Claude P. Genain

Autoantibody responses against conformational epitopes of myelin/oligodendrocyte glycoprotein (MOG) possess myelin destructive potential, as demonstrated in the marmoset model of human multiple sclerosis (MS) and in some rodent models of experimental allergic encephalomyelitis. We have previously characterized monoclonal Fab fragments specific for conformational epitopes of MOG that were derived from a combinatorial antibody library generated from a MOG-immune marmoset. In this paper, we address the molecular heterogeneity of humoral responses against MOG in this outbred model of MS by studying additional antibody clones derived from a genetically unrelated animal. We find that all MOG-specific IgGκ Fab fragments, unrelated to genetic make-up, utilize a restricted set of variable region genes, IGHV1 and IGHV3 for the H chain and IGKV1, IGKV3, and IGKV5 for the L chain. Despite these restricting factors, diversity within these antibody repertoires can be observed, predominantly within the H-chain CDR3 regions. Our findings suggest that only a limited set of Ig genes is necessary to launch a diverse, destructive humoral immune response against a single CNS antigen in primates. These results are the first to contribute to a better understanding of how myelin-directed and potentially destructive autoantibody responses may develop in human MS.

Myelin oligodendrocyte glycoprotein (MOG) is an integral membrane protein expressed in CNS oligodendrocytes and outermost myelin lamellae. Anti-MOG Abs cause myelin destruction (demyelination) in animal models of multiple sclerosis (MS); however, such pathogenic Abs have not yet been characterized in humans. Here, a method that specifically detects IgG binding to human MOG in its native, membrane-embedded conformation on MOG-transfected mammalian cells was used to evaluate the significance of these auto Abs. Compared with healthy controls, native MOG-specific IgGs were most frequently found in serum of clinically isolated syndromes (P< 0.001) and relapsing-remitting MS (P< 0.01), only marginally in secondary progressive MS (P< 0.05), and not at all in primary progressive MS. We demonstrate that epitopes exposed in this cell-based assay are different from those exposed on the refolded, extracellular domain of human recombinant MOG tested by solid-phase ELISA. In marmoset monkeys induced to develop MS-like CNS inflammatory demyelination, IgG reactivity against the native membrane-bound MOG is always detected before clinical onset of disease (P< 0.0001), unlike that against other myelin constituents. We conclude that (i) epitopes displayed on native, glycosylated MOG expressedin vivoare early targets for pathogenic Abs; (ii) these Abs, which are not detected in solid-phase assays, might be the ones to play a pathogenic role in early MS with predominant inflammatory activity; and (iii) the cell-based assay provides a practical serologic marker for early detection of CNS autoimmune demyelination including its preclinical stage at least in the primate MS model.

Preliminary observations of humoral immunity against the myelin oligodendrocyte glycoprotein (MOG) in experimental allergic encephalomyelitis (EAE) and human multiple sclerosis (MS) suggest that a subset of anti‐MOG autoantibodies directed against conformational epitopes is of pathogenic predominance. Here, we provide proof that in marmoset EAE, autoantibodies reactive against conformational epitopes of MOG are not only responsible for aggravating demyelination, but also an essential factor for disease dissemination in space within the central nervous system, a hallmark for typicalforms of human MS. In terms of effector mechanisms, IgG deposition and complement activation occur exclusively in association with presence of these conformational antibodies, while microglial/macrophage activation appears to be a common immunopathological finding regardless of the fine determinant specificity of anti‐MOG antibodies. These findings highlight for the first time the complex heterogeneity of function and pathogenicity in the polyclonal anti‐MOG antibody repertoire of outbred species. Because the linear and conformational antibody determinants of MOG are shared between marmosets and humans, these results are directly relevant to understanding effector mechanisms of organ damage in MS.See accompanying Commentary http//dx.doi.org/10.1002/eji.200425291

