Studies on the mechanism of T cell-mediated lysis at the single effector cell level. I. Kinetic analysis of lethal hits and target cell lysis in multicellular conjugates.
Studies on the mechanism of T cell-mediated lysis at the single effector cell level. I. Kinetic analysis of lethal hits and target cell lysis in multicellular conjugates.
复制标题
在单效应细胞水平上研究 T 细胞介导的裂解机制。
作者：
D. Zagury;J. Bernard;P. Jeannesson;N. Thiernesse;J. Cerottini
先前已经开发了一种程序，以允许在小鼠同种免疫腹膜渗出液淋巴细胞（PEL）和悬浮液中的相关肿瘤细胞之间形成的单个缀合物中的靶细胞裂解的动力学分析。在本工作中使用的条件下，60%的缀合物由一个淋巴细胞与一个靶细胞结合组成，而其余的是多细胞的，即，含有一个淋巴细胞与两个（24%）、三个（12%）或四个（4%）靶细胞结合。与个别多细胞共轭物的超微结构研究和显微操作实验表明，靶细胞结合到相同的PEL的程度的差异。在超过80%的多细胞缀合物中，所有缀合的靶细胞的裂解在37°C下的3小时孵育期内发生。然而，靶细胞裂解的动力学分析表明，靶细胞死亡所需的时间是相当可变的，在同一多细胞缀合物内和不同缀合物之间，从缀合后几分钟到3小时不等。通过比较含有一个淋巴细胞与三个靶细胞结合的缀合物中50%靶细胞裂解所需的经过时间，可以估计，平均而言，第一、第二和第三靶细胞的裂解分别在30、60和90分钟后发生。
通过在不同的时间间隔向单个多细胞缀合物的孵育培养基中加入EDTA来测量每个缀合的靶细胞的致死命中所需的时间。可以获得CTL一次仅在一个缀合的靶细胞中诱导不可逆损伤的证据，即，致命打击是按顺序而不是同时进行的。
A procedure has been previously developed to allow kinetic analysis of target cell lysis in individual conjugates formed between murine alloimmune peritoneal exudate lymphocytes (PEL) and relevant tumor cells in suspension. Under the conditions used in the present work, 60% of the conjugates consisted of one lymphocyte bound to one target cell, whereas the remaining were multicellular, i.e., contained one lymphocyte bound to either two (24%), three (12%), or four (4%) target cells. Both ultrastructural studies and micromanipulation experiments with individual multicellular conjugates indicated differences in the degree of target cell binding to the same PEL. In more than 80% of the multicellular conjugates, lysis of all conjugated target cells occurred within a 3-hr incubation period at 37°C. Kinetic analysis of target cell lysis, however, showed that the time required for target cell death was quite variable, both within the same multicellular conjugate and between different conjugates, ranging from a few minutes to 3 hr after conjugation. By comparing the elapsed time necessary for lysis of 50% of the target cells in conjugates containing one lymphocyte bound to three target cells, it could be estimated that, on the average, lysis of the first, second, and third target cells occurred after 30, 60, and 90 min, respectively.
The time required for lethal hit of each conjugated target cell was measured by adding EDTA to the incubation medium of individual multicellular conjugates at various time intervals. Evidence could be obtained that a CTL induced irreversible lesions in only one of the conjugated target cells at a time, i.e., lethal hits were given sequentially rather than simultaneously.