immunity to the extracellular domain of nogo-a modulates...

Immunity to the Extracellular Domain of Nogo-A ModulatesExperimental Autoimmune Encephalomyelitis1

Paulo Fontoura,2*¶ Peggy P. Ho,* Jason DeVoss,* Binhai Zheng,† Byung J. Lee,*Brian A. Kidd,§ Hideki Garren,� Raymond A. Sobel,‡ William H. Robinson,§

Marc Tessier-Lavigne,† and Lawrence Steinman*

Nogo-66, the extracellular 66 aa loop of the Nogo-A protein found in CNS myelin, interacts with the Nogo receptor and has beenproposed to mediate inhibition of axonal regrowth. It has been shown that immunization with Nogo-A promotes recovery inanimal models of spinal cord injury through induction of Ab production. In this report, studies were performed to characterizethe immune response to Nogo-66 and to determine the role of Nogo in experimental autoimmune encephalomyelitis (EAE).Immunization of EAE-susceptible mouse strains with peptides derived from Nogo-66 induced a CNS immune response withclinical and pathological similarities to EAE. The Nogo-66 peptides elicited strong T cell responses that were not cross-reactive toother encephalitogenic myelin Ags. Using a large scale spotted microarray containing proteins and peptides derived from a widespectrum of myelin components, we demonstrated that Nogo-66 peptides also generated a specific Ab response that spreads toseveral other encephalitogenic myelin Ags following immunization. Nogo-66-specific T cell lines ameliorated established EAE, viaNogo-66-specific Th2 cells that entered the CNS. These results indicate that some T cell and B cell immune responses to Nogo-66are associated with suppression of ongoing EAE, whereas other Nogo-66 epitopes can be encephalitogenic. The Journal ofImmunology, 2004, 173: 6981–6992.

T he Nogo-A protein is a member of the reticulon familypresent in myelin that inhibits neurite regrowth (1, 2).Nogo-A is encoded by the longest of three alternativelyproduced transcripts of the nogo gene, has 1163 aa residues and isfound mainly in the CNS. All three isoforms contain a predictedextracellular 66 aa loop in their C terminus end, and a major in-hibitory activity has been attributed to this extracellular loop,called Nogo-66 (2). Nogo-66 interacts with a GPI-anchored Nogoreceptor protein belonging to the family of leucine-rich repeat pro-teins and signals through the neurotrophin receptor p75NTR co-receptor, mediating neurite growth inhibition by antagonistic reg-ulation of intracellular signaling through RhoA and Rac1 (3–7).Other myelin proteins that have been implicated in inhibition of

axonal regrowth, such as myelin-associated glycoprotein (MAG)3

and oligodendrocyte-myelin glycoprotein also appear to act by sig-naling through the Nogo receptor system (8–10). Besides theNogo-66 region, it has recently been shown that two other regionsof Nogo-A might also be implicated in inhibition of neurite out-growth and collapse of growth cones (11).

Several therapeutic strategies aimed at improving axonal regen-eration have been directed toward blocking interactions in theNogo-Nogo receptor system (12); these have included using therecombinant mAb IN-1 against Nogo-A (13, 14), Nogo receptorantagonist peptide NEP1–40 (15, 16), and truncated soluble Nogoreceptor (17). More recently, studies of different Nogo knockoutmouse strains have yielded conflicting results about the role ofNogo in regeneration (18–20). Other groups have had some suc-cess in increasing neurite regrowth or enhancing neuroprotectionin models of spinal cord injury by inducing a broad-based immuneresponse against myelin-associated axonal regrowth inhibitors(21), or specifically against Nogo-A-derived peptide 472 (22).

Experimental autoimmune encephalomyelitis (EAE) is an ani-mal model that has been intensely investigated as a source ofpathophysiological and therapeutic insight into the human diseasemultiple sclerosis (MS) (23–28). EAE is mainly mediated by amyelin Ag-specific T cell response that coordinates the immuneattack against the CNS (29). The role of B cell responses is not aswell defined in EAE and MS, although Abs targeting myelin oli-godendrocyte glycoprotein (MOG) are thought to be pathogenic inboth EAE and MS, and other anti-myelin Abs have also been de-tected (30–33). We have recently shown that epitope spreading ofthe B cell response occurs after active EAE induction and that theextent of spreading of the immune response to a variety of myelincomponents correlates with relapse rate in chronic models (34). In

Departments of *Neurology and Neurological Sciences, School of Medicine, †Bio-logical Sciences, Howard Hughes Medical Institute, ‡Pathology, School of Medicine,and §Division of Immunology and Rheumatology, Department of Medicine, StanfordUniversity, Stanford, CA 94305; ¶Gulbenkian Science Institute, Oeiras, Portugal; and�Bayhill Therapeutics, Palo Alto, CA 94303

Received for publication July 8, 2004. Accepted for publication August 27, 2004.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by grants from the Fulbright Foundation and ChristopherReeve Paralysis Foundation (to P.F.), by the National Institutes of Health, NationalResearch Service Award 5F32NS11115 from the National Institute of NeurologicalDisorders and Stroke (to P.P.H.), by a Helen Hay Whitney Fellowship (to B.Z.), bythe National Institutes of Health Grant K08 AR02133, an Arthritis Foundation Chap-ter grant and Investigator award, and National Institutes of Health, National Heart,Lung, and Blood Institute contract N01 HV 28183 (to W.H.R.), by National Institutesof Health Grant NS 046414 (to R.A.S.), by a grant from the International SpinalResearch Trust (to M.T.-L.), and by a National Institutes of Health, National Instituteof Neurological Disorders and Stroke Grant 5R01NS18235, National Institutes ofHealth Grant U19 DK61934, and National Heart, Lung, and Blood Institute contractN01-HV-28183 (to L.S.). M.T.-L. is a Howard Hughes Medical Institute Investigator.2 Address correspondence and reprint requests to Dr. Paulo Fontoura, InflammationGroup, Gulbenkian Science Institute, Rua da Quinta Grande 6, P-2780-156 Oeiras,Portugal. E-mail address: [email protected]

3 Abbreviations used in this paper: MAG, myelin-associated glycoprotein; EAE, exper-imental autoimmune encephalomyelitis; MS, multiple sclerosis; MOG, myelin oligoden-drocyte glycoprotein; MBP, myelin basic protein; PLP, proteolipid protein; OSP, oligo-dendrocyte-specific protein; CNPase, 2�,3�-cyclic nucleotide 3�-phosphodiesterase.

The Journal of Immunology

Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00

recent years, axonal pathology has emerged as a major determinantof neurological deficit in MS (35), leading to the concept thatblocking the action of axonal regrowth inhibitors might also bebeneficial. In the present study, we sought to determine how T celland B cell responses to Nogo affect the pathophysiology of EAEand to ascertain the characteristics of responses that might be use-ful for therapy in CNS diseases with axonal injury.

Materials and MethodsAnimals

Female SJL/J and C57BL/6 mice were obtained from The Jackson Labo-ratory (Bar Harbor, ME) at 5 wk of age. Mice were between 6 and 8 wk ofage when experiments were initiated. Nogo-A/B/C knockout mice weregenerated as described by Zheng et al. (19); these H-2b mice C57BL/6 �129S7 mice have been backcrossed to C57BL/6, with three of four of themice bred to homozygosity on C57BL/6 for these experiments ((C57BL/6 � 129S7) N2 background). SJL/J Thy1.1a congenic mice were a kind giftfrom H. Y. Tse at Wayne State University (Detroit, MI) (36). All animalexperiments were conducted according to protocols reviewed and approvedby the Institutional Animal Care and Use Committee at Stanford University(Palo Alto, CA).