Myelin/oligodendrocyte glycoprotein (MOG) is a target antigen for myelin-destructive Abs in autoimmune central nervous system demyelinating disorders. Little is known about the molecular and structural basis of these pathogenic Ab responses. Here, we have characterized anti-MOG Ab specificities in the marmoset model of experimental allergic encephalomyelitis, by means of a combinatorial IgG-Fab library. We found that a diverse population of Ig genes encodes for auto-Abs that exclusively recognize conformation-dependent antigenic targets on MOG. These antigenic domains correspond to exposed epitopesin vivo, as the Fab fragments recognize native MOGin situin marmoset brain tissue. The Ab fragments described here represent Ab specificities that are common constituents of the humoral immune repertoire against MOG in outbred populations, as demonstrated by their ability to displace native anti-MOG Abs present in sera from MOG-immune marmosets and patients with multiple sclerosis. Furthermore, neuropathological analysis and characterization of Ab epitope specificities in animals immunized with MOG or MOG-derived peptides revealed that only conformation-dependent Abs are associated with demyelinating activity, suggesting that epitope recognition is an important factor for Ab pathogenicity. Our findings provide novel and unexpected knowledge on the diversity of anti-MOG Ab responses in nonhuman primates and humans, and will permit the dissection of pathogenic auto-Ab properties in multiple sclerosis.

Summary: Models that adequately reflect the complexity of human multiple sclerosis (MS) are needed, especially for preclinical testing of immunomodulatory drugs. Our group has created a unique experimental system in a New World outbred primate, the common marmoset Callithrix jacchus (C. jacchus). Following immunization with myelin, these monkeys develop a chronic, relapsing‐remitting form of experimental allergic encephalomyelitis (EAE), which pathologically recapitulates the hallmark features of lesions of human MS. The MS‐like lesion in C. jacchus results from a complex immune response against myelin antigens and requires both T cells and pathogenic antibodies. Studies of C. jacchus EAE have permitted the identification of a major target for pathogenic autoantibodies in MS, the myelin/oligodendrocyte glycoprotein. Other advantages of the model include a natural bone‐marrow chimerism, which permits T‐cell adoptive transfers between siblings, and the possibility of using different antigens to produce either inflammatory or demyelinating phenotypes of EAE. Despite their small size, sequential in vivo imaging and immunological studies are possible in these monkeys, and have been used to monitor efficacy in preclinical trials. Due to close phylogeny and high homology of immune and nervous system genes with humans, this model should fast‐track the development of novel therapeutics for MS.

Auto-reactive T cells present in healthy subjects remain in a state of unresponsiveness, but may trigger autoimmunity under various situations. Although myelin oligodendrocyte glycoprotein (MOG) is a potential target antigen in multiple sclerosis (MS), MOG-reactive T cell responses are present in the blood of both healthy subjects and MS-affected individuals. To investigate the disease-inducing potential and regulation of these autoreactive T cells in healthy outbred populations, we have characterized MOG-reactive T cell clones obtained by limiting dilution from peripheral blood ofunimmunized C. jacchus marmosets. We report an extraordinarily high prevalence of circulating MOG-reactive T cells in these naive animals (2.6 ± 1.4 / 10⁵ PBMC), and a broadly diverse repertoire of epitope recognition encompassing at least three regions within the extracellular domain of MOG. Adoptive transfer of a MOG_{21 – 40}-specific T cell clone resulted in mild clinical experimental allergic encephalomyelitis, characterized pathologically by rare foci of inflammation and minimal demyelination. We conclude that MOG-reactive T cells are present in healthy primates at a highly prevalent frequency, and are potentially capable of triggering central nervous system autoimmunity. Expansion of these autoreactive T cells must be tightly controlled to maintain immune homeostasis in healthy individuals.

Multiple sclerosis is a demyelinating disorder of the central nervous system (CNS), in which an immune attack directed against myelin constituents causes myelin destruction and death of oligodendrocytes, the myelin-producing cells. Here, the efficacy of nerve growth factor (NGF), a growth factor for neurons and oligodendrocytes, in promoting myelin repair was evaluated using the demyelinating model of experimental allergic encephalomyelitis (EAE) in the common marmoset. Surprisingly, we found that NGF delayed the onset of clinical EAE and, pathologically, prevented the full development of EAE lesions. We demonstrate by immunocytochemistry that NGF exerts its antiinflammatory effect by downregulating the production of interferon γ by T cells infiltrating the CNS, and upregulating the production of interleukin 10 by glial cells in both inflammatory lesions of EAE and normal-appearing CNS white matter. Thus, NGF, currently under investigation in human clinical trials as a neuronal trophic factor, may be an attractive candidate for therapy of autoimmune demyelinating disorders.