MECHANISMS OF AUTOIMMUNE ARTHRITIS

自身免疫性关节炎的机制

• 批准号: 2899929
• 负责人: Edward F Rosloniec
• 金额: $ 18.02万
• 依托单位: UNIVERSITY OF TENNESSEE HEALTH SCI CTR
• 依托单位国家: 美国
• 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/2899929
• 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

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（改编自研究者摘要）：本申请是 重点是表征HLA-DR 1和HLA-DR 4 MHC II类基因的作用 在对人II型胶原蛋白（hCII）的自身免疫反应的发展中。 在先前发表的研究和初步数据中，研究人员 描述了表达嵌合MHC II类的转基因小鼠的产生 其中β亚基的肽结合结构域衍生的分子 来自DRB 1或DRB 4，而β亚基的其余部分是鼠源的， 起源 通过共注射这种嵌合体来产生转基因小鼠。 小鼠/人基因构建体以及编码该cDNA的基因。 这些 转基因随后被证明赋予对胶原蛋白的易感性， 在正常抗性B10中诱导关节炎。这些研究人员建议， 利用这种“人源化小鼠”系统来表征分子 影响胶原诱导性关节炎易感性的机制。 他们 列出四个具体目标：1）表征关节炎和 HLA-DR 1和HLA-DR 4转基因小鼠的自身免疫应答， 用人II型胶原免疫。 这些研究涉及使用 一组跨越hCII的重叠肽，以识别识别的表位 通过应答CD 4 + T细胞。 2)以鉴定氨基酸替换， 产生改变的肽配体的hCII抗原决定簇， 抑制DR 1和DR 4限制的T细胞刺激，并防止 在DR转基因小鼠中自身免疫性关节炎的发展。 这些 实验将建立在具体目标1的结果，以确定改变 通过修饰抑制DR 1和DR 4限制性T细胞的肽配体 破坏TCR相互作用或降低与DR 1结合亲和力的残基 或DR 4。 3)以确定单个T细胞决定因素在 介导对hCii的DR 1和DR 4限制性免疫应答， 在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表达系统来产生突变体 hCII，其中单个抗原决定簇的序列已被 要么完全删除或改变的程度，决定因素没有 与DR 1或DR 4的结合时间更长。 然后，他们将免疫转基因小鼠， 这些突变的hCII分子，并评估对免疫应答的影响 和关节炎。 4)为了确定可溶性DR 1或DR 4与 共价连接的hCII肽破坏野生型T细胞识别 hCII呈递和预防自身免疫性关节炎的发展。 这些研究将产生可溶性DR分子， hCII的肽（在特定目的1和2中鉴定）在遗传上是 被工程化到DR β亚基的氨基末端。 可溶性 分子将使用DR在果蝇细胞表达系统中产生 在细胞膜上被截短的α和β亚基基因 结构域以允许可溶性产生α/β-CII肽二聚体。 这些重组DR分子将被测试其改变 体外T细胞对hCII的反应，并防止自身免疫性疾病的发展 DR转基因小鼠的关节炎。