenic CNS myelin Ags such as MBP, PLP, MOG, and MOBP, which are considered as primary candidate target Ags for the pathogenic autoreactivity in MS. However, because OSP is a relatively newly uncovered CNS myelin protein (13) and its relevance to MS pathogenesis has only recently been suggested, the pathogenic autoimmune reactivity against OSP has not yet been characterized. It is therefore of significance to understand in detail the T and B cell autoimmunity associated with OSP-related pathogenesis in laboratory animals.

OSP-induced experimental autoimmune encephalomyelitis (EAE) is a relatively new model of MS that has been the subject of only three previous reports (7, 8, 11). The aim of this study was to thoroughly characterize the autoimmune T and B cell responses and the clinical and pathological manifestations associated with OSP-induced EAE in SJL/J mice. Using a recombinant preparation of soluble mouse OSP (smOSP) deleted of hydrophobic putative transmembrane domains (8), and synthetic peptides spanning smOSP (pmOSP), the T and B cell epitopes of OSP were defined and partially delineated. We show that SJL/J mice immunized with smOSP develop EAE and optic neuritis; the disease is associated with B cell autoreactivity primarily directed against the immunodominant OSP22–46 and with T cell reactivity directed against three codominant epitope clusters. The studies presented also define a major and a minor encephalitogenic epitope of OSP, each eliciting pathogenic T cells with diverse expression of TCRVβ genes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Female SJL/J (H-2s) and C3H.SW (H-2b) mice were purchased from The Jackson Laboratory or obtained from the Weizmann Institute of Science colony. (SJL/J x C3H.SW)F1 mice were bred at the Weizmann Institute of Science. All mice were 2- to 3-mo old when used in the experiments. The Institutional Animal Care and Use Committee of the Weizmann Institute of Science has approved the experiments, which were performed in accordance to its relevant guidelines and regulations.

smOSP and OSP peptides

smOSP was prepared as previously described (8). The amino acid sequences of synthetic peptides spanning smOSP, pmOSP, and truncated peptides spanning pmOSP55–80 or pmOSP179–207 are listed in Table I. All peptides were synthesized in the laboratory of Prof. M. Fridkin (Department of Organic Chemistry, Weizmann Institute of Science), using the Fmoc technique with an automated peptide synthesizer (AMS422; Abimed).


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  		Table I. Peptides used in this study

 
T cell lines and T cell proliferative response

Ag-specific T cell lines were selected in vitro as described previously (19) from lymph node cells (LNC) of mice that had been primed 9 days earlier with Ag (150 µg of smOSP or 100 µg of pmOSP) emulsified in CFA containing 150 µg of Mycobacterium tuberculosis H37Ra (catalog no. 3114-25; Difco Laboratories). All T cell lines were maintained in vitro in medium containing IL-2 with alternate stimulation by the Ag every 10–14 days as previously described (19). Proliferation assays of selected T cell lines were performed exactly as previously described (19, 20).

Cytokine analysis

IL-2, IFN-{gamma}, IL-4, and IL-10 were measured by ELISA according to standard protocols from BD Pharmingen as described previously (21). The capture Abs were rat anti-mouse IL-4 (18191D; BD Pharmingen), rat anti-mouse IL-2 (18161D; BD Pharmingen), rat anti-mouse IL-10 (AMC0102; BioSource International), and rat anti-mouse IFN-{gamma} (AMC4834; BioSource International). The biotinylated Abs used were rat anti-mouse IL-4 (18042D), rat anti-mouse IL-2 (18172D), rat anti-mouse IL-10 (18152D), and rat anti-mouse IFN-{gamma} (18112D; all from BD Pharmingen). IL-17 was measured by ELISA using a DuoSet ELISA development kit (DY421; R&D Systems). TGF-β was measured by ELISA according to the standard protocol from R&D Systems using recombinant human TGF-β sRII/Fc chimera as capture reagent (341-BR; R&D Systems) and biotinylated anti-human TGF-β1 Ab (BAF240; R&D Systems). Recombinant human TGF-β1 (240-B; R&D Systems) was used to construct the standard curve.

Adoptive transfer of EAE

Cell transfer experiments were conducted as previously described (19, 20). Briefly, T cells were stimulated in vitro with the relevant Ag for three days and injected into the tail vein of irradiated (400 rad) or nonirradiated naive syngeneic SJL/J mice. Mice were observed and scored daily as previously described (8).

Active induction of EAE

SJL/J mice were injected subcutaneously at one site in the flank with 200 µl of emulsion containing 300 µg of smOSP or 200 µg of peptide in CFA with 300 µg of M. tuberculosis H37Ra. Mice received 300 ng of pertussis toxin in 500 µl of PBS in the tail vein 24 and 72 h after the immunization. Following the encephalitogenic challenge, mice were observed and scored as previously described (8).

PCR analysis of TCR Vβ gene expression by T cell lines

Analysis of TCR Vβ gene usage by encephalitogenic T cell lines was conducted by PCR amplification of expressed TCR genes as described previously (22) on total cellular RNA extracted with guanidium thiocyanate phenol (TRI-Reagent, catalog no. TR118; Molecular Research Center) from T cell lines 14–16 days after the sixth antigenic stimulation, when most accessory cells had disintegrated.

Pathological examination

Mice were perfused with 4% paraformaldehyde in PBS, and the tissues were postfixed in the same fixative for 24 h at 4°C. Histological evaluation was performed on paraffin-embedded sections of brains and spinal cords that were sampled at various time points after immunization or after T cell transfer. Paraffin sections were stained with H&E, Luxol fast blue, and Bielschowsky silver impregnation to assess inflammation, demyelination, and axonal pathology, respectively. In consecutive sections, immunohistochemistry was performed with Abs directed against the following targets: macrophages/activated microglia (MAC3; BD Pharmingen), T cells (CD3; Serotec), complement C9 (23), and β-amyloid precursor protein (βAPP; Chemicon International) (5). For staining, paraffin sections were pretreated with a steamer for 60 min. Bound primary Ab was detected with a biotin-avidin technique as previously described in detail (5).

