Multiple sclerosis (MS) is a common inflammatory/demyelinating disease of the central nervous system (CNS) in Europe and current treatments are only modestly beneficial. Myelin oligodendrocyte glycoprotein (MOG) is potentially relevant as target auto-antigen in MS. It can recruit both an encephalitogenic T cell response and a demyelinating B cell response. It is therefore of interest to explore the immunopathogenesis of MOG induced experimental autoimmune encephalomyelitis (EAE), its potential therapy and the role of MOG directed autoimmunity in MS.To reveal mechanisms of importance for MS, EAE models should mimic human disease as close as possible. The project therefore aimed at establishing such models with the use of MOG as inducing auto-antigen in rats. We established MOG induced EAE in different rat strains, being more MS like than any previously described rodent model. These rats displayed neuropathological lesions mimicking those found in MS and a chronic relapsing disease course. We have documented that the immunopathogenesis depend on both a pathogenic T and B cell response against critical epitopes of MOG, in turn controlled by the MHC haplotype. Therapeutic strategies need to take care of both arms of the autoimmunity. Thus, tolerogenic strategies dampening a pathogenic T cell response against MOG, under certain circumstances enhanced the B cell response, with aggravated disease outcome as result. Molecular mimicry with a milk protein, butyrophilin, suggested potential effects of milk products on MOG autoimmunity. Experiments in Fc receptor gene deleted mice demonstrated a crucial role of this receptor in autoimmune neuro-inflammation.
Tolerogenic strategies to limit disease progression in MOG induced EAE:
The results obtained during the three year period strengthen our initial hypothesis that the development of an MS-like pathology in MOG-induced EAE is dependent on the induction of both MOG-specific T and B cell responses. Functionally, the pathogenic T cell response initiates inflammation in the CNS, recruiting immune effector cells and disrupting the blood brain barrier, allowing antibody and other serum proteins to enter the CNS compartment. MOG-specific antibodies then initiate demyelination and further amplify the local inflammatory response by the production of complement derived pro-inflammatory factors. As a consequence of this antibody/complement driven inflammatory cascade, severe clinical disease and extensive demyelination develop in MOG-EAE even if the MOG-specific T cell response is minimal and by itself unable to induce disease (partner 2 and 3, Stefferl et al,1999). Analysis of the fine specificity of the demyelinating MOG-specific antibody response demonstrated that it recognises a restricted repertoire of conformation dependent, non-linear epitopes that are conserved between species (partners 1, 2 and 3, Brehm et al 1999, Lindert et al,1999). In contrast, the identity of the epitopes recognized by the encephalitogenic Th T cell response is defined by the MHC class II haplotype and is therefore species/strain specific (partners 1, 2 and 3, Weissert et al 1998, Stefferl et al 1999, Abdul-Majid et al,2000). The duality of the pathogenic autoimmune response to MOG imply that successful tolerogenic and/or therapeutic strategies must efficiently control both T cell and antibody dependent effector mechanisms (Partners 1, 2 and 3).
Ongoing experiments are investigating the tolerogenic potential of treatment with soluble full length MOG and MOG-derived peptides in both prophylactic and therapeutic settings. The data obtained so far indicate that regardless of the route of application (intravenous, intra peritoneal, nasal or oral) exposure to the intact antigen can down modulate (but not abolish) the encephalitogenic T cell response, but paradoxically stimulate a pathogenic antibody response. Cessation of treatment is then followed by an increase in disease activity that is often fulminant and lethal. However the use of truncated recombinant MOG proteins or synthetic peptides avoids the induction/enhancement of pathogenic antibody responses and can completely inhibit disease induction by specifically targeting the encephalitogenic T cell response (Borquin et al2000; Lobell et al. 2000).
The importance of avoiding the induction/enhancement of pathogenic anti-MOG antibody responses is also seen in studies analysing the therapeutic potential of DNA vaccination in MOG-EAE. In SJL/J mice DNA vaccination with constructs encoding the full length protein resulted in the induction of a high titer pathogenic antibody response that exacerbated EAE induced by either MOG or irrelevant myelin antigens such as PLP (Borquin et al,2000). In contrast, constructs encoding the immuno-dominant T cell epitopes of MOG for Lew.1AV1 and Lew.1N rats specifically targeted the effector T cell response in these rat strains and protected them against MOG peptide induced disease (Partners 1, 2 and 3, Lobell et al,2000). These results imply that universal strategies to re-establish self-tolerance to MOG and suppress disease activity in MOG-EAE will be successful if they efficiently suppress the effector T cell response while avoiding the induction/enhancement of a demyelinating antibody responses. In view of the importance of the antibody response in potentiating disease activity we are currently using site directed mutagenesis to identify those amino acid residues responsible for the maintaining the structure of the conformational epitopes targeted by demyelinating antibodies. We anticipate that this will lead to the development of a generation of modified MOG derivatives that can be used to block the effector T cell response across a broad range of MHC class II haplotypes (Partners 1, 2 and 3).
