This application is focused on characterizing the role of HLA-DR1 and HLA-DR4 MHC class II genes in the development of autoimmune responses to human type II collagen (hCII). In previously published studies and preliminary data, the investigators describe the production of transgenic mice expressing chimeric MHC class II molecules in which the peptide binding domain of the beta subunit is derived from DRB1 or DRB4, while the remainder of the beta subunit is of murine origin. Transgenic mice were produced by the co-injection of this chimeric mouse/human gene construct together with a gene encoding the DRA. These transgenes were subsequently shown to confer susceptibility to collagen induced arthritis in normally resistant B10.M. These investigators propose to utilize this """"""""humanized mouse"""""""" system to characterize the molecular mechanisms effecting susceptibility to collagen-induced arthritis. They list four specific aims: 1) to characterize the arthritogenic and autoimmune responses of HLA-DR1 and HLA-DR4 transgenic mice, elicited by immunization with human type II collagen. These studies involve the use of a set of overlapping peptides spanning hCII to identify epitopes recognized by responding CD4+ T-cells. 2) to identify amino acid substitutions within the hCII antigenic determinants that generate altered peptide ligands that inhibit DR1 and DR4-restricted T-cell stimulation and prevent the development of autoimmune arthritis in the DR transgenic mice. These experiments will build on the results of Specific Aim 1 to identify altered peptide ligands that inhibit DR1 and DR4-restricted T-cells via modification of residues that disrupt TCR interaction or reduce binding affinity to DR1 or DR4. 3) to determine the role of individual T-cell determinants in mediating the DR1 and DR4-restricted immune responses to hCii and the development of autoimmune arthritis in the DR transgenic mice. These studies will utilize a recombinant hCII expression system to produce mutant hCII in which the sequence of an individual antigenic determinant has been either entirely deleted or altered to the degree that the determinant no longer binds to DR1 or DR4. They will then immunize transgenic mice with these mutant hCII molecules and assess the effect on immune responsiveness and arthritis. 4) to determine the efficacy of soluble DR1 or DR4 with covalently linked hCII peptide in disrupting T-cell recognition of wild type hCII presentation and preventing the development of autoimmune arthritis. These studies will produce soluble DR molecules in which the immunodominant peptides of hCII (identified in Specific Aims 1 and 2) are genetically engineered into the amino terminus of the DR beta subunit. Soluble molecules will be produced in the drosophila cell expression system using DR alpha and beta subunit genes that have been truncated at the cell membrane domain to allow soluble production of the alpha/beta-CII peptide dimer. These recombinant DR molecules will be tested for their ability to alter T-cell responses to hCII in vitro, and prevent the development of autoimmune arthritis in the DR transgenic mice.
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