Paul A. Smith

High incidence of spontaneous disease in an HLA-DR15 and TCR transgenic multiple sclerosis model

Multiple sclerosis (MS) is thought to involve CD4 T cell recognition of self myelin, many studies focusing on a pathogenic role for anti-myelin, HLA-DR15-restricted T cells. In experimental allergic encephalomyelitis, it is known which epitopes trigger disease and that disease is associated with determinant spread of T cell reactivity. Characterization of these events in human MS is critical for the development of peptide immunotherapies, but it has been difficult to define the role of determinant spread or define which epitopes might be involved. In this study, we report humanized transgenic mice, strongly expressing HLA-DR15 with an MS-derived TCR; even on a RAG-2 wild-type background, mice spontaneously develop paralysis. Disease, involving demyelination and axonal degeneration, correlates with inter- and intramolecular spread of the T cell response to HLA-DR15-restricted epitopes of myelin basic protein, myelin oligodendrocyte glycoprotein, and αB-crystallin. Spread is reproducible and progressive, with two of the epitopes commonly described in responses of HLA-DR15 patients. The fact that this pattern is reiterated as a consequence of CNS tissue damage in mice demonstrates the value of the transgenic model in supplying an in vivo disease context for the human responses. This model, encompassing pathologically relevant, spontaneous disease with the presentation of myelin epitopes in the context of HLA-DR15, should offer new insights and predictions about T cell responses during MS as well as a more stringent test bed for immunotherapies.

Native myelin oligodendrocyte glycoprotein promotes severe chronic neurological disease and demyelination in Biozzi ABH mice

Myelin oligodendrocyte glycoprotein (MOG) is a powerful encephalitogen for experimental autoimmune demyelination. However, the use of MOG peptides or recombinant proteins representing part of the protein fails to fully address the possible pathogenic role of the full‐length myelin‐derived protein expressing post‐translational modifications. Immunization of mice with central nervous system tissues from wild‐type (WT) and MOG‐deficient (MOG–/–) mice demonstrates that MOG in myelin is necessary for the development of chronic demyelinating experimental autoimmune encephalomyelitis (EAE) in mice. While immunization with WT spinal cord homogenate (SCH) resulted in a progressive EAE phenotype, MOG–/– SCH induced a mild self‐limiting acute disease. Following acute EAE with MOG–/– SCH, mice developed T cell responses to recombinant mouse MOG (rmMOG), indicating that MOG released from myelin is antigenic; however, the lack of chronic disease indicates that such responses were not pathogenic. Chronic demyelinating EAE was observed when MOG–/– SCH was reconstituted with a dose of rmMOG comparable to MOG in myelin (2.5% of total white matter‐derived protein). These data reveal that while immunization with the full‐length post‐translational modified form of MOG in myelin promotes the development of a more chronic autoimmune demyelinating neurological disease, MOG (and/or other myelin proteins) released from myelin during ongoing disease do not induce destructive autoimmunity.

Cross-reactivity between related sequences found in Acinetobacter sp., Pseudomonas aeruginosa, myelin basic protein and myelin oligodendrocyte glycoprotein in multiple sclerosis

To investigate the possible role of molecular mimicry to bacterial components in multiple sclerosis (MS) pathogenesis we examined antibody responses to mimicry peptide sequences of Acinetobacter, Pseudomonas aeruginosa and myelin components. Antibodies to mimicry peptides from Acinetobacter (p<0.001), P. aeruginosa (p<0.001), myelin basic protein (MBP) (p<0.001) and myelin oligodendrocyte glycoprotein (MOG) (p<0.001) were significantly elevated in MS patients compared to controls. Antisera against MBP (residues 110-124) reacted with both Acinetobacter and Pseudomonas peptides from 4- and gamma-carboxymuconolactone decarboxylase, respectively. MOG (residues 43-57) antisera reacted with Acinetobacter peptide from 3-oxo-adipate-CoA-transferase subunit A. The role of these bacteria in MS is unclear but demonstrates that molecular mimicry is not restricted to viruses suggesting bacterial infections could play a role in MS pathogenesis. Further work is required to evaluate the relevance of these cross-reactive antibodies to the neuropathology of MS.