Determination of anti-OSP Abs and isotypes

Anti-OSP Abs in sera sampled 40–57 days after immunization were measured by ELISA. Polystyrene 96-well polyvinyl chloride plates (Maxisorp; Nunc) were coated overnight at 4°C with 10 µg/ml smOSP or synthetic peptides in PBS. The plates were washed with PBS containing 0.05% Tween 20 and blocked with 1% low-fat milk in PBS. The plates were again washed with PBS-Tween 20 and incubated for 2 h at 37°C with 100 µl of normal or test serum diluted 1/100 in PBS containing 1% low-fat milk and 0.05% Tween 20. The plates were washed and incubated with HRP-conjugated goat anti-mouse IgG (catalog no. 115-035-003; Jackson ImmunoResearch Laboratories) for 1 h at 37°C. The plates were washed and developed with 100 µl of 3,3',5,5'-tetramethylbenzidine (catalog no. ES001; Chemicon International). The color reaction was stopped with 50 µl of 0.2 M H2SO4.

To determine the Ig isotypes of anti-smOSP-, anti-OSP22–46-, and anti-OSP35–55-specific Abs, plates were coated with smOSP, pmOSP22–46, or pmOSP35–55, blocked with PBS containing 1% low-fat milk, incubated with diluted serum, and washed as described above. The plates were then incubated for 1 h at 37°C with alkaline phosphatase-conjugated goat anti-mouse IgG1, IgG2a, IgG2b, or IgG3 (catalog nos. 1070-04, 1080-04, 1990-04, and 1100-04, respectively; Southern Biotechnology Associates) diluted 1/1,000 and developed with 100 µl of phosphatase substrate (catalog no. SD942; Sigma-Aldrich). Alternatively, the plates were incubated for 1 h at 37°C with biotinylated rat anti-mouse IgE (kindly provided by Prof. Z. Eshhar, Dept. of Immunology, The Weizmann Institute of Science), washed, and incubated with HRP-conjugated streptavidin (catalog no. 016-030-084; Jackson ImmunoResearch Laboratories) for 20 min. The plates were then processed as described above.

Optical densities were measured in an ELISA reader (Pharmatec Instrumentation; Tecan Spectra).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Epitope-specificity of autoimmune encephalitogenic T cells involved in smOSP-induced chronic EAE in SJL/J mice

We previously showed that our soluble recombinant preparation smOSP is encephalitogenic for H-2b and H-2s mice (8). Fig. 1A shows the clinical course of EAE in four mice immunized with smOSP/CFA. As can be seen, the onset of smOSP-induced clinical EAE is relatively delayed (mean day of onset 26 ± 9 in three different experiments) as compared with EAE induced in SJL/J mice by MBP or PLP, which develops 12–15 days after encephalitogenic challenge. To determine the epitopes against which the encephalitogenic autoreactivity to smOSP is directed, LNC isolated from SJL/J mice immunized with smOSP were tested for their in vitro primary responses to a set of synthetic overlapping peptides, pmOSP, spanning smOSP. As shown in Fig. 1B, reactivities against three main regions encompassed by pmOSP22–46, pmOSP55–80, and pmOSP179–207 were consistently observed. The primary response to the OSP55–80 region (stimulation index = 11) was reproducibly higher than that against the OSP22–46 and OSP179–207 regions (stimulation index = 4 and 4.8, respectively), and was comparable to that against smOSP (stimulation index = 9.6), indicating a higher frequency of OSP55–80-reactive precursors and suggesting the possibility that OSP55–80 encompasses the immunodominant OSP T cell epitope(s). However, after five rounds of in vitro selection with smOSP, the resulting smOSP-specific T cell line reacted to a comparable extent against pmOSP55–80, pmOSP179–207, and pmOSP22–46; in addition, reactivity against pmOSP45–66, which overlaps the OSP55–80 region, was observed (Fig. 1C). The cytokine patterns of the smOSP-reactive line T cells upon stimulation with smOSP were extremely mixed, suggesting the presence of both anti-inflammatory and proinflammatory T cells (Fig. 1D). Nevertheless, the smOSP-specific CD4+ line T cells were highly encephalitogenic (Fig. 1E; 94% CD4+ T cells, staining not shown), transferring lethal disease in all four mice injected. Cytokine analysis upon stimulation of encephalitogenic smOSP-specific line T cells by pmOSP22–46, pmOSP55–80, and pmOSP179–207 also yielded a mixed pattern of anti-inflammatory and proinflammatory cytokine secretion (Fig. 1D) that did not help in determining which of these stimulating epitopes imparts the major encephalitogenic activity to the smOSP-specific line T cells. Nevertheless, the smOSP-specific line T cells secreted the highest amounts of IL-17 upon stimulation by pmOSP55–80, which could suggest that the OSP55–80 region contains epitope(s) of the highest encephalitogenic potential as compared with the other two epitopic regions, OSP22–46 and OSP179–207.


