MECHANISMS OF AUTOIMMUNE ARTHRITIS

自身免疫性关节炎的机制

• 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
• 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

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（改编自研究者的摘要）：该应用程序是 专注于表征 HLA-DR1 和 HLA-DR4 MHC II 类基因的作用 参与对人类 II 型胶原蛋白 (hCII) 的自身免疫反应的发展。 在之前发表的研究和初步数据中，研究人员 描述表达嵌合 MHC II 类转基因小鼠的产生 衍生出β亚基的肽结合结构域的分子 来自 DRB1 或 DRB4，而其余的 β 亚基来自小鼠 起源。 通过共同注射这种嵌合体产生了转基因小鼠 小鼠/人类基因构建体以及编码 DRA 的基因。 这些 随后证明转基因赋予胶原蛋白敏感性 在正常耐药的 B10.M 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-DR1 和 HLA-DR4 转基因小鼠的自身免疫反应，由 人II型胶原蛋白免疫。 这些研究涉及使用 一组跨越 hCII 的重叠肽，用于识别已识别的表位 通过响应 CD4+ T 细胞。 2) 鉴定氨基酸替换 hCII 抗原决定簇产生改变的肽配体 抑制 DR1 和 DR4 限制性 T 细胞刺激并防止 DR 转基因小鼠发生自身免疫性关节炎。 这些 实验将建立在特定目标 1 的结果的基础上，以确定改变的 通过修饰抑制 DR1 和 DR4 限制性 T 细胞的肽配体 破坏 TCR 相互作用或降低与 DR1 结合亲和力的残基 或 DR4。 3) 确定个体T细胞决定因素在 介导针对 hCii 的 DR1 和 DR4 限制性免疫反应以及 DR 转基因小鼠发生自身免疫性关节炎。 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，要么改变到决定因素不存在的程度 与 DR1 或 DR4 的结合时间更长。 然后他们将对转基因小鼠进行免疫接种 这些突变的 hCII 分子并评估对免疫反应的影响 和关节炎。 4) 确定可溶性DR1或DR4的功效 共价连接的 hCII 肽破坏 T 细胞对野生型的识别 hCII 的呈现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，其中免疫显性分子 hCII 的肽（在具体目标 1 和 2 中确定）在遗传上是 工程化到 DR β 亚基的氨基末端。 可溶 分子将使用 DR 在果蝇细胞表达系统中产生 α和β亚基基因在细胞膜处被截短 结构域允许可溶性产生 α/β-CII 肽二聚体。 这些重组 DR 分子将被测试其改变的能力 T 细胞在体外对 hCII 作出反应，并预防自身免疫性疾病的发展 DR 转基因小鼠的关节炎。