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Myelin-Associated Oligodendrocytic Basic Protein: Identification of an Encephalitogenic Epitope and Association with Multiple Sclerosis This information is current as of September 24, 2021. Andreas Holz, Bibiana Bielekova, Roland Martin and Michael B. A. Oldstone J Immunol 2000; 164:1103-1109; ; doi: 10.4049/jimmunol.164.2.1103 http://www.jimmunol.org/content/164/2/1103 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Myelin-Associated Oligodendrocytic Basic Protein: Identiﬁcation of an Encephalitogenic Epitope and Association with Multiple Sclerosis1

Andreas Holz,2* Bibiana Bielekova,† Roland Martin,† and Michael B. A. Oldstone*

Myelin-associated oligodendrocytic basic protein (MOBP) is an abundant myelin constituent expressed exclusively by oligodendrocytes, the myelin-forming cells of the CNS. We report that MOBP causes experimental allergic encephalomyelitis and is associated with multiple sclerosis. First, we note that purified recombinant MOBP inoculated into SJL/J mice produces CNS disease. Tests of overlapping peptides spanning the murine MOBP molecule map the encephalitogenic site to amino acids 37–60. MOBP-induced experimental allergic encephalomyelitis shows a severe clinical course and is characterized by a prominent CD4؉ T lymphocyte infiltration and a lesser presence of CD8؉ T cells and microglia/macrophages around vessels and in the white matter Downloaded from of the CNS. Second, PBL obtained from patients with relapsing/remitting multiple sclerosis mount a proliferative response to human MOBP, especially at amino acids 21–39. This response equals or exceeds the response to myelin basic protein and an influenza virus hemagglutinin peptide, both serving as internal controls. Thus, a novel myelin Ag, MOBP aa 37–60, plays a role in rodent autoimmune CNS disease, and its human MOBP counterpart is associated with the human demyelinating disease multiple sclerosis. The Journal of Immunology, 2000, 164: 1103–1109. http://www.jimmunol.org/ lymphocytes and their products play a pivotal role in the (2). Currently, the major myelin Ags known are myelin basic pro- pathogenesis of multiple sclerosis (MS),3 the most com- tein (MBP) (10–13), proteolipid protein (PLP) (14–18), and my- T mon demyelinating disease of humans, and in its labora- elin oligodendrocyte glycoprotein (19–21), although other neural tory model, experimental allergic encephalomyelitis (EAE) (re- Ags can also cause inflammation of the CNS (22). viewed in Ref. 1). A distinguishing theme for both MS and EAE To search for other genes specifically expressed by oligoden- is that T cells recognize and respond to specific peptides from drocytes that may be involved in demyelinating disease, myelin- myelin presented by selected MHC molecules. However, in MS associated oligodendrocytic protein (MOBP) was identified from a the environmental event(s) responsible for triggering such T cell rat spinal cord library (23, 24). By alternative splicing, a single-

