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The Journal of Immunology, 2004, 173: 6981-6992.
Copyright © 2004 by The American Association of Immunologists

Immunity to the Extracellular Domain of Nogo-A Modulates Experimental Autoimmune Encephalomyelitis1

Paulo Fontoura2,*,¶, Peggy P. Ho*, Jason DeVoss*, Binhai Zheng{dagger}, Byung J. Lee*, Brian A. Kidd§, Hideki Garren||, Raymond A. Sobel{ddagger}, William H. Robinson§, Marc Tessier-Lavigne{dagger} and Lawrence Steinman*

Departments of * Neurology and Neurological Sciences, School of Medicine, {dagger} Biological Sciences, Howard Hughes Medical Institute, {ddagger} Pathology, School of Medicine, and § Division of Immunology and Rheumatology, Department of Medicine, Stanford University, Stanford, CA 94305; Gulbenkian Science Institute, Oeiras, Portugal; and || Bayhill Therapeutics, Palo Alto, CA 94303


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Nogo-66, the extracellular 66 aa loop of the Nogo-A protein found in CNS myelin, interacts with the Nogo receptor and has been proposed to mediate inhibition of axonal regrowth. It has been shown that immunization with Nogo-A promotes recovery in animal models of spinal cord injury through induction of Ab production. In this report, studies were performed to characterize the 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 with clinical and pathological similarities to EAE. The Nogo-66 peptides elicited strong T cell responses that were not cross-reactive to other encephalitogenic myelin Ags. Using a large scale spotted microarray containing proteins and peptides derived from a wide spectrum of myelin components, we demonstrated that Nogo-66 peptides also generated a specific Ab response that spreads to several other encephalitogenic myelin Ags following immunization. Nogo-66-specific T cell lines ameliorated established EAE, via Nogo-66-specific Th2 cells that entered the CNS. These results indicate that some T cell and B cell immune responses to Nogo-66 are associated with suppression of ongoing EAE, whereas other Nogo-66 epitopes can be encephalitogenic.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The Nogo-A protein is a member of the reticulon family present in myelin that inhibits neurite regrowth (1, 2). Nogo-A is encoded by the longest of three alternatively produced transcripts of the nogo gene, has 1163 aa residues and is found mainly in the CNS. All three isoforms contain a predicted extracellular 66 aa loop in their C terminus end, and a major inhibitory activity has been attributed to this extracellular loop, called Nogo-66 (2). Nogo-66 interacts with a GPI-anchored Nogo receptor protein belonging to the family of leucine-rich repeat proteins and signals through the neurotrophin receptor p75NTR coreceptor, mediating neurite growth inhibition by antagonistic regulation of intracellular signaling through RhoA and Rac1 (3, 4, 5, 6, 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 signaling through the Nogo receptor system (8, 9, 10). Besides the Nogo-66 region, it has recently been shown that two other regions of Nogo-A might also be implicated in inhibition of neurite outgrowth and collapse of growth cones (11).

Several therapeutic strategies aimed at improving axonal regeneration have been directed toward blocking interactions in the Nogo-Nogo receptor system (12); these have included using the recombinant mAb IN-1 against Nogo-A (13, 14), Nogo receptor antagonist peptide NEP1–40 (15, 16), and truncated soluble Nogo receptor (17). More recently, studies of different Nogo knockout mouse strains have yielded conflicting results about the role of Nogo in regeneration (18, 19, 20). Other groups have had some success in increasing neurite regrowth or enhancing neuroprotection in models of spinal cord injury by inducing a broad-based immune response against myelin-associated axonal regrowth inhibitors (21), or specifically against Nogo-A-derived peptide 472 (22).

