DiSanto J P, Terry L A, Flomenberg N
Effector Lymphocyte Biology Laboratory, Sloan-Kettering Institute for Cancer Research, New York, NY 10021.
J Immunol Methods. 1991 Jul 26;141(1):123-31. doi: 10.1016/0022-1759(91)90218-5.
The CD8 glycoprotein is a lymphocyte differentiation antigen comprised of two distinct polypeptide chains, alpha and beta, which have the capacity to form homodimeric (CD8 alpha/alpha) or heterodimeric (CD8 alpha/beta) cell surface complexes. The majority of monoclonal antibodies which recognize the human CD8 antigen react with the CD8 alpha chain, while a single mAb, referred to as T8/2T8-5H7 (or 2ST8-5H7), has been identified which binds to the CD8 alpha/beta heterodimer. In order to generate antibodies specific for CD8 beta, murine fibroblast transfectants were constructed which express the human CD8 beta chain in combination with either the human CD8 alpha chain or the murine CD8 alpha homologue, the Lyt-2 molecule. These transfectants were used to raise polyclonal heteroantisera in mice. Transfectants expressing human CD8 alpha/beta heterodimers induced moderate anti-CD8 alpha titers, but were weakly effective in generating anti-CD8 beta titers, despite high level cell surface expression of this protein. In contrast, transfectants expressing mixed-species CD8 heterodimers (murine CD8 alpha and human CD8 beta) induced high anti-CD8 beta titers in immunized mice. Following fusion of splenocytes from mice immunized with mixed-species CD8 transfectants, the mAb 5F2 was isolated which specifically recognizes the human CD8 beta chain. Unlike T8/2T8-5H7, the mAb 5F2 can bind the CD8 beta chain irrespective of its pairing partner, and can immunoprecipitate the CD8 beta protein from cells transfected with the CD8 beta gene in the absence of the human or mouse CD8 alpha gene product. Anti-CD8 beta antibodies should help elucidate the extent of biochemical heterogeneity of the CD8 beta protein, and will also be useful in defining the role of the CD8 beta protein in thymocyte and lymphocyte development, recognition and activation.
CD8糖蛋白是一种淋巴细胞分化抗原，由两条不同的多肽链α和β组成，它们能够形成同二聚体（CD8α/α）或异二聚体（CD8α/β）细胞表面复合物。大多数识别人类CD8抗原的单克隆抗体与CD8α链发生反应，而已鉴定出一种称为T8/2T8 - 5H7（或2ST8 - 5H7）的单克隆抗体，它可结合CD8α/β异二聚体。为了产生针对CD8β的特异性抗体，构建了小鼠成纤维细胞转染体，其表达与人类CD8α链或小鼠CD8α同源物Lyt - 2分子结合的人类CD8β链。这些转染体用于在小鼠中产生多克隆异种抗血清。表达人类CD8α/β异二聚体的转染体诱导了中等水平的抗CD8α滴度，但尽管该蛋白在细胞表面高水平表达，在产生抗CD8β滴度方面效果较弱。相比之下，表达混合物种CD8异二聚体（小鼠CD8α和人类CD8β）的转染体在免疫小鼠中诱导了高抗CD8β滴度。在用混合物种CD8转染体免疫的小鼠的脾细胞融合后，分离出了特异性识别人类CD8β链的单克隆抗体5F2。与T8/2T8 - 5H7不同，单克隆抗体5F2可以与CD8β链结合而不考虑其配对伙伴，并且可以在不存在人类或小鼠CD8α基因产物的情况下，从用CD8β基因转染的细胞中免疫沉淀CD8β蛋白。抗CD8β抗体应有助于阐明CD8β蛋白生化异质性的程度，并且在确定CD8β蛋白在胸腺细胞和淋巴细胞发育、识别和激活中的作用方面也将是有用的。
