POLYMORPHISM AND TRANSPLANTATION BIOLOGY OF NOVEL HLA CLASS I GENES

新型HLA I类基因的多态性和移植生物学

• 批准号: 3810509
• 负责人: DANIEL GERAGHTY
• 金额: --
• 依托单位: FRED HUTCHINSON CANCER RESEARCH CENTER
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3810509
• 关键词: MHC class I antigen; T lymphocyte; alleles; antibody formation; bone marrow transplantation; cell mediated lymphocytolysis test; clone cells; gene expression; genetic polymorphism; genetically modified animals; graft versus host disease; laboratory mouse; major histocompatibility complex; monoclonal antibody; natural gene amplification; polymerase chain reaction; transfection

The overall objective of this proposal is to shed light on some of the basic questions concerning the biology of the new class I proteins. Because these genes were discovered after cloning and sequencing all of the class I homologous sequences isolated from a human cell line, no antibodies or sera specifically reactive with the protein products of these genes are known. While expression can be detected at the mRNA level in various cells and tissues, protein products have only been detected in HLA class I deficient cell lines transfected with these genes. One characteristic of the classical HLA-A,-B and -C antigens of importance to transplantation biology is the striking polymorphism found at each locus. The first specific aim of this proposal is to determine if, like the classical antigens, the HLA-E, F(5.4) and G(6.0) antigens exhibit polymorphism. We will use the polymerase chain reaction to specifically amplify E, F and G cDNA and genomic sequences from a set of genetically diverse individuals and determine their nucleotide sequence. If polymorphism is present at one or more of these loci, we will use the same methods to correlate that polymorphism with the outcome of an unrelated bone marrow transplant. The second specific aim is to isolate monoclonal antibodies specifically reactive with the HLA-E, -F(5.4) and G(6.0) molecules. We have constructed a set of cell lines expressing these proteins and are planning construction of additional lines. We will use these cells as a source of antigen for the production of monoclonal antibodies. In order to optimize the possibility of producing specific antibodies we are using transgenic mice expressing human beta2 microglobulin and HLA-B27 for immunization. The third specific aim is to determine if the HLA-E and G(6.0) gene products are recognized by T cells. We will first attempt to generate T cell clones reactive with transfectants of the class I deficient LCL .221 expressing the HLA-E and G(6.0) proteins. If polymorphism exists, we will attempt to demonstrate alloreactivity using a cell line from one individual expressing an allo E or G(6.0) antigen using an in vitro CML test to generate T cell clones reactive with the alloantigen. Learning the phenotype of these clones as well as their reactivity with a panel of transfected cell lines will be a fundamental step towards understanding the function of HLA-E and G(6.0). In specific aim four we will investigate the potential of these antigens to function as histocompatibility determinants. This work will help to answer whether these antigens contribute to the problems encountered in transplantation. This study will also provide important information and reagents which will significantly add to our knowledge of the expression and function of these new class I molecules.

本提案的总体目标是阐明一些 关于新I类蛋白质的生物学基本问题。 因为这些基因是在克隆和测序了所有 从人细胞系分离的I类同源序列，无抗体 或与这些基因的蛋白产物特异性反应的血清， 知道的 虽然可以在各种细胞中在mRNA水平上检测到表达， 和组织，蛋白质产物仅在HLA I类中检测到， 用这些基因转染的缺陷细胞系。 的一个特征 对移植重要的经典HLA-A、-B和-C抗原 生物学是在每个位点发现的惊人的多态性。 第一 这个建议的具体目的是确定，如果像经典的 抗原，HLA-E，F（5.4）和G（6.0）抗原表现出多态性。 我们 将使用聚合酶链反应特异性扩增E，F和G 来自一组遗传多样性个体的cDNA和基因组序列 并确定它们的核苷酸序列。 如果多态性存在于一个 或更多的这些位点，我们将使用相同的方法来关联， 多态性与无关骨髓移植的结果。 的 第二个具体目标是特异性分离单克隆抗体， 与HLA-E、-F（5.4）和G（6.0）分子反应。 我们已经构建 一组表达这些蛋白质的细胞系， 更多的线。 我们将用这些细胞作为抗原来源， 单克隆抗体的生产。 为了优化 我们使用转基因小鼠产生特异性抗体的可能性 表达人β 2微球蛋白和HLA-B27用于免疫。 的 第三个具体目标是确定HLA-E和G（6.0）基因产物是否 被T细胞识别。 我们将首先尝试产生T细胞克隆 与表达I类缺陷型LCL .221的转染子反应， HLA-E和G（6.0）蛋白。 如果多态性存在，我们将尝试 使用来自一个个体的细胞系证明同种异体反应性， 使用体外CML测试产生T细胞的同种异体E或G（6.0）抗原 与同种异体抗原反应的克隆。 学习这些基因的表型 克隆以及它们与一组转染细胞系的反应性 将是理解HLA-E功能的基础性步骤， G（6.0）. 在第四个具体目标中，我们将研究这些目标的潜力。 抗原作为组织相容性决定簇发挥作用。 这项工作将 有助于回答这些抗原是否导致了这些问题 在移植过程中遇到的 这项研究还将提供重要的 信息和试剂，这将大大增加我们的知识 这些新的I类分子的表达和功能。