HLA CLASS I ON NK CELL SUBSETS, REPERTOIRE AND FUNCTION

我们的中心假设是宿主的HLAI类分子调节NK亚群的表型和功能以及NK受体的谱系。我们建议检验以下假设：(1)宿主HLAI类表型影响NK亚群的表型；(2)宿主HLAI表型影响NK受体细胞表面密度；(3)原代细胞中的NK细胞表型不同于NK克隆。这些假说将通过三种创新的方法进行验证：(A)四色流式细胞术检测NK细胞上复杂受体的表达；(2)单细胞RT-PCR检测单个NK细胞中NK受体的表达；(3)通过定量FACS检测NK细胞亚群上NK受体的表面表达。最后，我们将比较原代外周血细胞的NK谱系和从人类白细胞抗原B或C/MICA特异性纯合子或杂合子的捐献者的细胞中产生的NK克隆。我们还将测试NK克隆对一组自身HLAI类基因转染靶细胞的细胞毒活性，以确定NK受体表型和NK功能之间是否存在关联。这些研究数据将为人类白细胞抗原依赖的特定NK细胞亚群大小、特异性NK受体和NK克隆功能的改变提供确凿的证据。了解影响NK亚群发育的遗传因素及其受体对于分配骨髓移植后排斥反应或移植物抗宿主反应的风险或NK介导的病毒感染细胞杀伤具有重要意义。

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.