Full-length Myelin Oligodendrocyte Glycoprotein Research Articles

Electrophysiological and Histological Correlations of Optic Neuritis in the Dark Agouti Rat Model of Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is an animal model of Inflammatory central nervous system (CNS) disease. Dark agouti (DA) rats immunized with full-length myelin oligodendrocyte glycoprotein (MOG1-125) typically develop a relapsing-remitting EAE form characterized by predominant demyelinating involvement of the spinal cord and optic nerve. Visually evoked potentials (VEP) are a useful objective tool to assess the optic nerve function and monitor electrophysiological changes in optic neuritis (ON). The current study aimed to assess the VEP changes in MOG-EAE DA rats using a minimally invasive recording device and to correlate them with histological findings. Twelve MOG-EAE DA rats and four controls underwent VEP recording at day 0, 7, 14, 21, and 28 post-EAE induction. Tissue samples were obtained on days 14, 21, and 28 from two EAE rats and one control. The median VEP latencies were significantly higher on days 14, 21, and 28 compared to baseline, with maximal latencies observed on day 21. The histological analyses on day 14 demonstrated inflammation with largely preserved myelin and axonal structures. Inflammation and demyelination with largely preserved axons were evident on days 21 and 28, which correlated with prolonged VEP latencies. These findings suggest that VEPs may be a reliable biomarker reflecting the optic nerve involvement in EAE. Moreover, the use of a minimally invasive device enables observation of VEP changes over time in MOG-EAE DA rats. Our findings may have important implications for testing the potential neuroprotective and regenerative effects of new therapies for CNS demyelinating diseases.

MOG-specific T cells skew high affinity during severe demyelinating disease after recombinant protein induction

Abstract Demyelinating diseases can be driven by CD4 T cells specific for self-antigen derived from myelin in the central nervous system (CNS). Mouse models utilizing peptide induction protocols may restrict the breadth of T cell specificity. Here, we compare mice induced with a peptide fragment of myelin oligodendrocyte glycoprotein (MOG), recombinant extracellular domain (ECD), and recombinant full-length MOG protein, which includes the transmembrane domain. We sought to understand why mice induced with recombinant MOG suffer a more severe demyelinating disease than those induced with peptide by focusing on MOG-specific CD4 T cells in the CNS. Fluorescently-labeled peptide-MHC tetramers revealed that mice induced with ECD and protein have a greater frequency of tetramer-positive T cells than peptide induction. To more precisely define the MOG-specific T cell response, we employed the two-dimensional micropipette adhesion frequency assay (2D-MP), which, unlike tetramer, identifies low-affinity antigen-specific T cells. We confirm that 2D-MP identifies more MOG-specific T cells in all three induction protocols; however, mice induced with protein had a lower frequency of MOG-specific T cells than those induced with ECD and peptide, likely due to the epitope within the transmembrane region. 2D-MP also unveiled that T cells from mice primed with protein skew high-affinity as compared to mice primed with peptide. Ultimately, our data indicate that disease after ECD and protein induction is driven by a greater frequency of high-affinity T cells than those from mice induced with peptide. We posit that this is due to a change in antigen presentation after recombinant protein priming. 5R01NS071518-09 5R01AI110113-05

Effect of Vitamin D on Experimental Autoimmune Neuroinflammation Is Dependent on Haplotypes Comprising Naturally Occurring Allelic Variants of CIITA (Mhc2ta)

An increasing body of evidence associates low vitamin D levels with increased risk of multiple sclerosis (MS), suggesting the possibility of a gene-environment interaction for this environmental factor in MS pathogenesis. Moreover, it has been shown that vitamin D downregulates major histocompatibility complex (MHC) class II expression in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. We here report about the impact of a dietary vitamin D supplementation on EAE in the rat strains having functionally relevant allelic variations in the CIITA (Mhc2ta) gene, a master regulator of MHC class II expression. Full length myelin oligodendrocyte glycoprotein (MOG)-EAE was induced in DA.PVGav1-Vra4 congenic rats harboring the Vra4 locus from PVG strain in the EAE- susceptible DA background, and compared to the parental strains. The congenic rats fed with either vitamin D supplemented, deprived or regular diet developed an intermediate clinical EAE phenotype, in contrast to DA and PVG strains. Immunopathological studies revealed vitamin D dose-dependent effect on demyelination and inflammatory infiltration of the central nervous system (CNS), expression of MHC class II and CIITA, as well as downregulation of a range of pro-inflammatory genes. Taken together, our findings demonstrate an impact of vitamin D on the target tissue pathology and peripheral immune response during EAE in DA.PVGav1-Vra4 congenic strain. Thereby, our data provide evidence of a modulatory effect of vitamin D in context of genetic variances in the Vra4 locus/Mhc2ta gene in MS-like neuroinflammation, with potential relevance for the human demyelinating disease.

