Rex Elizabeth B, Kim Suzie, Wiener Jake, Rao Navin L, Milla Marcos E, DiSepio Daniel
Discovery Sciences, Janssen Research and Development LLC, La Jolla, CA, USA
Discovery Sciences, Janssen Research and Development LLC, La Jolla, CA, USA.
J Biomol Screen. 2015 Aug;20(7):876-86. doi: 10.1177/1087057115585724. Epub 2015 May 6.
An EPIC label-free phenotypic platform was developed to explore B cell receptor (BCR) and CD40R-mediated B cell activation. The phenotypic assay measured the association of RL non-Hodgkin's lymphoma B cells expressing lymphocyte function-associated antigen 1 (LFA-1) to intercellular adhesion molecule 1 (ICAM-1)-coated EPIC plates. Anti-IgM (immunoglobulin M) mediated BCR activation elicited a response that was blocked by LFA-1/ICAM-1 specific inhibitors and a panel of Bruton's tyrosine kinase (BTK) inhibitors. LFA-1/ICAM-1 association was further increased on coapplication of anti-IgM and mega CD40L when compared to individual application of either. Anti-IgM, mega CD40L, or the combination of both displayed distinct kinetic profiles that were inhibited by treatment with a BTK inhibitor. We also established a FLIPR-based assay to measure B cell activation in Ramos Burkitt's lymphoma B cells and an RL cell line. Anti-IgM-mediated BCR activation elicited a robust calcium response that was inhibited by a panel of BTK inhibitors. Conversely, CD40R activation did not elicit a calcium response in the FLIPR assay. Compared to the FLIPR, the EPIC assay has the propensity to identify inhibitors of both BCR and CD40R-mediated B cell activation and may provide more pharmacological depth or novel mechanisms of action for inhibition of B cell activation.
开发了一种EPIC无标记表型平台，以探索B细胞受体（BCR）和CD40R介导的B细胞活化。该表型分析测量了表达淋巴细胞功能相关抗原1（LFA-1）的RL非霍奇金淋巴瘤B细胞与细胞间粘附分子1（ICAM-1）包被的EPIC板的结合。抗IgM（免疫球蛋白M）介导的BCR活化引发了一种反应，该反应被LFA-1/ICAM-1特异性抑制剂和一组布鲁顿酪氨酸激酶（BTK）抑制剂阻断。与单独应用抗IgM或巨型CD40L相比，抗IgM和巨型CD40L共同应用时，LFA-1/ICAM-1的结合进一步增加。抗IgM、巨型CD40L或两者的组合显示出不同的动力学曲线，这些曲线被BTK抑制剂处理所抑制。我们还建立了一种基于FLIPR的分析方法，以测量Ramos Burkitt淋巴瘤B细胞和RL细胞系中的B细胞活化。抗IgM介导的BCR活化引发了强烈的钙反应，该反应被一组BTK抑制剂抑制。相反，在FLIPR分析中，CD40R活化未引发钙反应。与FLIPR相比，EPIC分析有潜力识别BCR和CD40R介导的B细胞活化的抑制剂，并可能为抑制B细胞活化提供更多的药理学深度或新的作用机制。