Experimental autoimmune encephalomyelitis (EAE) is an animal model that has been intensely investigated as a source of pathophysiological and therapeutic insight into the human disease multiple sclerosis (MS) (23, 24, 25, 26, 27, 28). EAE is mainly mediated by a myelin Ag-specific T cell response that coordinates the immune attack against the CNS (29). The role of B cell responses is not as well defined in EAE and MS, although Abs targeting myelin oligodendrocyte glycoprotein (MOG) are thought to be pathogenic in both EAE and MS, and other anti-myelin Abs have also been detected (30, 31, 32, 33). We have recently shown that epitope spreading of the B cell response occurs after active EAE induction and that the extent of spreading of the immune response to a variety of myelin components correlates with relapse rate in chronic models (34). In recent years, axonal pathology has emerged as a major determinant of neurological deficit in MS (35), leading to the concept that blocking the action of axonal regrowth inhibitors might also be beneficial. In the present study, we sought to determine how T cell and B cell responses to Nogo affect the pathophysiology of EAE and to ascertain the characteristics of responses that might be useful for therapy in CNS diseases with axonal injury.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Female SJL/J and C57BL/6 mice were obtained from The Jackson Laboratory (Bar Harbor, ME) at 5 wk of age. Mice were between 6 and 8 wk of age when experiments were initiated. Nogo-A/B/C knockout mice were generated as described by Zheng et al. (19); these H-2b mice C57BL/6 x 129S7 mice have been backcrossed to C57BL/6, with three of four of the mice bred to homozygosity on C57BL/6 for these experiments ((C57BL/6 x 129S7) N2 background). SJL/J Thy1.1a congenic mice were a kind gift from H. Y. Tse at Wayne State University (Detroit, MI) (36). All animal experiments were conducted according to protocols reviewed and approved by 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{beta}5.1 (KGERSILKCIPISGHLSVA) peptides were synthesized by standard 9-fluorenylmethoxycarbonyl chemistry in our own facilities on a peptide synthesizer (model 9050; MilliGen, Burlington, MA). Peptides were HPLC purified, structures confirmed by amino acid analysis and mass spectroscopy and resuspended in PBS.

EAE induction

For Nogo immunizations, SJL/J and C57BL/6 female mice were injected s.c. with Nogo-peptide (from 100 to 500 µg per injection) emulsified in CFA, consisting of IFA (Difco, Detroit, MI) and 1 mg/ml heat-inactivated Mycobacterium tuberculosis (strain H37RA; Difco). Animals received two s.c. injections a week apart in different sites (flank and groin); at the time of the second immunization and 48 h later, mice were injected i.v. with Bordetella pertussis toxin (List Laboratories, Campbell, CA) in PBS, 500 ng/animal. For Nogo-A/B/C knockouts, female mice were immunized s.c. with 100 µg 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 and 48 h later. Chronic EAE was induced in SJL/J mice by s.c. injection of 100 µg of PLP139–151 peptide emulsion in CFA. Animals were clinically scored daily according to the following scale: 0, normal; 1, tail paralysis; 2, hind limb paraparesis; 3, complete hind limb paralysis; 4, forelimb paresis 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 samples were stained with H&E and Luxol fast blue stains and Bielschowsky impregnation. For semiquantitative analysis, meningeal and parenchymal inflammatory foci were counted by an observer blinded to the immunization and clinical status of the mice. For Thy1.1a immunohistochemistry, CNS samples were snap-frozen in tissue Tek OCT (Sakura Finetek, Torrance, CA) after partial immersion in liquid N2. Cryosections (8-µm thick) were fixed in acetone and immunostained with a biotinylated anti-Thy1.1a Ab conjugate (BD Pharmingen, San Diego, CA) followed by avidin-biotin complex reagent (Vector Laboratories, Burlingame, CA). Reaction product was 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 x 106 cells/well. Culture medium consisted of RPMI 1640 supplemented with L-glutamine (2 mM), sodium pyruvate (1 mM), nonessential amino acids (0.1 mM), penicillin (100 U/ml), streptomycin (0.1 mg/ml), 2-ME (5 x 10–5 M), and 10% FBS. Wells were pulsed with 1 µCi of [3H]TdR (ICN Pharmaceuticals, Irvine, CA) for the final 16 h of culture, and incorporated 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 in the literature. These T cells were then tested for the production of various cytokines. T cells (5 x 104) were incubated with 2.5 x 106 irradiated syngenic APC/ml in enriched RPMI 1640 and 10% FCS. After 48 h of culture, supernatants were collected and tested by sandwich ELISA using standard ELISA kits (BD Pharmingen).

Primary structure analysis

The Nogo-66 sequence was obtained from GenBank (accession number AAM77068) and analyzed using freely available software at http://www.expasy.org for the Kyte-Doolittle plot of hydropathicity (37), Zimmerman polarity (38), and bulkiness and recognition factors. Homology analysis was done using ClustalW with a Blosum matrix (http://www.ch.embnet.org/software/ClustalW.html). Computer modeling for Nogo peptide primary structure was performed using Insight II (Accelerys, San Diego, CA).

Myelin arrays

Myelin arrays and the associated methods used in this work were previously described in detail (34). Ordered Ag arrays were produced using a robotic microarrayer to attach myelin and control peptides and proteins to poly-L-lysine-coated microscopic slides. For the studies described, additional 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 derived from oligodendrocyte-specific protein (OSP), and golli-MBP. Reactive Abs were detected using Cy3-conjugated goat anti-mouse IgG/IgM secondary Ab before scanning. Individual arrays were probed with 1/150 dilutions of serum from individual animals. Data analysis was performed using Statistical Analysis for Microarrays (SAM) software (http://www.stat-class.stanford.edu/SAM/servlet/SAMServlet). The reported Ag lists are SAM-identified features with q values <5% (see Fig. 3E) and <10% (Fig. 3F) and absolute fluorescence levels above a numerator threshold of 1. Heat maps (see Fig. 3, G and H) of array reactivities are represented for a select subset of myelin Ag features. Cluster software was used to hierarchically order the mice and Ag features based on a pairwise similarity function, and TreeView software to display the results as a heat map (http://rana.lbl.gov/EisenSoftware.htm).



