STRUCTURE AND FUNCTION OF CYTOTOXIC T LYMPHOCYTE GRANULES

用Percoll梯度离心法制备克隆CTL的胞浆颗粒 离心通过氮气空化制备的匀浆。 此外 对于溶细胞素，这些颗粒含有能够 从细胞核释放125 I-UdR。 这种核酸酶活性在远 少量的非细胞毒性淋巴细胞颗粒， 克隆的CTL中的活性比LGL肿瘤细胞中观察到的活性更大。 这 酶在中性pH下有活性，并且显示出切割高分子 DNA重量 因此，似乎快速靶细胞DNA的报道 CTL介导的细胞毒性过程中的裂解可以由CTL解释， 颗粒DNA酶而不假定靶细胞中存在内源性DNA酶。 在 为了测试淋巴细胞毒性的颗粒胞吐模型， 既不是LGL也不是经典CTL的淋巴细胞，胞质颗粒 从小鼠LAK细胞、脾淋巴细胞培养物和 产生细胞毒活性的重组IL-2。 结果发现 颗粒中的细胞溶解活性在培养的第三天左右出现 并在接下来的6天继续增加。 这种模式大致 遵循LAK细胞毒活性，但开始较晚， 时间 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细胞溶素显示类似的 靶细胞杀伤谱和对钙的类似依赖性， LGL和CTL的细胞溶解作用也是如此。 LAK细胞溶素 也被抗LGL颗粒抗体中和。 这些结果支持 颗粒胞吐模型的LAK细胞介导的溶解。

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.