Figure 1
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  		FIGURE 1. smOSP-induced chronic EAE in SJL/J mice is associated with pathogenic CD4+ T cells primarily reactive against three epitopes of OSP. A, Examples of the clinical course of active EAE induced with smOSP as detailed in Materials and Methods. B, Primary proliferative response of smOSP-primed LNC to smOSP and synthetic peptides spanning smOSP. Draining LNC (5 x 105/well) isolated from SJL/J mice immunized 9 days earlier with smOSP/CFA were tested for their reactivity to smOSP (10 µg/ml), to synthetic peptides spanning the smOSP sequence, (pmOSP, 10 µg/ml), and to a purified protein derivative (PPD) (5 µg/ml), which is used as positive control. The mean background was 1309 ± 295 cpm. S. I, stimulation index. C, Epitope-specificity of smOSP-specific line T cells. After five rounds of in vitro selection with smOSP, the line T cells (1.2 x 104 T cells in the presence of 5 x 105 irradiated syngeneic spleen cells per well) were tested for their proliferative reactivity against pmOSP at indicated concentrations. The mean background was 157 ± 17 cpm. S. I, stimulation index. D, Cytokine profile of smOSP-specific line T cells. smOSP-specific line T cells were cultured in the presence of 5 µg/ml smOSP and APC as described in Materials and Methods. Culture supernatants were measured for cytokine after 48 h (IL-2, IFN{gamma}, IL-4, IL-10, and IL-17) and 72 h (TGFβ) as described in Materials and Methods. E, smOSP-specific line T cells are highly encephalitogenic. Activated smOSP-specific line T cells (5 x 106) were injected i.v. into the tail vein of naive SJL/J mice that had been irradiated with 400 rad. I, incidence; M, mortality. Clinical signs developed on day 6 after cell transfer; all mice had died by day 10.

 
Defining encephalitogenic epitopes of smOSP in SJL/J mice

SJL/J mice were immunized for EAE with synthetic peptides spanning smOSP, including epitopes recognized by the encephalitogenic smOSP-specific line T cells. Table II shows that pmOSP55–80, which strongly stimulated primed LNC (Fig. 1B), also had the strongest encephalitogenic activity, inducing EAE in four of five mice injected, whereas pmOSP45–66 or pmOSP179–207 induced clinical EAE only in one of the five immunized mice. Although pmOSP22–46 was strongly stimulatory for smOSP-primed LNC and encephalitogenic smOSP-specific line T cells, SJL/J mice immunized with this peptide did not develop EAE. pmOSP35–55, pmOSP103–123, and pmOSP142–161, which were nonstimulatory for primed LNC or for encephalitogenic smOSP-specific line T cells, were also nonencephalitogenic upon active immunization (Table II).


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  		Table II. Induction of EAE in SJL/J mice with peptides spanning smOSP

 
These results suggested that the major encephalitogenic activity of smOSP for SJL/J mice resides within OSP55–80 and that pmOSP179–207 contains a minor encephalitogenic epitope of smOSP. Because pmOSP45–66 overlaps with pmOSP55–80 by 12 aa, both peptides may contain the major encephalitogenic epitope; however, it appears that the shared epitope is a far more potent encephalitogen when delivered via pmOSP55–80 rather than via pmOSP45–66. These possibilities were further investigated by fine epitope specificity analysis of the T cell autoreactivity against the epitopes encompassed by pmOSP55–80 and pmOSP179–207 and by further analysis of their encephalitogenic potentials.

Fine epitope specificity analysis of the encephalitogenic OSP55–80-specific T cells further delineates the major encephalitogenic epitope to OSP55–66

The primary proliferative response by LNC isolated from mice immunized 9 days earlier with smOSP to a set of overlapping peptides with N- and/or C-terminal truncations of the encephalitogenic pmOSP55–80 suggested the possibility that the encephalitogenic epitope within OSP55–80 is located at the N-terminal part of OSP55–80. Thus, none of the peptides truncated at the N terminus could stimulate smOSP-primed LNC (Fig. 2A). Analysis of the encephalitogenic smOSP-specific line T cells for their proliferative response to an expanded set of truncated OSP55–80 peptides supported this possibility, and the epitope was further defined to OSP55–66 and is likely to be contained within OSP57–66 (Fig. 2B). However, the encephalitogenic OSP55–80-specific line T cells (Fig. 2C), which were selected from SJL/J mice immunized with pmOSP55–80/CFA, reacted against both the peptides truncated at the C terminus (OSP55–71, OSP57–68, OSP45–66, OSP51–66, and OSP55–66) and the peptides truncated at the N terminus (OSP60–76 and OSP62–76) of OSP55–80 (Fig. 2C). These contrasting results suggest that the C-terminal region of OSP55–80 contains an additional cryptic epitope or epitopes located within OSP62–76. We therefore investigated the encephalitogenic potential of all the N- and C-terminal OSP55–80-truncated overlapping peptides that could stimulate the encephalitogenic smOSP-specific and/or OSP55–80-specific line T cells. Table III shows that none of the N-terminal truncated OSP55–80 peptides tested (pmOSP62–81, pmOSP62–76, or pmOSP60–76) could induce EAE in SJL/J mice. In contrast, the C-terminal truncated peptides that were tested, pmOSP55–74, pmOSP55–71, and pmOSP55–66, induced clinical EAE with an incidence of 2/5, 4/4, and 4/4, respectively. Fig. 3 shows the clinical course in another experiment where active EAE induced in SJL/J mice with pmOSP55–71 or pmOSP55–66 was compared with that of active EAE induced by pmOSP55–80 (Fig. 3A) or to passive EAE induced by the encephalitogenic OSP55–80-specific line T cells (Fig. 3B). As shown in Fig. 3C, the encephalitogenic pmOSP55–80-specific line T cells displayed a mostly proinflammatory cytokine profile, secreting large amounts of IL-2 (119,000 pg/ml culture supernatant), IFN{gamma} (98,000 pg/ml culture supernatant), and IL-17 (4,896 pg/ml supernatant), but no detectable IL-10.