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  		FIGURE 3. Autoreactive B cell response characterization using myelin microarrays. A–D, Individual arrays incubated with normal preimmune SJL/J mouse serum (A), postimmune 60-day serum from individual SJL/J mice immunized with Nogo-1–22 peptide (B) or Nogo-45–66 peptide (C). The majority of green features are prelabeled marker features used to orient the arrays. Features highlighted illustrate high reactivities observed in two Nogo peptide-immunized animals (MOG, MAG, MBP, and {alpha}-B-crystallin ({alpha}BCrys)). D, Quantitation of the fluorescence intensities of highlighted features in A–C displayed in table format. E and F, Heat maps representing cluster analysis of Ag reactivities with statistically significant differences in reactivity between groups of SJL (E) and C57BL/6 (F) mice induced for EAE with different Nogo peptides. Animals were immunized with selected Nogo peptides, and sera collected from SJL mice at day 30 and from C57BL/6 mice at day 60 postimmunization for myelin array analysis. Each column represents results from a single control or immunized animal, and each row, fluorescent reactivity against a myelin peptide or protein based on the displayed color scale. Prefixes denoted the species from which each peptide was taken: h, human; gp, guinea pig; r, rat; m, mouse; SCH, spinal cord homogenate. Peptides are as found in Materials and Methods. G and H, Heat maps of a select subset of myelin peptide reactivities from myelin array analysis of SJL and C57BL/6 mice induced for EAE with different Nogo peptides as described in E and F.

(Figure continues)

 


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  		FIGURE 3A. continued

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Induction of EAE in Nogo-A/B/C mutant mice

To study the possible contribution of the Nogo-A protein to axonal regeneration in the EAE model, we induced disease in Nogo-A/B/C knockout mice on an H-2b background ((C57BL/6 x 129S7) NE2), generated by targeted mutation of the common C-terminal region that contains Nogo-66. This mutant has been shown to be a null for Nogo-C and a null or severe hypomorph for Nogo-A and Nogo-B (19). Animals were observed for 6 wk postinduction for disease phenotype and incidence. As shown in Fig. 1, Nogo knockout mice have reduced disease severity compared with wild-type C57BL/6 controls or controls with a similar genetic background, though this does not reach statistical significance at the end of the experiment (p = 0.043 at day 33, but otherwise p > 0.05 throughout). There is, however, a statistically significant difference in cumulative mortality and survival rates between both groups (Fig. 1, A and B, {chi}2 value p < 0.002). Histological assessment of sections comprising the entire CNS at the end of the experiment did not reveal a significant difference in numbers of inflammatory/demyelinating lesions between the knockout and wild-type mice or an appreciable difference in the appearance of the lesions or extent of axonal pathology (Fig. 1, C–F). Based on these data we hypothesized that Nogo-66 might have a specific role as an autoantigen capable of modifying the disease course in EAE.



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  		FIGURE 1. Induction of EAE in Nogo-A/B/C knockout mice. A, Mean disease scores (±SD) over time after induction of EAE with MOG35–55 peptide in CFA. Combined results from two different experiments comparing wild-type C57BL/6 mice ({diamond}, dashed line) and Nogo knockdown mice ({blacktriangleup}, solid line) (total n = 16 for each group). B, Cumulative EAE mortality rate and Kaplan-Meier survival curves for wild-type (WT) and Nogo knockout (KO) mice from previous experiment. There is a statistically significant difference in survival between knockdown and wild-type groups ({chi}2 value p < 0.002). C–F, Paraffin sections of illustrative CNS lesions in MOG peptide-induced EAE. C and D, Typical mononuclear cell infiltrates (right side of fields) in spinal cord of wild-type mouse. Myelin is intact (C, blue) and axons are preserved (D, black fibers) in the intact portions (left side of fields). E and F, Meningeal and parenchymal inflammatory lesion in spinal cord of Nogo knockout mouse. Spinal nerve root myelin and axons (upper left of fields) are preserved in the unaffected portions. There is demyelination and axonal loss in portions infiltrated by mononuclear cells. C and E, Luxol fast blue-H&E stain; D and F, Bielschowsky silver impregnation for axons. All pictures are at magnification of x228.

