Garner R, Helgason C D, Atkinson E A, Pinkoski M J, Ostergaard H L, Sorensen O, Fu A, Lapchak P H, Rabinovitch A, McElhaney J E
Department of Biochemistry, University of Alberta, Edmonton, Canada.
J Immunol. 1994 Dec 15;153(12):5413-21.
The mechanism(s) by which CTL induce target cell lysis have not been clearly elucidated. Perforin and the cytotoxic cell proteinases (granzymes) contained within the granules of CTL and NK, have been implicated, but abundant evidence for the existence of alternate lytic pathways has accumulated. In this report we characterize the mechanism of killing used by two cytolytic hybridomas (PMM-1 and MD90) that express neither perforin nor the granzymes. These characteristics are compared with results obtained by using a representative Ag-dependent, granule-containing T cell clone in cytolysis assays. The major differences were that the granule-negative hybridomas could lyse a variety of target cells in the presence of cyclosporin and the absence of calcium. All the effectors could kill in the presence of protein synthesis inhibitors (cycloheximide and emetine) and induced DNA fragmentation in the target cells. The cytolytic hybridomas had to be stimulated to be cytolytic and this activation required the presence of calcium, was dependent on protein synthesis, and inhibited by the addition of cyclosporin. Although TNF was shown not be involved, the sensitivity of the target cells to lysis by the granule-negative killers correlated with the level of expression of Fas Ag. With the use of L1210 and an L1210 cell line transfected with Fas cDNA we demonstrated that these MD90 and PMM-1 kill the latter much more effectively and that this increase was effectively inhibited with anti-Fas Ab. Furthermore the lack of sensitivity to cyclosporin, cycloheximide, emetine, and EGTA was confirmed with these targets. We conclude that these two cytolytic hybridomas use the Fas lytic pathway to induce lysis in target cells.
细胞毒性T淋巴细胞（CTL）诱导靶细胞裂解的机制尚未完全阐明。穿孔素以及CTL和自然杀伤细胞（NK）颗粒中所含的细胞毒性蛋白酶（颗粒酶）被认为与此有关，但越来越多的证据表明还存在其他裂解途径。在本报告中，我们描述了两种既不表达穿孔素也不表达颗粒酶的溶细胞杂交瘤（PMM-1和MD90）的杀伤机制。将这些特性与在细胞裂解试验中使用代表性的抗原依赖性、含颗粒的T细胞克隆所获得的结果进行了比较。主要差异在于，颗粒阴性杂交瘤在存在环孢菌素且无钙的情况下能够裂解多种靶细胞。所有效应细胞在存在蛋白质合成抑制剂（环己酰亚胺和依米丁）时均可杀伤靶细胞，并诱导靶细胞DNA片段化。溶细胞杂交瘤必须被刺激才能具有溶细胞活性，这种激活需要钙的存在，依赖于蛋白质合成，并可被添加环孢菌素所抑制。虽然已证明肿瘤坏死因子（TNF）不参与其中，但靶细胞对颗粒阴性杀伤细胞裂解的敏感性与Fas抗原的表达水平相关。通过使用L1210细胞以及转染了Fas cDNA的L1210细胞系，我们证明MD90和PMM-1对后者的杀伤效果更佳，且这种增强效应可被抗Fas抗体有效抑制。此外，这些靶细胞对环孢菌素、环己酰亚胺、依米丁和乙二醇双乙酸酯（EGTA）不敏感也得到了证实。我们得出结论，这两种溶细胞杂交瘤利用Fas裂解途径诱导靶细胞裂解。