The pathology of central nervous system inflammatory demyelinating disease accompanying myelin oligodendrocyte glycoprotein autoantibody

We sought to define the pathological features of myelin oligodendrocyte glycoprotein (MOG) antibody associated disorders (MOGAD) in an archival autopsy/biopsy cohort. We histopathologically analyzed 2 autopsies and 22 brain biopsies from patients with CNS inflammatory demyelinating diseases seropositive for MOG-antibody by live-cell-based-assay with full length MOG in its conformational form. MOGAD autopsies (ages 52 and 67) demonstrate the full spectrum of histopathological features observed within the 22 brain biopsies (median age, 10 years; range, 1–66; 56% female). Clinical, radiologic, and laboratory characteristics and course (78% relapsing) are consistent with MOGAD. MOGAD pathology is dominated by coexistence of both perivenous and confluent white matter demyelination, with an over-representation of intracortical demyelinated lesions compared to typical MS. Radially expanding confluent slowly expanding smoldering lesions in the white matter as seen in MS, are not present. A CD4+ T-cell dominated inflammatory reaction with granulocytic infiltration predominates. Complement deposition is present in all active white matter lesions, but a preferential loss of MOG is not observed. AQP4 is preserved, with absence of dystrophic astrocytes, and variable oligodendrocyte and axonal destruction. MOGAD is pathologically distinguished from AQP4-IgG seropositive NMOSD, but shares some overlapping features with both MS and ADEM, suggesting a transitional pathology. Complement deposition in the absence of selective MOG protein loss suggest humoral mechanisms are involved, however argue against endocytic internalization of the MOG antigen. Parallels with MOG-EAE suggest MOG may be an amplification factor that augments CNS demyelination, possibly via complement mediated destruction of myelin or ADCC phagocytosis.

Devic's index case: A critical reappraisal – AQP4-IgG-mediated neuromyelitis optica spectrum disorder, or rather MOG encephalomyelitis?

In 1894, Eugène Devic (1858–1930) and his doctoral student Fernand Gault (1873–1936) reported on a patient with optic neuritis (ON) and myelitis and proposed the name “neuro-myélite optique” for this syndrome. Subsequently, Devic became the eponym of neuromyelitis optica (NMO), which was then referred to as “Devic's syndrome”, “Devic's disease” or “Morbus Devic”. Thereby, the case became a historical index case of NMO. For many decades little attention was paid to NMO, which most authors considered a clinical variant of multiple sclerosis. However, the discovery of pathogenic antibodies to aquaporin-4 at the beginning of the 21st century revived interest in the syndrome, and AQP4-IgG-positive NMO spectrum disorders (NMOSD) are now studied as prototypical autoimmune diseases. More recently, antibodies to full-length myelin oligodendrocyte glycoprotein (MOG) have been detected in patients with ON as well as in patients with myelitis, some of whom exhibit a clinical phenotype very similar to that described by Devic. This raises the question of whether Devic's patient might have suffered from MOG encephalomyelitis rather than classic NMOSD. In this article, we summarise and discuss the available evidence for and against that hypothesis. We also discuss differential diagnoses and the question whether Devic's patient, who worked as a hatter and had initially been admitted for nervous hyperexcitability and tremor, might have suffered from co-existing erethism (‘mad hatter disease’), which is caused by chronic occupational exposure to mercury.

