TRANSMEMBRANE SIGNALLING IN THE ACTIVATION OF NK CELLS

自然杀伤(NK)细胞是大颗粒淋巴细胞(LGL) 自发地溶解有限范围的靶细胞，并且 可能在宿主抵抗恶性肿瘤和病毒方面很重要 感染。NK细胞缺乏T细胞抗原受体(Ti)， 表面免疫球蛋白(Ig)检查跨膜 涉及NK细胞激活的信号转导事件并识别 我们已经适应了激活所需的细胞表面结构 体外生长大鼠LGL肿瘤，RNK-16，具有以下特征 NK细胞。我们已经证明了RNK-16细胞的暴露 产生肌醇磷酸盐(INSPs)和一种 RNK-16细胞内游离钙((Ca~(2+))i)升高。单克隆 抗体OX-34，它识别大鼠的同源物 当OX-34被交叉激活时，人类CD2也刺激这些事件。 已链接。在没有交联剂的情况下，OX-34阻止了 InsP3和RNK-16细胞的细胞毒作用。这些研究提供了 跨膜信号传递事件的首次演示 与NK细胞激活有关，并且它们涉及CD2在 对这些信号的调节。我们建议研究的目标 是为了定义导致激活的生化事件 NK细胞和鉴定启动的细胞表面结构 激活。我们提议的研究将测试三个主要的 假设。 1.NK细胞需要CD2的细胞表面表达 活动。这些研究将检查一组肿瘤细胞，如 纯化的LGL对CD2和NK细胞活性的影响 抗CD2抗体的抑制作用。一个主要的焦点是 RNK-16 CD2突变体的获得 为了重建CD2的表达和突变体的杀伤，通过 CD2基因转染法。 2.CD2单独或与其他因素联合扰动 信号，可以激活NK细胞，通过脱颗粒来评估。 RNK-16脱颗粒释放丝氨酸酯酶。实验 将在5个区域检查脱颗粒所需的细胞信号， 聚焦于CD2的微扰和INSP的贡献， (Ca~(2+))i和蛋白激酶C的激活。 3.NK细胞表达CD2以外的细胞表面分子 可以刺激这一代的Insps。RNK-16的CD2突变体， 和任何其他CD2-大鼠LGL肿瘤，将检查是否 产生对凝集素和靶细胞作出反应的Insps。单抗 将针对RNK-16开发，以测试InsP3的刺激 通过CD2以外的表面结构。

Natural Killer (NK) cells are large granular lymphocytes (LGL) that spontaneously lyse a limited range of target cells and that may be important in host defense against malignancy and viral infections. NK cells lack the T cell antigen receptor (Ti) and surface immunoglobulin (Ig). To examine transmembrane signalling events involved in NK cell activation and to identify the cell surface structures required for activation we have adapted for in vitro growth a rat LGL tumor, RNK-16, with features of NK cells. We have demonstrated that exposure of RNK-16 cells to susceptible targets generates inositol phosphates (InsPs) and a rise in intracellular free calcium ((Ca2+)i) in RNK-16. Monoclonal antibody (MAb) OX-34, which recognizes the rat homologue for human CD2, also stimulates these events, when OX-34 is cross- linked. Without cross-linking, OX-34 blocks the generation of InsP3 and cytotoxicity by RNK-16 cells. These studies provide the first demonstration of transmembrane signalling events associated with NK cell activation, and they implicate CD2 in the regulation of these signals. The objectives of our proposed studies are to define the biochemical events that lead to the activation of NK cells and to identify the cell surface structures that initiate activation. Our proposed studies will test three primary hypothesis. 1. The cell surface expression of CD2 is required for NK cell activity. These studies will examine a panel of tumor cells as well as purified LGL for the association of CD2 with NK activity and the inhibitory effect of anti-CD2. A major focus is the derivation of CD2- mutants of RNK-16, with collaborative efforts to reconstitute CD2 expression and killing by the mutants, by transfection with the CD2 gene. 2. Perturbation of CD2, alone or in combination with other signals, can activate NK cells, as assessed by degranulation. Degranulation of RNK-16 releases serine esterase. Experiments in 5 areas will examine the cell signals required for degranulation, focusing on perturbation of CD2 and the contribution of InsPs, (Ca2+)i, and the activation of protein kinase C. 3. NK cells express cell-surface molecules other than CD2 that can stimulate that generation of InsPs. CD2- mutants of RNK-16, and any other CD2- rat LGL tumors, will be examined for the generation of InsPs in response to lectins and to target cells. Mab will be developed against RNK-16 to test for stimulation of InsP3 through surface structure other than CD2.