Björck P, Braesch-Andersen S, Paulie S
Department of Immunology, Stockholm University, Sweden.
Immunology. 1994 Nov;83(3):430-7.
The B-cell surface protein, CD40, belongs to the tumour necrosis factor/nerve growth factor (TNF/NGF) receptor family and plays a crucial role in T cell-dependent B-cell activation. Ligation of this receptor with antibodies or its recently defined ligand, gp39, generates an intracellular signal that, when combined with triggering of surface immunoglobulin or the interleukin-4 (IL-4) receptor, induces a variety of stimulatory effects in B cells. In this study we provide further evidence for the importance of receptor cross-linking in generating this signal and we also report on the presence of a soluble form of CD40. A new CD40 monoclonal antibody (mAb), 17:40, was found to synergize with other CD40 antibodies (mAb89 and S2C6) in inducing proliferation as well as IgE synthesis in IL-4-treated tonsillar B cells. However, both this mAb and mAb89 failed to co-operate with a soluble construct of the CD40 ligand, whereas such co-operation was seen with the S2C6 antibody. Cross-inhibition experiments showed that the 17:40 mAb recognized an epitope that was clearly distinct from that seen by S2C6 and mAb89. Although directed to separate epitopes, both 17:40 and mAb89 completely blocked binding of gp39 to its receptor, while the S2C6 mAb only partially interfered with this binding. The findings suggest a close relationship between the degree of receptor clustering and the strength of the delivered signal. With the access to antibodies recognizing distinct structures on CD40 we also established a sandwich enzyme-linked immunosorbent assay for quantitative determinations of the antigen. With this assay we could demonstrate the presence of a soluble form of CD40 (sCD40) in culture supernatants. The fact that sCD40 also retained its ligand-binding capacity indicates that it may have an important regulatory role and modulate the T cell-dependent stimulation via CD40. Both the finding of soluble receptors and the need for receptor clustering are features that CD40 share with other members of the TNF/NGF receptor family.
B细胞表面蛋白CD40属于肿瘤坏死因子/神经生长因子（TNF/NGF）受体家族，在T细胞依赖性B细胞激活过程中起关键作用。用抗体或其最近确定的配体gp39连接该受体，会产生一种细胞内信号，当与表面免疫球蛋白或白细胞介素-4（IL-4）受体的触发相结合时，会在B细胞中诱导多种刺激效应。在本研究中，我们为受体交联在产生该信号中的重要性提供了进一步证据，并且我们还报告了可溶性形式的CD40的存在。发现一种新的CD40单克隆抗体（mAb）17:40，在诱导IL-4处理的扁桃体B细胞增殖以及IgE合成方面，能与其他CD40抗体（mAb89和S2C6）协同作用。然而，该mAb和mAb89均未能与CD40配体的可溶性构建体协同作用，而S2C6抗体则能出现这种协同作用。交叉抑制实验表明，17:40 mAb识别的表位与S2C6和mAb89识别的表位明显不同。尽管针对不同的表位，但17:40和mAb89均完全阻断gp39与其受体的结合，而S2C6 mAb仅部分干扰这种结合。这些发现表明受体聚集程度与传递信号的强度之间存在密切关系。通过获得识别CD40上不同结构的抗体，我们还建立了一种夹心酶联免疫吸附测定法来定量测定该抗原。通过该测定法，我们能够证明培养上清液中存在可溶性形式的CD40（sCD40）。sCD40也保留其配体结合能力这一事实表明，它可能具有重要的调节作用，并通过CD40调节T细胞依赖性刺激。可溶性受体的发现以及受体聚集的必要性都是CD40与TNF/NGF受体家族其他成员共有的特征。