Peptides

Nogo-1–22 (RIYKGVIQAIQKSDEGHPFRAY), Nogo-23–44 (LESEVAISEELVQKYSNSALGH), Nogo-45– 66 (VNSTIKELRRLFLVDDLVDSLK), Nogo-11–30 (QKSDEGHPFRAYLESEVAIS), Nogo-31–52 (EELVQKYSNSALGHVNSTIKEL), proteolipid protein (PLP)139–151 (HSLGKWLGHPDKF), myelin basic protein (MBP)85–99 (ENPVVHFFKNIVTPR), MOG35–55 (MEVGWYRSPFSRVVHLYRNGK), and V�5.1(KGERSILKCIPISGHLSVA) peptides were synthesized by standard 9-flu-orenylmethoxycarbonyl chemistry in our own facilities on a peptide synthe-sizer (model 9050; MilliGen, Burlington, MA). Peptides were HPLC purified,structures confirmed by amino acid analysis and mass spectroscopy andresuspended in PBS.

EAE induction

For Nogo immunizations, SJL/J and C57BL/6 female mice were injecteds.c. with Nogo-peptide (from 100 to 500 �g per injection) emulsified inCFA, consisting of IFA (Difco, Detroit, MI) and 1 mg/ml heat-inactivatedMycobacterium tuberculosis (strain H37RA; Difco). Animals received twos.c. injections a week apart in different sites (flank and groin); at the timeof the second immunization and 48 h later, mice were injected i.v. withBordetella pertussis toxin (List Laboratories, Campbell, CA) in PBS, 500ng/animal. For Nogo-A/B/C knockouts, female mice were immunized s.c.with 100 �g of MOG35–55 peptide emulsion in CFA and received two i.v.injections of 300 ng of B. pertussis toxin, at the time of immunization and48 h later. Chronic EAE was induced in SJL/J mice by s.c. injection of 100�g of PLP139–151 peptide emulsion in CFA. Animals were clinicallyscored daily according to the following scale: 0, normal; 1, tail paralysis;2, hind limb paraparesis; 3, complete hind limb paralysis; 4, forelimb pa-resis or paralysis; 5, death.

Histology

Animals were killed by CO2 overdose and perfused with 10% formalin,after which brain and spinal cord were removed. Paraffin-embedded sam-ples were stained with H&E and Luxol fast blue stains and Bielschowskyimpregnation. For semiquantitative analysis, meningeal and parenchymalinflammatory foci were counted by an observer blinded to the immuniza-tion and clinical status of the mice. For Thy1.1a immunohistochemistry,CNS samples were snap-frozen in tissue Tek OCT (Sakura Finetek, Tor-rance, CA) after partial immersion in liquid N2. Cryosections (8-�m thick)were fixed in acetone and immunostained with a biotinylated anti-Thy1.1aAb conjugate (BD Pharmingen, San Diego, CA) followed by avidin-biotincomplex reagent (Vector Laboratories, Burlingame, CA). Reaction productwas visualized with 3.3�-diaminobenzidine (Sigma-Aldrich, St. Louis,MO) and the slides were counterstained with hematoxylin.

Lymph node cell proliferation assays

At termination of the experiments, draining lymph nodes were dissected,and lymph node cells were cultured in 96-well microtiter plates at 0.5 �106 cells/well. Culture medium consisted of RPMI 1640 supplementedwith L-glutamine (2 mM), sodium pyruvate (1 mM), nonessential aminoacids (0.1 mM), penicillin (100 U/ml), streptomycin (0.1 mg/ml), 2-ME

(5 � 10�5 M), and 10% FBS. Wells were pulsed with 1 �Ci of [3H]TdR(ICN Pharmaceuticals, Irvine, CA) for the final 16 h of culture, and incor-porated radioactivity was measured using a betaplate scintillation counter(Wallac, Turku, Finland).

Cytokine profile determination

T cell lines were established from Nogo immunized mice as described inthe literature. These T cells were then tested for the production of variouscytokines. T cells (5 � 104) were incubated with 2.5 � 106 irradiatedsyngenic APC/ml in enriched RPMI 1640 and 10% FCS. After 48 h ofculture, supernatants were collected and tested by sandwich ELISA usingstandard ELISA kits (BD Pharmingen).

Primary structure analysis

The Nogo-66 sequence was obtained from GenBank (accession numberAAM77068) and analyzed using freely available software at http://www.expasy.org for the Kyte-Doolittle plot of hydropathicity (37), Zim-merman polarity (38), and bulkiness and recognition factors. Homologyanalysis was done using ClustalW with a Blosum matrix (http://ww-w.ch.embnet.org/software/ClustalW.html). Computer modeling for Nogopeptide primary structure was performed using Insight II (Accelerys, SanDiego, CA).

Myelin arrays

Myelin arrays and the associated methods used in this work were previ-ously described in detail (34). Ordered Ag arrays were produced using arobotic microarrayer to attach myelin and control peptides and proteins topoly-L-lysine-coated microscopic slides. For the studies described, addi-tional Ags were added to the arrays including the Nogo-1–22, 23–44, 45–66, 11–30, and 31–52 peptides as well as overlapping peptides derivedfrom oligodendrocyte-specific protein (OSP), and golli-MBP. ReactiveAbs were detected using Cy3-conjugated goat anti-mouse IgG/IgM sec-ondary Ab before scanning. Individual arrays were probed with 1/150 di-lutions of serum from individual animals. Data analysis was performedusing Statistical Analysis for Microarrays (SAM) software (http://www.stat-class.stanford.edu/SAM/servlet/SAMServlet). The reported Ag listsare SAM-identified features with q values �5% (see Fig. 3E) and �10%(Fig. 3F) and absolute fluorescence levels above a numerator threshold of1. Heat maps (see Fig. 3, G and H) of array reactivities are represented fora select subset of myelin Ag features. Cluster software was used to hier-archically order the mice and Ag features based on a pairwise similarityfunction, and TreeView software to display the results as a heat map(http://rana.lbl.gov/EisenSoftware.htm).

ResultsInduction of EAE in Nogo-A/B/C mutant mice

To study the possible contribution of the Nogo-A protein to axonalregeneration in the EAE model, we induced disease in Nogo-A/B/C knockout mice on an H-2b background ((C57BL/6 � 129S7)NE2), generated by targeted mutation of the common C-terminalregion that contains Nogo-66. This mutant has been shown to be anull for Nogo-C and a null or severe hypomorph for Nogo-A andNogo-B (19). Animals were observed for 6 wk postinduction fordisease phenotype and incidence. As shown in Fig. 1, Nogo knock-out mice have reduced disease severity compared with wild-typeC57BL/6 controls or controls with a similar genetic background,though this does not reach statistical significance at the end of theexperiment ( p � 0.043 at day 33, but otherwise p � 0.05 through-out). There is, however, a statistically significant difference in cu-mulative mortality and survival rates between both groups (Fig. 1,A and B, �2 value p � 0.002). Histological assessment of sectionscomprising the entire CNS at the end of the experiment did notreveal a significant difference in numbers of inflammatory/demy-elinating lesions between the knockout and wild-type mice or anappreciable difference in the appearance of the lesions or extent ofaxonal pathology (Fig. 1, C–F). Based on these data we hypoth-esized that Nogo-66 might have a specific role as an autoantigencapable of modifying the disease course in EAE.