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

To ascertain the potential for Nogo-A to act as a target autoantigen for autoimmune demyelination, we focused on the putative extracellular 66 aa loop, Nogo-66. This extracellular domain would be a likely target for interactions with both the cellular and humoral arms of the immune system, and such interactions might therefore also modulate axonal regrowth. EAE susceptible SJL/J and C57BL/6 female mice were immunized with Nogo-66 derived peptides in CFA, as described in Materials and Methods. Three different peptides were synthesized, encompassing the entire sequence of Nogo-66 (peptides 1–22, 23–44, and 45–66). Beginning at day 10 postimmunization, all animals in both strains exhibited signs of photophobia, lethargy and stopped grooming, and some had a partial loss of tail tone. These findings lasted for ~3–4 days, and then subsided completely. Control animals immunized with PBS in CFA showed no abnormalities in behavior. A few of the SJL/J mice immunized with Nogo-45–66 peptide (2 of 10 in two combined experiments) also had typical signs of clinical EAE, including transient hind limb paralysis, loss of tail tonicity and bladder incontinence. Histological evaluation of brains and spinal cords of Nogo-immunized animals revealed inflammatory foci in the meninges and parenchyma, pathological findings characteristic of EAE (Fig. 2, A–C). Semiquantitative analysis of inflammatory lesions was done on CNS tissues of SJL/J and C57BL/6 mice that had been immunized with the Nogo peptides to which T cell responses could be generated. Small numbers of meningeal inflammatory foci were found in both strains and parenchymal lesions were more numerous in SJL/J than in C57BL/6 mice (Fig. 2D). The SJL/J mouse immunized with Nogo-45–66 with the most severe clinical disease (score = 2) had numerous meningeal (n = 65) and a few parenchymal (n = 11) foci, representing the high end of the pathological spectrum induced by immunization with this peptide. One SJL/J mouse immunized with Nogo-1–22 had 28 meningeal and 32 parenchymal inflammatory foci. These results indicate that Nogo-66 peptides may act as weak encephalitogens in EAE-susceptible mouse strains such as SJL/J.



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  		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 x228. C, Meningitis (arrow) adjacent to cerebellum of a C57BL/6 mouse that had been immunized with Nogo-1–22 in CFA and showed photophobia. Luxol fast blue/H&E stain. Magnification shown at x228. D, Semiquantitative analysis of CNS inflammatory foci in SJL/J and C57BL/6 mice immunized with Nogo-66 peptides and PBS or PBS alone in CFA. Meningeal inflammatory foci were present in both strains and parenchymal inflammatory foci are mainly in SJL/J mice with clinical disease. E, Immunization with Nogo peptides induces a T cell proliferative response. At day 30 postimmunization, draining lymph nodes were dissected and lymph node cell proliferation assays setup against Nogo peptides (Ng1–22, Ng23–44, Ng45–66), control peptide (V{beta}5.1), Con A (ConA), or medium. All peptides are at a concentration of 20 µg/ml, Con A at 5 µg/ml. Results shown are mean cpm ± SEM of triplicate samples. The experiments shown are representative of several independent experiments.

 
To evaluate T cell proliferative responses against Nogo peptides, we collected axillary and inguinal draining lymph nodes at the end of the experiments and performed proliferation assays against a panel of Nogo and control peptides. We found a strong and specific proliferative response to the immunizing peptide in Nogo-1–22 and Nogo-45–66 immunized in SJL/J mice and to a lesser degree to Nogo-1–22 and Nogo-23–44 in C57BL/6 animals (Fig. 2E), indicating that different strains respond to different Nogo epitopes.

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

To follow the induction of a specific Ab response to the immunizing Nogo peptides, and the possible spread to other myelin Ags, we used a 2304-feature myelin array developed in our laboratory that contains over 250 distinct Ags (Fig. 3). The array contains myelin proteins and overlapping peptides representing many EAE-relevant targets (34), and Ags including Nogo-66 overlapping peptides, which were added for these studies. After immunization with Nogo-45–66 or Nogo-1–22, Ab reactivity could be detected consistently against the immunizing Nogo peptide, and to several other myelin peptides present on the myelin array (Fig. 3, A–D). Sera from five immunized animals were obtained at different time points after EAE induction with all three Nogo peptides, and used to probe myelin arrays. Statistical analysis of array data revealed marked intra- and intermolecular epitope spreading of the Ab response to other myelin proteins and peptides. This response was most pronounced in SJL mice immunized with Nogo-45–66 and involved targeting of epitopes in myelin proteins with Abs directed against 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), {alpha}-B-crystallin (p121–140), 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) (p343–373), and Nogo (p1–22, p11–30, p45–66) (Fig. 3, E–H). Analysis of the spreading of the Ab response revealed distinct patterns for each immunizing Ag within each strain. The B cell response spread from Nogo-1–22 and Nogo-23–44 to a limited set of MBP, PLP, and MOG peptides. The greatest epitope spreading of autoreactive Ab responses was observed in SJL/J mice following immunization with Nogo-45–66 (Fig. 3, E and G), and increased spreading was associated with more severe clinical disease. In SJL/J mice, the group immunized with Nogo-1–22 demonstrated the second greatest degree of anti-myelin B cell epitope spreading, and mice from this group exhibited partial loss of tail tone and meningitis on histological examination. SJL/J mice immunized with Nogo-23–44 and C57BL/6 mice immunized with Nogo-1–22 or Nogo-23–44 exhibited relatively less spreading and did not manifest clinical disease. Our results show that Nogo-45–66 elicits broader Ab responses. In SJL/J mice Nogo-45–66 induced more severe paralytic disease than either of the other two synthesized peptides. These data suggest that Nogo-45–66 represents an immunodominant Nogo 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 not cross-react with other myelin Ags

