STRUCTURE AND FUNCTION OF CYTOTOXIC T LYMPHOCYTE GRANULES

用Percoll梯度法制备克隆的CTL胞质颗粒 氮气空化法制备的匀浆的离心法。此外 这些颗粒含有一种脱氧核糖核酸酶活性，能够 核内释放~(125)I-UdR。这种核酸酶活性在FAR中被发现 在非细胞毒性淋巴细胞颗粒中有少量存在，其水平 克隆的CTL比在LGL肿瘤细胞中的活性更强。这 酶在中性pH下是活性的，并被证明能裂解大分子 体重DNA。因此，关于快速靶细胞DNA的报道似乎 CTL可解释CTL介导的细胞毒作用中的裂解作用 在靶细胞中不假定内源性DNA酶的颗粒DNA酶。在……里面 为了验证颗粒胞吐模型对淋巴细胞毒性的影响 既不是LGL也不是经典的CTL的淋巴细胞，胞浆颗粒 从小鼠LAK细胞、脾淋巴细胞和 具有细胞毒活性的重组IL-2。结果发现， 颗粒中的细胞溶解活性大约在培养的第三天出现。 并在接下来的6天里继续增加。这种模式大体上 遵循LAK细胞毒活性，但开始较晚，且不随 时间到了。IL-2滴定显示需要类似数量的淋巴因子 产生颗粒溶解活性和LAK。LAK细胞溶血素表现出类似的 靶细胞杀伤光谱以及对钙和钙的类似依赖 与来自LGL和CTL的细胞质分裂一样，LGL和CTL的细胞质分裂也是如此。LAK细胞溶血素 也可被抗LGL颗粒抗体中和。这些结果支持 LAK介导裂解的颗粒胞吐模型。

Cytoplasmic granules from cloned CTL were prepared by Percoll gradient centrifugation of homogenates prepared by nitrogen cavitation. In addition to the cytolysin these granules contain a DNAse activity capable of releasing 125I-UdR from nuclei. This nuclease activity is found in far smaller amounts in granules of non-cytotoxic lymphocytes, and the level of activity in cloned CTL is greater that that seen in LGL tumor cells. This enzyme is active at neutral pH, and was shown to cleave high molecular weight DNA. Thus it appears that the reports of rapid target cell DNA cleavage during CTL-mediated cytotoxicity can ba accounted for by CTL granule DNAse without postulating endogenous DNAses in the target cell. In order to test the granule exocytosis model for lymphocyte toxicity on lymphocytes which are neither LGL nor classical CTL, cytoplasmic granules were prepared from mouse LAK cells, spleen lymphocytes cultures with recombinant IL-2 which develop cytotoxic activity. It was found that cytolytic activity in the granules appears on about day three of culture and continues to increase for the next 6 days. This pattern roughly follows LAK cytotoxic activity but begins later and does not plateau with time. IL-2 titration shows similar amounts of lymphokine are needed to generate granule lytic activity and LAK. The LAK cytolysin shows a similar spectrum of target cell killing and a similar dependence on calcium and rapid linetics as do the cytolysisns from LGL and CTL. The LAK cytolysin is also neutralized by anti-LGL granule antibodies. These results support the granule exocytosis model for LAK-mediated lysis.