MOG-IgG in primary and secondary chronic progressive multiple sclerosis: a multicenter study of 200 patients and review of the literature

BackgroundAntibodies to human full-length myelin oligodendrocyte glycoprotein (MOG-IgG) as detected by new-generation cell-based assays have recently been described in patients presenting with acute demyelinating disease of the central nervous system, including patients previously diagnosed with multiple sclerosis (MS). However, only limited data are available on the relevance of MOG-IgG testing in patients with chronic progressive demyelinating disease. It is unclear if patients with primary progressive MS (PPMS) or secondary progressive MS (SPMS) should routinely be tested for MOG-IgG.ObjectiveTo evaluate the frequency of MOG-IgG among patients classified as having PPMS or SPMS based on current diagnostic criteria.MethodsFor this purpose, we retrospectively tested serum samples of 200 patients with PPMS or SPMS for MOG-IgG using cell-based assays. In addition, we performed a review of the entire English language literature on MOG-IgG published between 2011 and 2017.ResultsNone of 139 PPMS and 61 SPMS patients tested was positive for MOG-IgG. Based on a review of the literature, we identified 35 further MOG-IgG tests in patients with PPMS and 55 in patients with SPMS; the only reportedly positive sample was positive just at threshold level and was tested in a non-IgG-specific assay. In total, a single borderline positive result was observed among 290 tests.ConclusionOur data suggest that MOG-IgG is absent or extremely rare among patients with PPMS or SPMS. Routine screening of patients with typical PPMS/SPMS for MOG-IgG seems not to be justified.

Gene Therapy-Induced Antigen-Specific Tregs Inhibit Neuro-inflammation and Reverse Disease in a Mouse Model of Multiple Sclerosis.

The devastating neurodegenerative disease multiple sclerosis (MS) could substantially benefit from an adeno-associated virus (AAV) immunotherapy designed to restore a robust and durable antigen-specific tolerance. However, developing a sufficiently potent and lasting immune-regulatory therapy that can intervene in ongoing disease is a major challenge and has thus been elusive. We addressed this problem by developing a highly effective and robust tolerance-inducing in vivo gene therapy. Using a pre-clinical animal model, we designed a liver-targeting gene transfer vector that expresses full-length myelin oligodendrocyte glycoprotein (MOG) in hepatocytes. We show that by harnessing the tolerogenic nature of the liver, this powerful gene immunotherapy restores immune tolerance by inducing functional MOG-specific regulatory T cells (Tregs) in vivo, independent of major histocompatibility complex (MHC) restrictions. We demonstrate that mice treated prophylactically are protected from developing disease and neurological deficits. More importantly, we demonstrate that when given to mice with preexisting disease, ranging from mild neurological deficits to severe paralysis, the gene immunotherapy abrogated CNS inflammation and significantly reversed clinical symptoms of disease. This specialized approach for inducing antigen-specific immune tolerance has significant therapeutic potential for treating MS and other autoimmune disorders.

CLIC1 regulates dendritic cell antigen processing and presentation by modulating phagosome acidification and proteolysis.

ABSTRACTIntracellular chloride channel protein 1 (CLIC1) participates in inflammatory processes by regulating macrophage phagosomal functions such as pH and proteolysis. Here, we sought to determine if CLIC1 can regulate adaptive immunity by actions on dendritic cells (DCs), the key professional antigen presenting cells. To do this, we first generated bone marrow-derived DCs (BMDCs) from germline CLIC1 gene-deleted (CLIC1−/−) and wild-type (CLIC1+/+) mice, then studied them in vitro and in vivo. We found phagocytosis triggered cytoplasmic CLIC1 translocation to the phagosomal membrane where it regulated phagosomal pH and proteolysis. Phagosomes from CLIC1−/− BMDCs displayed impaired acidification and proteolysis, which could be reproduced if CLIC1+/+, but not CLIC1−/− cells, were treated with IAA94, a CLIC family ion channel blocker. CLIC1−/− BMDC displayed reduced in vitro antigen processing and presentation of full-length myelin oligodendrocyte glycoprotein (MOG) and reduced MOG-induced experimental autoimmune encephalomyelitis. These data suggest that CLIC1 regulates DC phagosomal pH to ensure optimal processing of antigen for presentation to antigen-specific T-cells. Further, they indicate that CLIC1 is a novel therapeutic target to help reduce the adaptive immune response in autoimmune diseases.

Anti-MOG antibodies are frequently associated with steroid-sensitive recurrent optic neuritis.

