ANTIGEN PRESENTATION IN INSULIN DEPENDENT DIABETES MELLITUS

胰岛素依赖型糖尿病中的抗原呈递

• 批准号: 6268246
• 负责人: EMIL Raphael UNANUE
• 金额: $ 7.97万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-06-01 至 1999-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6268246
• 关键词: B lymphocyte; MHC class II antigen; NOD mouse; SDS polyacrylamide gel electrophoresis; T lymphocyte; antigen presentation; antigen presenting cell; autoimmunity; binding proteins; biochemistry; cell cell interaction; cellular immunity; chemical association; crosslink; diabetes mellitus genetics; genetically modified animals; immune tolerance /unresponsiveness; immunogenetics; insulin dependent diabetes mellitus; leukocyte activation /transformation; pancreatic islets; peptides; protein sequence; synthetic peptide; tissue /cell culture

We propose to examine the peptide-binding properties of the class II MHC molecule (I-Ag7) from NOD mice. Our proposal is based on the following findings; i) Ag7 molecules isolated from a variety of Antigen Presenting Cells are unstable when examined by SDS. PAGE, ii) this unstability does not seem to be caused by a lack of association with Invariant chain or by a lack of processing and assembly of intracellular Ag7, iii) all peptides so far examined bind very weakly to Ag7 either from purified molecules or in APC, iv) the rate of dissociation of all peptides tested is very fast and t his increased off-rate is dramatically evident in T cell assays. Indeed T cells fail to respond if the peptide is not in the culture all of the time, v) preliminary studies indicate that the beta cell diabetogenic antigen behaves the same. These results led us to speculate that the weak binding of peptides by Ag7 is, contrary to expectations, a feature that does not allow for tolerance of autoreactive T cells. The proposal called for, first, an extensive biochemical characterization of Ag7 and its interaction with peptides, including the use of photoaffinity labeled peptides. Second, we will examine how APC bind peptides, their immunogenicity and their time of persistence. For both these issues we select foreign peptides, known autoreactive peptides, and peptides that are isolated from Ag7 molecules. A third goal examines the immune response to peptides bound weakly to Ag7. We propose two experiments, one is an analysis of a peptide processed by APC from Ealpha chain and which interacts in dramatically different ways when binding to Ag7 or Ab. We will test the immune response in (NOD X B6) F1 mice expressing the E chain (and, in principle, tolerant to it). Another experiment attempts to engineer the Aalpha chain of NOD so as to produce a stable, high affinity binding Ag7. If successful we will compare the reaction of T cells to it, both in culture and in in vivo experiment. Finally, we plan to examine these phenomena on human IDDM susceptible class II molecules DQ2 and DQ8.

我们建议检查 II 类 MHC 的肽结合特性 来自 NOD 小鼠的分子 (I-Ag7)。 我们的建议基于以下内容 发现; i) 从多种抗原呈递中分离出的 Ag7 分子 通过 SDS 检查时，细胞不稳定。 PAGE，ii）这种不稳定性确实 似乎不是由于缺乏与不变链的关联或由 缺乏细胞内 Ag7 的加工和组装，iii) 所有肽 到目前为止，已检测到与来自纯化分子的 Ag7 或来自纯化分子的 Ag7 的结合非常弱。 在 APC 中，iv) 所有测试的肽的解离速度都非常快 这种解离率的增加在 T 细胞检测中非常明显。 事实上，如果培养物中没有该肽，T 细胞就无法做出反应。 时间，v) 初步研究表明，β 细胞会导致糖尿病 抗原的行为相同。 这些结果使我们推测弱者 与预期相反，Ag7 与肽的结合是一个特征 不允许自身反应性 T 细胞耐受。 该提案首先要求进行广泛的生化表征 Ag7 及其与肽的相互作用，包括使用 光亲和标记的肽。 其次，我们将研究APC如何绑定 肽、其免疫原性及其持续时间。 对于两者 这些问题我们选择外源肽、已知的自身反应肽，以及 从 Ag7 分子中分离出来的肽。 第三个目标检查 对与 Ag7 微弱结合的肽的免疫反应。 我们建议两个 实验，一个是对来自 Ealpha 的 APC 加工的肽进行分析 链，并且在绑定到时以截然不同的方式相互作用 Ag7 或抗体。 我们将测试 (NOD X B6) F1 小鼠的免疫反应 表达E链（并且原则上容忍它）。 其他 实验试图改造 NOD 的 Aalpha 链，从而产生 稳定、高亲和力结合 Ag7。 如果成功，我们将比较 T 细胞在培养物和体内实验中对其的反应。 最后，我们计划在人类 IDDM 易感人群中研究这些现象 II 分子 DQ2 和 DQ8。