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类抗原发挥独特性和免疫反应中的基本作用。扮演的基本角色经典的I类基因已在人类中鉴定出来。这些基因，命名为HLA-E，-f和-g，每个都有表达方式区分的模式它们彼此以及HLA -A，-b和-c抗原。最多最近，已经证明了HLA-G在人类中表达通过滋养细胞细胞胎盘，可以以两种形式表达膜结合和一个水溶性。在此提案中，我们提出可以证明HLA-G可以在所谓的免疫的那些组织中表达私密的包括胎盘，前眼和精子。我们目前的证据表明HLA-G蛋白可能存在替代形式，由主要成绩单的替代剪接产生。基于我们证明了对HLA-E蛋白的特异性CTL响应，并具有检查了转染的细胞系中蛋白质表达的模式，我们提出HLA-E确实在体内发挥作用，是经典的限制元素，可能在体内具有另一个未知功能。提出的实验该应用程序旨在通过进一步检验上述假设描述和解释表达和测试非古典抗原。实验设计和方法如下。特定的目标1专注于扩展组织和细胞的知识表达HLA -E，-f和-g抗原。我们将通过连续三个分析级别：a）识别组织表达HLA -E，-f和-g mRNA。 b）鉴定抗原表达使用单克隆抗体在特定的细胞子集上。 c）表征每种细胞类型表达的不同抗原形式。具体 AIM 2我们采取的目标是更充分地表征不同的蛋白质形式由HLA-G基因表示，允许进一步测试其功能。这将通过实现以下目标来实现：a）表达HLA-G的替代形式的细胞系的结构。 b）这些替代品反应产生血清和单克隆抗体表格。 c）HLA-G的可溶性形式的纯化。研究概述了在特定目标中提出了研究影响表面的机制 HLA-E的运输。我们将通过：a）对HLA-E进行比较蛋白质集中在那些将其与A区分开的特征上古典抗原。 b）检查中氨基酸残基 HLA_E蛋白影响细胞表面表达。最后，特定目标4 提议分析与HLA-E和HLA-G反应的T细胞群体在两种方式：a）我们将通过其T细胞表面标记的表征包括分子其T细胞受体的表征。 b）我们将集中精力通过试图测量可溶性HLA-G蛋白的细胞反应性在体外对正常T细胞功能的影响。