Optic neuritis (ON) is an inflammatory disease of the optic nerve characterized by pain and visual loss and often associated with multiple sclerosis (MS) or neuromyelitis optica spectrum disorders (NMOSD). Recent evidence suggests that certain forms of ON are associated with anti–myelin oligodendrocyte glycoprotein (MOG) antibodies.1,–4 A distinct clinical subset of ON is characterized by multiple episodes that involve one or both optic nerves, occur within months or weeks, and do not involve any other associated clinical or radiologic findings. This entity, defined as either recurrent optic neuritis (rON) or chronic relapsing inflammatory optic neuritis (CRION),5 is typically corticosteroid-responsive and corticosteroid-dependent, often requiring immunosuppressive therapy for corticosteroid-sparing effect. Our aim was to determine whether aquaporin-4 (AQP4)-negative patients with ON harbor antibodies to MOG and whether anti-MOG antibodies are clinically relevant. Methods. We examined sera from 111 patients initially referred to our diagnostic service for AQP4 testing. These patients had at least one episode of unilateral or bilateral ON, as reported by their referring physicians. All 111 patients were APQ4-negative. Twelve patients with primary progressive MS and 30 patients with relapsing-remitting MS (RRMS) were used as disease controls. The Ethics Committee of the University of Athens granted ethical approval. Anti-MOG screening was performed using a cell-based-assay (CBA). Patient sera (1:60 dilution) were applied on live human embryonic kidney 293T cells and transiently transfected with full-length MOG enhanced green fluorescent protein followed by a goat anti-human secondary antibody (Alexa Fluor 568). Positive samples were retested and titrated in a blinded fashion by M.R. To investigate optic nerve specificity, anti-MOG–positive sera were applied onto 10 μm nonfixed or 2% paraformaldehyde-fixed sections of fresh frozen human optic nerves (Netherlands Brain Bank, Amsterdam). A commercial monoclonal anti-MOG antibody was used as a positive control (Millipore, Billerica, MA; clone 8–18C5). Results. Anti-MOG antibodies were detected by CBA in 8/111 AQP4-seronegative patients and in 0/42 MS disease controls (figure 1, B–D). All 8 MOG-positive patients had at least one episode of ON, and 5 of them fulfilled the criteria for rON/CRION (defined as ≥3 episodes of ON within a period of a few months to a year). Fifteen of 30 patients with RRMS had at least one episode of ON, and 10 of the 15 had a relapsing ON course. Figure 1 Clinical characteristics and antibody profile of patients The 5 patients with rON/CRION (followed by M.C.D. for 2–11 years) had recurrent disease confined to optic nerves, often associated with or preceded by pain. Brain/orbital MRI or CSF analyses were normal except for optic nerve enhancement in 2 patients. Spinal MRI depicted one small subclinical chronic lesion in the cervical spine in one of the patients who never developed any clinical symptoms of myelitis. Antibody titers did not correlate with disease severity or number of relapses (figure 1A). All received IV steroids during the early attacks, but they were subsequently maintained on a low-dose oral regimen. In 2 patients, the attacks occurred very frequently when the oral corticosteroid dose was lowered below 15–20 mg every other day, necessitating the concurrent administration of an immunosuppressant such as mycophenolate mofetil. One patient with multiple episodes over many years who eventually developed optic atrophy in one eye also received rituximab without success; relative stability (only 1 or 2 episodes yearly) was induced by bimonthly plasmapheresis (figure e-1 at Neurology.org/nn). As human MOG antibodies do not recognize paraffin-fixed denaturated epitopes,6 we used fresh-frozen, nonfixed, or lightly fixed human optic nerve tissue as substrate. No specific binding of the patients' sera or IgG was observed (figure e-2A). Discussion. We have characterized and longitudinally followed up 5/8 patients with anti-MOG–positive rON/CRION without spinal cord or brain symptomatology These patients do not fall within the rubric of NMOSD, and our follow-up revealed that relapses were confined to the optic nerves and were highly sensitive to even low doses of oral corticosteroids. Our results confirm recent observations3,4 that anti-MOG antibodies are frequently associated with recurrent forms of ON. Anti-MOG antibodies have also been associated with pediatric inflammatory demyelinating diseases, including acute disseminated encephalomyelitis.7 A comparative epitope mapping of MOG antibodies from different syndromes identified a dominant MOG epitope but failed to elucidate a causative relationship or define distinct clinical phenotypes.6 No binding of patients' sera was observed in our optic nerve preparations, in which native epitopes are preserved. Whether the rON-associated anti-MOG antibodies are pathogenic, causing conduction block in the optic nerve, demyelination, or edema, remains unclear. Our observations suggest that anti-MOG antibodies are frequently associated with the rON/CRION phenotype, which is a highly steroid-responsive disorder. Although the rON series is small, the finding is supported by the observation that all of the RRMS control patients with ON were MOG-seronegative.

