Steenbakkers P G, Boots A M, Rijnders A W
Department of Immunology, N.V. Organon, Oss, The Netherlands.
J Immunol Methods. 1997 Dec 15;210(1):51-64. doi: 10.1016/s0022-1759(97)00176-2.
Monoclonal antibodies (mAb) directed against the clonotypic structure of the T-cell receptor (TCR) may be useful reagents in the study and therapy of T-cell-mediated diseases. In contrast to several reports concerning the generation of anti-clonotype mAb to mouse TCR, only very limited numbers of anti-clonotype mAb to human TCR have been described. So far, a suitable method for the generation of anti-clonotype mAb to a given TCR has not been available and in this report we describe a novel strategy for the generation of such mAb. Mice were immunized with intact human T-cells. Then. spleen cell populations were precleared from B-cells reactive to CD3 and the constant region of the TCR by adsorption to TCR/CD3 complexes derived from an irrelevant T-cell clone. Subsequently, clonotype-specific B-cells were selected with TCR/CD3 complexes from the T-cell clone of interest. The small number of B-cells resulting from this selection were clonally expanded in a B-cell culture system and then immortalized by mini-electrofusion. Ten clonotype-specific mAb were generated against a DRB1*0401-restricted T-cell clone recognizing an epitope of the human cartilage glycoprotein 39 (HC gp-39). All mAb immunoprecipitated a heterodimeric 85 kDa protein. Absolute specificity was demonstrated in a T-cell agglutination test with the T-cell clone of interest compared to a set of 16 defined, irrelevant T-cell clones or lines. In functional assays, the mAb were found to inhibit or block antigen-specific T-cell stimulation. In addition, crosslinked mAb were found to stimulate proliferation of the specific clone in the absence of antigen and antigen presenting cells (APC). Such mAb may have clinical relevance in deleting or modulating autoreactive T-cells in a clonotype-specific manner.
针对T细胞受体（TCR）克隆型结构的单克隆抗体（mAb）可能是研究和治疗T细胞介导疾病的有用试剂。与关于产生抗小鼠TCR克隆型mAb的若干报道相反，针对人TCR的抗克隆型mAb的描述非常有限。到目前为止，尚未有适用于产生针对给定TCR的抗克隆型mAb的方法，在本报告中，我们描述了一种产生此类mAb的新策略。用完整的人T细胞免疫小鼠。然后，通过吸附到源自无关T细胞克隆的TCR/CD3复合物上，从对CD3和TCR恒定区有反应的B细胞中预先清除脾细胞群体。随后，用来自感兴趣的T细胞克隆的TCR/CD3复合物选择克隆型特异性B细胞。通过这种选择产生的少量B细胞在B细胞培养系统中进行克隆扩增，然后通过微电融合使其永生化。针对识别人类软骨糖蛋白39（HC gp-39）表位的DRB1*0401限制性T细胞克隆产生了十种克隆型特异性mAb。所有mAb均免疫沉淀出一种85 kDa的异二聚体蛋白。与一组16个明确的无关T细胞克隆或系相比，在与感兴趣的T细胞克隆进行的T细胞凝集试验中证明了绝对特异性。在功能测定中，发现这些mAb可抑制或阻断抗原特异性T细胞刺激。此外，发现交联的mAb在没有抗原和抗原呈递细胞（APC）的情况下可刺激特异性克隆增殖。此类mAb可能在以克隆型特异性方式清除或调节自身反应性T细胞方面具有临床相关性。