6982 NOGO-A MODULATES EAE

Induction of EAE and T cell responses with Nogo-66 peptides insusceptible mouse strains

To ascertain the potential for Nogo-A to act as a target autoantigenfor autoimmune demyelination, we focused on the putative extra-cellular 66 aa loop, Nogo-66. This extracellular domain would bea likely target for interactions with both the cellular and humoralarms of the immune system, and such interactions might thereforealso modulate axonal regrowth. EAE susceptible SJL/J andC57BL/6 female mice were immunized with Nogo-66 derivedpeptides in CFA, as described in Materials and Methods. Threedifferent peptides were synthesized, encompassing the entire se-quence of Nogo-66 (peptides 1–22, 23–44, and 45–66). Beginningat day 10 postimmunization, all animals in both strains exhibitedsigns of photophobia, lethargy and stopped grooming, and somehad a partial loss of tail tone. These findings lasted for �3–4 days,and then subsided completely. Control animals immunized withPBS in CFA showed no abnormalities in behavior. A few of theSJL/J mice immunized with Nogo-45–66 peptide (2 of 10 in twocombined experiments) also had typical signs of clinical EAE,including transient hind limb paralysis, loss of tail tonicity andbladder incontinence. Histological evaluation of brains and spinal

cords of Nogo-immunized animals revealed inflammatory foci inthe meninges and parenchyma, pathological findings characteristicof EAE (Fig. 2, A–C). Semiquantitative analysis of inflammatorylesions was done on CNS tissues of SJL/J and C57BL/6 mice thathad been immunized with the Nogo peptides to which T cell re-sponses could be generated. Small numbers of meningeal inflam-matory foci were found in both strains and parenchymal lesionswere more numerous in SJL/J than in C57BL/6 mice (Fig. 2D).The SJL/J mouse immunized with Nogo-45–66 with the most se-vere clinical disease (score � 2) had numerous meningeal (n � 65)and a few parenchymal (n � 11) foci, representing the high end ofthe pathological spectrum induced by immunization with this pep-tide. One SJL/J mouse immunized with Nogo-1–22 had 28 men-ingeal and 32 parenchymal inflammatory foci. These results indi-cate that Nogo-66 peptides may act as weak encephalitogens inEAE-susceptible mouse strains such as SJL/J.

To evaluate T cell proliferative responses against Nogo pep-tides, we collected axillary and inguinal draining lymph nodes atthe end of the experiments and performed proliferation assaysagainst a panel of Nogo and control peptides. We found a strongand specific proliferative response to the immunizing peptide in

FIGURE 1. Induction of EAE in Nogo-A/B/Cknockout mice. A, Mean disease scores (�SD) overtime after induction of EAE with MOG35–55 peptide inCFA. Combined results from two different experimentscomparing wild-type C57BL/6 mice (�, dashed line)and Nogo knockdown mice (Œ, solid line) (total n � 16for each group). B, Cumulative EAE mortality rate andKaplan-Meier survival curves for wild-type (WT) andNogo knockout (KO) mice from previous experiment.There is a statistically significant difference in survivalbetween knockdown and wild-type groups (�2 valuep � 0.002). C–F, Paraffin sections of illustrative CNSlesions in MOG peptide-induced EAE. C and D, Typi-cal mononuclear cell infiltrates (right side of fields) inspinal cord of wild-type mouse. Myelin is intact (C,blue) and axons are preserved (D, black fibers) in theintact portions (left side of fields). E and F, Meningealand parenchymal inflammatory lesion in spinal cord ofNogo knockout mouse. Spinal nerve root myelin andaxons (upper left of fields) are preserved in the unaf-fected portions. There is demyelination and axonal lossin portions infiltrated by mononuclear cells. C and E,Luxol fast blue-H&E stain; D and F, Bielschowsky sil-ver impregnation for axons. All pictures are at magni-fication of �228.

6983The Journal of Immunology

FIGURE 2. EAE induction with Nogo peptides in SJL/J and C57BL/6 mice. A and B, Meningeal (A, arrow) and perivascular parenchymal (B, arrow)mononuclear cell infiltrates in the cerebellum of an SJL/J mouse that had been immunized with Nogo-45–66 in CFA and showed clinical signs of EAE.Luxol fast blue/H&E stain. Magnification shown at �228. C, Meningitis (arrow) adjacent to cerebellum of a C57BL/6 mouse that had been immunizedwith Nogo-1–22 in CFA and showed photophobia. Luxol fast blue/H&E stain. Magnification shown at �228. D, Semiquantitative analysis of CNSinflammatory foci in SJL/J and C57BL/6 mice immunized with Nogo-66 peptides and PBS or PBS alone in CFA. Meningeal inflammatory foci were presentin both strains and parenchymal inflammatory foci are mainly in SJL/J mice with clinical disease. E, Immunization with Nogo peptides induces a T cellproliferative response. At day 30 postimmunization, draining lymph nodes were dissected and lymph node cell proliferation assays setup against Nogopeptides (Ng1–22, Ng23–44, Ng45–66), control peptide (V�5.1), Con A (ConA), or medium. All peptides are at a concentration of 20 �g/ml, Con A at5 �g/ml. Results shown are mean cpm � SEM of triplicate samples. The experiments shown are representative of several independent experiments.


Nogo-1–22 and Nogo-45–66 immunized in SJL/J mice and to alesser degree to Nogo-1–22 and Nogo-23–44 in C57BL/6 animals(Fig. 2E), indicating that different strains respond to different Nogoepitopes.

Nogo-66 peptide immunization induces a B cell response withepitope spreading to other myelin Ags

To follow the induction of a specific Ab response to the immu-nizing Nogo peptides, and the possible spread to other myelin Ags,we used a 2304-feature myelin array developed in our laboratorythat contains over 250 distinct Ags (Fig. 3). The array containsmyelin proteins and overlapping peptides representing many EAE-relevant targets (34), and Ags including Nogo-66 overlapping pep-tides, which were added for these studies. After immunization withNogo-45–66 or Nogo-1–22, Ab reactivity could be detected con-sistently against the immunizing Nogo peptide, and to severalother myelin peptides present on the myelin array (Fig. 3, A–D).Sera from five immunized animals were obtained at different timepoints after EAE induction with all three Nogo peptides, and usedto probe myelin arrays. Statistical analysis of array data revealedmarked intra- and intermolecular epitope spreading of the Ab re-sponse to other myelin proteins and peptides. This response wasmost pronounced in SJL mice immunized with Nogo-45–66 andinvolved targeting of epitopes in myelin proteins with Abs directedagainst MAG (p193–208, p313–328), PLP (p10–29, p103–116,p190–209), MBP (p85–99, p121–139, p131–149), MOG (p19–36,p35–55), OSP (p32–51), �-B-crystallin (p121–140), 2�,3�-cyclicnucleotide 3�-phosphodiesterase (CNPase) (p343–373), and Nogo(p1–22, p11–30, p45–66) (Fig. 3, E–H). Analysis of the spreadingof the Ab response revealed distinct patterns for each immunizingAg within each strain. The B cell response spread from Nogo-1–22and Nogo-23–44 to a limited set of MBP, PLP, and MOG pep-tides. The greatest epitope spreading of autoreactive Ab responseswas observed in SJL/J mice following immunization with Nogo-45–66 (Fig. 3, E and G), and increased spreading was associatedwith more severe clinical disease. In SJL/J mice, the group immu-nized with Nogo-1–22 demonstrated the second greatest degree ofanti-myelin B cell epitope spreading, and mice from this groupexhibited partial loss of tail tone and meningitis on histologicalexamination. SJL/J mice immunized with Nogo-23–44 andC57BL/6 mice immunized with Nogo-1–22 or Nogo-23–44 ex-hibited relatively less spreading and did not manifest clinical dis-ease. Our results show that Nogo-45–66 elicits broader Ab re-sponses. In SJL/J mice Nogo-45–66 induced more severe paralyticdisease than either of the other two synthesized peptides. Thesedata suggest that Nogo-45–66 represents an immunodominantNogo epitope for the B cell response in SJL/J and C57BL/6 mice.