Figure 2
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  		FIGURE 2. Delineation of the epitope within the major encephalitogenic region stimulatory for pathogenic OSP55–80-reactive T cells. Proliferative responses to pmOSP55–80 and to progressively N-terminal and/or C-terminal truncated peptides spanning OSP55–80 were assessed. A, Proliferative response of smOSP-primed LNC (5 x 105 cells/well). The mean background was 1309 ± 295 cpm. PPD, purified protein derivative; S.I., stimulation index. B, Proliferative response of line T cells (1.2 x 104 T cells/well) selected in vitro with smOSP from smOSP-primed LNC. The mean background was 179 ± 15 cpm. PPD, purified protein derivative; S.I., stimulation index. C, Proliferative response of line T cells selected in vitro with pmOSP55–80 from pmOSP55–80-primed LNC. The mean background was 826 ± 94 cpm. S.I., stimulation index.

 

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  		Table III. Encephalitogenic activity of truncated pmOSP55–80 peptides

 

Figure 3
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  		FIGURE 3. The clinical course of EAE induced by pmOSP55–80 and OSP55–80-truncated peptides in SJL/J mice. A, Active EAE was induced by s.c. injection of 200 µl of emulsion containing 200 µg of pmOSP55–66, pmOSP55–71, or pmOSP55–80 and 300 µg of M. tuberculosis H37Ra. Mice were injected in the tail vein with 300 ng of pertussis toxin immediately and 48 h after immunization. I, incidence; M, mortality. B, Passive EAE transferred by pmOSP55–80-specific line T cells. Naive syngeneic SJL/J mice were irradiated (400 rad) and injected i.v. with 2 x 106 activated pmOSP55–80-specific line T cells. I, incidence; M, mortality. C, Cytokine profile of pmOSP55–80-specific line T cells. Line T cells were cultured in the presence of 5 µg/ml pmOSP55–80 and APC, and supernatants were measured for cytokine after 48 h as described in Materials and Methods.

 
Altogether, these in vitro and in vivo analyses strongly indicate that the N-terminal part of OSP55–80, OSP55–66, harbors the major encephalitogenic epitope of OSP in SJL/J mice and that the C-terminal cryptic epitope for the encephalitogenic OSP55–80-specific line T cells, OSP62–76, is not encephalitogenic.

Analysis of the T cell response to OSP179–207 and delineation of a minor encephalitogenic epitope to OSP194–207

Although a highly significant T cell reactivity against OSP179–207 was elicited following encephalitogenic inoculation with smOSP, and although pmOSP179–207 strongly stimulated the encephalitogenic smOSP-specific line T cells in vitro (Fig. 1, B and C), pmOSP179–207 was only weakly encephalitogenic (Table II). Therefore, we analyzed the fine epitope specificity of T cells reactive against OSP179–207 by using N- and/or C-terminal truncated peptides of OSP179–207, and we investigated the encephalitogenic potential of these truncated peptides. Fig. 4A shows that LNC isolated from SJL/J mice immunized with smOSP/CFA reacted only against one N-terminal truncated peptide, pmOSP194–207, to a level comparable to that against pmOSP179–207. Similar results were obtained when the set of truncated peptides was analyzed for the ability to stimulate encephalitogenic smOSP-specific line T cells (data not shown). Line T cells that were in vitro selected from LNC of SJL/J mice immunized with pmOSP179–207/CFA were also only reactive against the N-terminal truncated pmOSP194–207 (Fig. 4B). A demonstration that the epitope recognized by the encephalitogenic T cells is located at the C terminus of OSP179–207 was corroborated by the in vivo studies (Table IV) showing that of the N-terminal and/or C-terminal truncated OSP179–207 peptides tested, only pmOSP194–207 could induce clinical EAE, albeit only in one of the five mice immunized. Although the pmOSP179–207-specific line T cells were pathogenic, causing mild EAE in four of four naive syngeneic recipients (Fig. 4C), OSP194–207 can be considered only as a minor encephalitogenic epitope of OSP for SJL/J mice in view of the low incidence and relatively late onset of actively induced clinical EAE (Tables II and IV). Analysis of the cytokines secreted by the pmOSP179–207-specific line T cells upon stimulation with their epitope revealed a mixed cytokine profile (Fig. 4D), albeit with more anti-inflammatory cytokines secreted (IL-4 = 40,256 pg/ml and IL-10 = 590 pg/ml) than by pmOSP55–80-specific line T cells (IL-4 = 1,602 pg/ml and IL-10 < 15 pg/ml; Fig. 3C). These data likely reflect the lower encephalitogenic potential of pmOSP179–207 as compared with the highly encephalitogenic pmOSP55–80.


Figure 4
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  		FIGURE 4. Delineation of the epitope within the minor encephalitogenic region OSP179–207 stimulatory for pathogenic OSP179–207-specific T cells. A and B, smOSP-primed LNC (A) or pmOSP179–207-specific line T cells (B) selected from pmOSP179–207-primed LNC were analyzed for their proliferative response to progressively N-terminal and/or C-terminal truncated peptides spanning OSP179–207. C, Passive EAE transferred by pmOSP179–207-specific line T cells (6.6 x 106/mouse) into naive irradiated (400 rad) syngeneic SJL/J mice. I, incidence; M, mortality. D, Cytokine profile of pmOSP179–207-specific line T cells. Line T cells were cultured in the presence of 5 µg/ml pmOSP179–207 and APC, and supernatants were measured for cytokine after 48 h as described in Materials and Methods.

