HEAT SHOCK PROTEIN GP96 AND IMMUNITY

该项目提出了一种假设，即gp96是一种与哺乳动物抗原递呈有关的热休克蛋白，它是一种祖先途径的一部分，该途径先于使用MHC分子的抗原递呈途径，并且独立于抗原递呈途径。我们将通过探索gp96在非洲爪蟾免疫过程中的作用来验证这一假设。此外，由于两栖类动物在脊椎动物的进化过程中占有举足轻重的地位，因此我们预计，如果我们的假设是正确的，gp96应该参与青蛙和哺乳动物的免疫过程。由于前变质免疫能力的爪蟾蝌蚪不表达细胞表面MHC I类分子，因此青蛙模型允许我们探索gp96在MHC I类依赖呈递途径缺失（幼虫）和存在（成虫）的免疫过程中的作用。为了开始确定gp96是否参与爪蟾的免疫过程，将解决四个具体目标。目的1讨论了从正常爪蟾组织中纯化的gp96能结合多肽的假设。这一假设将通过检测爪蟾gp96装载水疱性口炎病毒和卵清蛋白肽的能力来验证。除了确定Xenopus gp96是否通常与天然肽相关外，我们还将克隆Xenopus gp96同源物，并确定其初级结构，特别是其推定的肽结合结构域的保守程度。此外，克隆爪蟾的gp96同源基因将为确定gp96基因是否与MHC基因（如hsp70）或编码其他hsp90家族成员的基因相关提供有用的工具。并确定其启动子是否含有ifn - γ响应元件。目的2的重点是验证假设，如果爪蟾gp96参与抗原呈递和/或免疫调节，它可能作为细胞表面分子被检测到。最近的数据显示，在成年非洲爪蟾b细胞亚群、幼虫淋巴细胞、硬骨鱼和盲鳗淋巴细胞上存在细胞表面gp96。我们建议进一步研究这种细胞表面gp96在MHC I+类成虫和MHC I-类幼虫淋巴细胞上的表达模式。目的3和4提出了一种新的假设，即在体内，gp96的免疫原性可能是通过其在成人中引起对少量h抗原不同的同种异体皮肤移植物的加速排斥和幼虫对同种移植物的耐受能力来揭示的。评估这些假定的同种免疫反应的特异性和肽复合物依赖性的实验，以及揭示所涉及的效应细胞性质的实验。

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.