Shivnan E, Clayton L, Allridge L, Keating K E, Gullberg M, Alexander D R
Department of Immunology, The Babraham Institute, Cambridge, United Kingdom.
J Immunol. 1996 Jul 1;157(1):101-9.
The effects of a pan-CD45 mAb (CD45.2) on TCR-mediated signaling pathways were investigated in Jurkat T cells. The simultaneous addition of CD45 mAb with an activating OKT3 mAb had little effect on TCR-stimulated signals. However, when Jurkat cells were exposed to the CD45 mAb for 10 to 20 min before the addition of OKT3, a partial uncoupling of the TCR from intracellular signals was observed. The maximal increase in intracellular calcium was inhibited 47 +/- 10% (n = 11, range 33-67%), whereas no inhibition of inositol trisphosphate production was detected. The transient TCR-mediated activation of the Ca2+/calmodulin-activated kinase IV/Gr was also inhibited by the CD45 mAb, and this was reflected in a 50 to 60% inhibition in the TCR-stimulated generation of the p21 and p23 phosphoisomers of oncoprotein 18, a Ca2+/calmodulin-activated kinase IV/Gr substrate recently implicated in cell cycle regulatory events. Oncoprotein 18 is also a substrate for mitogen- activated protein kinase, but no inhibition by the CD45 mAb of TCR-triggered mitogen-activated protein kinase activation was observed. The CD45 mAb was therefore selective in causing the uncoupling of the TCR from calcium signals and calcium-regulated events without promoting a general inhibition of all TCR-mediated signals. Confocal microscopy revealed that binding of the CD45 mAb caused patching of CD45 molecules at the cell surface and, unexpectedly, a marked redistribution of intracellular CD45. However, no change was observed in the total level of CD45 expressed at the cell surface. Aggregation of CD45 at the cell surface may result in its sequestration from its tyrosine kinase substrates, with a consequent selective uncoupling of the TCR from intracellular signaling pathways.
在Jurkat T细胞中研究了泛CD45单克隆抗体（CD45.2）对TCR介导的信号通路的影响。将CD45单克隆抗体与激活型OKT3单克隆抗体同时添加对TCR刺激的信号几乎没有影响。然而，当在添加OKT3之前将Jurkat细胞暴露于CD45单克隆抗体10至20分钟时，观察到TCR与细胞内信号部分解偶联。细胞内钙的最大增加被抑制了47±10%（n = 11，范围33 - 67%），而未检测到肌醇三磷酸生成的抑制。CDCa2+/钙调蛋白激活激酶IV/Gr的短暂TCR介导的激活也被CD45单克隆抗体抑制，这反映在TCR刺激的癌蛋白18的p21和p23磷酸异构体生成中50%至60%的抑制，癌蛋白18是一种Ca2+/钙调蛋白激活激酶IV/Gr底物，最近涉及细胞周期调节事件。癌蛋白18也是丝裂原活化蛋白激酶的底物，但未观察到CD45单克隆抗体对TCR触发的丝裂原活化蛋白激酶激活的抑制。因此，CD45单克隆抗体在导致TCR与钙信号和钙调节事件解偶联方面具有选择性，而不会对所有TCR介导的信号产生普遍抑制。共聚焦显微镜显示，CD45单克隆抗体的结合导致细胞表面CD45分子的斑块形成，并且出乎意料的是，细胞内CD45有明显的重新分布。然而，在细胞表面表达的CD45总水平未观察到变化。CD45在细胞表面的聚集可能导致其与酪氨酸激酶底物隔离，从而使TCR与细胞内信号通路选择性解偶联。