responses as well as the specific myelin Ags recognized in vivo by copy Mobp gene generated in rodents and humans several different by guest on September 24, 2021 these T cells are largely unknown. Thus, it is important to identify transcripts that encoded MOBP isoforms with identical amino ter- the potential disease-causing Ags (peptides) in myelin and use that mini but different carboxyl ends. Analysis of the MOBP proteins data to determine whether such myelin peptides mimic the se- revealed their structure to be highly basic with isoelectric points quence or confirmations of microbial peptides. Further, different larger than 10 and insignificant homologies with other myelin con- myelin Ags (peptides) may be associated with different subsets of stituents like MBP or PLP. The most abundant MOBP isoform was humans having MS (2, 3). Hence, the recognition of all the myelin the 81 aa constituting MOBP81 protein (23, 25). Biologic analysis Ags that cause disease are important and may shed light on strat- of MOBP indicated three major differences from MBP and PLP. egies to suppress Ag-specific immunopathologic T cell responses First, MOBP was located exclusively in oligodendrocytes of the (4, 5), give leads for identifying and testing whether a specific CNS, whereas MBP and PLP appeared both in the CNS and pe- infectious agent is involved in the triggering of disease (6–9), and ripheral nervous systems (23, 24, 26). Second, in rodents, tran- assist in identifying different subgroups of patients with disease scription of the Mobp gene occurred 2–3 days later than expression of the MBP or PLP genes (23, 27). Third, MOBP was transcrip- tionally active at the time of myelin compaction (23), and MOBP *Viral-Immunobiology Laboratory, Division of Virology, Department of Neurophar- protein was noted at the major dense line of myelin (24), suggest- macology, The Scripps Research Institute, La Jolla, CA 92037; and †Neuroimmunol- ing a role for this protein in myelin structure. MOBP was estimated ogy Branch, National Institute of Neurologic Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892 as the third most abundant myelin constituent of the CNS (24). Here we analyzed the role of MOBP in CNS autoimmune dis- Received for publication July 27, 1999. Accepted for publication November 1, 1999. ease. We find that this new myelin constituent causes EAE in 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 susceptible mice and map its encephalitogenic peptide sequence. Fur- with 18 U.S.C. Section 1734 solely to indicate this fact. thermore, we demonstrate that MOBP is involved in the pathogenesis 1 This work was supported in part by U.S. Public Health Service Grants NS38719 and of MS, and T cells isolated from MS patients show significantly AI09484 and the J. D. and Iva Leiper Trust. A.H. is funded by fellowships of the elevated proliferative responses to human MOBP peptides. Swiss National Foundation and the National Multiple Sclerosis Society. 2 Address correspondence and reprint requests to Dr. Andreas Holz, Division of Virology, Department of Neuropharmacology, The Scripps Research Institute, 10550 Materials and Methods N. Torrey Pines Road, La Jolla, CA 92037. E-mail address: [email protected] Peptides 3 Abbreviations used in this paper: MS, multiple sclerosis; EAE, experimental allergic encephalomyelitis; MOBP, myelin-associated oligodendrocytic basic protein; PLP, Peptide libraries covering the entire murine MOBP81 peptide (25) proteolipid protein; MBP, myelin basic protein; Flu-HA, influenza-hemagglutinin; and human MOBP aa 1–70 (24) were synthesized at Chiron Technologies IPTG, isopropyl ␤-D-thiogalactoside. (San Diego, CA; see Table I and Fig. 3). The encephalitogenic PLP aa

