HEAT SHOCK PROTEIN GP96 AND IMMUNITY

该项目解决了这样一个假设，即gp 96，一种与哺乳动物抗原呈递有关的热休克蛋白，是一种祖先途径的一部分，该途径是使用MHC分子的抗原呈递途径的前因，并且独立于该途径。 我们将通过探索gp 96在蛙（非洲爪蟾）免疫过程中的作用来验证这一假设。 此外，由于两栖类在脊椎动物的进化中占据着关键的地位，如果我们的假设是正确的，那么gp 96应该参与青蛙和哺乳动物的免疫过程。 由于premetamorphic免疫活性爪蟾蝌蚪不表达细胞表面的MHC I类分子，青蛙模型使我们能够探索gp 96在免疫过程中的作用，在没有（幼虫）和存在（成人）的MHC I类依赖的介绍途径。为了开始确定gp 96是否参与爪蟾的免疫过程，将提出四个具体目标。 目的1处理的假设，从正常爪蟾组织中纯化的gp 96可以结合肽。 这一假设将通过检查负载有来自水泡性口炎病毒和卵清蛋白的肽的爪蟾gp 96引发小鼠CTL克隆的能力来检验。 除了确定非洲爪蟾gp 96是否通常与天然肽，我们将克隆非洲爪蟾gp 96同源物，并确定其一级结构，特别是其推定的肽结合结构域，已被保守的程度。 此外，克隆非洲爪蟾的gp 96同源物将提供有用的工具，以确定该gp 96基因是否与MHC（如hsp 70）的基因或编码hsp 90家族的其他成员的基因相连;并确定其启动子是否包含IFN-γ应答元件。 目的2的重点是测试假设，如果爪蟾gp 96参与抗原呈递和/或免疫调节，它可能是可检测的细胞表面分子。 最近的数据显示，细胞表面gp 96的一个子集的成年非洲爪蟾B细胞，幼虫的淋巴细胞，和硬骨鱼和盲鳗的淋巴细胞。实验提出了进一步表征这种细胞表面gp 96的图案的淋巴细胞从MHC I类+成人和MHC I类幼虫。 目的3和4发展了一种新的假设，即在体内，gp 96的免疫原性可以通过其在成人中引起较小H抗原不同皮肤同种异体移植物的加速排斥反应和在幼虫中引起相同移植物的耐受性的能力来揭示。 实验，以评估这些推定的同种异体免疫反应性的特异性和肽复合物依赖性的实验，以揭示所涉及的效应细胞的性质。

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.