Real-time analysis of the detailed sequence of cellular events in mAb-mediated complement-dependent cytotoxicity of B-cell lines and of chronic lymphocytic leukemia B-cells
Real-time analysis of the detailed sequence of cellular events in mAb-mediated complement-dependent cytotoxicity of B-cell lines and of chronic lymphocytic leukemia B-cells
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DOI：
10.1016/j.molimm.2015.12.007
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发表时间：
2016-02-01
影响因子：
3.6
通讯作者：
Taylor, Ronald P.
中科院分区：
文献类型：
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作者：
Lindorfer, Margaret A.;Cook, Erika M.;Taylor, Ronald P.
补体依赖性细胞毒性是用于癌症免疫治疗的某些mAb（包括奥法木单抗和利妥昔单抗）的重要作用机制。然而，在被mAb和补体攻击的有核细胞中发生的细胞变化的详细序列尚未被描述。最近开发的CD 20单克隆抗体，经工程改造，在结合细胞时形成六聚体，与血清中的B细胞反应，螯合C1 q，然后激活补体并促进细胞杀伤，比其野生型前体更有效。我们使用这些工程化的mAb作为模型来研究当mAb结合B细胞系和来自慢性淋巴细胞白血病患者的原代细胞然后激活补体时发生的事件的顺序。基于四色共聚焦显微镜实时电影和高分辨率数字成像，我们发现在CD 20 mAb结合和C1 q摄取后，C3 b沉积在细胞上，随后是Ca 2+内流，通过用钙指示剂FLUO-4标记的细胞上产生的明亮的绿色信号显示。明亮的FLUO-4/Ca 2+信号消失，被线粒体中的点状绿色信号取代，表明Ca 2+定位。这一步导致线粒体中毒，随后细胞死亡。整个序列是在
Complement-dependent cytotoxicity is an important mechanism of action of certain mAbs used in cancer immunotherapy, including ofatumumab and rituximab. However, the detailed sequence of cellular changes that occur in nucleated cells attacked by mAb and complement has not been delineated. Recently developed CD20 mAbs, engineered to form hexamers on binding to cells, react with B-cells in serum, chelate C1q, and then activate complement and promote cell killing considerably more effectively than their wild-type precursors. We used these engineered mAbs as a model to investigate the sequence of events that occur when mAbs bind to B-cell lines and to primary cells from patients with chronic lymphocytic leukemia and then activate complement. Based on four-color confocal microscopy real-time movies and high resolution digital imaging, we find that after CD20 mAb binding and C1q uptake, C3b deposits on cells, followed by Ca2+ influx, revealed by bright green signals generated on cells labeled with FLUO-4, a Ca2+ indicator. The bright FLUO-4/Ca2+ signal fades, replaced by punctate green signals in mitochondria, indicating Ca2+ localization. This step leads to mitochondrial poisoning followed by cell death. The entire sequence is completed in