The analysis of costimulatory receptor signaling cascades in normal T lymphocytes using in vitro gene transfer and reporter gene analysis
The analysis of costimulatory receptor signaling cascades in normal T lymphocytes using in vitro gene transfer and reporter gene analysis
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DOI：
10.1038/nm1001-1155
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发表时间：
2001-10-01
期刊：
影响因子：
82.9
通讯作者：
McKean, DJ
中科院分区：
文献类型：
--
作者：
Bell, MP;Huntoon, CJ;McKean, DJ
T淋巴细胞表面抗原受体和共刺激受体的连接启动了调节细胞周期进程和细胞分化的细胞内信号。为了有效地操纵T细胞的激活用于免疫治疗，了解这些信号通路是如何整合以控制特定的基因转录事件将是重要的。在这里，我们描述了一种新的瞬时转基因程序，该程序有效地将DNA导入未分裂的正常人类和小鼠T淋巴细胞，同时保持高细胞活力。利用这项技术，可以引入报告基因来表征细胞内调节正常T淋巴细胞群体中特定基因转录事件的信号通路。我们发现，在不同的T淋巴细胞群体中，CD28受体可以差异地偶联到下游的信号通路。此外，我们证明了编码标记的有丝分裂原激活的激酶-1蛋白的基因可以被转染并快速表达，以调节正常胸腺细胞中Bcl-2的表达。
Ligation of the antigen receptor and costimulatory receptors on the surface of T lymphocytes initiates intracellular signals that regulate cell-cycle progression and cell differentiation. To effectively manipulate the activation of T cells for immunotherapeutic applications, it will be important to understand how these signaling pathways are integrated to control specific gene transcription events. Here we describe a novel transient transfection procedure that efficiently introduces DNA into non-dividing normal human and murine T lymphocytes while maintaining high cell viability. Using this technique, reporter genes can be introduced to characterize intracellular signaling pathways that regulate specific gene transcription events in normal T-lymphocyte populations. We show that the CD28 receptor can be differentially coupled to downstream signaling pathways in different T-lymphocyte populations. In addition, we demonstrate that a gene encoding a tagged constitutively active mitogen-activated kinase kinase-1 protein can be transfected and rapidly expressed to regulate the expression of Bcl-2 in normal thymocytes.