We asked whether the clinical EAE seen in SJL/J mice, and histological evidence of demyelination in SJL/J and C57BL/6 mice following immunization with Nogo-66 peptides could result from cross-reactivity between these Nogo peptides and other encephalitogenic myelin Ags such as PLP139–151 or MOG35–55, which are capable of eliciting EAE in SJL/J and C57BL/6 mice, respectively. We analyzed the specificity of the T cell responses using Nogo-1–22-, Nogo-45–66-, and PLP139–151-specific T cell lines from SJL/J animals. The PLP139–151-specific T cell line proliferates against its cognate Ag, but show no reactivity to any of the three Nogo-66 peptides. Nogo-specific T cell lines also show reactivity only against their specific peptide, and do not proliferate against PLP139–151, MBP85–99, or MOG35–55 (Fig. 4A). Therefore, we conclude that Nogo immunization generates a specific T cell response that does not show cross-reactivity to other myelin autoantigens. Because the secondary structure of the Nogo protein is currently unknown, we compared the primary structure of 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 find any relevant similarities between Nogo-66 peptides and the other myelin Ags studied. We also could predict the existence of potential antigenic regions in all three of the arbitrarily selected Nogo peptides, namely in the C terminus region of Nogo-1–22 and Nogo-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 program ClustalW did not reveal significant similarities, leading us to conclude that the presence of an immune response against Nogo is highly unlikely to be related to cross-reactivity to other myelin constituents.



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  		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 PLP139–151 immunized SJL/J mice and maintained in vitro. Proliferation assays were set up using 2 x 104 T cells and 5 x 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 against their cognate Ag and all Nogo peptides. All peptide concentrations at 20 µg/ml, Con A at 5 µg/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 acid number, showing the existence of potential antigenic sites for Nogo-1–22 (negative hydropathicity, low bulkiness, and high polarity), Nogo-23–44 (negative hydropathicity, high recognition factor, low bulkiness) and Nogo-45–66 (negative hydropathicity, high recognition factor, high polarity), highlighted in yellow. C, Computer modeling of primary structure for Nogo-1–22, Nogo-23–44, Nogo-45–66, PLP139–151, and MOG35–55. Stick models generated from the sequence used for peptide synthesis.

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

Given the difficulty in inducing EAE with Nogo peptides despite strong T cell and B cell responses, the expression of several activation markers and costimulatory molecules for both T cells and APCs (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 expression profile for these molecules (data not shown), which excluded defects in Ag presentation or T cell activation that might cause the mild clinical phenotype after immunization (39). Therefore, we hypothesized that adoptive transfer of purified T cell lines might be more efficient at disease induction. Naive mice were immunized with Nogo-1–22 or Nogo-45–66 emulsified in CFA and 10 days later, draining lymph nodes were collected, whole lymph node cell cultures were established and restimulated in vitro with the Nogo peptide. From these we established Ag-specific T cell lines, which were used for adoptive transfer and cytokine secretion profile evaluation. For disease induction, we transferred up to 5 x 107 T cell lines that were either reactive against Nogo-1–22 or Nogo-45–66 i.v. into naive SJL mice. In some of the experiments, animals received 107 T cells and were boosted 15 days later with 100 µg of Nogo-peptide emulsion in CFA injected s.c. and an i.v. injection of pertussis toxin. Although we could demonstrate the presence of Nogo-reactive T cells in adoptively transferred animals up to 3 mo postimmunization or T cell transfer in in vitro proliferative responses (data not shown), these animals never developed clinical signs of disease. In view of the fact that Nogo-66 might be a cryptic autoantigen, we postulated that inefficient induction of disease might be overcome by transferring Nogo-reactive cells to animals that already have EAE. In such mice, Nogo-66 might be available for presentation to T cells in acute demyelinating foci. Therefore, we induced EAE in SJL/J mice by immunization with 100 µg of PLP139–151 peptide in CFA; at the peak of second relapse (day 31), animals were randomized into three groups and given either weekly i.v. administration of PBS, Nogo-1–22 or Nogo-45–66 reactive T cells in PBS (average of 1.5 x 107 cells) i.v. for 3 wk. Four days after T cell line transfer, animals receiving Nogo-reactive T cell lines showed a statistically significant reduction in clinical score; this reduction was most noticeable in mice receiving Nogo45–66 T cell lines (PBS score day 35 and 36 of 2.44 ± 0.72, day 37 of 2.78 ± 0.97, and day 38 of 2.56 ± 0.88 vs Nogo-45–66 PBS score 1.40 ± 0.96 throughout the same period with respective p values of 0.035, 0.035, 0.013, and 0.028; PBS score 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 throughout the next 45 days of the experiment, up to 30 days after the last T cell line administration. For Nogo-1–22 the effect was more transient, 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 administration but not to levels seen with PBS controls. At the conclusion of the experiment, we could still demonstrate the existence of both PLP139–151 and Nogo reactive T cells by in vitro proliferation assays (Fig. 5B). In fact, proliferation against Nogo-45–66 was much stronger than against the EAE-inducing Ag, suggesting that there might be cross-suppression by these cells of PLP reactive T cells.



