MECHANISMS OF AUTOIMMUNE ARTHRITIS

自身免疫性关节炎的机制

基本信息

批准号：
2593298
负责人：
Edward F Rosloniec
金额：
$ 17.5万
依托单位：
UNIVERSITY OF TENNESSEE HEALTH SCI CTR
依托单位国家：
美国
项目类别：
财政年份：
1998
资助国家：
美国
起止时间：
1998-05-15 至 2002-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2593298
关键词：
MHC class II antigen T cell receptor T lymphocyte collagen disease /disorder model epitope mapping gene expression genetically modified animals histocompatibility gene laboratory mouse leukocyte activation /transformation molecular cloning pathologic process receptor binding recombinant proteins rheumatoid arthritis

项目摘要

DESCRIPTION (Adapted from Investigator's abstract): 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.

描述（根据调查员的摘要改编）：此应用程序是专注于表征HLA-DR1和HLA-DR4 MHC II类基因的作用在开发对人类II型胶原蛋白（HCII）的自身免疫反应中。在先前发表的研究和初步数据中，研究人员描述表达嵌合MHC II的转基因小鼠的产生 β亚基的肽结合结构域的分子得出来自DRB1或DRB4，而其余的beta亚基则为鼠起源。转基因小鼠是通过共同注射而产生的小鼠/人类基因与编码DRA的基因构建。这些随后显示转基因以赋予胶原蛋白的敏感性正常抗性B10.M.诱导关节炎。这些调查人员建议利用这种“人性化小鼠”系统来表征分子影响胶原蛋白诱导的关节炎的机制。他们列出四个具体目的：1）表征关节化和 HLA-DR1和HLA-DR4转基因小鼠的自身免疫反应，由人类II型胶原蛋白的免疫。这些研究涉及使用一组跨越HCII的重叠肽，以识别已识别的表位通过响应CD4+ T细胞。 2）确定氨基酸的取代产生改变肽配体的HCII抗原决定因素抑制DR1和DR4限制的T细胞刺激，并防止 DR转基因小鼠中自身免疫性关节炎的发展。这些实验将基于特定目标1的结果以确定变化通过修饰抑制DR1和DR4限制的T细胞的肽配体破坏TCR相互作用或减少与DR1结合亲和力的残基或DR4。 3）确定单个T细胞决定因素在介导DR1和DR4限制的免疫反应对HCII和 DR转基因小鼠中自身免疫性关节炎的发展。这些研究将利用重组HCII表达系统产生突变体 HCII，其中单个抗原决定因素的序列已完全删除或更改为决定符号更长的结合DR1或DR4。然后他们将用这些突变的HCII分子并评估对免疫反应性的影响和关节炎。 4）确定可溶性DR1或DR4的功效共价连接的HCII肽在破坏T细胞识别野生型中 HCII表现并防止自身免疫性关节炎的发展。这些研究将产生可溶性的DR分子，其中免疫主导 HCII的肽（在特定目的1和2中鉴定）是遗传的设计到博士Beta亚基博士的氨基终点站。可溶分子将使用DR在果蝇细胞表达系统中产生在细胞膜上已截断的α和β亚基基因域可允许α/β-CII肽二聚体的可溶性产生。这些重组DR分子的变化能力将测试在体外对HCII的T细胞反应，并防止自身免疫的发展 DR转基因小鼠的关节炎。