 

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  		Table IV. Encephalitogenic activity of pmOSP179–207 truncated peptides

 
Diverse TCRVβ gene expression by encephalitogenic OSP-specific T cells

The pathogenic T cell lines specific for smOSP, pmOSP55–80, and pmOSP179–207 (Figs. 1E, 3B, and 4C, respectively), were analyzed for their TCRVβ gene expression. RT-PCR analysis, using a set of 17 Vβ gene family-specific 5' primers and a Cβ-specific reverse primer, showed that pathogenic autoreactivity against OSP is associated with T cells using a spectrum of TCRVβ genes for their TCR expression. As shown in Table V, the smOSP-specific line T cells expressed most of the TCRVβ genes relevant for H-2s mice that were tested in this study. The diverse TCRVβ gene usage by the encephalitogenic smOSP-specific line T cells was not surprising in view of their reactivity against several OSP epitopes located within aa 22–46, 55–80, and 179–207 (Fig. 1C). Similarly, the recognition of both N- and C-terminal epitopes by the pmOSP55–80-specific line T cells (Fig. 2C) could also explain their TCRVβ gene diversity (Table V). Less expected was the finding that the pmOSP179–207-specific line T cells also expressed extensively diverse TCRVβ genes (Table V) despite their focused reactivity against OSP194–207. These results altogether strongly suggest extensive TCR diversity associated with the pathogenic T cell autoreactivity against OSP in SJL/J mice.


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  		Table V. TCRVβ gene expression by OSP-reactive T cell lines in SJL/J micea

 
Predominant epitope-specificity of anti-OSP Abs in mice with smOSP-induced EAE

Sera from five SJL/J mice with clinical EAE were taken 43–57 days after immunization with smOSP and analyzed by ELISA for Abs against smOSP. Fig. 5A shows that all five sera had high levels of anti-smOSP Abs. Analysis of their epitope-specificity by ELISA with the set of synthetic peptides spanning smOSP indicated that the anti-smOSP Abs elicited were primarily directed against pmOSP22–46, as shown by the strong reactivity to this peptide in four of the five sera (Fig. 5A). No Ab binding could be detected against pmOSP103–123, pmOSP142–161, or pmOSP186–205. The Ab reactivities against pmOSP35–55, pmOSP51–72, or pmOSP179–207 were only sporadic. Hence, the Ab response against smOSP displayed a far more focused epitope-specificity than the T cell response. A similar predominant response to OSP22–46 was observed also in (SJL/J x C3H.SW)F1 mice (Fig. 5B) that developed clinical EAE following immunization with smOSP/CFA.


Figure 5
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  		FIGURE 5. Epitope-specificity and isotypes of anti-OSP Abs in mice with smOSP-induced chronic EAE. Sera were sampled 43–57 days after encephalitogenic challenge from mice displaying chronic EAE with clinical scores of 3–4. The values presented represent mean OD of tested serum duplicates minus mean OD of normal serum duplicates after background subtraction. A and B, Sera obtained from SJL/J (A) or (SJL/J x C3H.SW)F1 (B) mice displaying chronic EAE induced by smOSP/CFA were analyzed by ELISA for Abs against smOSP or synthetic peptides spanning smOSP. C, Ig isotyping of Abs reacting against smOSP and OSP22–46. Representative data from one mouse are presented.

 
The anti-OSP22–46 Abs in (SJL/J x C3H.SW)F1 mice with chronic EAE were primarily of the IgG1, IgG2a, and IgG2b isotypes; significant levels of anti-OSP22–46 Abs of the IgE isotype were also observed (Fig. 5C). These data suggest that the B cell response to OSP22–46 is regulated by both IL-4- and IFN-{gamma}-secreting T cells, albeit with a stronger trend to regulation by IL-4-secreting T cells (IgG1 and IgE), suggesting that the T cells reactive against pmOSP22–46 are likely to be of the Th2 type, which provides a possible explanation for the lack of encephalogenicity of pmOSP22–46. However, it should be noted that mouse strains with the Igh1-b allele, such as SJL/J mice, do not have the gene for IgG2a and instead express the IgG2c isotype (24, 25). Because there is a 16% difference in amino acid sequence between IgG2a and IgG2c (24, 26), the OD values obtained here for anti-IgG2a Ab binding to (SJL/J x C3H.SW)F1 sera may be underestimated (26).

OSP-induced histopathology associated with chronic EAE

Mice were sampled at various time points after immunization with smOSP for histopathological examination of the CNS. A summary of neuropathological findings in 10 (SJL/J x C3H.SW)F1 mice is shown in Table VI. Before disease onset, the pathology was marked mostly by inflammation only in the brain of immunized mice (mice 1–3). However, clinical signs were accompanied by evidence of demyelination as well as inflammation throughout the CNS, including the optic system. In the brain, there was no tangible difference in demyelination in mice early after EAE onset or with ongoing disease, whereas in the spinal cord the extent of demyelination tended to be more marked in mice with chronic EAE (mice 8–10) as compared with mice at an early stage after clinical onset (mice 4–7). Demyelination in the optic system was observed in all mice with clinical signs of EAE except in one mouse sampled at the onset of clinical disease and which showed only mild manifestations of EAE (mouse 5). Panel 1 of Fig. 6 shows typical pathological manifestations associated with severe ongoing disease. The pathological changes in the spinal cord of (SJL/JxC3H.SW)F1 mice with smOSP-induced chronic EAE were characterized by massive inflammation with confluent perivascular and subpial demyelination (Fig. 6, 1A and 1B). Apart from a large granulocyte population with a considerable eosinophilic component (Fig. 6, 1B and 1C), the inflammatory infiltrate consisted mainly of macrophages (Fig. 6, 1D) and T-lymphocytes (Fig. 6, 1E). In areas of ongoing demyelinating activity in the spinal cord, βAPP-positive axonal spheroids were abundant (Fig. 6, 1F), suggesting that OSP-related pathogenic autoimmunity is associated with substantial axonal injury and loss. A few perivascular and parenchymal complement deposits were detected in areas of demyelination (Fig. 6, 1G), which could reflect a potential pathogenic role for anti-OSP Abs in disease progression; however, unequivocal demonstration of activated complement deposition in the mouse system is difficult (27), and determining a role for Abs in the pathogenesis of murine OSP-induced EAE would necessitate a cotransfer T cell/Ab study with full immunological and immunohistopathological characterization, which is beyond the scope of our present report. Inflammation and demyelination were also observed in the brain, mainly in the cerebellum (Fig. 6, 1J), and to a lesser extent also in the periventricular white matter (data not shown) as well as in the optic nerves and tracts (Fig. 6, 1H and 1I). Panel 2 of Fig. 6 further details the inflammation and demyelination in the optic chiasm (Fig. 6, 2A and 2B, stained with H&E and Luxol blue, respectively) and optic nerve (Fig. 6, 2E and 2F, stained with H&E and Luxol blue, respectively) of EAE-affected mice as compared with a healthy optic chiasm (Fig. 6, 2C and 2D, stained with H&E and Luxol blue, respectively) and a healthy optic nerve (Fig. 6, 2G and 2H, stained with H&E and Luxol blue, respectively) in naive mice. As can be seen, the massive inflammation observed in both chiasm and optic nerve (Fig. 6, 2A and 2E) was accompanied by complete demyelination (Fig. 6, 2B and 2F) in OSP-induced EAE. Neither inflammation (Fig. 6, 2C and 2G) nor demyelination (Fig. 6, 2D and 2H) could be observed in the tissues isolated from control normal mice.