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  		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/J mice with PLP139–151 peptide in CFA. Animals were randomized into groups at peak of second relapse to receive PBS (n = 9) ({triangleup}, dashed line), Nogo-1–22 reactive T cells (n = 10) {square}, solid line), or Nogo-45–66 reactive T cells (n = 10) ({blacktriangleup}, solid line). Each animal received three i.v. injections of 1.5 x 107 T cells in PBS at weekly intervals for 3 wk (arrow). *, Statistically significant difference in disease score (p < 0.05 Mann-Whitney) between the 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 (PBS score 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). Axillary and inguinal lymph nodes were dissected at the termination of the above experiment, whole lymph node cells were harvested and proliferation tested in vitro against PLP139–151 and Nogo peptides. Values represent mean cpm ± SEM of triplicate samples of pooled cells from five animals per group. OVA was used as irrelevant peptide control. All peptides at 20 µg/ml, Con A (ConA) at 5 µg/ml. C–E, Anti-Nogo T cell lines penetrate the parenchyma of EAE-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 peptides for 4 days, after which 1 x 107 cells were injected i.v. into diseased SJL/J mice. Animals were killed 3 days after T cell transfer and their CNS collected, embedded and frozen for immunohistochemistry. Thy1.1a+ T cells (arrows) were detected in the perivascular spaces and penetrating the brain parenchyma in mice with EAE that received PLP139–151, Nogo-1–22 and Nogo-45–66 stimulated cells (C–E, respectively). F, Anti-Nogo T cell lines spontaneously develop different Th phenotypes. 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 were restimulated in vitro with Nogo peptides, PLP139–151 peptide, irrelevant peptide control (V{beta}5.1), negative (medium), and positive (ConA) controls. Levels of IL-4, IL-10, and IFN-{gamma} 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 ({blacksquare}) and Nogo-45–66 T cell lines ({square}) results are shown.

 
To demonstrate that adoptively transferred Nogo-reactive T cells could enter the CNS of animals with EAE, we immunized Thy1.1a SJL/J female mice with Nogo peptides or PLP139–151, collected draining lymph nodes 10 days later and restimulated whole lymphocytes in vitro with the immunizing Ag. We then i.v. administered 10 x 107 Thy1.1a T cells into wild type (Thy1.2) SJL/J females that had been induced with EAE and were at the peak of disease. Three days later we collected the CNS from these animals and detected the presence of Thy1.1a cells in the CNS by immunohistochemistry. We could demonstrate the presence of adoptively transferred anti-PLP and anti-Nogo peptide-stimulated cells in the CNS vasculature, perivascular infiltrates and infiltrating the parenchyma (Fig. 5, C–E). These results indicate that Nogo-reactive T cells are capable of migrating into the CNS of animals with active EAE, and therefore, they might modulate disease by acting locally at CNS inflammatory foci.

