EXPRESSION & FUNCTION OF THREE NEW HLA CLASS I ANTIGENS

表达

基本信息

批准号：
2066823
负责人：
DANIEL E. GERAGHTY
金额：
$ 26.18万
依托单位：
FRED HUTCHINSON CANCER RESEARCH CENTER
依托单位国家：
美国
项目类别：
财政年份：
1991
资助国家：
美国
起止时间：
1991-07-01 至 1995-06-30
项目状态：
已结题

项目摘要

The broad, long-term objectives of the proposal are aimed at testing the hypothesis that the nonclassical HLA class I antigens play unique and fundamental roles in the immune response. The fundamental role played by the classical class I genes have been identified in humans. These genes, named HLA-E,-F, and -G, each have patterns of expression distinguishing them from one another and from the HLA-A, -B, and -C antigens. Most recently, it has been demonstrated that HLA-G is expressed in the human placenta by trophoblast cells and may be expressed in two forms, one membrane bound and one water soluble. In this proposal, we present evidence that HLA-G may be expressed in those tissues so called immune priveledged including the placenta, the anterior eye, and sperm. We present evidence that alternate forms of the HLA-G protein may exist, resulting from alternate splicing of the primary transcript. Based on our having demonstrated a specific CTL response to the HLA-E protein and having examined the pattern of protein expression in transfected cell lines, we propose HLA-E does function in vivo as a classical restricting element and may have another unknown function in vivo. The experiments proposed in this application are designed to test the above hypothesis by further describing and explaining the expression and testing the function of the nonclassical antigens. The experimental design and methods are as follows. Specific aim 1 is focused on expanding the knowledge of tissues and cells expressing the HLA-E, -F, and -G antigens. We will accomplish this through three successive levels of analysis: a) identification of tissues expressing HLA-E, -F, and -G mRNA. b) identification of antigen expression on specific cell subsets using monoclonal antibodies. c) characterization of the different antigen forms expressed by each cell type. In specific aim 2 we undertake to more fully characterize the different protein forms expressed by the HLA-G gene, allowing for further testing of their function. this will be accomplished by achieving the following goals: a) construction of cell lines expressing the alternate forms of HLA-G. b) generation of sera and monoclonal antibodies reactive with these alternate forms. c) Purification of the soluble form of HLA-G. The studies outlined in specific aim 3 propose to study the mechanisms affecting the surface transport of HLA-E. We will do this by: a) a comparison of the HLA-E protein concentrating on those features which distinguish it from a classical antigen. b) an examination of the amino acid residues in the HLA_E protein affecting cell surface expression. Finally, specific aim 4 proposes to analyze the T cell populations reacting with HLA-E and HLA-G in two ways: a) We will analyze the HLA-E reactive cells by the characterization of their T cell surface markers including a molecular characterization of their T cell receptor. b) We will concentrate on the T cell reactivity of the soluble HLA-G protein by attempting to measure it's effect on normal T cell function in vitro.

该提案的广泛、长期目标旨在测试假设非经典 HLA I 类抗原发挥独特的作用在免疫反应中发挥重要作用。所发挥的基础性作用经典的 I 类基因已在人类中被发现。这些基因，命名为 HLA-E、-F 和 -G，各自具有区分的表达模式它们彼此之间以及HLA-A、-B 和-C 抗原之间的区别。最多最近，研究表明HLA-G在人类中表达。胎盘由滋养层细胞表达，可能以两种形式表达，一种是膜结合的和一种水溶性的。在本提案中，我们提出有证据表明 HLA-G 可能在这些所谓的免疫组织中表达特权包括胎盘、前眼和精子。我们提供证据表明可能存在 HLA-G 蛋白的替代形式，由初级转录本的交替剪接产生。基于我们的已证明对 HLA-E 蛋白具有特异性 CTL 反应，并且具有检查转染细胞系中蛋白质表达的模式，我们提出 HLA-E 在体内确实起到经典限制元件的作用，并且体内可能还有另一种未知的功能。中提出的实验该应用程序旨在通过进一步测试上述假设描述和解释表达式并测试其功能非经典抗原。实验设计和方法如下。具体目标 1 侧重于扩展组织和细胞的知识表达HLA-E、-F和-G抗原。我们将通过以下方式实现这一目标三个连续的分析级别：a) 组织识别表达 HLA-E、-F 和 -G mRNA。 b) 抗原表达的鉴定使用单克隆抗体针对特定细胞亚群。 c) 表征每种细胞类型表达的不同抗原形式。具体来说目标 2 我们致力于更全面地表征不同蛋白质形式由 HLA-G 基因表达，可以进一步测试其功能。这将通过实现以下目标来实现：a) 表达 HLA-G 替代形式的细胞系的构建。 b) 产生与这些替代物反应的血清和单克隆抗体形式。 c) 可溶形式的HLA-G的纯化。研究概述具体目标3建议研究影响表面的机制 HLA-E 的运输。我们将通过以下方式做到这一点： a) HLA-E 的比较蛋白质集中于那些区别于其他蛋白质的特征经典抗原。 b) 氨基酸残基的检查 HLA_E 蛋白影响细胞表面表达。最后具体目标4 提议分析与 HLA-E 和 HLA-G 反应的 T 细胞群有两种方法： a) 我们将通过以下方法分析 HLA-E 反应性细胞： T 细胞表面标记物的表征，包括分子他们的T细胞受体的特征。 b) 我们将专注于T 通过尝试测量可溶性 HLA-G 蛋白的细胞反应性体外对正常 T 细胞功能的影响。