139–151 peptide (VSLGKWLGHPDKF) (28) was used as a positive con- munomodulatory therapy within 3 mo prior testing. Between groups were trol for EAE induction. The immunodominant human MBP aa 83–99 pep- no significant age or MHC class II differences. tide (10, 11, 29) and influenza-hemagglutinin (Flu-HA) aa 306–318 pep- The T lymphocyte proliferation assay was performed as described (34). tide (PKYVKQNTLKLAT) (30, 31) served as control Ags for proliferation A total of 6 ϫ 106 PBMC were tested for each Ag in 96-well microtiter studies using human T cells. plates. PBMC from each individual were plated in 60 wells each containing 1 ϫ 105 PBMC. PBMC were stimulated with 1 ␮M of each peptide in Induction of EAE and clinical scoring IL-7-enriched (10 ng/ml) T cell medium. On day 7, cells were pulsed with [3H]thymidine for 6 h, and incorporated radioactivity was determined by EAE induction was performed according to established protocols (18, 32). scintillation counting. Sixty negative control wells (seeded without Ag) Sixty micrograms MOBP fusion protein, 100 ␮g MOBP peptides, or 150 were included for each patient. An unrelated Ag, Flu-HA aa 306–318, ␮g PLP aa 139–151 peptide were used as inducing agents. These Ags were which induces T cell recall responses in almost all individuals (30, 31), was emulsified in CFA containing 5 mg/ml Mycobacterium tuberculosis included as a positive control and served as an internal standard. Wells H37RA (Difco Laboratories, Detroit, MI) and injected s.c. at the base of were considered positive when both of the following criteria were reached: the tail of 6- to 8-wk-old female SJL/J mice (The Jackson Laboratory, Bar 1) the stimulation index is Ͼ2 (cpm greater than twice the average cpm of Harbor, ME). Simultaneously, 150 ng pertussis toxin (List Biological Lab- all negative control wells); and 2) the cpm is greater than the average cpm oratories, Campbell, CA) was given i.v. and 48 h later. Animals were of negative control wells ϩ 3 SD. The combination of these criteria have scored and weighed daily. Clinical scoring was according to the following been reported to be optimal to ensure a high degree of specificity while scale: 0, normal animal, no clinical signs of EAE; 1, completely limp tail; maintaining the sensitivity of the assay (34). 2, impaired righting reflex with completely limp tail; 3, partial hind limb To compare proliferative responses of PBMC from MS patients directly paralysis; 4, complete hind limb paralysis; 5, hind and fore limb paralysis, with those from controls, stimulation indexes of each Ag were calculated moribund. individually and normalized against the proliferative response of the Generation of MOBP-GST fusion protein Flu-HA peptide. Downloaded from The open reading frame encoding rat MOBP81 protein (23) was PCR am- plified using the primers 5Ј-CGGAATTCTGATGAGTCAAAAAGTG- Results Ј GCC and 5 -CATCAGCAGGTGTCCAC. The PCR product was EcoRI/ Recombinant rodent MOBP81 protein is encephalitogenic in XhoI digested and ligated unidirectionally into the EcoRI/XhoI sides of plasmid pGEX-KG (33) (kindly provided by Dr. John Patterson, The SJL/J mice Scripps Research Institute, La Jolla, CA). DNA sequencing confirmed the To characterize MOBP functionally, we engineered a cDNA ex- integrity of the resulting GST-MOBP plasmid expressing rat MOBP aa 1–81 in frame with the GST protein. Recombinant fusion protein was made pressing a fusion protein of the major rodent MOBP (MOBP81) http://www.jimmunol.org/ in Escherichia coli strain BL21 after induction with 0.5 mM isopropyl isoform and the GST protein. The fusion gene was expressed in E. ␤-D-thiogalactoside (IPTG) and affinity-purified with glutathione beads as coli, and the soluble product was purified by affinity chromatog- described (33). In brief, exponentially growing bacterial cultures were raphy (Fig. 1). Subsequently, several MOBP-specific bands were IPTG induced, and after a 2-h incubation period cells were collected by observed by using anti-MOBP Ab (Fig. 1A). This result likely centrifugation. Bacteria were lysed with lysozyme (10 ␮g/ml) in 25 mM sodium-HEPES buffer, pH 7.5, containing 0.1% Nonidet P-40, 1 mM indicates an altered migration behavior of the highly basic fusion ETDA, and a mixture of various protease inhibitor (Boehringer Mannheim, protein or its partial degradation. However, a unique protease Mannheim, Germany). After sonication and centrifugation at 10,000 ϫ g, cleavage site within the fusion protein (33) allowed the enzymatic soluble proteins of the supernatant were incubated with glutathione beads cleavage and removal of MOBP from GST after incubation with

