Complement activation on B lymphocytes opsonized with rituximab or ofatumumab produces substantial changes in membrane structure preceding cell lysis
Complement activation on B lymphocytes opsonized with rituximab or ofatumumab produces substantial changes in membrane structure preceding cell lysis
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DOI：
10.4049/jimmunol.181.1.822
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发表时间：
2008-07-01
影响因子：
4.4
通讯作者：
Taylor, Ronald P.
中科院分区：
文献类型：
--
作者：
Beum, Paul V.;Lindorfer, Margaret A.;Taylor, Ronald P.
CD20 mAb 利妥昔单抗 (RTX) 与正常人血清 (NHS) 中的 B 淋巴细胞结合可激活补体 (C)，并促进 C3b 沉积在细胞结合的 RTX 上或附近。基于转盘共聚焦显微镜分析，我们首次报告了 C3b 沉积和 C 介导的 RTX 调理 B 细胞杀伤的实时可视化。 RTX 调理的 Daudi B 细胞激活 C 会诱导膜快速起泡，并产生从细胞表面突出的长而薄的结构，我们称之为流光。 Ofatumumab 是一种独特的 mAb，针对 CD20 Ag 的独特结合位点（小环表位），与 RTX 相比，它能更快地杀死 Daudi 细胞并促进其流动。与 RTX 相比，奥法木单抗可促进慢性淋巴细胞白血病患者 ARH77 细胞和原代 B 细胞的流注形成和杀伤。流光的生成需要C激活；在培养基、NHS-EDTA 或 C5 或 C9 耗尽的血清中不会发生流化。可以通过相位成像在明场中可视化流光，荧光染色图案表明它们含有膜脂和聚合肌动蛋白。如果细胞在培养基中与蜂毒蜂毒肽发生反应，也会发生流光，蜂毒蜂毒肽会穿透细胞并以类似于 C 的膜攻击复合物的方式形成膜孔。当 Ab 调理的绵羊红细胞（无核细胞）与 NHS 反应时，可以证明类似于流光的结构。综上所述，我们的研究结果表明膜攻击复合体是流的关键介质。因此，流光的形成可能代表补体诱导的细胞死亡前不久发生的膜结构变化。
Binding of the CD20 mAb rituximab (RTX) to B lymphocytes in normal human serum (NHS) activates complement (C) and promotes C3b deposition on or in close proximity to cell-bound RTX. Based on spinning disk confocal microscopy analyses, we report the first real-time visualization of C3b deposition and C-mediated killing of RTX-opsonized B cells. C activation by RTX-opsonized Daudi B cells induces rapid membrane blebbing and generation of long, thin structures protruding from cell surfaces, which we call streamers. Ofatumumab, a unique mAb that targets a distinct binding site (the small loop epitope) of the CD20 Ag, induces more rapid killing and streaming on Daudi cells than RTX. In contrast to RTX, ofatumumab promotes streamer formation and killing of ARH77 cells and primary B cells from patients with chronic lymphocytic leukemia. Generation of streamers requires C activation; no streaming occurs in media, NHS-EDTA, or in sera depleted of C5 or C9. Streamers can be visualized in bright field by phase imaging, and fluorescence-staining patterns indicate they contain membrane lipids and polymerized actin. Streaming also occurs if cells are reacted in medium with bee venom melittin, which penetrates cells and forms membrane pores in a manner similar to the membrane-attack complex of C. Structures similar to streamers are demonstrable when Ab-opsonized sheep erythrocytes (non-nucleated cells) are reacted with NHS. Taken together, our findings indicate that the membrane-attack complex is a key mediator of streaming. Streamer formation may, thus, represent a membrane structural change that can occur shortly before complement-induced cell death.