Finally, we examined the phenotype of anti-Nogo T cell line by in vitro cytokine production assays. Briefly, Nogo-1–22 and Nogo-45–66 T cell lines were stimulated in vitro with their specific Ag in the presence of syngeneic irradiated APCs. Supernatants were collected after 48 h and cytokine production measured by ELISA. Results show that anti-Nogo 45–66 T cell lines spontaneously developed a Th2 phenotype, producing large amounts of IL-4 and IL-10, whereas Nogo-1–22 T cell lines show increased production of IFN-{gamma} (Fig. 5F). Based on these results, it appears that adoptively transferred Nogo-reactive cells might modulate ongoing EAE by migrating into active demyelinating foci, where they encounter their specific Ag and differentiate into potentially beneficial Th2 phenotypes, which are capable of regulating the autoimmune response.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We hypothesized that Nogo plays a central role in axonal pathology in MS and also in CNS trauma, and predicted that there might be an advantage in immunizing against the extracellular 66 loop domain of Nogo to block interactions with Nogo, therefore potentially improving axonal regrowth. We first attempted to induce EAE in Nogo knockout mice to ascertain the role that this molecule might have in a chronic nonrelapsing model of disease. Our results show that there is a slight reduction in disease severity in the knockout mice, but without a clear difference from the wild-type mice in histological assessment of axonal pathology. Surprisingly, we found that Nogo knockout animals showed a markedly reduced mortality rate using our protocol for induction of EAE and interpreted this to imply that Nogo might also have a role as an autoantigen in EAE, akin to the dual role that MAG has as both an axonal regrowth inhibitor and encephalitogenic Ag, capable of eliciting EAE (40).

There have been a few other studies that attempted immunization with Nogo-A-derived peptides in EAE-susceptible rat and mouse strains, and these could not find evidence for demyelination or clinical paralysis (21, 22, 41, 42). However, in one case, the immunization protocol used IFA and aluminum hydroxide, which are insufficient to induce EAE although being able to generate a strong Ab response (41). A similar strategy, but using whole myelin immunization in IFA, was successful in generating an Ab response and promoting regeneration, but did not result in EAE (21). Immunization of EAE-susceptible Lewis rats with a CFA emulsion of peptide p472, which is part of the Nogo-A-specific region (11), resulted in a T cell response that was also not encephalitogenic, but no Abs could be detected (22); our own results show that in the SJL/J mouse strain T cell responses could not be generated against this peptide, and that the B cell response does not show 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 used to specifically induce a T cell-independent Ab response, to prevent the induction of EAE, and as might be expected no T cell activation could be demonstrated (42). More recently, evidence has been provided that immunization against the N terminus Nogo-A sequence 623–640 may be protective in EAE models (43). Our results show that this is the case when a Th2 response is generated, but that in other conditions immunization with Nogo may induce meningoencephalitis with clinical and histological characteristics of EAE. We also show that after immunization with Nogo-66 we can detect Abs against a number of other myelin components, with potential consequences for the inflammatory response. In comparison with that study, for this work we chose to focus on a different region of the Nogo-A molecule, the extracellular 66-aa loop that interacts with the Nogo receptor, which in our view is a more likely target for the immune response.

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

Similar to the susceptibility of different mouse strains to EAE induction by different encephalitogens, antigenic domains in Nogo-66 appear to be strain specific. SJL/J (H-2s) animals made T cell responses only to peptides Nogo-1–22 and Nogo-45–66, and C57BL/6 (H-2b) animals responded to Nogo-1–22 and Nogo-23–44. At present, we do not know whether the fine specificity of recognition of the Nogo-1–22 epitope is the same for both strains. We demonstrated the existence of a strong T cell response that is both specific and noncross-reactive with other known autoantigens for EAE.

We also detected specific Ab responses and marked epitope spreading to several other myelin Ags. Abs against several myelin components have been described in both MS and EAE, and there is evidence both for correlation between Ab responses to myelin proteins and disease progression, and direct anti-MOG Ab-induced demyelination (30, 31, 32, 33). We have previously demonstrated temporal spreading of anti-myelin B cell responses in EAE based on myelin array analysis of serial serum samples collected during the acute and chronic phases of the disease, and found that epitope spreading of the Ab response correlated with increased relapse rate (34). The diversification observed in Nogo-induced EAE is similar in nature and extent to that which we previously described in PLP- and MBP-induced EAE; this diversification could be due to the local release of myelin Ags, which may in fact be the basis for the phenomenon of epitope spreading. In either case, classical epitope spreading or local priming from released myelin debris, the targeting of multiple myelin Ags likely contributes to autoimmunity.

Recently, Abs against the N-terminal domain of Nogo-A have been identified in the serum and cerebrospinal fluid of MS patients as well as in other inflammatory and noninflammatory acute CNS diseases (44). The role of these Abs in disease pathogenesis is so far unknown, and they could either represent an epiphenomenon related to tissue injury that causes minor Ag presentation and Ab production, or alternatively they could be implicated in immune-mediated demyelination or contribute to a protective or regenerating response to injury. In this study, we found that increased spreading of the Ab response after immunization with Nogo-45–66 was related to an increased capacity to induce demyelination and clinical paralysis leading us to assume a contributory role of anti-Nogo Abs to the pathogenesis of EAE.

