TARGET CELL DAMAGE BY IMMUNE MECHANISMS

为了验证淋巴细胞颗粒胞吐模型， 细胞毒性，我们已经检查了RBL细胞的细胞毒性活性 用细胞毒性淋巴细胞颗粒成分的基因转染。 RBL， 大鼠粘膜肥大细胞肿瘤系，其响应于 交联其IgE Fc受体不是细胞毒性的，但获得了有效的免疫抑制。 当转染时，对IgE包被的红细胞的溶解活性 小鼠溶细胞素基因。 这种效力相当于克隆的 CTL这个目标。 我们已经发现电穿孔给了很多 比钙离子转染效率高（2个数量级） 最初使用的磷酸盐技术。 此外，克隆的RBL 电穿孔转染子（RBL-cy）产生甚至更有效的裂解 针对红细胞靶点的活性比钙产生的活性高 磷酸盐 使用肿瘤靶细胞，RBL-cy具有良好的细胞毒性， 尽管不如克隆的CTL有效。 当靶DNA降解 用RBL-cy效应细胞检查，在显著的 与CTL诱导的相同实验结果相反。 因此细胞-- 递送的溶细胞素模拟加入到细胞中的纯化溶细胞素的作用。 介质 我们构建了双和单RBL转染子 表达溶细胞素和颗粒丝氨酸蛋白酶颗粒酶A（gza）。 正如预期的，具有蛋白质表达水平的RBL-gza转染子 与克隆的CTL相当，没有显示出细胞毒活性。 RBL-cy-gza 显示这两种颗粒组分良好表达的转染子 在RBC和肿瘤上显示出与RBL-cy相当的细胞溶解活性 目标的 当检测肿瘤靶点的DNA断裂时， 用RBL-cygza效应物清楚地观察到，尽管不如 CTL效应器。 这些初步结果支持了我们先前的研究结果。 有证据表明，颗粒酶A触发靶DNA分解后， 从而进入靶细胞的细胞质。

In order to test the granule exocytosis model for lymphocyte cytotoxicity, we have examined the cytotoxic activity of RBL cells transfected with genes for cytotoxic lymphocyte granule components. RBL, a rat mucosal mast cell tumor line which degranulates in response to cross-linking its IgE Fc receptor is not cytotoxic but acquires a potent lytic activity against IgE coated red cells when transfected with the mouse cytolysin (cy) gene. This potency is equivalent to that of cloned CTL with this target. We have found that electroporation gives much higher (2 orders of magnitude) transfection efficiency than the calcium phosphate technique originally used. Furthermore the cloned RBL electroporation transfectants (RBL-cy) give even more potent lytic activity against RBC targets than did those generated with calcium phosphate. Using tumor target cells, the RBL-cy give good cytotoxicity, although not as potent as cloned CTL. When target DNA degradation was examined with RBL-cy effector cells, it was not detectable, in striking contrast to that induced by CTL in the same experiment. Thus the cell-- delivered cytolysin mimics the effects of purified cytolysin added to the medium. We have constructed double and single RBL transfectants expressing cytolysin and the granule serine protease granzyme A (gza). As expected, RBL-gza transfectants with protein expression levels comparable to cloned CTL show no cytotoxic activity. RBL-cy-gza transfectants showing good expression of both these granule components showed cytolytic activity comparable to RBL-cy on both RBC and tumor targets. When DNA breakdown in tumor targets was examined, it was clearly observed with RBL-cygza effectors, although not as potently as with CTL effectors. These preliminary results support our earlier evidence suggesting that granzyme A triggers target DNA breakdown after gaining access to the target cell cytoplasm.