(Pharmacia, Piscataway, NJ) for 30 min. Beads were washed several times by guest on September 24, 2021 with phosphate buffer. GST-MOBP was released from the beads by incu- thrombin protease (Fig. 1B); the resulting recombinant 12-kDa bation with 10 mM reduced glutathione (Sigma, St. Louis, MO) in 50 mM MOBP was consistent with the full-length MOBP molecule (23), Tris, pH 8.0. GST protein that was used as a control protein for animal and no MOBP-specific degradation products were detected. inoculations was prepared following the same procedure. Inoculation of GST-MOBP (60 ␮g/mouse) induced EAE in fe- Protein fractions were analyzed by 16% PAGE and Western blotting as male SJL/J 6- to 8-wk-old mice (Fig. 1C; five of five mice inoc- described (23). For GST-MOBP cleavage, fusion protein was incubated with 1 U thrombin protease (Pharmacia) for 15 min at room temperature. ulated). Tissues obtained 14 days after immunization contained mononuclear cell infiltrates within the CNS, around blood vessels, Histology and immunohistochemistry and at both subventricular and white matter areas of the paren- Tissues used for histologic analysis were fixed in 10% Bouin’s solution and chyma. Immunohistochemical study revealed that these infiltrates embedded in paraffin. Tissue sections were stained with either hematoxy- were primarily CD4ϩ T lymphocytes with a minority of CD8ϩ T lin-eosin or Luxol fast blue before mounting and photography. cells. Control animals (four mice per group) inoculated with PBS/ For immunohistochemistry, brains and spinal cords of SJL/J mice were CFA or GST/CFA lacked any such mononuclear cell infiltration rapidly dissected and fresh frozen in OCT Tissue Tek compound (Fisher Scientific, Pittsburgh, PA). Next, 8-␮m thin cryomicrotome sections were into the CNS. collected on SuperFrost/Plus slides (Fisher Scientific, Pittsburgh, PA), postfixed in ice-cold 95% ethanol and air-dried. Slides were blocked with 2% FBS/phosphate buffer for 1 h and with avidin/biotin solutions for 10 MOBP peptides spanning aa 37–60 induce severe clinical EAE min each (Vector Laboratories, Burlingame, CA). Tissue sections were in SJL/J mice incubated for1hatroom temperature with primary Abs directed against several cell-surface markers (rat anti-mouse CD4ϩ, rat anti-mouse CD8ϩ, We next sought the peptide sequence(s) of the MOBP responsible rat anti-mouse B220 (all obtained from PharMingen, San Diego, CA, and for the encephalitogenic activity. Overlapping peptides, each con- used at 1:200 dilutions), and rat anti-mouse F4/80 (1:100 diluted; Serotec, taining 15 aa, were synthesized that covered the entire rodent Oxford, U.K.)). After washes with phosphate buffer, slides were covered MOBP molecule (25). These peptides were combined into six sep- for 30 min with FBS/phosphate buffer solution containing 1:200 diluted arate pools (Table I); individual pools were emulsified in CFA and biotinylated anti-rat secondary Ab (Vector Laboratories). Tissue sections were washed again and incubated with a streptavidine-HRP complex for 30 the separate emulsions injected into SJL/J mice (seven to eight min (Vector Laboratories). After PBS washes, slides were stained for 5 min mice per group; two separate experiments). Of these six pools, with diaminobenzidine (Zymed, South San Francisco, CA), counterstained only pool 4, consisting of MOBP aa 37–60, induced acute EAE with hematoxylin (Sigma), and embedded in AquaMount before photog- (Table I). The pool 4 inoculum produced characteristic clinical raphy. Control experiments omitting the primary Ab produced no staining, indicating the absence of background. signs (Table I) of weight loss, limp tail, paresis, ataxia, and para- plegia. The mean time of disease onset was 10 days after the initial T cell proliferation studies inoculation, and the mean maximum severity of disease was 3.5 on PBMC were obtained from MS patients and healthy donors. All patients a scale of 5 (Table I). The incidence rate of EAE induced by the had relapsing/remitting MS and did not receive immunosuppressive or im- MOBP peptides was 75% (six of eight mice). The Journal of Immunology 1105 Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 1. Recombinant GST-MOBP induces lymphocytic infiltration in the CNS of SJL/J mice. A, Bacteria containing a GST-MOBP-expressing plasmid were induced with IPTG, and recombinant fusion protein was affinity purified using glutathione beads (33). During the purification process, fractions were monitored by PAGE and Western blotting using an anti-MOBP peptide Ab (23). Lane 1, No induction. Lane 2, Induction with 0.5 mM IPTG. Lane 3, Total soluble proteins of the induced fraction. Lane 4, Affinity-purified GST-MOBP. B, GST-MOBP fusion protein incubated with or without 1 U of thrombin protease. A 12-kDa protein corresponding to full-length MOBP was detectable by Western blotting (23). C, Photomicrograph of CNS tissue taken from an SJL/J mouse 14 days after inoculation with 60 ␮g GST-MOBP emulsified in CFA. A monoclonal anti-murine CD4ϩ Ab shows perivascular accumulation of CD4ϩ T lymphocytes in the white matter region of the cerebellum (counterstained with hematoxylin). Similar results were obtained in all five mice inoculated with purified MOBP.

