HEAT SHOCK PROTEIN GP96 AND IMMUNITY

热休克蛋白 GP96 与免疫

• 批准号: 6133518
• 负责人: Nicholas Cohen
• 金额: $ 31.85万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6133518
• 关键词: MHC class I antigen; Xenopus; antigen presentation; cytotoxic T lymphocyte; developmental genetics; gene expression; heat shock proteins; histocompatibility; histocompatibility antigens; immature animal; immune tolerance /unresponsiveness; immunity; mature animal; protein binding; receptor; receptor expression; skin transplantation; transplant rejection

This project addresses the hypothesis that gp96, a heat shock protein that has been implicated in antigen presentation in mammals, is part of an ancestral pathway that is antecedent to, and independent of, the antigen presentation pathway that uses MHC molecules. We will test this hypothesis by exploring the role of gp96 in immune processes in the frog, Xenopus. Moreover, since the Amphibia occupy a pivotal position in the evolution of vertebrates, it is anticipated that if our hypothesis is correct, gp96 should be involved in immune processes in frogs as well as mammals. Since premetamorphic immunocompetent Xenopus tadpoles do not express cell surface MHC class I molecules, the frog model allows us to explore the role of gp96 in immune processes in the absence (larvae) and presence (adults) of MHC class I-dependent presentation pathways. To begin to determine whether gp96 is involved in immune processes in Xenopus, four specific aims will be addressed. Aim 1 deals with the hypothesis that gp96 purified from normal Xenopus tissues can bind peptides. This hypothesis will be tested by examining the ability of Xenopus gp96 loaded with peptides from vesicular stomatitis virus and ovalbumin to prime murine CTL clones. In addition to determining whether Xenopus gp96 is normally associated with native peptides, we will clone the Xenopus gp96 homologue and determine the degree to which its primary structure, particularly its putative peptide-binding domain, has been conserved. In addition, cloning the gp96 homologue of Xenopus will provide useful tools to determine whether this gp96 gene is linked to genes of the MHC (like hsp70) or to genes encoding other members of the hsp90 family; and determine whether its promoter contains an IFN-gamma-responsive element. Aim 2 focuses on testing the hypothesis that if Xenopus gp96 is involved in antigen presentation and/or immunomodulation, it may be detectable as a cell surface molecule. Recent data reveal cell surface gp96 on a subset of adult Xenopus B-cells, on larval lymphocytes, and on lymphocytes from teleosts and hagfish. Experiments are proposed to further characterize the pattern of this cell surface gp96 on lymphocytes from MHC class I+ adults and MHC class I- larvae. Aims 3 and 4 develop the novel hypothesis that in vivo, the immunogenicity of gp96 may be revealed by its capacity to evoke accelerated rejection of minor H-antigen disparate skin allografts in adults, and tolerance of the same grafts in larvae. Experiments to evaluate the specificity and peptide complex-dependency of these putative alloimmune reactivities are presented as are experiments to reveal the nature of effector cells involved.

该项目解决了以下假设：GP96是一种与哺乳动物抗原相关的热休克蛋白，是祖先途径的一部分，该途径是使用MHC分子的抗原呈递途径，并且与使用MHC分子的抗原呈递途径无关。 我们将通过探索GP96在青蛙爪蟾中的免疫过程中的作用来检验这一假设。 此外，由于两栖动物在脊椎动物的进化中占据关键位置，因此预计如果我们的假设是正确的，则GP96应参与青蛙和哺乳动物的免疫过程。 由于预成态免疫能力曲to骨t不会表达细胞表面MHC I类分子，因此Frog模型使我们能够探索GP96在不存在（幼虫）和MHC I类依赖性依赖性依赖性表现途径的情况下GP96在免疫过程中的作用。为了开始确定Xenopus中的GP96是否参与免疫过程，将解决四个具体目标。 AIM 1处理以下假设：从正常爪蟾组织中纯化的GP96可以结合肽。 该假设将通过检查带有囊泡口腔炎病毒和椭圆蛋白到基质鼠CTL克隆的肽的异武GP96的能力来检验。 除了确定爪蟾GP96是否通常与天然肽相关联，我们还将克隆Xenopus gp96同源物，并确定其主要结构（尤其是其推定的肽结合结构域）的程度。 此外，克隆Xenopus的GP96同源物将提供有用的工具，以确定该GP96基因是将MHC基因（如HSP70）还是与编码HSP90家族其他成员的基因相关的；并确定其启动子是否包含IFN-GAMMA响应元件。 AIM 2侧重于检验以下假设：如果Xenopus gp96参与抗原表现和/或免疫调节，则可以检测为细胞表面分子。 最近的数据揭示了成人B细胞子集的细胞表面GP96，幼虫淋巴细胞以及定蛋白和Hagfish的淋巴细胞上的细胞表面。提出了实验，以进一步表征该细胞表面GP96在MHC I类+成人和MHC I-arrvae的淋巴细胞上的模式。 目的3和4提出了新的假设，即在体内，GP96的免疫原性可以通过唤起成年人中较小的H抗原皮肤异常的较小H抗原皮肤的能力来揭示，并且在幼虫中耐受同样的移植物的耐受性。 提出了评估这些推定的同种免疫反应率的特异性和肽复合依赖性的实验，并进行了实验，以揭示涉及效应细胞的性质。