Modulation of MOG specific autoimmune responses by environmental factors:
The etiology of multiple sclerosis (MS) is believed to involve environmental factors that disrupt immunological self-tolerance to CNS myelin in genetically susceptible individuals, but the identity and mode of action of the factors involved remain unknown. Intriguingly, sequence homologies involving the extracellular immunoglobulin-like domain of MOG (MOGIgd) identified MOG as a member of an extended family of "B7-like"proteins. Of particular interest was the observation that the highest level of sequence identity was with a homologous extracellular Ig domain of butyrophilin (BTN) (Stefferl et al,2000), a major protein of the milk fat globule membrane (MFGM). This observation led us to speculate that immunological cross-reactivity or "molecular mimicry" with BTN may influence the function of the MOG-reactive autoimmune repertoire. We demonstrated that this was indeed the case, identifying for the first time a dietary antigen that can have a significant impact on the functional activity of the autoimmune response to an immunodominant myelin antigen (Stefferl et al., 2000). In the Dark Agouti (DA) rat active immunization with native BTN triggers an inflammatory response in the CNS characterized by the formation of scattered meningeal and perivascular infiltrates of T cells and macrophages. This pathology was mediated by a MHC class II restricted T cell response that recognizes a homologous peptide sequence in MOG and BTN. Conversely, molecular mimicry with BTN can also be exploited to suppress disease activity in MOG-induced EAE. MOG-induced EAE can be suppressed in DA rats by both i.v. and trans-mucosal treatment with BTN (Stefferl et al 2000).
This observation suggests that dietary exposure to BTN may modulate the MOG-specific autoimmune response in man. We therefore initiated an investigation of molecular mimicry between MOG and BTN in normal healthy controls and patients with MS. Western blotting and ELISA studies identified anti-BTN IgG antibodies in approximately 30% of all donors irrespective of their disease status. Isolating MOGIgd-specific antibodies from seropositive donors and testing their reactivity with a panel of BTN peptides by ELISA determined whether or not this response cross-reacted with MOG. We found that MOG-specific antibodies isolated from approximately 60% of the MS patients cross-reacted with one or more BTN peptides. In contrast, anti-MOG antibodies isolated from the pooled sera of approximately 3000 healthy donors failed to bind to any BTN peptides, although they recognized a variety of MOG peptide epitopes (Guggenmos et al, 2000). This result identifies for the first time an enhanced level of immunological cross-reactivity between a candidate auto-antigen and a common environmental factor in multiple sclerosis. The functional relevance of this enhanced cross-reactivity is now being investigated at the level of the T cell response to BTN.
Key regulatory immunological mechanisms as examined in gene deleted mice Immunisation of DBA/1 mice with MOG1-125 /CFA induces EAE after 9 days. Pathology reveals extensive demyelination with infiltration of macrophages and T cells and a MOG-specific antibody response. We assessed the in vivo consequences of deficiency of receptors for immunoglobulin (FcgRs) which are important for antibody-mediated immunity in this mouse model of multiple sclerosis. were immunised id with MOG/CFA. Clinical EAE scores were monitored in female DBA/1 mice lacking FcgRI/ FcgRIII or FcgRII, serum levels of anti-MOG antibodies were determined and MOG-specific T cell function addressed in vitro by cellular recall assays and in vivo by adoptive transfer. Degree of CNS pathology was determined histologically. All mice developed comparable T and B cell specific immune responses following MOG immunisation, as assessed by anti-MOG ELISA, cell proliferation and the ability of T cells from knockout mice to transfer EAE to naive wild-type recipients. Mice lacking FcgRI / FcgRIII did not develop EAEas assessed clinically and histologically. Mice lacking FcgRII had a trend to a worsened disease. We conclude that distinct FcgRs differentially regulate MOG-EAE susceptibility in DBA/1 mice (Abdul-Majid et al 2000).
In the same DBA/1 mouse model, we had access to mice deleted of their CD 4 and CD8 genes. This is of interest since the vast majority of studies in MS and EAE has focused on pathogenic CD4 + cells. Recently there are genetic evidence suggesting that the MHC class I genotype in humans may modulate the risk/course of MS. Similarly, there are scattered reports in the literature on MHC class I regulation of EAE. Interestingly, we could document a regulation of MOG EAE by CD 8+ cells, since CD4 -/- mice as well as CD8-/- mice developed an ameliorated form of EAE as compared to their littermate wild type controls. Antibody depletions confirmed this conclusion (Wefer et al. 2000).