STRUCTURE AND FUNCTION OF CYTOTOXIC T LYMPHOCYTE GRANULES

细胞毒性T淋巴细胞胞浆颗粒的研究进展 以评估它们在这些细胞的溶解功能中的作用。颗粒剂来自 克隆的CTL用Percoll梯度离心法纯化，并显示 含有两种能够裂解胰酶底物的丝氨酸酯酶 BLT除了裂解细胞溶血素蛋白和核DNA释放 活动。CTL中主要的BLT酯酶是一个60kd的蛋白质，包括 两条30kd的蛋白质链通过二硫键相连。这种酶是 二异丙基氟磷酸盐(DFP)和苯甲基磺酰基灭活 氟化物(PMSF)，但这些试剂在完整细胞中与酶反应 效率低下。这被证明是由于体内pH值较低引起的。 细胞内的完整颗粒，因为已知的药物可以提高细胞内的pH值 酸性细胞器(氨、氯喹、莫能菌素和黑素)起作用 PMSF协同灭活体内BLT酯酶的研究 细胞，而不是溶解的酶。同样的选择性协同效应 在用放射性标记标记该蛋白条带时观察到了效果 DFP。BLT酯酶活性大于95%的CTL的裂解功能 通过这些处理灭活了它，发现它实际上 未受影响，表明BLT酯酶可能不是 CTL裂解过程。CTL-BLT酯酶在培养液中的分泌 已被证明是一种方便的颗粒胞吐的标志物 进程。利用这种方法，我们研究了活体产生的CTL。 异体肿瘤最近被排斥的腹膜渗出物。二 已证实定向异种特异性BLT酯酶分泌是从 这样的CTL，证实了颗粒胞吐机制在这些 细胞在裂解过程中。除了这些研究外，还有 经典CTL，我们发现克隆的辅助性T淋巴细胞含有 胞质颗粒中含有高水平的BLT酯酶。至少有一部分 这些辅助克隆具有强大的裂解功能。我们发现了 BLT酯酶是由特定的抗原性和 MHC限制性刺激。

Cytoplasmic granules of cytotoxic T lymphocytes have been studied in order to assess their role in the lytic function of these cells. Granules from cloned CTL have been purified by Percoll gradient centrifugation and shown to contain two serine esterases capable of cleaving the trypsin substrate BLT in addition to the lytic cytolysin protein and a nuclear DNA releasing activity. The major BLT esterase in CTL is a 60kd protein, comprised of two 30kd protein chains linked by disulfide bonds. The enzyme is inactivated by diispropylfluorophosphage (DFP) and phenylmethylsulfonyl fluoride (PMSF), but these reagents react with the enzyme in intact cells inefficiently. This was shown to be caused by a low internal pH of the intact granules inside the cells, since agents known to raise the pH of acidic organelles (ammonia, chloroquine, monensin and nigericin) acted synergistically with PMSF in the inactivation of BLT esterase in intact cells but not the solubilized enzyme. The same selective synergistic effect was observed in the labeling of this protein band with radiolabelled DFP. When lytic function of CTL whose BLT esterase was greater than 95% inactivated by these treatments was examined, it was found to be virtually unaffected, indicating that the BLT esterase is probably not required for the CTL lytic process. The secretion of CTL BLT esterase into the medium has been shown to be a convenient marker for the granule exocytosis process. Using this assay, we have studied in vivo generated CTL from peritoneal exudates in which allo tumors have been recently rejected. Two directional allo-specific BLT esterase secretion has been demonstrated from such CTL, confirming the granule exocytosis mechanism operates in these cells during the lytic process. In addition to these studies with classical CTL, we have found that cloned helper T lymphocytes contain cytoplasmic granules with high levels of BLT esterase. At least some of these helper clones are capable of potent lytic function. We have found that BLT esterase is secreted into the medium by specific antigenic and MHC-restricted stimuli.