Peter M. Mathisen

The epitope spreading cascade during progression of experimental autoimmune encephalomyelitis and multiple sclerosis

Summary: We have made the following observations regarding self‐recognition during the development and progression of murine experimental autoimmune encephalomyelitis (EAE) and human multiple sclerosis (MS); I) chronic progression of EAE is accompanied by a sequential, predictable cascade of neo‐autoreactivity, commonly referred to as epitope spreading, presumably caused by endogenous self‐priming during autoimmune‐mediated tissue damage; 2) there is an invariant relationship between the progression of EAE and the emergence of epitope spreading; 3) progression of EAE can be inhibited by the induction of antigen‐specific tolerance to spreading determinants after onset of initial neurologic symptoms; 4) CD4+ Th1 cells responding to spreading determinants are autonomously encephalitogenic; 5) epitope spreading occurs during the development of MS and in some cases involves HLA‐DP class II‐restricted self‐recognition; and 6) progression of both EAE and MS is accompanied by the decline of primary T‐cell autoreactivity associated with disease onset and by the concurrent emergence of the epitope spreading cascade. Our studies directly challenge the traditional view that EAE and MS are initiated and maintained by autoreactivity directed against a single predominant myelin protein or determinant. Our results indicate that progression of EAE and MS involves a shifting of T‐cell autoreactivity from primary initialing self‐determinants to defined cascades of secondary determinants that sustain the inflammatory self‐recognition process during disease progression.

Modulation of the IL-10/IL-12 cytokine circuit by interferon-β inhibits the development of epitope spreading and disease progression in murine autoimmune encephalomyelitis

IFN-beta has been shown to be effective in the treatment of multiple sclerosis (MS). However, the primary mechanism by which IFN-beta mediates its therapeutic effect remains unclear. Recent studies indicate that under defined conditions, IFN-beta may downregulate DC expression of IL-12. We and others have shown that IFN-beta may also downregulate IL-10. In light of the recently proposed paradigm that an IL-10/IL-12 immunoregulatory circuit controls susceptibility to autoimmune disease, we examined the effect of IFN-beta on the development and behavior of the autoreactive T cell repertoire during experimental autoimmune encephalomyelitis (EAE), an animal model sharing many features with MS. SWXJ mice were immunized with the immunodominant p139-151 determinant of myelin proteolipid protein (PLP), and at onset of EAE were treated every other day with IFN-beta. After eight weeks of treatment, we assessed autoreactivity and observed no significant IFN-beta effect on splenocyte proliferation or splenocyte production of IFN-gamma, IL-2, IL-4, or IL-5 in response to the priming determinant used to initiate disease. However, in IFN-beta treated mice, the cytokine profile in response to the priming immunogen was significantly skewed toward an increased production of IL-10 and a concurrent decreased production of IL-12. Moreover, the in vivo modulation of the IL-10/IL-12 immunoregulatory circuit in response to the priming immunogen was accompanied by an aborted development of epitope spreading. Our results indicate that IFN-beta induces a reciprocal modulation of the IL-10/IL-12 cytokine circuit in vivo. This skewed autoreactivity establishes an inflammatory microenvironment that effectively prevents endogenous self-priming thereby inhibiting the progression of disease associated with epitope spreading.

Visinin-like protein (VILIP) is a neuron-specific calcium-dependent double-stranded RNA-binding protein.

Double-stranded RNA-binding proteins function in regulating the stability, translation, and localization of specific mRNAs. In this study, we have demonstrated that the neuron-specific, calcium-binding protein, visinin-like protein (VILIP) contains one double-stranded RNA-binding domain, a protein motif conserved among many double-stranded RNA-binding proteins. We showed that VILIP can specifically bind double-stranded RNA, and this interaction specifically requires the presence of calcium. Mobility shift studies indicated that VILIP binds double-stranded RNA as a single protein-RNA complex with an apparent equilibrium dissociation constant of 9.0 × 10−6 m. To our knowledge, VILIP is the first double-stranded RNA-binding protein shown to be calcium-dependent. Furthermore, VILIP specifically binds the 3′-untranslated region of the neurotrophin receptor, trkB, an mRNA localized to hippocampal dendrites in an activity-dependent manner. Given that VILIP is also expressed in the hippocampus, these data suggest that VILIP may employ a novel, calcium-dependent mechanism to regulate its binding to important localized mRNAs in the central nervous system.

GENE THERAPY IN THE TREATMENT OF AUTOIMMUNE DISEASE

Abstract The genetic modification of autoreactive memory T cells and autoimmune target cells to produce immunoregulatory cytokines and regenerative growth factors offers a promising new approach for the treatment of autoimmune disease.

