Elucidation of mechanism of NK cell activation

NK细胞在抗肿瘤免疫反应中发挥重要作用。然而，它们的确切激活机制仍不清楚。在这项研究中，我们利用CD3CD3ζ缺陷小鼠分析了NK细胞的激活机制，并开发了一种新的信号分子识别方法。通过对CD3ζ缺陷小鼠的分析，我们发现CD3ζ对NK细胞表面FcγRIII的表达和功能具有负性调节作用。接下来，我们利用NFAT-GFP转染的报告细胞和独特的CD8嵌合体cDNA文库，将逆转录病毒感染的报告细胞与抗CD8抗体交联，并对GFP阳性的报告细胞进行克隆。用从脾组织制备的文库，从这些细胞中回收的cDNA包括Igα、Igβ、CD3ε、ZAP70、Syk。另一方面，从NK细胞制备的文库中鉴定出FcRγ、DAP12、ZAP70。这些结果表明，该方法实际上是一种克隆淋巴细胞信号分子的有效方法。我们将这种方法命名为NFAT激活基因克隆系统(NACS)。除了这些已知的分子外，我们还鉴定了几个未知的分子，它们在交联时激活NFAT驱动的转录。在未来，我们将进一步分析这些新克隆的分子，以探索NK细胞的激活机制。

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.