TRANSMEMBRANE SIGNALLING IN THE ACTIVATION OF NK CELLS

自然杀伤（NK）细胞是大颗粒淋巴细胞（LGL） 其自发地裂解有限范围的靶细胞， 在宿主防御恶性肿瘤和病毒中可能是重要的 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 参与NK细胞活化的信号传导事件，并鉴定 我们已经适应了激活所需的细胞表面结构， 对于大鼠LGL肿瘤RNK-16的体外生长，其特征在于 NK细胞。 我们已经证明了RNK-16细胞的暴露 对敏感的目标产生肌醇磷酸（InsPs）和 RNK-16细胞内游离钙（（Ca 2+）i）升高。 单克隆 抗体（MAb）OX-34，它识别大鼠同源物， 人CD 2，也刺激这些事件，当OX-34是交叉- 有联系 没有交联，OX-34阻止了 InsP 3和RNK-16细胞的细胞毒性。 这些研究提供 第一次证明跨膜信号事件 与NK细胞活化相关，它们暗示CD 2参与了 这些信号的调控。 我们建议研究的目的 是为了定义导致激活的生化事件， NK细胞，并确定细胞表面结构，启动 activation. 我们提出的研究将测试三个主要的 假说. 1. NK细胞需要CD 2的细胞表面表达， 活动 这些研究将检查一组肿瘤细胞， 纯化的LGL用于CD 2与NK活性的关联 和抗CD 2的抑制作用。 一个主要的焦点是 RNK-16的CD 2突变体的衍生，与合作努力 为了重建突变体的CD 2表达和杀伤， 用CD 2基因转染。 2. CD 2的扰动，单独或与其他 信号，可以激活NK细胞，如通过脱粒评估的。 RNK-16的脱粒释放丝氨酸酯酶。 实验 在5个区域中将检查脱粒所需的细胞信号， 关注CD 2的扰动和InsPs的贡献， (Ca2+）i和蛋白激酶C的活化。 3. NK细胞表达除CD 2以外的细胞表面分子， 可以刺激InsPs的产生。 RNK-16的CD 2-突变体， 和任何其他CD 2-大鼠LGL肿瘤，将检查 响应于凝集素和靶细胞的InsP的产生。 mAb 将针对RNK-16开发以测试InsP 3的刺激 除了CD 2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.