Histologic and immunohistochemical evaluation of MOBP aa infiltration profile of MOBP-induced EAE was indistinguishable 37–60 induced EAE from that produced by inoculation with a PLP encephalitogenic Analysis of brains and spinal cords revealed widespread infiltra- peptide used as a control. None of the other five peptide pools tion of mononuclear cells throughout the CNS (Fig. 2), but no caused EAE when similarly inoculated into SJL/J female mice, nor infiltration in the peripheral nervous system or other peripheral did PBS/CFA produce disease (seven to eight mice per group; tissues. In the CNS, mononuclear cells were noted in perivenular, experiment confirmed on two occasions). periventricular, and subpial areas (Fig. 2, A and B), as well as in Four overlapping peptides of the encephalitogenic site were in- the CNS parenchyma, primarily in white matter. CNS inflamma- dividually administered to SJL/J mice (five mice per group). All tion was observed in all animals inoculated with the peptide pool four peptides initiated infiltration of mononuclear cells, primarily CD4ϩ, in the CNS. Although peptides aa 37–51 and aa 40–54 spanning MOBP aa 37–60. The amount of infiltration observed in ϩ animals correlated with the severity of clinical disease. A myelin- caused more CNS infiltration of CD4 T lymphocytes than MOBP specific stain (Luxol fast blue) revealed moderate myelin break- peptides aa 43–57 and aa 46–60, none of the mice inoculated down (data not shown). The degree of demyelination in MOBP- developed clinical EAE. inoculated animals was similar to that observed in mice in which EAE was induced with PLP aa 139–151. Immunohistochemical analysis showed that the infiltrates con- T cells from MS patients respond to human MOBP peptides by tained mostly CD4ϩ T lymphocytes (Fig. 2, C and F) accompanied specific proliferation by F480ϩ macrophages/microglia (Fig. 2D), fewer CD8ϩ T lym- Lastly, we turned our attention to whether MOBP is a potential phocytes (Fig. 2E), and very few B lymphocytes (not shown). The target Ag for lymphocytes from MS patients. Human MOBP (24) 1106 MOBP IS ASSOCIATED WITH EAE AND MS

Table I. MOBP aa 37-60 induces EAE in SJL/J micea

No. Mice with Mouse MOBP EAE/Total Maximum Pool Peptide Position Inoculated Score Inﬁltration Composition of Inﬁltrate

1 A3-1 MSQKMAKEGPRLSKN 1–15 0/8 0 No A4-1 KMAKEGPRLSKNQKF 4–18 A5-1 KEGPRLSKNQKFSEH 7–21 A6-1 PRLSKNQKFSEHFSI 10–24 2 A7-1 SKNQKFSEHFSIHCC 13–27 0/8 0 No A8-1 QKFSEHFSIHCCPPF 16–30 A9-1 SEHFSIHCCPPFTFL 19–33 A10-1 FSIHCCPPFTFLNSK 22–36 3 A11-1 HCCPPFTFLNSKREI 25–39 0/8 0 No A12-1 PPFTFLNSKREIVDR 28–42 A1-2 TFLNSKREIVDRKYS 31–45 A2-2 NSKREIVDRKYSICK 34–48 4 A3-2 REIVDRKYSICKSGC 37–51 6/8 3.5 ϩϩϩϩ CD4ϩ, CD8ϩ T cells, A4-2 VDRKYSICKSGCFYQ 40–54 B cells, macrophages/ A5-2 KYSICKSGCFYQKKE 43–57 activated microglia A6-2 ICKSGCFYQKKEEDW 46–60 Downloaded from 5 A7-2 SGCFYQKKEEDWICC 49–63 0/7 0 No A8-2 FYQKKEEDWICCACQ 52–66 A9-2 KKEEDWICCACQKTR 55–69 A10-2 EDWICCACQKTRLRR 58–72 6 A11-2 ICCACQKTRLRRRSR 61–75 0/8 0 No