Autoimmunity against myelin oligodendrocyte glycoprotein is dispensable for the initiation although essential for the progression of chronic encephalomyelitis in common marmosets.

To elucidate the pathogenetic significance of myelin/oligodendrocyte glycoprotein (MOG)-specific autoreactivity in a genetically and immunologically heterogeneous nonhuman primate model of multiple sclerosis, we analyzed experimental autoimmune encephalomyelitis (EAE) in the outbred common marmoset (Callithrix jacchus). One sibling each of 5 bone marrow chimeric marmoset twins was immunized with myelin derived from wild-type (WT) C57BL/6 mice (WT myelin); the other sibling was immunized with myelin from MOG-deficient C57BL/6 mice (MOG−/− myelin). One twin pair developed acute EAE simultaneously; the 4 remaining twin siblings immunized with WT myelin developed chronic progressive EAE, whereas siblings of these 4 monkeys remained free of clinical disease signs. Many EAE-related abnormalities were identified in the CNS of both groups by magnetic resonance imaging and histologic analysis, but mean percentages of spinal cord demyelination were lower in monkeys immunized with MOG−/− myelin (8.2%) than in WT myelin-immunized animals (40.5%). There was a strong correlation between the development of overt clinical EAE and seropositivity for anti-MOG antibodies, but blood and lymph node T-cell proliferative responses showed no relationship to disease. These results indicate that the initiation of CNS inflammation and demyelination can take place in the absence of detectable autoimmunity against MOG, but the clinical progression and histopathologic severity depends on the presence of antibodies against MOG in this multiple sclerosis model.

Disease-related epitope spread in a humanized T cell receptor transgenic model of multiple sclerosis

While EAE has been an invaluable model for the immunopathogenesis of multiple sclerosis, it has sometimes been difficult to bridge the gap between findings and therapies in the rodent models and the cellular and molecular interactions that can be studied in the human disease. Humanized transgenic models offer a means of achieving this, through the expression of disease‐implicated HLA class II molecules, co‐expressed with a cognate HLA‐class II‐restricted, myelin‐specific TCR derived from a human T cell clone implicated in disease. We have generated such a transgenic line, called line 8, that co‐expresses a high level of HLA‐DR15 and a human TCR specific for HLA‐DR15/MBP 85–99. T cells from the transgenic line are skewed to the CD4 single‐positive compartment and produce IFN‐γ in response to peptide from mylein basic protein. Mice develop a spontaneous disease phenotype, showing poverty of movement, although this rarely develops into paralysis except following immunization with peptide. On induction of paralysis by immunization with peptide, disease correlates with epitope spread to a number of additional, HLA‐DR15‐restricted myelin epitopes. This model should be valuable for analyzing epitope spread in a humanized immunogenetic environment and for the testing of specific immunotherapies.

Exposure to a dysfunctional glucocorticoid receptor from early embryonic life programs the resistance to experimental autoimmune encephalomyelitis via nitric oxide-induced immunosuppression.

Glucocorticoid (GC) hormones play a central role in the bidirectional communication between the neuroendocrine and the immune systems and exert, via GC receptors (GR), potent immunosuppressive and anti-inflammatory effects. In this study, we report that GR deficiency of transgenic mice expressing GR antisense RNA from early embryonic life has a dramatic impact in programming the susceptibility to experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. GR deficiency renders mice resistant to myelin oligodendrocyte glycoprotein-induced EAE, and such mice do not develop clinical or histological signs of disease compared with EAE-susceptible wild-type mice. Resistance to EAE in GR-deficient mice is associated not with endogenous GC levels, but with a significant reduction in spleen and lymph node cell proliferation. The use of NO inhibitors in vitro indicates that NO is the candidate immunosuppressor molecule. GR-deficient mice develop 3- to 6-fold higher nitrite levels in the periphery and are resistant to NO inhibition by GCs. Specific inhibition of NO production in vivo by treatment with the inducible NO synthase inhibitor, l-N6-(1-iminoethyl)-lysine, suppressed circulating nitrites, increased myelin oligodendrocyte glycoprotein-specific cell proliferation, and rendered GR-deficient mice susceptible to EAE. Thus, life-long GR deficiency triggers inducible NO synthase induction and NO generation with consequent down-regulation of effector cell proliferation. These findings identify a novel link among GR, NO, and EAE susceptibility and highlight NO as critical signaling molecule in bidirectional communication between the hypothalamic-pituitary-adrenocortical axis and the immune system.

