PROGRAMMED CELL DEATH IN LYMPHOCYTES

为了测试颗粒胞吐模型对淋巴细胞的细胞毒性， 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系RBL的研究 组件。我们已经构建了三重、双倍和单倍RBL 表达细胞溶素(Cy)和颗粒丝氨酸蛋白酶的转染体 颗粒酶A(GZA)和颗粒酶B(GZB)。RBL-Cy转染体仅显示 对肿瘤靶点的适度细胞毒性，没有伴随的靶DNA 退化。而RBL-GZA转染体在可比水平上表达GZA 为了克隆CTL并将其分泌以响应Iger交联物，他们已经 未检测到细胞毒活性。Rbl-Cy-GZA转基因克隆显示 这两种颗粒成分的良好表达显示了细胞溶解活性 在RBC靶点上与RBL-Cy相当，但比RBC靶点高3倍以上 三个不同的肿瘤靶点。这种细胞毒性伴随着 目标DNA碎片。为了确认杀伤细胞颗粒酶需要 进入靶细胞，我们将大分子加载到靶细胞中 蛋白水解酶抑制剂抑肽酶，通过渗透裂解胞质小体。与.相比 BSA加载靶向或未加载靶向，抑肽酶靶向较少 CTL和RBL转染体对裂解和DNA破坏敏感 然而，表达颗粒酶A的RBL转染体仅表达 细胞溶血素对牛血清白蛋白和抑肽酶靶点的裂解作用 效率。作为对蛋白酶诱导能力的直接测试 细胞毒性当被引入靶细胞的细胞质中时，我们有 利用渗透裂解将不同的蛋白水解酶“注入”肿瘤细胞 针小体。内切酶胰酶、胰凝乳酶和蛋白酶K分别为 一剂就能溶解几种不同类型的肿瘤细胞 依赖方式，通过51Cr释放来衡量。这起死亡事件总体上是 通过形态标准和DNA片段化来判断细胞的凋亡率。

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.