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  		Table VI. Neuropathological findings in (C3H.SW x SJL/J)F1 mice with smOSP-induced EAEa

 

Figure 6
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  		FIGURE 6. Histopathological analysis. Panel 1, Active EAE. CNS tissue was dissected from a (SJL/J x C3H.SW)F1 mouse displaying smOSP-induced EAE with a clinical score of 3.0 taken for pathology on day 42 after encephalitogenic challenge (mouse 9; Table VI). Pathology is characterized by widespread demyelination (A, Luxol fast blue) and massive inflammatory infiltration as demonstrated by H&E staining (B). The inflammatory infiltrate is composed of some granulocytes with a considerable eosinophilic component (B and C, H&E). The arrowhead in B indicates the region enlarged in C; the rectangle in B marks the region enlarged in (D–F). Numerous macrophages (D, MAC3) and T-lymphocytes (E, CD3) can also be found. βAPP-positive axonal spheroids are most abundant in the areas of ongoing demyelination at the border of the lesions (F, βAPP, arrowheads). Some demyelinated lesions show deposition of complement C9 in parenchyma (G, arrowheads). Demyelination (Luxol fast blue) in the brains of smOSP-immunized mice is apparent in the optic nerve (H and I, the arrowhead in H indicates the area enlarged in I) and the cerebellum (J, arrowheads). Scale bars: A and B, 625 µm; C, 30 µm; D and E, 220 µm; F and G, 25 µm; H, 1000 µm; I, 215 µm; and J, 385 µm. Panel 2, Inflammation and demyelination in the optic system in smOSP-induced EAE. A–D, Transverse sections of optic chiasm (magnification, x20) from a mouse with smOSP-induced EAE (A and B) or a control normal mouse (C and D) stained with H&E (A and C) or Luxol fast blue (B and D). E–H, Sections of optic nerve (magnification, x40) from a mouse with smOSP-induced EAE (E and F) or a control normal mouse (G and H) stained with H&E (E and G) or Luxol fast blue (F and H). Note the massive inflammation (A and E) and the complete demyelination (B and F) in both the chiasm and optic nerves of EAE-affected mice compared with normal tissue in control mice (C, D, G, and H). The blue spots observed in F are stained Schwann cell-derived myelin from the peripheral nerve of the eye. Panel 3, Passive EAE. CNS tissue was taken from a naive SJL/J mice that had received 5 x 106 smOSP-specific T cells by i.v. transfer (mouse 3-2; Table VII). Low magnification (x 40) showing inflammatory infiltrates (A, H&E) and some demyelination (B, Luxol fast blue) in the dorsal and lateral column of the spinal cord. Higher magnification (x200) of an inflammatory lesion (C, H&E) with axonal loss (D, Bielschowsky silver impregnation). Inflammation (E, H&E) and demyelination (F, Luxol fast blue) in the optic nerve (magnification, x30); note the pronounced reduction in axonal density within the inflammatory lesion seen in one of the optic nerves near the chiasm upon Bielschowsky silver impregnation (G, magnification, x240).

 
Examination of the CNS of mice transferred with large numbers of smOSP-specific line T cells (10 x 106 cells/mouse) indicated severe inflammation accompanied by confluent perivascular demyelination rather than the large plaques of demyelination often seen with active immunization with smOSP (Table VI), mostly in the spinal cord and optic system (Table VII). In mice transferred with fewer line T cells (2 or 5 x 106 cells/mouse), the clinical manifestation was associated with variable inflammation and demyelination in the spinal cord. In the optic system sparse inflammation was observed in mice with clinical EAE, but little or no demyelination could be seen (Table VII). Histopathological findings in passive EAE are shown in panel 3 of Fig. 6. Areas of inflammation accompanied by demyelination and axonal loss can be clearly seen both in the spinal cord (Fig. 6, 3A, 3B, and 3C) and in the optic system (Fig. 6, 3E, 3F, and 3G). However, although the inflammation in mice with passive transfer EAE (Table VII) appeared more severe than that in mice with active EAE (Table VI), the demyelinating lesions were smaller, not exceeding a score of 2 (++, confluent perivascular demyelination; Table VII), in contrast to the large plaques of demyelination (+++/++++; Table VI) seen after active EAE induction. Not unexpectedly, complement reactivity was not detected in the lesions in mice with passive EAE.


