HEAT SHOCK PROTEIN GP96 AND IMMUNITY

这个项目解决了这样的假设，即gp96，一种与哺乳动物抗原提呈有关的热休克蛋白，是使用MHC分子的抗原提呈途径之前且独立于其之前的祖先途径的一部分。我们将通过探索gp96在非洲爪蛙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有举足轻重的地位，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。由于变态前免疫活性非洲爪哇蝌蚪不表达细胞表面MHC I类分子，青蛙模型允许我们在缺乏(幼虫)和存在(成人)依赖MHC I类递送途径的情况下探索gp96在免疫过程中的作用。为了开始确定gp96是否参与非洲爪哇的免疫过程，将解决四个具体目标。目的1研究从正常非洲爪哇组织中提纯的gp96能与多肽结合的假说。这一假设将通过检测非洲爪哇gp96装载水泡性口炎病毒和卵清蛋白的多肽来启动小鼠CTL克隆的能力来检验。除了确定非洲爪哇gp96是否正常与天然多肽相关外，我们还将克隆非洲爪哇gp96同源物，并确定其一级结构，特别是其推测的肽结合结构域的保守程度。此外，克隆非洲爪哇的gp96同源物将提供有用的工具，以确定该gp96基因是否与MHC的基因(如HSP70)或编码HSP90家族其他成员的基因有关；并确定其启动子是否包含干扰素-伽马反应元件。目的2重点验证一种假说，即如果非洲爪蛙gp96参与抗原提呈和/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，细胞表面的gp96在成虫B细胞的亚群上，在幼虫淋巴细胞上，以及在硬骨鱼和角叉鱼的淋巴细胞上。为了进一步研究这种细胞表面gp96在MHC Class I+成虫和MHC Class I-幼虫淋巴细胞上的模式，建议进行实验。目标3和4提出了一个新的假设，即在体内，gp96的免疫原性可能是通过在成人体内引起对不同H抗原的微小异体皮肤移植物的加速排斥反应以及在幼虫对相同移植物的耐受性来揭示的。评价这些假定的同种异体免疫反应的特异性和多肽复合体依赖性的实验，以及揭示所涉及的效应细胞的性质的实验也被提出。

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.