Epitope spread is not critical for the relapse and progression of MOG 8-21 induced EAE in Biozzi ABH mice

Emerging autoimmunity (epitope-spreading) generated as a consequence of myelin damage is suggested to underlie the relapses in multiple sclerosis (MS). Myelin oligodendrocyte glycoprotein (MOG 8-21) induces relapsing EAE in ABH mice characterized by broadening of the autoimmune reportoire. Despite epitope spreading tolerance to the priming antigen, but not emerging epitope reactivities, resulted in long-term inhibition of clinical relapse. In contrast, spinal cord homogenate induced EAE was dominated by a proteolipid protein (PLP 56-70) autoreactivity despite the plethora of CNS antigens in the immunogen. This data suggests that during relapsing-remitting demyelinating disease the pathogenic process is dominated by the initiating antigen, with only a minor role played by emerging T-cell populations. These findings may have important implications for the efficacy of antigen-based immune therapies in autoimmune disorders.

MRI-guided immunotherapy development for multiple sclerosis in a primate

Multiple sclerosis is a serious neurological disease that affects 1 in 1000 young adults in Europe and the USA. The development of an effective therapy for this enigmatic disease is plagued by the failure of many treatments to reproduce in patients the promising effects observed in animal models. This review describes a new preclinical model in a non-human primate that might help to bridge the gap between currently used animal models and the patients.

Novel pathogenic epitopes of myelin oligodendrocyte glycoprotein induce experimental autoimmune encephalomyelitis in C57BL/6 mice

Myelin oligodendrocyte glycoprotein (MOG), a minor protein of the central nervous system myelin, is recognized as a potential target in multiple sclerosis and neuromyelitis optica. The extracellular domain of MOG is commonly used in a wide range of mouse strains and other animals to induce experimental autoimmune encephalomyelitis (EAE), an autoimmune animal model of multiple sclerosis, because it is a target for antibody‐mediated attack. Previous studies, using selected peptides, have indicated that MOG35–55 peptide is an encephalitogenic epitope in C57BL/6 (H‐2b) mice. A more systematic analysis of both T‐cell and B‐cell responses following immunization of C57BL/6 mice with either recombinant extracellular mouse MOG protein (1–116) or with overlapping peptides spanning the whole sequence of MOG, before assessment of responses to 15 mer and 23 mer peptides was undertaken. The studies identified T‐cell responses within the MOG35–55 (extracellular domain) but also two new immunogenic and encephalitogenic T‐cell epitopes within residues MOG113–127, MOG120–134 (localized in the transmembrane region) and MOG183–197 (in the second hydrophobic MOG domain). In addition, residue MOG113–127 was found to be a B‐cell epitope, suggesting that this may be a useful adjunct for the induction of EAE as well as for immunological studies in C57BL/6 mice, which are increasingly being used to study immune function through the use of transgenic and gene knockout technology.

Experimental Autoimmune Encephalomyelitis in Primates

Due to their genetic and immunological proximity to humans, the common marmoset, Callithrix jacchus, provides an important bridge between rodent-based research and the human disease. Experimental autoimmune encephalomyelitis (EAE) induced in the outbred common marmoset provides a highly reproducible model of multiple sclerosis (MS) phenotypically characterised by neurological deficits, inflammatory lesions of the cerebral white matter and spinal cord, primary demyelination and axonal destruction. A fused placental blood circulation in utero, unique amongst monkeys, results in an immune tolerance between chimeric twins which allows adoptive transfer experiments assessing the relative contributions of T- and B-cells to pathogenesis. Overall, the ability to perform serial blood sampling, quantitative magnetic resonance imaging (MRI) and histopathological techniques, which are comparable to the human MS situation, means that the common marmoset EAE model provides an excellent test system for investigating the relationship between neurological deficits and pathological processes and the evaluation of new therapeutics.