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Elucidation of mechanism of NK cell activation

阐明NK细胞活化机制

基本信息

批准号：
11670312
负责人：
TAKI Shinsuke
金额：
$ 2.5万
依托单位：
Chiba University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670312/
关键词：
NFAT retrovirus library NK cell lymphocyte activator signal transducer expression cloning reporter gene CD3ζ鎖 FcγRIII NKT細胞 NK1.1 diphthelia toxin マウス

项目摘要

NK cells play important roles in anti-tumor immune response. However, the exact mechanism for their activation still remains unclear. In this study, we have analyzed activation mechanism of NK cells using CD3ζ-deficient mice and a newly developed method for identifying signaling molecules. From the analysis of CD3ζ-deficient mice, we found that CD3ζ negatively regulate the expression and function of FcγRIII on NK cells. Next, we developed a new method using NFAT-GFP transfected reporter cells and unique CD8 chimera cDNA library, in which retrovirally infected reporter cells were crosslinked with anti-CD8 antibody and those become positive for GFP were cloned. cDNAs recovered from these cells included Igα, Igβ, CD3ε, ZAP70, Syk when a library prepared from spleen were used. On the other hand, FcRγ, DAP12, ZAP70 were identified from a library prepared from NK cells. These results show that this method is in fact a powerful method for cloning signaling molecules of lymphocytes. We named this method as NFAT Activating gene Cloning System (NACS). In addition to these already known molecules, we have identified several unknown molecules that activate NFAT-driven transcription upon crosslinking. In future, we will further analyze these newly cloned molecules to exploire the activation mechanism of NK cells.

NK细胞在抗肿瘤免疫反应中发挥重要作用。然而，它们激活的确切机制仍不清楚。在这项研究中，我们使用 CD3ze 缺陷小鼠和新开发的识别信号分子的方法分析了 NK 细胞的激活机制。通过对CD3δ缺陷小鼠的分析，我们发现CD3δ负向调节NK细胞上FcγRIII的表达和功能。接下来，我们开发了一种使用 NFAT-GFP 转染的报告细胞和独特的 CD8 嵌合 cDNA 文库的新方法，其中逆转录病毒感染的报告细胞与抗 CD8 抗体交联，并对 GFP 呈阳性的报告细胞进行克隆。当使用从脾脏制备的文库时，从这些细胞中回收的 cDNA 包括 Igα、Igβ、CD3ε、ZAP70、Syk。另一方面，从由NK细胞制备的文库中鉴定出FcRγ、DAP12、ZAP70。这些结果表明该方法实际上是克隆淋巴细胞信号分子的有力方法。我们将该方法命名为NFAT激活基因克隆系统（NACS）。除了这些已知的分子之外，我们还鉴定了几种在交联时激活 NFAT 驱动转录的未知分子。未来，我们将进一步分析这些新克隆的分子，探索NK细胞的激活机制。