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）及其毒性因子裂解的手段。CTL通过两种途径杀死靶标：涉及通过溶细胞素、穿孔素形成孔的神经性途径和涉及Fas分子在靶标表面上的连接的凋亡途径。CTL本身对其他CTL或穿孔素或分离的分泌颗粒的裂解具有高度抗性。它们的膜抵抗穿孔素通道的形成。这使得CTL能够在对靶细胞的攻击中存活。其他细胞，包括CD 4 + Th 1克隆，通过内吞作用去除穿孔素损伤和恢复跨膜电位来增强其存活能力。其他因素也调节靶细胞对CTL和穿孔素的敏感性;这些因素包括在细胞周期中的位置和暴露于热应激。具体目标1。目的之一是鉴定由CTL表达的基因及其产物，这些CTL保护它们免受穿孔素的侵害。虽然与CTL密切相关，但当ATP耗尽时，小鼠Th 1克隆屈服于穿孔素，而CTL则不然。为了富集负责CTL抗性的基因，我们将利用去除由Th 1克隆表达的基因的CTL基因的消减cDNA文库。基因将通过其赋予穿孔素敏感性淋巴瘤系抗性的能力来鉴定。此外，细胞毒性颗粒的组分在分泌后沉积在CTL表面，并可修饰膜。因此，我们将破碎颗粒，并评估各组分对细胞对穿孔素损伤的敏感性的影响。具体目标2。另一个目的是确定细胞周期的变化和暴露于热应激如何改变靶细胞对T细胞介导的细胞毒性的易感性。G1晚期的细胞对穿孔素的敏感性低于细胞周期其他阶段的细胞。我们将确定它们的耐药性是否是由于结合穿孔素的能力或修复膜的能力的变化。我们还将研究热应激如何改变靶细胞对CTL的易感性，以及热休克蛋白70的诱导型亚型i-hsp 70在防止同源CTL识别A20淋巴瘤中的作用。

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.