The T cell response to Nogo-66 peptides is specific and does notcross-react with other myelin Ags

We asked whether the clinical EAE seen in SJL/J mice, and his-tological evidence of demyelination in SJL/J and C57BL/6 micefollowing immunization with Nogo-66 peptides could result fromcross-reactivity between these Nogo peptides and other encepha-litogenic myelin Ags such as PLP139–151 or MOG35–55, whichare capable of eliciting EAE in SJL/J and C57BL/6 mice, respec-tively. We analyzed the specificity of the T cell responses usingNogo-1–22-, Nogo-45–66-, and PLP139–151-specific T cell linesfrom SJL/J animals. The PLP139–151-specific T cell line prolif-erates against its cognate Ag, but show no reactivity to any of thethree Nogo-66 peptides. Nogo-specific T cell lines also show re-activity only against their specific peptide, and do not proliferateagainst PLP139–151, MBP85–99, or MOG35–55 (Fig. 4A).Therefore, we conclude that Nogo immunization generates a spe-

cific T cell response that does not show cross-reactivity to othermyelin autoantigens. Because the secondary structure of the Nogoprotein is currently unknown, we compared the primary structureof the Nogo peptides with those of PLP139–151 and MOG35–55.Using well-known primary structure scales for hydropathicity (37),polarity (38), recognition factors, and bulkiness, we could not findany relevant similarities between Nogo-66 peptides and the othermyelin Ags studied. We also could predict the existence of poten-tial antigenic regions in all three of the arbitrarily selected Nogopeptides, namely in the C terminus region of Nogo-1–22 andNogo-23–44, and N terminus region for Nogo-45–66 (Fig. 4B).Furthermore, comparisons between Nogo-66 and MBP, PLP,MOG, and MAG using the general-purpose protein alignment pro-gram ClustalW did not reveal significant similarities, leading us toconclude that the presence of an immune response against Nogo ishighly unlikely to be related to cross-reactivity to other myelinconstituents.

Adoptive transfer of anti-Nogo reactive T cell lines amelioratesEAE in SJL/J mice

Given the difficulty in inducing EAE with Nogo peptides despitestrong T cell and B cell responses, the expression of several acti-vation markers and costimulatory molecules for both T cells andAPCs (CD4, CD8a, CD25, CD28, CTLA-4, CD45RB, CD40L,CD62 ligand, CD18, CD44. CD11c, CD80, CD86, CD40, CD69)was studied with flow cytometry, and revealed a normal expressionprofile for these molecules (data not shown), which excluded de-fects in Ag presentation or T cell activation that might cause themild clinical phenotype after immunization (39). Therefore, wehypothesized that adoptive transfer of purified T cell lines might bemore efficient at disease induction. Naive mice were immunizedwith Nogo-1–22 or Nogo-45–66 emulsified in CFA and 10 dayslater, draining lymph nodes were collected, whole lymph node cellcultures were established and restimulated in vitro with the Nogopeptide. From these we established Ag-specific T cell lines, whichwere used for adoptive transfer and cytokine secretion profile eval-uation. For disease induction, we transferred up to 5 � 107 T celllines that were either reactive against Nogo-1–22 or Nogo-45–66i.v. into naive SJL mice. In some of the experiments, animals re-ceived 107 T cells and were boosted 15 days later with 100 �g ofNogo-peptide emulsion in CFA injected s.c. and an i.v. injection ofpertussis toxin. Although we could demonstrate the presence ofNogo-reactive T cells in adoptively transferred animals up to 3 mopostimmunization or T cell transfer in in vitro proliferative re-sponses (data not shown), these animals never developed clinicalsigns of disease. In view of the fact that Nogo-66 might be acryptic autoantigen, we postulated that inefficient induction of dis-ease might be overcome by transferring Nogo-reactive cells to an-imals that already have EAE. In such mice, Nogo-66 might beavailable for presentation to T cells in acute demyelinating foci.Therefore, we induced EAE in SJL/J mice by immunization with100 �g of PLP139–151 peptide in CFA; at the peak of secondrelapse (day 31), animals were randomized into three groups andgiven either weekly i.v. administration of PBS, Nogo-1–22 orNogo-45–66 reactive T cells in PBS (average of 1.5 � 107 cells)i.v. for 3 wk. Four days after T cell line transfer, animals receivingNogo-reactive T cell lines showed a statistically significant reduc-tion in clinical score; this reduction was most noticeable in micereceiving Nogo45–66 T cell lines (PBS score day 35 and 36 of2.44 � 0.72, day 37 of 2.78 � 0.97, and day 38 of 2.56 � 0.88 vsNogo-45–66 PBS score 1.40 � 0.96 throughout the same periodwith respective p values of 0.035, 0.035, 0.013, and 0.028; PBSscore 2.67 � 0.86 day 41 vs Nogo-45–66 PBS score 1.60 � 0.96,p � 0.035; PBS score 2.78 � 0.13 day 45 vs Nogo-45–66 PBS


score 1.50 � 0.03, p � 0.035) ( p values are represented in Fig.5A). For these animals, this improvement was maintained through-out the next 45 days of the experiment, up to 30 days after the lastT cell line administration. For Nogo-1–22 the effect was moretransient, reaching statistical significance at a single time point(PBS score 2.78 � 0.97 day 37 vs Nogo-1–22 PBS score 1.60 �0.96, p � 0.043), and clinical scores worsened after the last ad-ministration but not to levels seen with PBS controls. At the con-clusion of the experiment, we could still demonstrate the existenceof both PLP139–151 and Nogo reactive T cells by in vitro prolif-eration assays (Fig. 5B). In fact, proliferation against Nogo-45–66was much stronger than against the EAE-inducing Ag, suggestingthat there might be cross-suppression by these cells of PLP reac-tive T cells.

To demonstrate that adoptively transferred Nogo-reactive Tcells could enter the CNS of animals with EAE, we immunizedThy1.1a SJL/J female mice with Nogo peptides or PLP139–151,collected draining lymph nodes 10 days later and restimulatedwhole lymphocytes in vitro with the immunizing Ag. We then i.v.administered 10 � 107 Thy1.1a T cells into wild type (Thy1.2)SJL/J females that had been induced with EAE and were at thepeak of disease. Three days later we collected the CNS from theseanimals and detected the presence of Thy1.1a cells in the CNS byimmunohistochemistry. We could demonstrate the presence ofadoptively transferred anti-PLP and anti-Nogo peptide-stimulated

cells in the CNS vasculature, perivascular infiltrates and infiltratingthe parenchyma (Fig. 5, C–E). These results indicate that Nogo-reactive T cells are capable of migrating into the CNS of animalswith active EAE, and therefore, they might modulate disease byacting locally at CNS inflammatory foci.

