HLA CLASS I ON NK CELL SUBSETS, REPERTOIRE AND FUNCTION

我们的中心假设是宿主HLA I类分子调节NK亚群和NK受体库的表型和功能。 我们提出以下假设：（1）宿主HLA I类表型影响NK亚群的库，（2）宿主HLA I类表型影响NK受体细胞表面密度和（3）原代细胞中的NK细胞库与NK克隆不同。 这些假设将通过三种创新方法进行检验：（a）用于NK细胞上复合受体表达的四色FACS，（2）用于确定单个NK细胞中NK受体库的单细胞RT-PCR，以及（3）通过定量FACS测定NK亚群上NK受体的表面表达。 最后，我们将比较原代外周血细胞的NK库与从HLA-B或C/云母特异性纯合或杂合的供体细胞产生的NK克隆库。 我们还将测试NK克隆对一组自身HLA I类转染的靶细胞的细胞毒性活性，以确定NK受体表型和NK功能之间是否存在关联。 拟开展的研究的数据应能为特定NK细胞亚群的大小、特定NK受体库和NK克隆功能的HLA依赖性变化提供确凿证据。 了解影响NK细胞亚群发育的遗传因素和定义其库的受体，对于骨髓移植或NK介导的病毒感染细胞杀伤后排斥反应或移植物抗宿主反应的风险分配至关重要。

Our central hypothesis is that host HLA Class I molecules regulate the phenotype and function of NK subsets and the repertoire of NK receptors. We propose to test the following hypotheses: (1) host HLA class I phenotype influences the repertoire of NK subsets, (2) host HLA class I phenotype affects NK receptor cell surface density and (3) NK cell repertoire in primary cells differs from NK clones. These hypotheses will be tested by three innovative methods: (a) four-color FACS for complex receptor expression on NK cells, (2) single cell RT-PCR to determine the repertoire of NK receptors in individual NK cells and (3) the surface expression of NK receptors on NK subsets by a quantitative FACS assay. Finally we will compare the NK repertoire of primary peripheral blood cells with that of NK clones generated from cells of donors who are homozygous or heterozygous for HLA-B or C/MICA specificities. We will also test NK clones for cytotoxic activity against a panel of self HLA class I-transfected target cells to determine whether there is an association between NK receptor phenotype and NK function. The data of the proposed studies should provide conclusive evidence for the HLA-dependent changes in the sizes of specific NK cell subsets, the repertoire of specific NK receptors and NK clone function. Understanding the genetic factors that influence the development of NK subsets and the receptors that define their repertoire will be important in the assignment of risks for rejection or for graft-versus-host reaction following bone marrow transplantation or NK-mediated killing of virus-infected cells.