CELLULAR RESISTANCE TO T CELL MEDIATED CYTOXICITY

本项目的目的是表征靶细胞抵抗细胞毒性T淋巴细胞(CTL)裂解的方式及其毒性因素。CTL通过两种途径杀伤靶细胞：一种是神经性途径，涉及细胞溶素、穿孔素的形成；另一种是细胞凋亡途径，涉及靶表面Fas分子的连接。CTL本身对其他CTL、穿孔素或分离的分泌颗粒的裂解具有高度的抵抗力。它们的膜抵抗穿孔素通道的形成。这使得CTL能够在受到靶细胞的攻击后存活下来。其他细胞，包括CD4+Th1克隆体，通过内吞作用和恢复跨膜电位来去除穿孔素损伤，从而增强其存活率。其他因素也调节靶细胞对CTL和穿孔素的敏感性；这些因素包括细胞周期中的位置和暴露于热应激。具体目标1.一个目标是鉴定由CTL表达的保护它们免受穿孔素攻击的基因及其产物。尽管与CTL密切相关，但小鼠Th1克隆在耗尽ATP时会屈服于穿孔素，而CTL则不会。为了丰富与CTL抗性有关的基因，我们将利用一个CTL基因的消减cDNA文库，该文库中的CTL基因没有Th1克隆表达的基因。基因将通过对穿孔素敏感的淋巴瘤株产生抗药性的能力来识别。此外，细胞毒性颗粒的成分在分泌时沉积在CTL表面，并可能修饰细胞膜。因此，我们将对颗粒进行分级，并评估分级对细胞对穿孔素损伤的敏感性的影响。另一个目的是确定细胞周期的变化和热应激如何改变靶细胞对T细胞介导的细胞毒性的敏感性。与处于细胞周期其他阶段的细胞相比，处于G1晚期的细胞对穿孔素的敏感性较低。我们将确定它们的耐药性是由于结合穿孔素的能力发生了变化，还是由于它们的膜修复能力发生了变化。我们还将研究热应激如何改变靶细胞对CTL的敏感性，以及热休克蛋白70的可诱导亚型I-HSP70在防止同源CTL识别A20淋巴瘤方面的作用。

The aim of this project is to characterize the means by which target cells resist lysis by cytotoxic T lymphocytes (CTLs) and their toxic factors. CTLs kill targets by two pathways: a neurotic pathway involving formation of pores by the cytolysin, perforin, and an apoptotic pathway involving ligation of Fas molecules on target surfaces. CTLs, themselves, are highly resistant to lysis by other CTLs or by perforin or isolated secretory granules. Their membrane resists formation of perforin channels. This enables CTLs to survive attacks on target cells. Other cells, including CD4+ Th1 clones, enhance their survivability by removing perforin lesions through endocytosis and restoration of transmembrane potentials. Other factors also modulate sensitivity of target cells to CTLs and to perforin; these include position in the cell cycle and exposure to heat stress. Specific Aim 1. One aim is to identify the genes, and their products, expressed by CTLs that protect them from perforin. Though closely related to CTLs, murine Th1 clones succumb to perforin when depleted of ATP while CTLs do not. To enrich for genes responsible for the resistance of CTLs, we will utilize a subtracted cDNA library of CTL genes depleted of genes expressed by a Th1 clone. Genes will be identify by their ability to confer resistance to a perforin-sensitive lymphoma line. Also, components of cytotoxic granules are deposited in CTL surfaces upon secretion and may modify the membrane. Thus, we will fractionate the granules and evaluate the effect of fractions on the susceptibility of cells to perforin damage. Specific Aim 2. Another aim is to determine how changes in the cell cycle and exposure to heat stress alter susceptibility of target cells to T cell mediate cytotoxicity. Cells in late G1 are less susceptible to perforin than those at other stages of the cell cycle. We will determine whether their resistance is due to changes in ability to bind perforin or ability to repair their membranes. We will also examine how heat stress alters the susceptibility of target cells to CTLs and the role of the inducible isoform of heat-shock protein 70, i-hsp70, in preventing recognition of A20 lymphoma by cognate CTLs.