Finally, we examined the phenotype of anti-Nogo T cell line byin vitro cytokine production assays. Briefly, Nogo-1–22 and Nogo-45–66 T cell lines were stimulated in vitro with their specific Agin the presence of syngeneic irradiated APCs. Supernatants werecollected after 48 h and cytokine production measured by ELISA.Results show that anti-Nogo 45–66 T cell lines spontaneously de-veloped a Th2 phenotype, producing large amounts of IL-4 andIL-10, whereas Nogo-1–22 T cell lines show increased productionof IFN-� (Fig. 5F). Based on these results, it appears that adop-tively transferred Nogo-reactive cells might modulate ongoingEAE by migrating into active demyelinating foci, where they en-counter their specific Ag and differentiate into potentially benefi-cial Th2 phenotypes, which are capable of regulating the autoim-mune response.

DiscussionWe hypothesized that Nogo plays a central role in axonal pathol-ogy in MS and also in CNS trauma, and predicted that there mightbe an advantage in immunizing against the extracellular 66 loopdomain of Nogo to block interactions with Nogo, therefore potentially

FIGURE 3. Autoreactive B cell response character-ization using myelin microarrays. A–D, Individual ar-rays incubated with normal preimmune SJL/J mouse se-rum (A), postimmune 60-day serum from individualSJL/J mice immunized with Nogo-1–22 peptide (B) orNogo-45–66 peptide (C). The majority of green fea-tures are prelabeled marker features used to orient thearrays. Features highlighted illustrate high reactivitiesobserved in two Nogo peptide-immunized animals(MOG, MAG, MBP, and �-B-crystallin (�BCrys)). D,Quantitation of the fluorescence intensities of high-lighted features in A–C displayed in table format. E andF, Heat maps representing cluster analysis of Ag reac-tivities with statistically significant differences in reac-tivity between groups of SJL (E) and C57BL/6 (F) miceinduced for EAE with different Nogo peptides. Animalswere immunized with selected Nogo peptides, and seracollected from SJL mice at day 30 and from C57BL/6mice at day 60 postimmunization for myelin array anal-ysis. Each column represents results from a single con-trol or immunized animal, and each row, fluorescentreactivity against a myelin peptide or protein based onthe displayed color scale. Prefixes denoted the speciesfrom which each peptide was taken: h, human; gp,guinea pig; r, rat; m, mouse; SCH, spinal cord homog-enate. Peptides are as found in Materials and Methods.G and H, Heat maps of a select subset of myelin peptidereactivities from myelin array analysis of SJL andC57BL/6 mice induced for EAE with different Nogopeptides as described in E and F.

(Figure continues)


improving axonal regrowth. We first attempted to induce EAE inNogo knockout mice to ascertain the role that this molecule mighthave in a chronic nonrelapsing model of disease. Our results show thatthere is a slight reduction in disease severity in the knockout mice, butwithout a clear difference from the wild-type mice in histologicalassessment of axonal pathology. Surprisingly, we found that Nogoknockout animals showed a markedly reduced mortality rate using

our protocol for induction of EAE and interpreted this to imply thatNogo might also have a role as an autoantigen in EAE, akin to thedual role that MAG has as both an axonal regrowth inhibitor andencephalitogenic Ag, capable of eliciting EAE (40).

There have been a few other studies that attempted immuniza-tion with Nogo-A-derived peptides in EAE-susceptible rat andmouse strains, and these could not find evidence for demyelination

FIGURE 3 continued


FIGURE 4. Legend continues


or clinical paralysis (21, 22, 41, 42). However, in one case, theimmunization protocol used IFA and aluminum hydroxide, whichare insufficient to induce EAE although being able to generate astrong Ab response (41). A similar strategy, but using whole my-elin immunization in IFA, was successful in generating an Abresponse and promoting regeneration, but did not result in EAE(21). Immunization of EAE-susceptible Lewis rats with a CFAemulsion of peptide p472, which is part of the Nogo-A-specificregion (11), resulted in a T cell response that was also not enceph-alitogenic, but no Abs could be detected (22); our own resultsshow that in the SJL/J mouse strain T cell responses could not begenerated against this peptide, and that the B cell response does notshow any appreciable spread to other myelin Ags (data not shown).In a recent report, a fusion protein of a Nogo-A-specific region(NiG, aa 174–979) and the C-fragment of tetanus toxin was usedto specifically induce a T cell-independent Ab response, to preventthe induction of EAE, and as might be expected no T cell activa-tion could be demonstrated (42). More recently, evidence has beenprovided that immunization against the N terminus Nogo-A se-quence 623–640 may be protective in EAE models (43). Our re-sults show that this is the case when a Th2 response is generated,but that in other conditions immunization with Nogo may inducemeningoencephalitis with clinical and histological characteristicsof EAE. We also show that after immunization with Nogo-66 wecan detect Abs against a number of other myelin components, withpotential consequences for the inflammatory response. In compar-ison with that study, for this work we chose to focus on a differentregion of the Nogo-A molecule, the extracellular 66-aa loop thatinteracts with the Nogo receptor, which in our view is a morelikely target for the immune response.

We demonstrate in this report that the Nogo-66 sequence con-tains at least three antigenically important domains that are capableof inducing and T cell and B cell autoimmune responses in EAE-susceptible mouse strains. Using an EAE induction protocol de-signed to break tolerance to minor Ags, we also show that a de-myelinating response can be generated after immunization withNogo-66 peptides, including clinical signs of paralysis in a smallpercentage of SJL/J animals.

Similar to the susceptibility of different mouse strains to EAEinduction by different encephalitogens, antigenic domains inNogo-66 appear to be strain specific. SJL/J (H-2s) animals made Tcell responses only to peptides Nogo-1–22 and Nogo-45–66, andC57BL/6 (H-2b) animals responded to Nogo-1–22 and Nogo-23–44. At present, we do not know whether the fine specificity ofrecognition of the Nogo-1–22 epitope is the same for both strains.We demonstrated the existence of a strong T cell response that isboth specific and noncross-reactive with other known autoantigensfor EAE.

We also detected specific Ab responses and marked epitopespreading to several other myelin Ags. Abs against several myelincomponents have been described in both MS and EAE, and thereis evidence both for correlation between Ab responses to myelinproteins and disease progression, and direct anti-MOG Ab-induced

demyelination (30–33). We have previously demonstrated tempo-ral spreading of anti-myelin B cell responses in EAE based onmyelin array analysis of serial serum samples collected during theacute and chronic phases of the disease, and found that epitopespreading of the Ab response correlated with increased relapse rate(34). The diversification observed in Nogo-induced EAE is similarin nature and extent to that which we previously described in PLP-and MBP-induced EAE; this diversification could be due to thelocal release of myelin Ags, which may in fact be the basis for thephenomenon of epitope spreading. In either case, classical epitopespreading or local priming from released myelin debris, the tar-geting of multiple myelin Ags likely contributes to autoimmunity.