A12-2 ACQKTRLRRRSRSTP 64–78 http://www.jimmunol.org/ A1-3 KTRLRRRSRSTPRKK 67–81

PLP139–151 8/8 4.5 ϩϩϩϩ CD4ϩ, CD8ϩ T cells, B cells, macrophages/ active microglia PBS/CFA 0 0 No

a A panel of overlapping peptides from mouse MOBP81 protein was synthesized and combined into six pools as indicated. Animals were inoculated with 100 ␮g of each peptide per pool. Only peptides in pool 4 induced clinical and histological EAE. The PBS/CFA control and the remaining MOBP peptide-injected mice failed to develop either clinical or histological evidence of disease. The encephalitogenic peptide PLP aa 139–151, used as a positive control, induced EAE. Similar results were obtained in two other experiments with four mice per group. by guest on September 24, 2021

is 97% homologous with the common amino-terminal part of mu- and 11-fold higher in the patients than in controls, respectively rine MOBP. Sequence analysis identified the presence of putative (Fig. 3B). peptide-binding motifs for the MS-associated HLA-DR alleles DRB5*0101 (DR2a), DRB1*0401, and also a partial motif for DRB1*1501 (also referred to as DR2b) in human MOBP aa 21–39 Discussion (data not shown) that mapped near the murine MOBP encephali- The main findings of this report are, first, the mapping of a MOBP togenic region. This observation of a shared encephalitogenic re- peptide sequence encephalitogenic in SJL/J mice, and, second, the gion in SJL/J mice and DR2-positive MS patients had also been association of human MOBP with MS. The link of MOBP with observed for MBP aa 83–99 (1, 10, 35). To test the immunoge- CNS autoimmune disease by these two lines of evidence might nicity of human MOBP, a set of six overlapping peptides spanning lead to further the understanding of the multifactorial disease MS, human MOBP aa 1–70 was made and used to assay the prolifer- the cause of which is unknown. ative response of lymphocytes obtained from eight MS patients The knowledge of association of MOBP (or any other autoan- suffering from active r/r disease and from seven healthy donors tigen) with CNS autoimmune disease may result in uncovering MS (Fig. 3). Except for one, each patient responded strongly to at least triggering factors/agents because self-Ags may act as molecular one of the MOBP peptides (Fig. 3A), and the overall response was mimics for a specific and critical pathogen involved in disease comparable to that of a standard recall Ag, Flu-HA aa 306–318 initiation (7). Studies of monozygotic twins, of case clusters, and (30, 31) (Fig. 3). Equal numbers of the healthy donors and patients of MHC types document that individuals who develop MS must with MS responded by specific proliferation to both MBP aa 83–99 have an appropriate immunogenetic background influenced by and Flu-HA aa 306–318. The proliferative response against MBP several MHC genes likely involved in presenting a limited number in the healthy control group was expected and agrees with previous of “self” myelin Ags (39–44). However, the discordance among findings (36–38). Accordingly, we detected proliferation against monozygotic twins (39, 45), the epidemiologic studies of suscep- MOBP peptides also in the control group. To directly compare the tibility or resistance to MS in individuals migrating to areas with MOBP-specific proliferation between MS patients and controls, high or low incidence of disease before their 15th year of age (46), we normalized the response to each peptide to that of Flu-HA aa the association of MS with viral infections (47, 48), as well as 306–318 (Fig. 3B). The difference in MOBP-specific proliferation outbreaks of MS in the Faroe Islands (49) indicate that environ- between MS patients and healthy donors was significant ( p ϭ mental factors, presumably infections, are also important for dis- 0.008; Fig. 3B), particularly the stimulation by MOBP aa 21–39, ease pathogenesis. Hence, it is important to know about all mol- MOBP aa 31–49, and MOBP aa 1–19. Responses were 15-, 12- ecules involved in MS. Our data links MOBP with MS. Therefore, The Journal of Immunology 1107 Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 2. Amino acids 37–60 is an encephalitogenic site on the murine MOBP. Photomicrographs of spinal cords taken from a SJL/J mouse inoculated with a pool of peptides containing MOBP aa 37–60. Hematoxylin-eosin staining documents infiltration of mononuclear cells near the ventricle (A) and around a blood vessel near the surface of the spinal cord (B). C–E, Immunohistochemical staining on adjacent sections of the white matter of the spinal cord counterstained with hematoxylin; C shows the presence of CD4ϩ T lymphocytes, D shows F4/80-positive macrophages/microglia, and E shows CD8ϩ T lymphocytes. F, Perivascular infiltration of CD4ϩ T cells in the white matter of the brain stem parenchyma is shown. Similar infiltration was noted in spinal cord, cerebellum, and cortex. In contrast, there was no infiltration in peripheral tissues. Corresponding results were seen in mice in two separate experiments. G, The lack of CD4ϩ T cells (or any other mononuclear cells; not shown) infiltrating into the CNS of animals inoculated with MOBP pool 3 (MOBP aa 25–48; histologic section obtained from the spinal cord; study of sections from brain stem, cerebellum, and cortex also failed to reveal any CD4ϩ T cell infiltration) is demonstrated. A similar absence of T cell infiltration was noted in SJL/J mice inoculated with peptides from other regions of MOBP comprising aa 1–24 (pool 1), 13–36 (pool 2), 49–72 (pool 5), and 61–81 (pool 6; see Table I).

