POLYMORPHISM AND TRANSPLANTATION BIOLOGY OF NOVEL HLA CLASS I GENES

这项提案的总体目标是阐明一些 关于新的I类蛋白质生物学的基本问题。 因为这些基因是在克隆和测序所有 从人类细胞系分离的I类同源序列，无抗体 或与这些基因的蛋白质产物发生特异性反应的血清 为人所知。而在不同的细胞中可以检测到mRNA水平的表达 和组织，蛋白质产物只在人类白细胞抗原I类中被检测到 转导这些基因的缺陷细胞系。的一个特点 对移植具有重要意义的经典人类白细胞抗原-A、B和C 生物学是在每个基因座上发现的惊人的多态。第一 这项提议的具体目的是确定是否像古典的 抗原中，人类白细胞抗原E、F(5.4)和G(6.0)呈多态分布。我们 将使用聚合酶链式反应来特异地扩增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。如果多态出现在一个 或者更多的这些基因座，我们将使用相同的方法来关联 多态与无关的骨髓移植的结果。这个 第二个特定目的是分离特异性的单抗。 与人类白细胞抗原-E、-F(5.4)和G(6.0)分子反应。我们已经建造了 一组表达这些蛋白质的细胞系，并计划构建 额外的行数。我们将利用这些细胞作为抗原来源 单抗的生产。为了优化 我们使用转基因小鼠生产特定抗体的可能性 表达人β2微球蛋白和人类白细胞抗原B27用于免疫。这个 第三个特定目的是确定人类白细胞抗原-E和G(6.0)基因产物 是由T细胞识别的。我们将首先尝试生成T细胞克隆 与表达第I类缺陷LCL.221的转染体的反应 人类白细胞抗原-E和G(6.0)蛋白。如果存在多态，我们将尝试 用一个人的细胞系显示同种异体反应性 利用体外CML试验产生T细胞的allo E或G(6.0)抗原 克隆与同种异体抗原发生反应。了解这些生物的表型 克隆及其与一组转基因细胞系的反应性 将是了解人类白细胞抗原-E功能的基本步骤 G(6.0)。在具体目标四中，我们将调查这些措施的潜力 作为组织相容性决定因素的抗原。这项工作将 帮助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 信息和试剂将显著增加我们对 这些新的I类分子的表达和功能。

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.