Pre-clustering of the B cell antigen receptor demonstrated by mathematically extended electron microscopy
Pre-clustering of the B cell antigen receptor demonstrated by mathematically extended electron microscopy
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DOI：
10.3389/fimmu.2013.00427
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发表时间：
2013-01-01
影响因子：
7.3
通讯作者：
Schamel, Wolfgang W. A.
中科院分区：
文献类型：
--
作者：
Fiala, Gina J.;Kaschek, Daniel;Schamel, Wolfgang W. A.
B 细胞抗原受体 (BCR) 在适应性免疫中起着至关重要的作用，因为 BCR 的抗原诱导信号传导会导致 B 细胞激活并在免疫反应过程中产生抗体。然而，在遇到抗原之前，BCR 在细胞表面的空间纳米级组织仍然存在争议。在这里，我们固定了小鼠 B 细胞，用免疫金对细胞表面的 BCR 进行染色，并通过透射电子显微镜观察金颗粒的分布。大约 30% 的金颗粒聚集在一起。然而，15% 的低染色效率无法得出有关 BCR 寡聚状态的定量结论。为了克服这一限制，我们使用蒙特卡罗模拟来包含或排除 BCR 的可能分布。我们的组合实验建模方法假设不同 BCR 大小的最低数量来解释观察到的金分布，表明 40% 的表面 IgD-BCR 以二聚体形式存在，60% 形成约 18 个受体的大层状簇。相反，mIgD 分子的跨膜突变体仅形成 IgD-BCR 二聚体。我们的方法补充了高分辨率荧光成像，并清楚地证明了静息 B 细胞上预形成的 BCR 簇的存在，对 BCR 激活的经典交联模型提出了质疑。
The B cell antigen receptor (BCR) plays a crucial role in adaptive immunity, since antigen-induced signaling by the BCR leads to the activation of the B cell and production of antibodies during an immune response. However, the spatial nano-scale organization of the BCR on the cell surface prior to antigen encounter is still controversial. Here, we fixed murine B cells, stained the BCRs on the cell surface with immuno-gold and visualized the distribution of the gold particles by transmission electron microscopy. Approximately 30% of the gold particles were clustered. However the low staining efficiency of 15% precluded a quantitative conclusion concerning the oligomerization state of the BCRs. To overcome this limitation, we used Monte-Carlo simulations to include or to exclude possible distributions of the BCRs. Our combined experimental-modeling approach assuming the lowest number of different BCR sizes to explain the observed gold distribution suggests that 40% of the surface IgD-BCR was present in dimers and 60% formed large laminar clusters of about 18 receptors. In contrast, a transmembrane mutant of the mIgD molecule only formed IgD-BCR dimers. Our approach complements high resolution fluorescence imaging and clearly demonstrates the existence of pre-formed BCR clusters on resting B cells, questioning the classical cross-linking model of BCR activation.