Pre-Emptive Targeting of the Epitope Spreading Cascade with Genetically Modified Regulatory T Cells During Autoimmune Demyelinating Disease

Epitope spreading or endogenous self-priming has been implicated in mediating the progression of autoimmune disease. In the present study we created an immune-deviated, epitope spreading response in SWXJ mice after the onset of experimental autoimmune encephalomyelitis, a prototypic autoimmune animal model widely used in multiple sclerosis research. We established an immunoregulatory spreading repertoire by transferring T cells genetically modified to produce high levels of IL-10 in response to a dominant epitope spreading determinant. Installation of a Th2/Tr1-like spreading repertoire resulted in a marked and prolonged inhibition of disease progression and demyelination characterized by 1) bystander inhibition of the recall response to the priming immunogen, and 2) a Th1→Tr1 immune-deviated spreading response involving a shift in the source of IL-10 production from the transferred regulatory population to the host-derived, endogenously primed repertoire. Thus, our data provide a rationale for cell-based therapeutic intervention in multiple sclerosis by showing that pre-emptive targeting of the epitope spreading cascade with regulatory T cells effectively induces an immune-deviated spreading response capable of inhibiting ongoing inflammatory autoreactivity and disease progression.

T cell design for therapy in autoimmune demyelinating disease

It has become increasingly more evident that a considerable refinement of currently used reagents and conditions will be needed before an effective gene therapy strategy can be used in the treatment of human autoimmune diseases. Such refinements will focus on optimizing three basic requirements for effective gene therapy, viz.: (1) targeted delivery of the therapeutic gene and/or its gene product in a reliable, efficient manner; (2) long-term expression of the therapeutic gene; and (3) regulated expression of the therapeutic gene so that it is activated only when needed. Using an experimental autoimmune encephalomyelitis mouse model, we have examined the potential for using the T cell as a gene therapy vector for targeted, long-term, regulated delivery of therapeutic transgene factors to the autoimmune inflammatory milieu. Our data indicate that the autoreactive T cell may serve as a useful endogenous vector for antigen-inducible, site-specific delivery of a variety of therapeutic transgene factors capable of mediating both inhibition of autoimmune inflammation and regeneration and/or protection of damaged tissue.

T-cell design: Optimizing the therapeutic potential of autoreactive T cells by genetic modification

Autoreactive CD4+ Th2 cells have been shown to be therapeutic in the treatment of EAE. However, their full therapeutic potential has yet to be realized. Genetic modification of autoreactive Th2 T cells may provide the means for delivering therapeutic transgene factors to autoimmune inflammatory lesions. Optimum therapeutic effects may be achieved by designing Th2 T cells in such a way that expression of transgene factors is regulated by antigen-inducible IL4, IL5 or IL10 transgene promoters. The innate antiinflammatory effects of the native autoreactive Th2 T cell may be enhanced by incorporating transgene regenerative growth factors in the T-cell design. Such factors may include remyelination growth factors (PDGF-A, bFGF, and IGF-I) that complement each other by acting predominantly at different stages in the development of mature myelinating oligodendrocytes. Moreover, in light of recent findings indicating extensive axonal damage during MS, neuroprotective transgene factors may prove to be therapeutic when delivered to EAE lesions by autoreactive Th2 T cells. Thus, optimum therapeutic effects may require multiple transfers of autoreactive Th2 T cells producing several distinct complementary transgene factors. In addition, the pathogenicity of epitope spreading and the inherent instability of self recognition during EAE may require serial transfer of genetically modified T cells reacting to multiple self determinants.

Stabilization of myelin mRNAs as measured in a brain slice system.

The stabilization and destabilization of myelin mRNA is increasingly recognized as a major control point in regulating myelin gene expression. A brain slice system was developed and characterized to study mRNA stability in actively myelinating oligodendrocytes. The mRNA half‐life of a major CNS myelin protein, proteolipid protein (PLP), was measured to be 5 hr. The half‐life of another CNS myelin protein mRNA, myelin basic protein (MBP), was measured to be greater than 12 hr. A long half‐life for MBP mRNA is consistent with MBP mRNA being stable long enough to be translocated to the myelin internode where it is then translated. Using semi‐quantitative reverse transcriptase‐PCR, it was determined that there was no differential stabilization between the two major PLP mRNA isoforms, PLP and DM20. It was also determined that protein synthesis was required for the specific stabilization of PLP/DM20 mRNAs. Inasmuch as PLP is a major structural protein of the CNS myelin, the PLP/DM20 mRNAs have relatively short half‐lives. However, the PLP/DM20 mRNAs half‐lives may be increased by the action of trans‐acting factors that are themselves very labile. J. Neurosci. Res. 50:1030–1039, 1997. © 1997 Wiley‐Liss, Inc.

Gene Therapy in Experimental Autoimmune Encephalomyelitis

Gene therapy traditionally has been associated with “gene replacement,” where exogenous recombinant DNA is introduced ex vivo into somatic cells that are then introduced back into the patient as a way to correct an inherited genetic defect. However, several novel gene therapy strategies for treating autoimmune diseases recently have emerged. Strategies involving the use of several types of DNA vaccines, the application of various viral vectors, and the use of diverse cellular vectors have shown promise in inhibiting autoimmune-mediated inflammation and repairing tissue damaged as a result of autoimmune attack. In the current review, we examine and discuss the development and proposed use of emerging gene therapy strategies for the treatment of autoimmune disease with specific emphasis on experimental autoimmune encephalomyelitis (EAE), an animal model widely used in multiple sclerosis (MS) research.