it will be of interest to identify sequences from microbial agents pathogenetic mechanisms may contribute to disease onset and its that have linear or conformational ﬁts (6–9) with human MOBP progression (2). The fact that autoreactivity against MBP, PLP, and testing their abilities to elicit cross-reactive immune responses. myelin oligodendrocyte glycoprotein, and, as demonstrated here, The disease patterns of MS are variable, and the pathology of MOBP, is observed during the course of MS indicates by itself the multiple sclerosis is highly diverse (50). Hence, several immuno- complexity of immunopathological mechanisms involved in CNS 1108 MOBP IS ASSOCIATED WITH EAE AND MS

FIGURE 3. T lymphocytes from several MS patients show a vigorous and specific proliferative response to MOBP. MOBP-, MBP-, and Flu-HA- peptide-specific T cell proliferation was analyzed in MS patients (n ϭ 8) and healthy donors (n ϭ 7) using IL-7-modified primary proliferation. This assay expands Ag-specific memory and in vivo-activated T cells (34). Briefly, a total of 60 ϫ 105 PBMC from each individual was tested per Ag (1 ␮M). PBMC Downloaded from were stimulated for 7 days in the presence of peptide and IL-7 (10 ng/ml). After this period, cells were pulsed with [3H]thymidine and cpm were measured by scintillation counting. A, Raw data for a representative MS patient. The lines for the cut-off and the background are indicated. The cut-off line represents two stringent criteria: first, a stimulation index larger than 2 and, second, a cpm (cpm) larger than background level plus 3 SD. Note that the cut-off for Ag-specific proliferation was high above background values. Between zero and five positive wells were detected for each Ag, which is consistent with precursor frequency estimates for these autoreactive cells in the range of one to five per million. These data are characteristic of those observed with the other six positively responding MS patients studied. B, Proliferation data for lymphocytes obtained from MS patients and healthy donors and normalized http://www.jimmunol.org/ for proliferation to Flu-HA (internal control). The differences of MOBP-specific proliferation between MS patients and healthy donors were statistically significant (␹2 calculated based on the number of positive wells per each Ag, p ϭ 0.008). Furthermore, although the proliferative response to MOBP peptides was comparable to or higher than those to Flu-HA in the MS patients, the responses to Flu-HA were greater in the control population (p Ͻ 0.01; Mann-Whitney rank sum test). autoimmune disease. To fully understand them is ultimately Met) (25). This amino acid substitution is located outside the iden- required for development of effective treatments. Therefore, it will tified encephalitogenic site in SJL/J mice (MOBP aa 37–60). Ac- be of great interest to evaluate and compare the immune responses cordingly, the synthesized mouse peptides at positions aa 37–60 to MOBP and other MS-associated autoantigens in the heteroge- and the bacterially expressed recombinant rat protein both caused by guest on September 24, 2021 neous disease phenotypes in both the animal model and MS (2, 22, EAE in SJL/J mice. 50) and the time when T lymphocytes recognize these molecules MS is an autoimmune disease affecting the CNS. Therefore, during