Bismuth G, Gouy H, Mariuzza R A, Vaquero C, Theodorou I, Debre P
Laboratoire d'Immunologie Cellulaire et Tissulaire, CNRS UA, Hôpital de la Pitié-Salpêtrière, Paris, France.
Mol Immunol. 1990 Nov;27(11):1127-36. doi: 10.1016/0161-5890(90)90101-5.
In a recent report, a construction containing the alpha chain-variable region (V alpha) coding sequence of a cDNA clone derived from a diphtheria toxoid-specific human T cell (P28), fused to a human immunoglobulin kappa light chain constant region (Ck), was used stably to transfect a murine myeloma cell. In the present study, these transfected cells were employed as an immunogen to raise a mAb, termed 1C5V alpha, specific both for the V alpha Ck chimeric protein secreted by the transfectant and the P28 T cell antigen receptor-V alpha region. mAb 1C5V alpha specifically immunoprecipitates the V alpha Ck protein as a family of 32-35 kDa bands present in the 35S-methionine-labeled culture supernatant from the transfected cells. It specifically binds clone P28. Surface molecules recognized by mAb 1C5V alpha are physically linked to the CD3 molecules since cell treatment with either 1C5V alpha or anti-CD3 mAbs caused the simultaneous down-regulation of the CD3/TCR molecular complex. This link is further supported by immunoprecipitation experiments. Thus, both the 1C5V alpha and the anti-CD3 mAbs precipitate the 16-28 kDa CD3 molecules and the disulfide-linked form of P28 TCR from 125I-labeled P28 T cells. Studies performed in order to define whether a stimulus directly acting on the TCR-V alpha region may trigger the intracellular events observed during human T cell activation showed that (a) mAb 1C5V alpha efficiently triggers the phospholipase C transduction pathway revealed by an accelerated phosphoinositides turn-over and an increased production of phosphorylated derivatives of inositol phosphates; (b) mAb 1C5V alpha induces an up-regulation of IL2R mRNA, accompanied by a slight increase of IL2 and IFN alpha mRNA transcripts evidently amplified in the presence of PMA; (c) soluble mAb 1C5V alpha is strongly mitogenic together with PMA. These results provide the first evidence for the structural authenticity of a secreted water-soluble chimeric form of the variable region of a human TCR alpha chain. They further demonstrate that such chimeric proteins may be valuable tool to further dissect the various functional structure of the human TCR.
在最近的一份报告中，一个构建体被用于稳定转染鼠骨髓瘤细胞，该构建体包含源自白喉类毒素特异性人T细胞（P28）的cDNA克隆的α链可变区（Vα）编码序列，并与人类免疫球蛋白κ轻链恒定区（Ck）融合。在本研究中，这些转染细胞被用作免疫原以产生一种单克隆抗体，称为1C5Vα，它对转染子分泌的VαCk嵌合蛋白和P28 T细胞抗原受体-Vα区均具有特异性。单克隆抗体1C5Vα能特异性免疫沉淀VαCk蛋白，其表现为转染细胞35S-甲硫氨酸标记培养上清中存在的一系列32 - 35 kDa条带。它能特异性结合克隆P28。单克隆抗体1C5Vα识别的表面分子与CD3分子物理相连，因为用1C5Vα或抗CD3单克隆抗体处理细胞会导致CD3/TCR分子复合物同时下调。免疫沉淀实验进一步支持了这种联系。因此，1C5Vα和抗CD3单克隆抗体都能从125I标记的P28 T细胞中沉淀出16 - 28 kDa的CD3分子和二硫键连接形式的P28 TCR。为了确定直接作用于TCR - Vα区的刺激是否能触发人类T细胞激活过程中观察到的细胞内事件而进行的研究表明：（a）单克隆抗体1C5Vα能有效触发磷脂酶C转导途径，表现为磷酸肌醇周转加速和肌醇磷酸磷酸化衍生物产量增加；（b）单克隆抗体1C5Vα诱导IL2R mRNA上调，同时伴有IL2和IFNαmRNA转录本略有增加，在PMA存在下明显放大；（c）可溶性单克隆抗体1C5Vα与PMA一起具有很强的促有丝分裂作用。这些结果为人类TCRα链可变区分泌型水溶性嵌合形式的结构真实性提供了首个证据。它们进一步证明，这种嵌合蛋白可能是进一步剖析人类TCR各种功能结构的有价值工具。
