PROGRAMMED CELL DEATH IN LYMPHOCYTES

为了测试颗粒胞吐模型的淋巴细胞毒性， 我们检测了大鼠粘膜肥大细胞瘤的细胞毒活性， 细胞毒性淋巴细胞颗粒基因转染后的RBL细胞 件. 我们已经构建了三重，双重和单一的RBL 表达溶细胞素（cy）和颗粒丝氨酸蛋白酶的转染子 颗粒酶A（gza）和颗粒酶B（gz B）。 RBL-cy转染子仅显示 对肿瘤靶点的中度细胞毒性，无伴随靶DNA 降解 而RBL-gza转染子表达的gza水平与 克隆的CTL并分泌它以响应IgER交联， 未检测到细胞毒活性。RBL-cy-gza转染子克隆显示 这两种颗粒组分的良好表达显示出细胞溶解活性 与RBC靶点上的RBL-cy相当，但溶解性超过3倍 三种不同的肿瘤靶点 这种细胞毒性伴随着 靶向DNA片段化。 为了证实杀伤细胞颗粒酶需要 进入靶细胞，我们在靶细胞上装载了大分子， 蛋白酶抑制剂抑肽酶通过渗透裂解的胞饮体。 相比 BSA负载靶点或未负载靶点，抑肽酶靶点较少 易受CTL和RBL转染子裂解和DNA断裂的影响 表达颗粒酶A。 然而，RBL转染子仅表达 溶细胞素裂解BSA负载的和抑肽酶负载的靶标， 效率 作为对蛋白酶诱导细胞凋亡能力的直接测试， 当引入靶细胞的细胞质中时， 使用渗透裂解法将各种蛋白酶“注射”到肿瘤细胞中。 胞饮体 内切蛋白酶胰蛋白酶、胰凝乳蛋白酶和蛋白酶K为 所有这些都被发现在一个剂量下溶解几种不同类型的肿瘤细胞 依赖的方式，如通过51 Cr释放测量的。 这种死亡通常是 通过形态学标准和DNA片段化检测细胞凋亡。

In order to test the granule exocytosis model for lymphocyte cytotoxicity, we have examined the cytotoxic activity of the rat mucosal mast cell tumor line RBL after transfection with genes for cytotoxic lymphocyte granule components. We have constructed triple, double, and single RBL transfectants expressing cytolysin (cy) and the granule serine proteases granzyme A (gza) and granzyme B (gzb). RBL-cy transfectants show only modest cytotoxicity on tumor targets, with no accompanying target DNA degradation. While RBL-gza transfectants express gza at levels comparable to cloned CTL and secrete it in response to IgER cross-linking, they have no cytotoxic activity detectable. RBL-cy-gza transfectant clones showing good expression of both these granule components showed cytolytic activity comparable to RBL-cy on RBC targets, but were greater than 3x more lytic on three different tumor targets. This cytotoxicity is accompanied by target DNA fragmentation. To confirm that killer cell granzymes need to enter the target cell, we loaded target cells with the macromolecular protease inhibitor aprotinin by osmotic lysis of pinosomes. Compared to BSA-loaded targets or unloaded targets, aprotinin-targets were less susceptible to lysis and DNA breakdown by CTL and RBL transfectants expressing granzyme A. However, RBL transfectants expressing only cytolysin lysed BSA-loaded and aprotinin-loaded targets with equal efficiency. As a direct test of the ability of proteases to induce cytotoxicity when introduced into the cytoplasm of a target cell, we have "injected" various proteases into tumor cells using osmotic lysis of pinosomes. The endoproteases trypsin, chymotrypsin, and proteinase K were all found to lyse several different types of tumor cells in a dose dependent manner, as measured by 51Cr release. This death was generally apoptotic by morphological criteria and DNA fragmentation.