Boyer C, Auphan N, Luton F, Malburet J M, Barad M, Bizozzero J P, Reggio H, Schmitt-Verhulst A M
Centre d'Immunologie, INSERM-CNRS de Marseille, France.
Eur J Immunol. 1991 Jul;21(7):1623-34. doi: 10.1002/eji.1830210707.
The fate of the T cell receptor (TcR)/CD3 complex was examined on a cytotoxic T lymphocyte (CTL) clone (KB5.C20) activated either via binding of an anti-TcR monoclonal antibody (mAb) or by a Ca2+ ionophore and phorbol 12-myristate 13-acetate (PMA). After binding of the anti-TcR mAb, electron microscopy revealed internalization through coated vesicles followed by slow degradation of the antibody as shown by use of radiolabeled mAb. The influence of activation on TcR/CD3 internalization was analyzed. The Ca2+ ionophore alone had no effect on internalization, whereas PMA induced an accelerated internalization of anti-TcR mAb. PMA-induced internalization was dependent on protein kinase C (PKC) as shown by its absence in PKC-depleted cells or in the presence of the PKC inhibitor staurosporine. Anti-TcR mAb-induced internalization was maintained in PKC-depleted cells, but unexpectedly remained sensitive to inhibition by staurosporine. The monovalent anti-TcR mAb Fab fragment is non-stimulatory for the CTL. It was poorly internalized but its internalization was induced by PMA. Surprisingly, on PKC-depleted cells, the Fab was internalized more readily than in untreated cells and this internalization was sensitive to inhibition by staurosporine. Inhibition of PMA-induced phosphorylation of gamma and epsilon subunits of CD3 was demonstrated after depletion of PKC or in the presence of staurosporine, confirming that PKC function was inhibited in those conditions. Cross-linking of the TcR via plastic-coated anti-TcR mAb led to phosphorylation of CD3 gamma and epsilon and also of zeta, known to be phosphorylated on tyrosines. All of these phosphorylation events were inhibited by treatment with staurosporine. Our results indicate that staurosporine inhibits the receptor internalization induced by anti-TcR mAb by means other than inhibition of PKC, suggesting that other kinases may control a step of this internalization process.
通过抗T细胞受体（TcR）单克隆抗体（mAb）结合或通过钙离子载体和佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯（PMA）激活细胞毒性T淋巴细胞（CTL）克隆（KB5.C20）后，研究了TcR/CD3复合物的命运。抗TcR mAb结合后，电子显微镜显示其通过被膜小泡内化，随后抗体缓慢降解，放射性标记的mAb实验证实了这一点。分析了激活对TcR/CD3内化的影响。单独的钙离子载体对内化没有影响，而PMA诱导抗TcR mAb加速内化。如在蛋白激酶C（PKC）缺失的细胞中或存在PKC抑制剂星形孢菌素时所显示的，PMA诱导的内化依赖于PKC。抗TcR mAb诱导的内化在PKC缺失的细胞中得以维持，但出乎意料的是，其仍对星形孢菌素的抑制敏感。单价抗TcR mAb Fab片段对CTL无刺激作用。它内化较差，但PMA可诱导其内化。令人惊讶的是，在PKC缺失的细胞中，Fab比未处理的细胞更容易内化，且这种内化对星形孢菌素的抑制敏感。在PKC缺失或存在星形孢菌素的情况下，证实了PMA诱导的CD3γ和ε亚基磷酸化受到抑制，这表明在这些条件下PKC功能被抑制。通过塑料包被的抗TcR mAb使TcR交联导致CD3γ和ε以及已知在酪氨酸上被磷酸化的ζ磷酸化。所有这些磷酸化事件都被星形孢菌素处理所抑制。我们的结果表明，星形孢菌素通过抑制PKC以外的其他方式抑制抗TcR mAb诱导的受体内化，这表明其他激酶可能控制这一内化过程的一个步骤。