Recently, Abs against the N-terminal domain of Nogo-A havebeen identified in the serum and cerebrospinal fluid of MS patientsas well as in other inflammatory and noninflammatory acute CNSdiseases (44). The role of these Abs in disease pathogenesis is sofar unknown, and they could either represent an epiphenomenonrelated to tissue injury that causes minor Ag presentation and Abproduction, or alternatively they could be implicated in immune-mediated demyelination or contribute to a protective or regener-ating response to injury. In this study, we found that increasedspreading of the Ab response after immunization with Nogo-45–66 was related to an increased capacity to induce demyelina-tion and clinical paralysis leading us to assume a contributory roleof anti-Nogo Abs to the pathogenesis of EAE.

We did not identify any significant structural similarities be-tween Nogo-66 and other known myelin autoantigens, or the im-munodominant Ags for both studied mouse strains using computeralgorithms for comparison. Although the structure of the Nogoreceptor ectodomain has been described (45, 46), until the three-dimensional structure of Nogo is known it will be difficult to com-pletely exclude the possibility of molecular cross-recognition be-tween Nogo and other myelin Ags.

Finally, we showed that adoptive transfer of T cells reactive toNogo Ags ameliorated ongoing EAE induced with other Ags. Themodulatory T cells were shown to migrate to inflammatory sites inthe CNS and presumably cross-regulate pathogenic Th1 phenotypecells, as well as promote the resting state of microglia after adopt-ing beneficial Th2 phenotypes, a phenomenon that has been pre-viously shown for myelin-specific Th2 cell lines (47), as well asnonmyelin Ags (48). In corroboration of this hypothesis, we wereable to demonstrate that Nogo-specific T cell lines are capable ofsurviving for long periods of time and they actively migrate to theCNS parenchyma in EAE animals after adoptive transfer.

Classical criteria for probable target autoantigens for multiplesclerosis, namely that humoral and cell-mediated immunity bedocumented in MS patients against a molecule, and that the samemolecule is capable of inducing EAE have been met by few myelincomponents, most notably MBP, PLP, MOG, MAG, and OSP(49). Based on the recent evidence of B cell responses and our owndata presented in this report, Nogo-A can potentially join that re-stricted group.

FIGURE 4. Specificity of the T cell response against Nogo peptides. A, T cell lines (TCL) were derived from Nogo-1–22, Nogo-45–66 and PLP139–151immunized SJL/J mice and maintained in vitro. Proliferation assays were set up using 2 � 104 T cells and 5 � 105 irradiated syngeneic APCs per well.Nogo reactive T cells were tested against all Nogo peptides, PLP139–151, MBP85–99, and MOG35–55 peptides. PLP139–151 T cells were tested againsttheir cognate Ag and all Nogo peptides. All peptide concentrations at 20 �g/ml, Con A at 5 �g/ml. B, Primary structure analysis of the Nogo-66 sequence.Calculated Kyte and Doolittle plot of hydropathicity (37), recognition factors, bulkiness, and Zimmerman polarity (38) scores plotted against amino acidnumber, showing the existence of potential antigenic sites for Nogo-1–22 (negative hydropathicity, low bulkiness, and high polarity), Nogo-23–44 (negativehydropathicity, high recognition factor, low bulkiness) and Nogo-45–66 (negative hydropathicity, high recognition factor, high polarity), highlighted inyellow. C, Computer modeling of primary structure for Nogo-1–22, Nogo-23–44, Nogo-45–66, PLP139–151, and MOG35–55. Stick models generatedfrom the sequence used for peptide synthesis.


FIGURE 5. Transfer of anti-Nogo T cells into SJL/J mice with PLP139–151 induced EAE ameliorates chronic disease. A, EAE was induced in SJL/Jmice with PLP139–151 peptide in CFA. Animals were randomized into groups at peak of second relapse to receive PBS (n � 9) (‚, dashed line),Nogo-1–22 reactive T cells (n � 10) �, solid line), or Nogo-45–66 reactive T cells (n � 10) (Œ, solid line). Each animal received three i.v. injections of1.5 � 107 T cells in PBS at weekly intervals for 3 wk (arrow). �, Statistically significant difference in disease score (p � 0.05 Mann-Whitney) betweenthe Nogo-45–66 or Nogo-1–22 group and PBS control. Final score at day 76 failed to reach statistical significance with Mann-Whitney p � 0.053 (PBSscore 3 � 1.5, Nogo-1–22 score 2.3 � 1.05, Nogo-45–66 score 1.6 � 1.07). B, Lymph node cell proliferation at termination of experiment (A). Axillaryand inguinal lymph nodes were dissected at the termination of the above experiment, whole lymph node cells were harvested and proliferation tested invitro against PLP139–151 and Nogo peptides. Values represent mean cpm � SEM of triplicate samples of pooled cells from five animals per group. OVAwas used as irrelevant peptide control. All peptides at 20 �g/ml, Con A (ConA) at 5 �g/ml. C–E, Anti-Nogo T cell lines penetrate the parenchyma ofEAE-induced animals. EAE was induced in SJL/J mice with PLP139–151 peptide in CFA. Thy1.1a congenic SJL/J mice were immunized with PLP139–151, Nogo-1–22 and Nogo-45–66. Draining lymph nodes were dissected 10 days after immunization, and T cells stimulated in vitro with the respective peptidesfor 4 days, after which 1 � 107 cells were injected i.v. into diseased SJL/J mice. Animals were killed 3 days after T cell transfer and their CNS collected, embeddedand frozen for immunohistochemistry. Thy1.1a T cells (arrows) were detected in the perivascular spaces and penetrating the brain parenchyma in mice with EAEthat received PLP139–151, Nogo-1–22 and Nogo-45–66 stimulated cells (C–E, respectively). F, Anti-Nogo T cell lines spontaneously develop different Thphenotypes. T cell lines were isolated and maintained in culture from SJL/J mice immunized with Nogo-1–22 and Nogo-45–66 peptides. These T cells wererestimulated in vitro with Nogo peptides, PLP139–151 peptide, irrelevant peptide control (V�5.1), negative (medium), and positive (ConA) controls. Levels ofIL-4, IL-10, and IFN-� in the supernatants after 48 h of culture were measured by sandwich ELISA. Results are expressed in picograms per milliliter (�SEM)of triplicate samples. Nogo-1–22 T cell lines (f) and Nogo-45–66 T cell lines (�) results are shown.


Nogo Ags thus appear to behave similarly to other known en-cephalitogenic myelin Ags for these mouse strains, such as themajor encephalitogenic determinants PLP139–151 and MOG35–55, or the minor determinants MBP85–99, MAG, MOB, OSP, andCNPase. Comparisons between these types of EAE are made dif-ficult by different induction protocols, but in our opinion Nogo-66belongs in the minor Ag group, although with a lesser degree ofencephalitogenicity (40, 49–51). Unlike these Ags, however, andapart from MAG, none of the other myelin proteins are thought toplay a role in axonal regrowth. The potential for oligodendrocyte-myelin glycoprotein to play a role as an encephalitogen is atpresent unknown. Therefore, the recognition of the possible con-tribution of Nogo-66 to CNS autoimmune demyelination is dis-tinct: first, as an encephalitogenic Ag Nogo might assume an im-portant role in the development of axonal pathology in this diseaseand in MS (44), where Abs to Nogo have been detected. Also,because modulation of the immune response to Nogo-66 appearsto be capable of ameliorating EAE, several Ag-specific therapeuticstrategies might be devised to take advantage of this phenomenonto treat human autoimmune demyelinating diseases. It is also pos-sible that Nogo immunization might worsen disease given the ca-pacity for inducing such strong T cell and B cell responses. In fact,we and others have found that Th2 type responses can be detri-mental in EAE, in that not only anaphylaxis to myelin proteins ispossible (52), but also that myelin-reactive Th2 cells can causeEAE (53).

