STRUCTURE AND FUNCTION OF CYTOTOXIC T LYMPHOCYTE GRANULES

细胞毒性T淋巴细胞的胞浆颗粒已被研究， 以评估它们在这些细胞的溶解功能中的作用。 颗粒从 克隆的CTL已通过Percoll梯度离心纯化， 含有两个能够切割胰蛋白酶底物的丝氨酸酯酶 除了溶解溶细胞素蛋白和核DNA释放之外，BLT 活动 CTL中主要的BLT酯酶是一种60kd的蛋白质，包括： 两条30kd的蛋白质链通过二硫键连接。 所述酶是 二异丙基氟磷酸（DFP）和苯甲磺酰基灭活 氟化物（PMSF），但这些试剂与完整细胞中的酶反应 效率低下。 这被证明是由低内部pH值的 细胞内的完整颗粒，因为已知的试剂可以提高细胞的pH值， 酸性细胞器（氨、氯喹、莫能菌素和尼日利亚菌素）起作用 与PMSF协同灭活BLT酯酶， 细胞，而不是溶解的酶。 相同的选择性协同 在用放射性标记的蛋白质条带标记中观察到效应 DFP。 当BLT酯酶活性大于95%时， 检查这些处理的灭活情况，发现实际上 不受影响，表明BLT酯酶可能不需要 CTL裂解过程。 CTL BLT酯酶分泌到培养基中 已被证明是颗粒胞吐作用的方便标记 过程 使用该试验，我们研究了从小鼠体内产生的CTL。 腹膜渗出物，其中同种异体肿瘤最近被排斥。 两 定向的同种异体特异性BLT酯酶分泌已经从 这种CTL，证实颗粒胞吐机制在这些细胞中起作用， 细胞在裂解过程中。 除了这些研究， 经典的CTL，我们发现克隆的辅助性T淋巴细胞含有 具有高水平BLT酯酶的细胞质颗粒。 中的至少一些 这些辅助克隆能够具有有效的裂解功能。 我们发现 BLT酯酶由特异性抗原分泌到培养基中， 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.