the course of MS. Current studies evaluate such responses potential autoantigens associated with MS must reside within brain prospectively in MS patients. and spinal cord. It is important to note that MOBP is specifically We report that EAE can be induced with recombinant rodent expressed by oligodendrocytes (23). Expression of MOBP outside MOBP81 protein and, by systematically assaying overlapping pep- the CNS has not been observed (23, 24). In contrast, the MS- tides spanning the entire murine MOBP81 protein, we mapped a associated autoantigens MBP and PLP both are expressed by oli- MOBP encephalitogenic epitope to aa 37–60. The clinical, histo- godendrocytes, but also in the peripheral nervous system (52–54) logical, and immunohistochemical evaluation of MOBP-induced as well as by cells in the thymus (55). In agreement with the CNS- EAE revealed no significant differences when compared with a specific expression of MOBP, we do not detect mononuclear cell PLP encephalitogenic peptide: clinical signs were acute, and dis- infiltration in peripheral organs or the peripheral nervous system ease included severe hind leg paralysis, CD4ϩ T lymphocytes and after inoculation of SJL/J mice with the encephalitogenic MOBP macrophages dominated the mononuclear cell infiltrate, and a focal peptide pool. The strict target-organ-specific expression of MOBP myelin breakdown was observed. Recently, it was shown that makes this protein an attractive autoantigen for MS, in particular another isoform of rat MOBP is able to cause clinical and histo- after the demonstration of an association of MOBP with CNS au- logical signs of EAE in SJL/J mice (51). Rat MOBP170 (e.g., toimmune disease. rOPRP1; Ref. 24) was expressed and purified in a baculovirus expression system, and, similar to our findings (Table I and Fig. 2), Acknowledgments EAE was observed when recombinant MOBP170 was adminis- The technical help of Bryan Coon and the assistance by Dr. Mathias Kalbus tered to SJL/J mice (51). We used recombinant rat MOBP81 pro- in identifying the DR-associated MOBP peptide motifs is acknowledged. tein for EAE induction in SJL/J mice (Fig. 1). Because the rodent This is publication number 12064-NP from the Division of Virology, De- MOBP81 and MOBP170 protein isoforms are identical in their partment of Neuropharmacology, The Scripps Research Institute, La Jolla, first amino-terminal 69 aa (23–25), it can be concluded that at least California. one encephalitogenic domain must be contained in MOBP aa 1–69. In agreement with this consideration, we find that an en- References cephalitogenic domain is located in the MOBP peptide sequence 1. Martin, R., H. F. McFarland, and D. E. McFarlin. 1992. Immunological aspects aa 37–60 (Table I and Fig. 2). In our and in Maata et al. (51) of demyelinating diseases. Annu. Rev. Immunol. 10:153. reports, recombinant rat protein was used for EAE induction in 2. Lassmann, H. 1998. Neuropathology in multiple sclerosis: new concepts. Multi- ple Sclerosis 4:93. mouse. Rat and mouse MOBP81 are highly conserved and show 3. Noseworthy, J. H. 1999. Progress in determining the causes and treatment of only a single conserved amino acid change at position 5 (Val 3 multiple sclerosis. Nature 399:A40. The Journal of Immunology 1109

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