STRUCTURE AND FUNCTION OF CYTOTOXIC T LYMPHOCYTE GRANULES

颗粒丝氨酸蛋白酶在CTL裂解功能中的作用是 通过用PMSF组合处理完整CTL来探索 与提高酸性细胞内 隔室，然后比较残留颗粒BLT 酯酶活性与残余裂解活性。 PMSF单独给药 导致裂解活性损失约50%，BLT损失约90 酯酶，而PMSF和氯化铵造成的损失， 约70%的裂解活性和99%的BLT酯酶。 这些 结果表明，低pH区室中的丝氨酸酯酶 不太可能直接参与溶解功能。 之一 主要的CTL颗粒蛋白酶，一种BLT酯酶，已经用作 用于测量胞吐作用的颗粒标记物。 使用小鼠CTL， 酶在被靶细胞抗原刺激后分泌，或 固定化的抗T细胞受体复合物的抗体，和 用人CTL，固定化抗T3刺激。 在这两个 系统，抗LFA-1抗体阻断分泌，显示这可以 直接作用于效应细胞，而不一定是阻断效应细胞。 靶细胞结合。 使用体内产生的CTL、BLT酯酶 在颗粒中发现了相同的方法用于克隆 CTL. 这种酶的分泌是由 肿瘤靶细胞和细胞毒性。 溶细胞素活性 在这些CTL的致密颗粒中发现，但约为100 × 低于克隆的CTL。 克隆的辅助性T细胞也 含有高水平BLT酯酶的颗粒， 用二聚体“主要”LGL丝氨酸蛋白酶的抗体。 使用BLT酯酶释放作为标志物，颗粒胞吐作用 这些细胞被抗原触发后， 2个匹配的呈递细胞，通过Con A、PMA和钙 离子载体和固定化的抗T细胞受体抗体。 在 后一种情况，分泌物可以在1小时内测量， 和分泌溶酶体酶β-N-乙酰基 还检测到氨基己糖苷酶。 这些辅助细胞的EM研究 已经揭示了细胞质颗粒含有醒目的， 不寻常的板层样结构正在发生胞吐作用。 使用β-N- 乙酰氨基己糖苷酶释放作为测定， 未从抗T3包被的刺激的正常T细胞中观察到 珠

The role of granule serine proteases in CTL lytic function was probed by treatment of intact CTL with PMSF in combination with agents which raise the pH of acidic intracellular compartments, and then compared the residual granule BLT esterase activity with the residual lytic activity. PMSF alone caused losses of about 50% in lytic activity and 90% in BLT esterase, while PMSF and ammonium chloride caused losses of about 70% in lytic activity and 99% in BLT esterase. These results suggest that serine esterases in low pH compartments are unlikely to be directly involved in the lytic function. One of the major CTL granule proteases, a BLT esterase, has been useful as a granule marker for measuring exocytosis. With mouse CTL, the enyzme is secreted after stimulation by target cell antigen or immobilized antibodies against the T cell receptor complex, and with human CTL, immobilized anti-T3 stimulate. In both these systems, anti LFA-1 antibodies block secretion, showing this can act directly on effector cells rather than necessarily blocking a target cell binding. Using in vivo generated CTL, BLT esterase was found in granules by the same approaches used for cloned CTL. Secretion of this enzyme was triggered specifically by tumor target cells and preceded cytotoxicity. Cytolysin activity was found in dense granules of these CTL, but was about 100 x lower than found in cloned CTL. Cloned helper T cells also contain granules with high levels of BLT esterase which reacts with antibodies to the dimeric "major" LGL serine protease. Using BLT esterase release as a marker, granule exocytosis from these cells has been triggered by antigen after processing by class 2 matched presenting cells, by Con A, by PMA and calcium ionophore, and by immobilized anti-T cell receptor antibodies. In the latter situation, secretion can be measured within on hour, and secretion of the lysosomal enzyme beta-N-acetyl hexosaminidase is also detected. EM studies of these helper cells have revealed cytoplasmic granules containing striking and unusual lamellar figures undergoing exocytosis. Using beta-N- acetyl hexosaminidase release as an assay, granule exocytosis was not observed from normal T cells stimulated by anti-T3 coated beads.