Finally, because immunization with Nogo peptides is capable ofgenerating such a broad based B cell response to other myelin Ags,including MAG, vaccination with these Ags might have a usefulrole in improving axonal regeneration in CNS traumatic models,such as spinal cord injury. The phenomenon of B cell epitopespreading after immunization with myelin Ags might allow a ther-apeutically important Ab response against several other potentiallyimportant neurite growth inhibitors even with single myelin Agvaccination, with important implications for ameliorating CNSinjury.

AcknowledgmentsWe thank Dennis Mitchell for peptide synthesis.

References1. Chen, M. S., A. B. Huber, M. E. van der Haar, M. Frank, L. Schnell,

A. A. Spillman, F. Christ, and M. E. Schwab. 2000. Nogo-A is a myelin-asso-ciated neurite outgrowth inhibitor and an antigen for monoclonal antibody IN-1.Nature 403:434.

2. GrandPré, T., F. Nakamura, T. Vartanian, and S. M. Strittmatter. 2000. Identi-fication of the Nogo inhibitor of axonal regeneration as a Reticulon protein.Nature 403:439.

3. Fournier, A. E., T. GrandPré and, S. M. Strittmatter. 2001. Identification of areceptor mediating Nogo-66 inhibition of axonal regeneration. Nature 409:341.

4. Brittis, P. A., and J. G. Flanagan. 2001. Nogo domains and a Nogo receptor:implications for axon regeneration. Neuron 30:11.

5. Wang, K. C., J. A. Kim, R. Sivasankaran, R. Segal, and Z. He. 2002. p75 interactswith the Nogo receptor as a co-receptor for Nogo, MAG and OMgp. Nature420:74.

6. Wong, S. T., J. R. Henley, K. C. Kanning, K. H. Huang, M. Bothwell, andM. M. Poo. 2002. A p75NTR and Nogo receptor complex mediates repulsivesignaling by myelin-associated glycoprotein. Nat. Neurosci. 5:1302.

7. Niederost, B., T. Oertle, J. Fritsche, R. A. McKinney, and C. E. Bandtlow. 2002.Nogo-A and myelin-associated glycoprotein mediate neurite growth inhibition byantagonistic regulation of RhoA and Rac1. J. Neurosci. 22:10368.

8. Liu B. P., A. Fournier, T. GrandPré, and S. M. Strittmatter. 2002. Myelin-asso-ciated glycoprotein as a functional ligand for the Nogo-66 receptor. Science297:1190.

9. Wang, K. C., V. Koprivica, J. A. Kim, R. Sivasankaran, Y. Guo, R. L. Neve, andZ. He. 2002. Oligodendrocyte-myelin glycoprotein is a Nogo receptor ligand thatinhibits neurite outgrowth. Nature 417:941.

10. Domeniconi, M., Z. Cao, T. Spencer, R. Sivasankaran, K. Wang, E. Nikulina,N. Kimura, H. Cai, K. Deng, Y. Gao, Z. He, and M. Filbin. 2002. Myelin-associated glycoprotein interacts with the Nogo66 receptor to inhibit neurite out-growth. Neuron 35:283.

11. Oertle, T., M. E. van der Haar, C. E. Bandtlow, A. Robeva, P. Burfeind, A. Buss,A. B. Huber, M. Simonen, L. Schnell, C. Brosamle, et al. 2003. Nogo-A inhibits

neurite outgrowth and cell spreading with three discrete regions. J. Neurosci.23:5393.

12. Karim, F., D. Volker, and M. E. Schwab. 2001. Improving axonal growth andfunctional recovery after experimental spinal cord injury by neutralizing myelinassociated inhibitors. Brain Res. Rev. 36:204.

13. Merkler, D., G. A. S. Metz, O. Raineteau, V. Dietz, M. E. Schwab, and K. Fouad.2001. Locomotor recovery in spinal cord-injured rats treated with an antibodyneutralizing the myelin-associated neurite growth inhibitor Nogo-A. J. Neurosci.21:3665.

14. Brosamle, C., A. B. Huber, M. Fiedler, A. Skerra, and M. E. Schwab. 2000.Regeneration of lesioned corticospinal tract fibers in the adult rat induced by arecombinant, humanized IN-1 antibody fragment. J. Neurosci. 20:8061.

15. GrandPré, T., S. Li, and S. M. Strittmatter. 2002. Nogo-66 receptor antagonistpeptide promotes axonal regeneration. Nature 417:547.

16. Li, S., and S. M. Strittmatter. 2003. Delayed systemic Nogo-66 receptor antag-onist promotes recovery from spinal cord injury. J. Neurosci. 23:4219.

17. Fournier, A. E., G. C. Gould, B. P. Liu, and S. M. Strittmatter. 2002. Truncatedsoluble Nogo receptor binds Nogo-66 and blocks inhibition of axon growth bymyelin. J. Neurosci. 22:8876.

18. Simonen, M., V. Pedersen, O. Weinmann, L. Schnell, A. Buss, B. Ledermann,F. Christ, G. Sansig, H. van der Putten and M. E. Schwab. 2003. Systemic de-letion of the myelin-associated outgrowth inhibitor Nogo-A improves regenera-tive and plastic responses after spinal cord injury. Neuron 38:201.

19. Zheng, B., C. Ho, S. Li, H. Keirstead, O. Steward, and M. Tessier-Lavigne. 2003.Lack of enhanced spinal regeneration in Nogo-deficient mice. Neuron 38:213.

20. Kim, J. E., S. Li, T. GrandPre, D. Qiu, and S. M. Strittmatter. 2003. Axonregeneration in young adult mice lacking Nogo-A/B. Neuron 38:187.

21. Huang, D. W., L. McKerracher, P. E. Braun, and S. David. 1999. A therapeuticvaccine approach to stimulate axon regeneration in the adult mammalian spinalcord. Neuron 34:639.

22. Hauben, E., A. Ibarra, T. Mizrahi, R. Barouch, E. Agranov, and M. Schwartz.2001. Vaccination with a Nogo-A-derived peptide after incomplete spinal cordinjury promotes recovery via a T-cell-mediated neuroprotective response: com-parison with other myelin antigens. Proc. Natl. Acad. Sci. USA 98:15173.

23. Steinman, L., R. Martin, C. C. Bernard, P. Conlon, and J. R. Oksenberg. 2002.Multiple sclerosis: deeper understanding of its pathogenesis reveals new targetsfor therapy. Annu. Rev. Neurosci. 25:491.

24. Seamons, A., A. Perchellet, and J. Goverman. 2003. Immune tolerance to myelinproteins. Immunol. Res. 28:201.

25. Steinman, L. 1999. Assessment of animal models for MS and demyelinatingdisease in the design of rational therapy. Neuron 24:511.

26. t Hart, B. A., and S. Amor. 2003. The use of animal models to investigate thepathogenesis of neuroinflammatory disorders of the central nervous system. Curr.Opin. Neurol. 16:375.

27. Steinman, L. 2003. Optic neuritis, a new variant of experimental encephalomy-elitis, a durable model for all seasons, now in its seventieth year. J. Exp. Med.197:1065.