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  		Table VII. Neuropathological findings in SJL/J mice with passive EAE transferred by smOSP-specific line T-cells

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The demonstration that OSP is encephalitogenic in H-2b and H-2s mice (7, 8), together with the detection of autoimmune B and T cells reactive against OSP in MS (9, 10), strongly support the possibility that OSP is a bona fide target Ag in MS. In this study, the T and B cell autoimmunity associated with the clinical and pathological manifestations of smOSP-induced chronic EAE in SJL/J mice were comprehensively characterized in view of the likely possibility that OSP-related autoimmunity may contribute to the pathogenesis of MS. We show that immunization of SJL/J mice with smOSP elicit CD4+ T cells that are primarily reactive against OSP22–46, OSP55–80, and OSP179–207. However, only T cells reactive against OSP55–80 and OSP179–207, which were found to represent the major and the minor encephalitogenic regions, respectively, could cause the development of chronic EAE. The disease was associated with CNS pathology, which involves intense perivascular and parenchymal inflammatory infiltrates, widespread demyelination, axonal loss, and a remarkable optic neuritis. The major and minor encephalitogenic epitopes were further defined to OSP55–66 and 0SP194–207, respectively. Molecular modeling of the interaction of the major encephalitogenic OSP55–80 region with I-As predicts OSP57–65 as the major nonameric core epitope recognized by the OSP55–80-reactive encephalitogenic T cells (data not shown). This prediction is supported by the fine epitope specificity pattern of the encephalitogenic line T cells (both the smOSP-T cells and the pmOSP55–80-T cells), which show reactivity against pmOSP57–68 and pmOSP55–66 (Fig. 2, B and C). However, as was observed for MOG35–55 (28) or MOBP15–36 (5), the T cells specific for the major encephalitogenic epitope apparently require peptides longer than the core sequence for their optimal stimulation. Accordingly, OSP55–71 (this study) or OSP52–71 (7) represents the optimal stimulatory and encephalitogenic epitope for OSP-reactive encephalitogenic T cells in SJL/J mice. As observed with T cells reactive against the major encephalitogenic epitopes of MOG (20, 28) or MOBP (5), the encephalitogenic T cells reactive against smOSP or against the major encephalitogenic region OSP55–80 express extensively diverse TCRVβ genes, strongly suggesting that a TCR-targeting approach to immune-specific modulation of OSP-related pathogenesis in unlikely to be effective.

Our study and that of Stevens et al. (7) agree on the encephalitogenic potential of OSP52–71 and OSP194–207, which, as demonstrated in the present study, represent major and minor encephalitogenic epitopes of OSP for SJL/J mice, respectively. However, Stevens et al. reported additional encephalitogenic regions at amino acids 82–101, 102–121, 142–161, and 182–201 upon immunization of SJL/J mice with a panel of 20-mer overlapping peptides spanning mouse OSP. We did not test the encephalitogenic potential of the OSP82–101 region that spans the second hydrophobic putative transmembrane domain of OSP (13), which was deleted in our recombinant smOSP (8). In our study, pmOSP103–123 and pmOSP142–161 were not stimulatory for the encephalitogenic smOSP-specific line T cells, and pmOSP182–201 was stimulatory neither for the encephalitogenic smOSP-specific line T cells nor for the encephalitogenic pmOSP179–207-specific line T cells. As only weak encephalitogenic potential was reported by Stevens et al. for the OSP102–121, OSP142–161, and OSP182–201 regions (7), it is possible that our failure to demonstrate their encephalitogenic potential may be related to subtle differences in mode of immunization or maintenance of the mice, which may affect the susceptibility of the mice to EAE induction. It should also be noted that these regions may only represent cryptic encephalitogenic epitopes, as they were not stimulatory for encephalitogenic smOSP-reactive line T cells in our study and could not elicit proliferative T cell responses in the study by Stevens et al. (7).

The multiepitope specificity pattern of the encephalitogenic CD4+ smOSP-specific line T cells is worth noting. Upon repeated in vitro stimulation of smOSP-primed LNC with smOSP, the selected smOSP-specific T cell line would be expected to show dominant reactivity to OSP55–80 in view of the ex vivo recall proliferative response where OSP55–80 appeared as the major stimulatory epitope of smOSP. Instead, the line T cells gained equal reactivity to pmOSP22–46, pmOSP45–56, pmOSP55–80, and pmOSP179–207. The increased reactivity to pmOSP45–56 harboring the predicted OSP55–66 core epitope for OSP55–80 is not unexpected, nor is the increased reactivity to OSP179–207, which contains the minor encephalitogenic epitope. However, it is unclear why the OSP22–46-reactive T cells were enriched upon in vitro selection; this may be related to the presence of the major B cell epitope contained within OSP22–46. Another possibility for maintenance of the reactivity against OSP22–46 in the long-term smOSP-line T cells is suggested by our preliminary data whereby in vitro selected pmOSP55–80-reactive T cells cross-react with pmOSP22–46 (data not shown) despite the lack of sequence homology between pmOSP55–80 and pmOSP22–46. Such heteroclite cross-reactivity may also account for the relatively high levels of IL-17 secreted upon stimulation of encephalitogenic smOSP-specific line T cells by pmOSP22–46 (Fig. 1D) despite the fact that pmOSP22–46 by itself is not encephalitogenic (Table II). The characteristics of the intramolecular cross-reactivity between OSP22–46 and OSP55–80 are now under investigation.

