One of the goals of this project is to examine mechanisms of activation of autoreactive T lymphocytes in the animal model for multiple sclerosis (MS), i.e. experimental allergic encephalomyelitis (EAE). EAE is induced in susceptible animals either by injection of myelin proteins or peptides or via adoptive transfer of myelin-specific T cells from EAE animals into naive animals. Many important aspects of the pathogenesis and treatment of demyelinating diseases such as MS have been studied in the EAE model. With respect to an early event in disease pathogenesis, i.e. the activation of autoreactive T cells, it has been shown that viruses and bacteria can lead to the activation and expansion of autoreactive T cells via molecular mimicry or antigenic similarity between foreign agent and autoantigen. In order to study these questions, we have chosen to develop humanized transgenic mice that express chimeric receptors combining transmembrane regions of mouse class II with the antigen-binding domain of the MS-associated HLA-DR alleles DRB1*1501 and DRB1*0401, and on the T cell receptor (TCR) side, the TCR constant region from the mouse with the antigen-specific domains from human MBP-specific TCRs. Three double transgenic mouse lines have been generated: Designation: DR restriction Specificity MS2-3C8 DRB1*0401 MBP (111-129); 1113-1C2 DRB1*0401 MBP (111-129); TL 3A6 DRB5*0101 MBP (83-99). The first mouse created was shown to possess a unique phenotype and display clinical symptoms which have been observed in MS patients, and particularly had been observed in the MS patient from whom this TCC was originally isolated. A second transgenic mouse was completed in the last year which has a different specificity and HLA restriction element. Thus far, experiments have shown that these mice carrying an autoreactive, myelin-specific TCR and the MS-associated MHC class II molecule have a low incidence of spontaneous autoimmunity. Current research efforts are aimed at examining the degeneracy of antigen recognition by autoreactive T cells, and identifying bacterial or viral pathogens which may serve as ?molecular mimics? and activate these auto-reactive T cells through their homology to myelin antigens. Positional scanning combinatorial libraries (PSCL) have been used to identify amino acids in peptide sequences which are predicted to be stimulatory for these myelin-reactive T cells. The next steps will be to test the peptides contained in these pathogens to test their stimulatory potential for myelin reactive T cells a) in vitro and b) in vivo in the transgenic mice to assess their ability to induce CNS autoimmunity. This will require testing their ability to induce and score T cell proliferative responses and cytokine production both in the T cells isolated from the transgenic mice as well as T cell clones isolated from MS patients which recognize myelin proteins. Once candidate antigens with cross reactive potential to myelin antigens have been validated in vitro, their ability to bind to the disease associated MHC in vivo and to be recognized by the autoreactive T cells in vivo will be tested and gauged by monitoring their activation status in vivo as well as examining their ultimate potential to induce EAE in the transgenic mice. In addition, a collaboration is currently ongoing with Dr. Roy Mariuzza, University of Maryland, to obtain the crystal structure of this TCR-antigen-MHC complex, which will provide structural insight into the nature of this degenerate antigen recognition. The initial results of ths collaboration have now been published Finally, a model for a chronic bacterial infection of the CNS is currently being studied in order to understand better the molecular events involved in the development of chronic central nervous system Lyme disease. This project is being pursued in collaboration with the National Institute of Allergy and Infectious Diseases. Publications about the humanized transgenic mice and the tolerizing capacity of dendritic cells have been submitted or have been published in this year.
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