28. Dal Canto, M. C., R. W. Melvold, B. S. Kim, and S. D. Miller. 1995. Two modelsof multiple sclerosis: experimental allergic encephalomyelitis (EAE) and Thei-ler’s murine encephalomyelitis virus (TMEV) infection: a pathological and im-munological comparison. Microsc. Res. Tech. 32:215.

29. Zamvil, S. S., and L. Steinman. 1990. The T lymphocyte in experimental allergicencephalomyelitis. Annu. Rev. Immunol. 8:579.

30. Berger, T., P. Rubner, F. Schautzer, R. Egg, H. Ulmer, I. Mayringer, E. Dilitz,F. Deisenhammer, and M. Reindl. 2003. Antimyelin antibodies as a predictor ofclinically definite multiple sclerosis after a first demyelinating event. N. Engl.J. Med. 349:139.

31. Genain, C. P., B. Cannella, S. L. Hauser, and C. S. Raine. 1999. Identification ofautoantibodies associated with myelin damage in multiple sclerosis. Nat. Med.5:170.

32. Steinman, L. 1996. Multiple sclerosis: a coordinated immunological attackagainst myelin in the central nervous system. Cell 85:299.

33. Warren, K. G., I. Catz, and L. Steinman. 1995. Fine specificity of the antibodyresponse to myelin basic protein in the central nervous system in multiple scle-rosis: the minimal B cell epitope and a model of its unique features. Proc. Natl.Acad. Sci. USA 92:11061.

34. Robinson, W. H., P. Fontoura, B. J. Lee, H. E. de Vegvar, J. Tom, R. Pedotti,C. D. DiGennaro, D. J. Mitchell, D. Fong, P. P. Ho, et al. 2003. Protein microar-rays guide tolerizing DNA vaccine treatment of autoimmune encephalomyelitis.Nat. Biotechnol. 21:1033.

35. Trapp, B. D., J. Peterson, R. M. Ransohoff, R. Rudick, S. Mork, and L. Bo. 1998.Axonal transection in the lesions of multiple sclerosis. N. Engl. J. Med. 338:278.

36. Skundric, D. S., C. Kim, H. Y. Tse, and C. S. Raine. 1993. Homing of T cells tothe central nervous system throughout the course of relapsing experimental au-toimmune encephalomyelitis in Thy-1 congenic mice. J. Neuroimmunol. 46:113.

37. Kyte, J., and R. F. Doolittle. 1982. A simple method for displaying the hydro-pathic character of a protein. J. Mol. Biol. 157:105.

38. Zimmerman J. M., N. Eliezer, and R. Simha. 1968. Polarity. J. Theor. Biol.21:170.

39. Chang, T. T., C. Jabs, R. A. Sobel, V. K. Kuchroo, and A. H. Sharpe. 1999.Studies in B7-deficient mice reveal a critical role for B7 costimulation in bothinduction and effector phases of experimental autoimmune encephalomyelitis.J. Exp. Med. 190:733.


40. Morris-Downes, M. M., K. McCormack, D. Baker, D. Sivaprasad,J. Natkuranajah, and S. Amor. 2002. Encephalitogenic and immunogenic poten-tial of myelin-associated glycoprotein (MAG), oligodendrocyte-specific glycop-rotein (OSP) and 2�,3�-cyclic nucleotide 3�-phosphodiesterase (CNPase) in ABHand SJL mice. J. Neuroimmunol. 122:20.

41. Sicotte, M., O. Tsatas, S. Y. Jeong, C. Q. Cai, Z. He, and S. David. 2003.Immunization with myelin or recombinant Nogo-66/MAG in alum promotesaxon regeneration and sprouting after corticospinal tract lesions in the spinalcord. Mol. Cell. Neurosci. 23:251.

42. Merkler, D., T. Oertle, A. Buss, D. D. Pinschewer, L. Schnell, F. M. Bareyre,M. Kerschensteiner, B. S. Buddeberg, and M. E. Schwab. 2003. Rapid inductionof autoantibodies against Nogo-A and MOG in the absence of an encephalito-genic T cell response: implication for immunotherapeutic approaches in neuro-logical diseases. FASEB J. 17:2275.

43. Karnezis, T., W. Mandemakers, J. L. McQualter, B. Zheng, P. P. Ho,K. A. Jordan, B. M. Murray, B. Barres, M. Tessier-Levigne, andC. C. A. Bernard. 2004. The neurite outgrowth inhibitor Nogo A is involved inautoimmune-mediated demyelination. Nat. Neurosci. 7:736.

44. Reindl, M., S. Khantane, R. Ehling, K. Schanda, A. Lutterotti, C. Brinkhoff,T. Oertle, M. E. Schwab, F. Deisenhammer, T. Berger, and C. E. Bandtlow. 2003.Serum and cerebrospinal fluid antibodies to Nogo-A in patients with multiplesclerosis and acute neurological disorders. J. Neuroimmunol. 145:139.

45. He, X. L., J. F. Bazan, G. McDermott, J. B. Park, K. Wang, M. Tessier-Lavigne,Z. He, and K. C. Garcia. 2003. Structure of the Nogo receptor ectodomain: arecognition module implicated in myelin inhibition. Neuron 38:177.

46. Barton, W. A., B. P. Liu, D. Tzvetkova, P. D. Jeffrey, A. E. Fournier, D. Sah,R. Cate, S. M. Strittmatter, and D. B. Nikolov. 2003. Structure and axon out-

growth inhibitor binding of the Nogo-66 receptor and related proteins. EMBO J.22:3291.

47. Gimsa, U., S. A. Wolf, D. Haas, I. Bechmann, and R. Nitsch. 2001. Th2 cellssupport intrinsic anti-inflammatory properties of the brain. J. Neuroimmunol.119:73.

48. Falcone, M., and B. R. Bloom. 1997. A T helper cell 2 (TH2) immune responseagainst non-self antigens modifies the cytokine profile of autoimmune T cells andprotects against experimental autoimmune encephalomyelitis. J. Exp. Med.185:901.

49. Stevens, D. B., K. Chen, R. S. Seitz, E. E. Sercarz, and J. M. Bronstein. 1999.Oligodendrocyte-specific protein peptides induce experimental autoimmune en-cephalomyelitis in SJL/J mice. J. Immunol. 162:7501.

50. Holz, A., B. Bielekova, R. Martin, and M. B. Oldstone. 2000. Myelin-associatedoligodendrocytic basic protein: identification of an encephalitogenic epitope andassociation with multiple sclerosis. J. Immunol. 164:1103.

51. Sakai, K., S. S. Zamvil, D. J. Mitchell, M. Lim, J. B. Rothbard, and L. Steinman.1988. Characterization of a major encephalitogenic T cell epitope in SJL/J micewith synthetic oligopeptides of myelin basic protein. J. Neuroimmunol. 19:21.

52. Pedotti, R., D. Mitchell, J. Wedemeyer, M. Karpuj, D. Chabas, E. M. Hattab,M. Tsai, S. J. Galli, and L. Steinman. 2001. An unexpected version of horrorautotoxicus: anaphylactic shock to a self-peptide. Nat. Immunol. 2:216.

53. Lafaille, J. J., F. V. Keere, A. L. Hsu, J. L. Baron, W. Haas, C. S. Raine and S.Tonegawa. 1997. Myelin basic protein-specific T helper 2 (Th2) cells cause ex-perimental autoimmune encephalomyelitis in immunodeficient hosts rather thanprotect them from the disease. J. Exp. Med. 186:307.


immunity to the extracellular domain of nogo-a modulates...

Documents