The complex pattern of cytokine secretion by the encephalitogenic smOSP-specific T cell line reflected its multiple reactivity against pmOSP55–80, pmOSP22–46, and pmOSP179–207 with different encephalitogenic potential, accounting for the secretion of both proinflammatory and anti-inflammatory cytokines in the response of smOSP-specific line T cells to smOSP. However, the different levels of the proinflammatory and anti-inflammatory cytokines secreted by the smOSP-specific line T cells in response to the different epitopes as compared with their response to smOSP could suggest a differential pathogenic role for autoimmunity to these epitopes. Thus, the enhanced secretion of IL-2 and IFN{gamma} and particularly of IL-17 by smOSP-specific line T cells upon stimulation with pmOSP55–80 is likely to be related to the high encephalitogenic potential of this OSP region, whereas the enhanced secretion of IL-10 upon stimulation of smOSP-specific line T cells with pmOSP179–207 could reflect the low encephalitogenic potential of this region. Analysis of the cytokine profile of the T cell lines raised to the major and minor encephalitogenic epitopes (Figs. 3C and 4D) corroborates these observations, with pmOSP55–80-specific line T cells secreting the highest amounts of proinflammatory Th1 cytokines (IL-2 and IFN{gamma}) and IL-17, but no IL-10, whereas pmOSP179–207-specific line T cells secreted the highest amounts of anti-inflammatory Th2-type cytokines, IL-4 and IL-10. Nevertheless, the presence of even a few IL-17-secreting T cells within the pmOSP179–207-specific T cells may be sufficient for their encephalitogenic activity. Thus, as little as 1.5 x 105 CNS PLP-specific IL-17-producing CD4+ T cells could transfer EAE to naive syngeneic recipients (29), suggesting that IL-17-producing Th cells, which are apparently essential in promoting autoimmune inflammation of the brain (29, 30, 31, 32, 33, 34) and joint (35, 36), are highly encephalitogenic. In this respect, however, the secretion of IL-17 by smOSP-specific line T cells in response to pmOSP22–46 (Fig. 1D) is bothersome, because neither active EAE induction with pmOSP22–46 nor passive transfer of pmOSP22–46-specific line T cells resulted in disease. Nevertheless, the potential cross-stimulation of encephalitogenic OSP55–80-specific T cells by pmOSP22–46, which we mentioned above, may account for the secretion of relatively high levels of IL-17 by the smOSP-specific line T cells in response to this nonencephalitogenic epitope.

The epitope-specificity of the Ab response against OSP appears more focused than the T cell reactivity. The B cell autoimmunity against smOSP was predominantly directed against OSP22–46, and the Abs were mostly of IgG1, IgG2a, and IgG2b isotypes. Anti-OSP22–46 Abs of the IgGE isotype were also detected. Whether anti-OSP Abs play a pathogenic role in OSP-induced EAE is not yet known. To date, MOG is the only myelin protein known to initiate a demyelinating autoantibody response in EAE, as demonstrated by numerous studies (reviewed in Refs. 37, 38); however, although linear MOG epitopes have been associated with autoantibodies over vesiculated myelin (39), only Abs to conformational epitopes were shown to be demyelinating (40, 41). In contrast to smOSP-induced EAE, the autoantibody response in animals immunized for EAE with recombinant MOG is generally not focused to a single region of the molecule, in particular in outbred species of non-human primates (42, 43, 44), and unlike OSP the encephalitogenic region always contains a B cell epitope (reviewed in Ref. 42). In the PLP-specific B cell response by SJL/J mice immunized with whole PLP, determinant-specific Ab populations were not detectable (45), which may suggest that the Abs recognize only conformational determinants. In rats immunized with whole PLP, however, reactivity to peptide PLP265–276 representing the C-terminal region of the molecule was detected; B cell response to peptides spanning other regions was not observed (46). The B cell response in rats immunized for EAE with MBP was broad, albeit to immunodominant epitopes that did not correspond to the encephalitogenic region (47); in (SJL x PL)F1 mice, predominant recognition of the encephalitogenic region was also not seen (48). The histopathological analysis of OSP-induced chronic EAE showing large numbers of granulocytes and eosinophils in the inflammatory infiltrates may indicate the involvement of B cells regulated by Th2-type cytokines in the disease pathogenesis. This observation, together with the detection of some complement fixation in demyelinated lesions in the spinal cord, may suggest a potential pathogenic role for anti-OSP22–46 Abs. However, to determine whether these Abs do have demyelinating activity, a more extensive study should be conducted that would include in vivo cotransfer of myelin-specific inflammatory T cells and Abs to be tested and a full analysis of the associated neuropathology. Such a study should be conducted preferably in the rat model where immunohistochemical detection of an activated complement is known to be more effective than in the mouse.

Overall, the clinical progression and the neuropathology showing extensively demyelinated areas, axonal loss, and the involvement of optic neuritis in OSP-induced EAE reflect MS to a large extent. The comprehensive characterization of T and B cells involved in the disease is essential to understand pathogenic mechanisms and to define the means for immune-specific intervention in OSP-related autoimmunity. Thus, the extensive TCRVβ diversity of the pathogenic T cells strongly suggest that a TCR-targeting approach to immune-specific modulation of OSP-related pathogenesis is unlikely to be effective. The multiepitope specificity of the OSP-specific T cells may also be a significant obstacle in the epitope-directed approach to immune-specific modulation of OSP-related pathogenesis.


   Acknowledgments
 
We thank Dr. Lydia Cohen for preparing smOSP.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by research grants from the Israel Science Foundation, the National Multiple Sclerosis Society of New York, the Israel Ministry of Health, and the William Sahm Foundation. A.B.-N. is the incumbent of the Eugene and Marcia Applebaum Professorial Chair. Back

2 N.K. and M.-C.Z. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Avraham Ben-Nun, Department of Immunology, The Weizmann Institute of Science, P.O. Box 26, Rehovot 76000, Israel. E-mail address: avraham.ben-nun{at}weizmann.ac.il Back

4 Abbreviations used in this paper: MS, multiple sclerosis; βAPP, β-amyloid precursor protein; CNS, central nervous system; EAE, experimental autoimmune encephalomyelitis; LNC, lymph node cells; MBP, myelin basic protein; MOBP, myelin-associated oligodendrocytic basic protein; MOG, myelin oligodendrocyte glycoprotein; OSP, oligodendrocyte-specific protein; smOSP, recombinant soluble mouse OSP; pmOSP, mouse OSP peptide; PLP, proteolipid protein. Back

Received for publication November 15, 2005. Accepted for publication August 1, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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