MEASUREMENT OF LIGAND-INDUCED ACTIVATION IN SINGLE VIABLE T-CELLS USING THE LACZ REPORTER GENE
MEASUREMENT OF LIGAND-INDUCED ACTIVATION IN SINGLE VIABLE T-CELLS USING THE LACZ REPORTER GENE
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DOI：
10.1073/pnas.88.9.3972
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发表时间：
1991-05-01
影响因子：
11.1
通讯作者：
SHASTRI, N
中科院分区：
文献类型：
--
作者：
KARTTUNEN, J;SHASTRI, N
我们使用细菌β-半乳糖苷酶基因（lacZ）作为报告基因，用于快速测量T细胞抗原受体（TCR）介导的单个T细胞活化。 报道构建体含有在人白细胞介素2增强子的活化T细胞核因子（NF-AT）元件控制下的lacZ基因[Fiering，S.，诺斯罗普，J. P.，诺兰湾，澳-地P.的人，Matilla，P.，克拉布特里湾R. & Herzenberg，L. A. 05 The Dog of the Woman（1990）4，1823-1834]。 细胞内lacZ酶的活性通过流式细胞术测量荧光素在加载有荧光β-半乳糖苷酶底物荧光素二-β-D-吡喃半乳糖苷的细胞中的积累来分析。 作为模型系统，用NF-AT-lacZ构建体转染对溶菌酶肽（氨基酸74-88）/A（B）复合物具有特异性的T细胞杂交瘤BO 4 H 9. 1。 在暴露于药理学试剂、生理肽/主要组织相容性复合物配体或其它TCR特异性刺激物后，在50-100%的转染细胞中诱导lacZ活性。 有趣的是，增加刺激浓度增加lacZ+细胞的分数，但不是每个细胞的lacZ活性水平。 即使在广泛变化的刺激水平下，单个lacZ+细胞中的lacZ活性水平仍保持在非常窄的范围内。 这些结果表明，TCR介导的活化可以在单个T细胞中容易地测量，并强烈表明，一旦致力于活化，单个T细胞中的NF-AT转录活性水平与刺激的形式或浓度无关。 该测定可能被证明可用于研究单个T细胞和TCR配体中的早期活化事件。
We have used the bacterial beta-galactosidase gene (lacZ) as a reporter gene for the rapid measurement of T-cell antigen receptor (TCR)-mediated activation of individual T cells. The reporter construct contained the lacZ gene under the control of the nuclear factor of activated T cells (NF-AT) element of the human interleukin 2 enhancer [Fiering, S., Northrop, J. P., Nolan, G. P., Matilla, P., Crabtree, G. R. & Herzenberg, L. A. (1990) Genes Dev. 4, 1823-1834]. The activity of the intracellular lacZ enzyme was analyzed by flow cytometric measurement of fluorescein accumulation in cells loaded with the fluorogenic beta-galactosidase substrate fluorescein di-beta-D-galactopyranoside. As a model system, the T-cell hybridoma BO4H9.1, which is specific for the lysozyme peptide (amino acids 74-88)/A(b) complex, was transfected with the NF-AT-lacZ construct. lacZ activity was induced in 50-100% of the transfectant cells following exposure to pharmacological agents, to the physiological peptide/major histocompatibility complex ligand, or to other TCR-specific stimuli. Interestingly, increasing concentrations of the stimulus increased the fraction of lacZ+ cells, but not the level of lacZ activity per cell. Even under widely varying levels of stimulus, the level of lacZ activity in individual lacZ+ cells remained within a remarkably narrow range. These results demonstrate that TCR-mediated activation can be readily measured in single T cells and strongly suggest that once committed to activation, the level of NF-AT transcriptional activity in individual T cells is independent of the form or concentration of stimulus. This assay is likely to prove useful for the study of early activation events in individual T cells and of TCR ligands.