Zumla A, Marguerie C, So A, Yokoyama W M, Saito T, Batchelor J R, Lechler R I
Department of Immunology, Royal Postgraduate Medical School, Hammersmith Hospital, London, UK.
J Immunol Methods. 1992 Apr 27;149(1):69-76. doi: 10.1016/s0022-1759(12)80050-0.
In this study we demonstrate that human T cell receptor (TcR) chains can be co-expressed with murine CD3 on the cell surface of a murine T cell hybridoma. Human TcR alpha and beta genes from the Jurkat leukaemic cell line were transfected into a TcR-negative mouse T cell hybridoma, TG40. The Jurkat TcR was successfully co-expressed at the cell surface with mouse CD3 components. Brightly staining transfectants were selected by fluorescence-activated cell sorting, and levels of expression comparable to a normal T cell line were achieved suggesting that the human TcR dimer assembled efficiently with the mouse CD3 complex. Northern blot analysis demonstrated similar levels of TcR messenger RNA to those found in the parental Jurkat line. Although we have not formally demonstrated surface expression of the Jurkat TcR alpha chain, the residual alpha gene transcript which is present in murine TG40 line is non-expressible. In order to test the signalling capacity of this human/mouse complex, the transfectants were stimulated with an anti-V beta 8 monoclonal antibody. This stimulus led to interleukin-2 production by the transfectants, demonstrating the delivery of a transmembrane signal. In addition, B10.A mice were immunised with the transfectants, and the antisera from these mice stained the transfectant and the Jurkat cell line, but not the parental T cell hybridoma. This interspecies transfection approach should now permit us to explore the requirements for T cell activation, the constraints on TcR alpha beta chain pairing, and creates ideal reagents for inducing a mouse anti-human TcR-specific response with a view to producing panels of anti-human TcR monoclonal antibodies.
在本研究中，我们证明人T细胞受体（TcR）链可与鼠CD3在鼠T细胞杂交瘤的细胞表面共表达。将来自Jurkat白血病细胞系的人TcRα和β基因转染到TcR阴性的小鼠T细胞杂交瘤TG40中。Jurkat TcR成功地与小鼠CD3成分在细胞表面共表达。通过荧光激活细胞分选选择染色明亮的转染子，实现了与正常T细胞系相当的表达水平，这表明人TcR二聚体与小鼠CD3复合物有效组装。Northern印迹分析显示TcR信使RNA水平与亲本Jurkat系中的水平相似。虽然我们尚未正式证明Jurkat TcRα链的表面表达，但存在于鼠TG40系中的残留α基因转录本是不可表达的。为了测试这种人/鼠复合物的信号传导能力，用抗Vβ8单克隆抗体刺激转染子。这种刺激导致转染子产生白细胞介素-2，证明了跨膜信号的传递。此外，用转染子免疫B10.A小鼠，并从这些小鼠获得的抗血清可染色转染子和Jurkat细胞系，但不能染色亲本T细胞杂交瘤。这种种间转染方法现在应该使我们能够探索T细胞激活的要求、TcRαβ链配对的限制，并创造理想的试剂来诱导小鼠产生抗人TcR特异性反应，以期生产抗人TcR单克隆抗体库。