Immunodominant T-cell epitopes of MOG reside in its transmembrane and cytoplasmic domains in EAE

Objective:Studies evaluating T-cell recognition of myelin oligodendrocyte glycoprotein (MOG) in multiple sclerosis (MS) and its model, experimental autoimmune encephalomyelitis (EAE), have focused mostly on its 117 amino acid (aa) extracellular domain, especially peptide (p) 35-55. We characterized T-cell responses to the entire 218 aa MOG sequence, including its transmembrane and cytoplasmic domains.Methods:T-cell recognition in mice was examined using overlapping peptides and intact full-length mouse MOG. EAE was evaluated by peptide immunization and by adoptive transfer of MOG epitope-specific T cells. Frequency of epitope-specific T cells was examined by ELISPOT.Results:Three T-cell determinants of MOG were discovered in its transmembrane and cytoplasmic domains, p119–132, p181–195, and p186–200. Transmembrane MOG p119-132 induced clinical EAE, CNS inflammation, and demyelination as potently as p35-55 in C57BL/6 mice and other H-2b strains. p119-128 contained its minimal encephalitogenic epitope. p119-132 did not cause disease in EAE-susceptible non-H-2b strains, including Biozzi, NOD, and PL/J. MOG p119-132–specific T cells produced Th1 and Th17 cytokines and transferred EAE to wild-type recipient mice. After immunization with full-length MOG, a significantly higher frequency of MOG-reactive T cells responded to p119-132 than to p35-55, demonstrating that p119-132 is an immunodominant encephalitogenic epitope. MOG p181-195 did not cause EAE, and MOG p181-195–specific T cells could not transfer EAE into wild-type or highly susceptible T- and B-cell–deficient mice.Conclusions:Transmembrane and cytoplasmic domains of MOG contain immunodominant T-cell epitopes in EAE. A CNS autoantigen can also contain nonpathogenic stimulatory T-cell epitopes. Recognition that a myelin antigen contains multiple encephalitogenic and nonencephalitogenic determinants may have implications for therapeutic development in MS.

MOG transmembrane and cytoplasmic domains contain highly stimulatory T-cell epitopes in MS.

Objective:Recently, we reported that the 218 amino acid murine full-length myelin oligodendrocyte glycoprotein (MOG) contains novel T-cell epitopes p119-132, p181-195, and p186-200, located within its transmembrane and cytoplasmic domains, and that p119-132 is its immunodominant encephalitogenic T-cell epitope in mice. Here, we investigated whether the corresponding human MOG sequences contain T-cell epitopes in patients with multiple sclerosis (MS) and healthy controls (HC).Methods:Peripheral blood T cells from patients with MS and HC were examined for proliferation to MOG p119-130, p181-195, p186-200, and p35-55 by fluorescence-activated cell sorting analysis using carboxylfluorescein diacetate succinimidyl ester dilution assay. Intracellular production of proinflammatory cytokines was analyzed by flow cytometry.Results:MOG p119-130, p181-195, and p186-200 elicited significantly greater T-cell responses than p35-55 in patients with MS. T cells from patients with MS proliferated significantly more strongly to MOG p119-130 and p186-200 than did T cells from HC. Further, MOG p119-130–specific T cells exhibited Th17 polarization, suggesting this T-cell epitope may be relevant to MS pathogenesis.Conclusions:Transmembrane and cytoplasmic MOG domains contain potent T-cell epitopes in MS. Recognition of these determinants is important when evaluating T-cell responses to MOG in MS and may have implications for development of myelin antigen-based therapeutics.