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

Finally, we showed that adoptive transfer of T cells reactive to Nogo Ags ameliorated ongoing EAE induced with other Ags. The modulatory T cells were shown to migrate to inflammatory sites in the CNS and presumably cross-regulate pathogenic Th1 phenotype cells, as well as promote the resting state of microglia after adopting beneficial Th2 phenotypes, a phenomenon that has been previously shown for myelin-specific Th2 cell lines (47), as well as nonmyelin Ags (48). In corroboration of this hypothesis, we were able to demonstrate that Nogo-specific T cell lines are capable of surviving for long periods of time and they actively migrate to the CNS parenchyma in EAE animals after adoptive transfer.

Classical criteria for probable target autoantigens for multiple sclerosis, namely that humoral and cell-mediated immunity be documented in MS patients against a molecule, and that the same molecule is capable of inducing EAE have been met by few myelin components, most notably MBP, PLP, MOG, MAG, and OSP (49). Based on the recent evidence of B cell responses and our own data presented in this report, Nogo-A can potentially join that restricted group.

Nogo Ags thus appear to behave similarly to other known encephalitogenic myelin Ags for these mouse strains, such as the major encephalitogenic determinants PLP139–151 and MOG35–55, or the minor determinants MBP85–99, MAG, MOB, OSP, and CNPase. Comparisons between these types of EAE are made difficult by different induction protocols, but in our opinion Nogo-66 belongs in the minor Ag group, although with a lesser degree of encephalitogenicity (40, 49, 50, 51). Unlike these Ags, however, and apart from MAG, none of the other myelin proteins are thought to play a role in axonal regrowth. The potential for oligodendrocyte-myelin glycoprotein to play a role as an encephalitogen is at present unknown. Therefore, the recognition of the possible contribution of Nogo-66 to CNS autoimmune demyelination is distinct: first, as an encephalitogenic Ag Nogo might assume an important role in the development of axonal pathology in this disease and in MS (44), where Abs to Nogo have been detected. Also, because modulation of the immune response to Nogo-66 appears to be capable of ameliorating EAE, several Ag-specific therapeutic strategies might be devised to take advantage of this phenomenon to treat human autoimmune demyelinating diseases. It is also possible that Nogo immunization might worsen disease given the capacity for inducing such strong T cell and B cell responses. In fact, we and others have found that Th2 type responses can be detrimental in EAE, in that not only anaphylaxis to myelin proteins is possible (52), but also that myelin-reactive Th2 cells can cause EAE (53).

Finally, because immunization with Nogo peptides is capable of generating such a broad based B cell response to other myelin Ags, including MAG, vaccination with these Ags might have a useful role in improving axonal regeneration in CNS traumatic models, such as spinal cord injury. The phenomenon of B cell epitope spreading after immunization with myelin Ags might allow a therapeutically important Ab response against several other potentially important neurite growth inhibitors even with single myelin Ag vaccination, with important implications for ameliorating CNS injury.


   Acknowledgments
 
We thank Dennis Mitchell for peptide synthesis.


   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 grants from the Fulbright Foundation and Christopher Reeve Paralysis Foundation (to P.F.), by the National Institutes of Health, National Research Service Award 5F32NS11115 from the National Institute of Neurological Disorders and Stroke (to P.P.H.), by a Helen Hay Whitney Fellowship (to B.Z.), by the National Institutes of Health Grant K08 AR02133, an Arthritis Foundation Chapter 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 Institutes of Health Grant NS 046414 (to R.A.S.), by a grant from the International Spinal Research Trust (to M.T.-L.), and by a National Institutes of Health, National Institute of Neurological Disorders and Stroke Grant 5R01NS18235, National Institutes of Health Grant U19 DK61934, and National Heart, Lung, and Blood Institute contract N01-HV-28183 (to L.S.). M.T.-L. is a Howard Hughes Medical Institute Investigator. Back

2 Address correspondence and reprint requests to Dr. Paulo Fontoura, Inflammation Group, Gulbenkian Science Institute, Rua da Quinta Grande 6, P-2780-156 Oeiras, Portugal. E-mail address: pfontoura{at}igc.gulbenkian.pt Back

3 Abbreviations used in this paper: MAG, myelin-associated glycoprotein; EAE, experimental autoimmune encephalomyelitis; MS, multiple sclerosis; MOG, myelin oligodendrocyte glycoprotein; MBP, myelin basic protein; PLP, proteolipid protein; OSP, oligodendrocyte-specific protein; CNPase, 2',3'-cyclic nucleotide 3'-phosphodiesterase. Back

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


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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