Gu J J, Harriss J V, Ozato K, Gottlieb P D
Department of Microbiology, University of Texas at Austin 78712.
J Immunol. 1994 Nov 15;153(10):4408-17.
A previous report from this laboratory described the production of CD8+, class I-specific T cell hybridomas which developed specific cytolytic activity and the ability to secrete IL-2 upon Con A or specific Ag stimulation. Unlike normal lymphocytes or long-term CTL lines for which exposure to Ag triggers both differentiation and proliferation, T cell hybridoma lines can be activated functionally against a background of continuous proliferation. They therefore provide a unique system with which to study the molecular events involved in the induction of cytolytic function. The expression of mRNA from a series of genes was evaluated by Northern hybridization at various times after Con A stimulation of the H-2Ld-specific CD8+ 3D9 hybridoma. Induction of the c-fos proto-oncogene by 45 min poststimulation was followed shortly by c-myc induction. Perforin mRNA was expressed at a low level in the unstimulated hybridomas, but was down-regulated upon Con A stimulation to levels undetectable by PCR. Interestingly, production of granzyme A mRNA was strongly induced by 45 min after Con A stimulation. In the CD8+ RT-1.3G3 hybridoma, which is nonlytic and specific for the HIV-1 envelope glycoprotein, c-fos but not granzyme A mRNA was induced by 45 min poststimulation, and no granzyme A mRNA was detectable at any time. Thus, a significant role for granzyme A in the induction of cytolytic activity is suggested. Cytolysis by the 3D9 hybridoma involved both target cell membrane damage and DNA fragmentation, and both Ca(2+)-dependent and Ca(2+)-independent cytolysis were observed. Although TNF-alpha mRNA was induced by 4 h poststimulation, Ab to TNF-alpha failed to inhibit the Ca(2+)-independent lysis observed, leaving the basis for the observed Ca(2+)-independent lysis unexplained.
本实验室之前的一份报告描述了CD8+、I类特异性T细胞杂交瘤的产生，这些杂交瘤在受到刀豆蛋白A（Con A）或特异性抗原刺激后，产生了特异性细胞溶解活性以及分泌白细胞介素-2（IL-2）的能力。与正常淋巴细胞或长期细胞毒性T淋巴细胞（CTL）系不同，正常淋巴细胞或长期CTL系暴露于抗原会触发分化和增殖，而T细胞杂交瘤系在持续增殖的背景下可被功能性激活。因此，它们提供了一个独特的系统，用于研究诱导细胞溶解功能所涉及的分子事件。在对H-2Ld特异性CD8+ 3D9杂交瘤进行Con A刺激后的不同时间，通过Northern杂交评估了一系列基因的mRNA表达。刺激后45分钟，原癌基因c-fos被诱导，随后不久c-myc也被诱导。穿孔素mRNA在未刺激的杂交瘤中表达水平较低，但在Con A刺激后被下调至PCR检测不到的水平。有趣的是，Con A刺激后45分钟，颗粒酶A mRNA的产生被强烈诱导。在对HIV-1包膜糖蛋白具有特异性且无细胞溶解活性的CD8+ RT-1.3G3杂交瘤中，刺激后45分钟诱导了c-fos而非颗粒酶A mRNA，且在任何时候都检测不到颗粒酶A mRNA。因此，提示颗粒酶A在诱导细胞溶解活性中起重要作用。3D9杂交瘤的细胞溶解涉及靶细胞膜损伤和DNA片段化，并且观察到了钙依赖性和非钙依赖性细胞溶解。尽管刺激后4小时诱导了肿瘤坏死因子-α（TNF-α）mRNA，但抗TNF-α抗体未能抑制观察到的非钙依赖性细胞溶解，因此观察到的非钙依赖性细胞溶解的机制尚不清楚。