Secondary B Cell Receptor Diversification Is Necessary for T Cell Mediated Neuro-Inflammation during Experimental Autoimmune Encephalomyelitis

BackgroundClinical studies of B cell depletion in Multiple Sclerosis (MS) have revealed that B Lymphocytes are involved in the neuro-inflammatory process, yet it remains unclear how B cells can exert pro- and anti-inflammatory functions during MS. Experimental Autoimmune Encephalomyelitis (EAE) is an animal model of MS whereby myelin-specific T cells become activated and subsequently migrate to the Central Nervous System (CNS) where they perform pro-inflammatory functions such as cytokine secretion. Typically EAE is induced by immunization of mice of a susceptible genetic background with peptide antigen emulsified in Complete Freund's Adjuvant. However, novel roles for B-lymphocytes in EAE may also be explored by immunization with full-length myelin oligodendrocyte glycoprotein (MOG) that contains the B cell conformational epitope. Here we show that full length MOG immunization promotes a chronic disease in mice that depends on antigen-driven secondary diversification of the B cell receptor.MethodsActivation-Induced Deaminase (AID) is an enzyme that is essential for antigen-driven secondary diversification of the B cell receptor. We immunized AID−/− mice with the extracellular domain (amino acids 1–120) of recombinant human MOG protein (rhMOG) and examined the incidence and severity of disease in AID−/− versus wild type mice. Corresponding with these clinical measurements, we also evaluated parameters of T cell activation in the periphery and the CNS as well as the generation of anti-MOG antibodies (Ab).ConclusionsAID−/− mice exhibit reduced severity and incidence of EAE. This suggests that the secondary diversification of the B cell receptor is required for B cells to exert their full encephalogenic potential during rhMOG-induced EAE, and possibly also during MS.

T Cell Epitope 119-132, but Not 35-55, Is the Immunodominant Encephalitogenic Determinant of the CNS Autoantigen, Myelin Oligodendrocyte Glycoprotein (P05.118)

Objective: To evaluate native full-length MOG for novel T cell epitopes. Background Amino acid (aa) residues 35-55 within the extracellular domain 1-117 of myelin oligodendrocyte glycoprotein (MOG) contain its only known encephalitogenic T cell determinant for C57BL/6 (H-2^b) mice, a strain frequently studied in the multiple sclerosis (MS) model, experimental autoimmune encephalomyelitis (EAE). Based upon studies showing that processing of native MOG is required for presentation of 35-55, we predicted that MOG contains additional T cell epitopes. Design/Methods: C57BL/6 mice were immunized with full-length (218 aa) murine (self) MOG or overlapping peptides encompassing its entire sequence in complete Freund9s adjuvant. Proliferation was measured by 3^H-thymidine incorporation. ELISPOT was used to measure frequency of epitope-specific IFN-γ-producing T cells that were generated by in vivo priming with full-length MOG. Results: Three novel T cell epitopes were discovered, an encephalitogenic determinant within transmembrane domain residues 119-132, and two discrete nonencephalitogenic determinants, p181-195 and p186-200, in the cytoplasmic domain. Clinical EAE, CNS inflammation and demyelination induced by p119-132 were equal, or greater, in severity than with p35-55. T cell recognition of MOG p119-132 was restricted by MHC II (I-A^b) molecules and p119-132 induced EAE in other H-2^b strains, but not in mice of other haplotypes. MOG p119-132-primed T cells, which produced higher levels of pathogenic Th1 and Th17 cytokines than p35-55-primed T cells, adoptively transferred EAE to naive recipients. Frequency of T cells that responded to p119-132 after immunization with full-length MOG was greater than to p35-55 or the non-encephalitogenic determinants, demonstrating that p119-132 is the immunodominant determinant. Conclusions: MOG p119-132, not p35-55, is the immunodominant encephalitogenic determinant of MOG. Not all T cell epitopes of a self antigen are pathogenic. Recognizing that MOG contains multiple encephalitogenic and non-encephalitogenic determinants permits testing tolerogenic approaches that target individual non-pathogenic or pathogenic T cell epitopes. Supported by: NIH and Boehringer-Ingelheim. Disclosure: Dr. Shetty has received research support from Boehringer Ingelheim. Dr. Gupta has received research support from Boehringer Ingelheim Pharmaceuticals, Inc. Dr. Weber has received personal compensation for activities with Hoffmann-LaRoche, Genentech, Inc., Novartis, Bayer Pharmaceuticals Corporation, Merck Serono, Biogen Idec and Teva Neuroscience as a speaker and/or participant on an advisory board. Dr. Weber has received research support from Teva Neuroscience. Dr. Molnarfi has nothing to disclose. Dr. Forsthuber has received research support from the National Institutes of Health. Dr. Sobel has received research support from the NIH. Dr. Bernard has nothing to disclose. Dr. Slavin has received personal compensation for activities with Boehringer Ingelheim as an employee. Dr. Slavin holds stock and/or stock options in Boehringer Ingelheim. Dr. Slavin has received research support from Boehringer Ingelheim. Dr. Zamvil has received personal compensation for activities with Biogen Idec, Teva Neuroscience and EMD Serono-Pfizer. Dr. Zamvil has received research support from the National Institutes of Health, National Multiple Sclerosis Society, the Maisin Foundation, the Guthy Jackson Charitable Foundation, Teva Neuroscience, and Boehringer Ingelheim Pharmaceuticals, Inc.

