TARGET CELL LYSIS BY CYTOLYTIC EFFECTOR CELLS

细胞毒性T细胞(CTL)是免疫的基石之一 系统。因此，有很多人有兴趣了解 他们分解目标的机制，这是的总体目标 这项提议。尽管我们在这方面取得了很大进展 对CTL裂解的理解仍有许多问题尚未解决。吸附量 CTL通过受体对靶细胞的作用触发极化 CTL中的细胞骨架元素。这会导致重新定位 高尔基体(GA)朝向目标结合部位 分泌通道形成蛋白-穿孔素(PF)-它们是 被认为会导致靶细胞溶解。最近的证据表明，S 用体外CTL克隆建立的这一事件序列确实 不能在体内操作诱导的CTL。证据也在不断积累 通过通道形成的靶向裂解可能不是通用的 CTL裂解机制和靶子本身发挥的活性 在它的分解过程中扮演着重要的角色。我们建议从几个方面探讨 体内诱导的CTL的并发CTL裂解模型，因为 它们在体外还没有获得异常的性质。CTL将 高度提纯并测试它们是否显示GA重定向 当与目标结合时，因为这种反应被认为是 正确的目标识别。下一个CTL将测试他们的 将PF通道插入红细胞膜的能力。至 诱导这一反应，我们将使用双功能抗靶抗T 细胞受体抗体偶联。如果CTL无法插入通道 我们将测试从红血球中去除细胞核 目标抑制目标裂解。在药物抑制研究中，我们将 一方面检测CTL的持续裂解活性，另一方面 靶点对另一端的溶解敏感性需要蛋白质 CTL-靶标结合前的合成。初步数据支持 靶点参与CTL诱导裂解的观点。去探索 目标如何参与裂解我们提出了几种方法，一种 这是基于蛋白质合成抑制剂的能力 干扰CTL裂解。另一种是以相伴为基础 发生对CTL裂解的抗性和对 一个细胞系中的糖皮质激素。我们建议研究是否 糖皮质激素抵抗确实存在相关性 和CTL敏感性。这将为公共的 两种细胞裂解机制的途径。我们还建议 选择CTL抗性目标以了解他们是否获得了 对PF和/或糖皮质激素耐药。这些变种将 为研究假定的自杀途径提供了工具。最后， 我们将探讨体内诱导的CTL是否改变了它们的 体外长时间培养裂解与细胞激活途径 已描述的克隆CTL的介导性裂解。

Cytotoxic T cells (CTL) are one of the cornerstones of the immune system. There is therefore much interest in understanding the mechanism by which they lyse targets, which is the overall goal of this proposal. Although significant strides have been made in our understanding of CTL lysis many questions remain open. Adsorption of CTL to targets via receptors triggers polarization of cytoskeletal elements in the CTL. This leads to reorientation of the Golgi apparatus (GA) towards the target binding site and secretion of channel forming proteins -perforins (PF) - which are thought to cause target cell lysis. Recent evidence suggest s that this sequence of events established with in vitro CTL clones does not operate in vivo induced CTL. Evidence is also accumulating that target lysis by channel formation may not be the universal mechanism of CTL lysis and that the target itself plays an active role in its lysis. We propose to explore several aspects of the concurrent models for CTL lysis in in vivo induced CTL, because they have not yet acquired abnormal properties in vitro. CTL will be highly purified and tested whether they show GA reorientation when bound to targets because this reaction is thought to signal proper target recognition. Next CTL will be tested as to their ability to insert PF channels into erythrocyte membranes. To induce this reaction we will use bifunctional anti-target anti-T cell receptor antibody conjugates. If CTL fail to insert channels into erythrocytes we will test whether removal of nuclei from targets inhibits target lysis. In drug inhibition studies we will examine whether continued lytic activity of the CTL on one hand and lytic susceptibility of the target on the other require protein synthesis prior to CTL-target binding. Preliminary data support the view that targets participate in CTL induced lysis. To explore how targets participate in lysis we propose several approaches, one of which is based on the ability of protein synthesis inhibitors to interfere with CTL lysis. Another is based on the concomitant occurrence of resistance to CTL lysis and resistance to glucocorticoids in one cell line. We propose to examine whether there indeed exists a correlation between glucocorticoid resistance and CTL sensitivity. This would provide support for a common pathway for both mechanisms of cell lysis. We also propose to select CTL resistant targets to explore whether they acquire resistance to PF and/or glucocorticoids. These variants will provide the tool to study the postulated suicide pathway. Finally, we will explore whether in vivo induced CTL change their mode of lysis upon prolonged culture in vitro and activate pathways of cell mediated lysis that have been described for cloned CTL.