CD8 T Cell Responses to Myelin Oligodendrocyte Glycoprotein-Derived Peptides in Humanized HLA-A*0201-Transgenic Mice

Multiple sclerosis (MS) is a demyelinating inflammatory disease of the CNS. Though originally believed to be CD4-mediated, additional immune effector mechanisms, including myelin-specific CD8(+) T cells, are now proposed to participate in the pathophysiology of MS. To study the immunologic and encephalitogenic behavior of HLA-A*0201-binding myelin-derived epitopes in vivo, we used a humanized HLA-A*0201-transgenic mouse model. Eight HLA-A*0201-binding peptides derived from myelin oligodendrocyte glycoprotein (MOG), an immunodominant myelin self-Ag, were identified in silico. After establishing their relative affinity for HLA-A*0201 and their capacity to form stable complexes with HLA-A*0201 in vitro, their immunological characteristics were studied in HLA-A*0201-transgenic mice. Five MOG peptides, which bound stably to HLA-A*0201 exhibited strong immunogenicity by inducing a sizeable MOG-specific HLA-A*0201-restricted CD8(+) T cell response in vivo. Of these five candidate epitopes, four were processed by MOG-transfected RMA target cells and two peptides proved immunodominant in vivo in response to a plasmid-encoding native full-length MOG. One of the immunodominant MOG peptides (MOG(181)) generated a cytotoxic CD8(+) T cell response able to aggravate CD4(+)-mediated EAE. Therefore, this detailed in vivo characterization provides a hierarchy of candidate epitopes for MOG-specific CD8(+) T cell responses in HLA-A*0201 MS patients identifying the encephalitogenic MOG(181) epitope as a primary candidate.

Myelin/oligodendrocyte glycoprotein is a unique member of the immunoglobulin superfamily.

Myelin/oligodendrocyte glycoprotein (MOG) is a primary target autoantigen in experimental autoimmune encephalomyelitis, a widely used animal model for autoimmune demyelinating diseases such as multiple sclerosis. We have isolated several rat MOG cDNAs and confirmed their identity by comparison with MOG N-terminal peptide sequence. As expected, MOG mRNA expression is CNS-specific and peaks during active myelination. Our studies show that full length MOG mRNA is approximately 1.6 kb and encodes a signal peptide of 27 amino acids, followed by 218 residues for mature MOG (24,962 MW). A single site for N-glycosylation is found at Asn-31. Rather than the ubiquitous AAUAAA polyadenylation signal, a series of three overlapping, rare poly A signals were identified. The N-terminal half of mature MOG shares 52% identity with bovine butyrophilin, a possible lipid receptor. This same region has 39% identity with chicken B-G antigen, a major histocompatibility complex antigen involved in B cell selection and immune repertoire development. We show that both MOG and butyrophilin, each exhibiting a single Ig-like variable region domain, meet criteria for inclusion in the immunoglobulin superfamily. Moreover, MOG appears to represent a unique member of this superfamily in that it possesses two potential transmembrane domains, in contrast to a single membrane-spanning domain or glycophospholipid anchor found in all other members of Ig superfamily members.