Chentouf Myriam, Ghannam Soufiane, Bès Cédric, Troadec Samuel, Cérutti Martine, Chardès Thierry
Centre National de la Recherche Scientifique (CNRS), Unité Mixte de Recherche 5236, Centre d'études d'agents Pathogènes et Biotechnologies pour la Santé, Faculté de Pharmacie, 15 Avenue Charles Flahault, 34093 Montpellier, France.
J Immunol. 2007 Jul 1;179(1):409-20. doi: 10.4049/jimmunol.179.1.409.
The biological effects of rIgG(1) 13B8.2, directed against the CDR3-like loop on the D1 domain of CD4, are partly due to signals that prevent NF-kappaB nuclear translocation, but the precise mechanisms of action, particularly at the level of membrane proximal signaling, remain obscure. We support the hypothesis that rIgG(1) 13B8.2 acts by interfering with the spatiotemporal distribution of signaling or receptor molecules inside membrane rafts. Upon cross-linking of Jurkat T lymphocytes, rIgG(1) 13B8.2 was found to induce an accumulation/retention of the CD4 molecule inside polyoxyethylene-20 ether Brij 98 detergent-resistant membranes at 37 degrees C, together with recruitment of TCR, CD3zeta, p56 Lck, Lyn, and Syk p70 kinases, linker for activation of T cells, and Csk-binding protein/phosphoprotein associated with glycosphingolipid adaptor proteins, and protein kinase Ctheta, but excluded Zap70 and its downstream targets Src homology 2-domain-containing leukocyte protein of 76 kDa, phospholipase Cgamma1, and p95(vav). Analysis of key upstream events such as Zap70 phosphorylation showed that modulation of Tyr(292) and Tyr(319) phosphorylation occurred concomitantly with 13B8.2-induced Zap70 exclusion from the membrane rafts. 13B8.2-induced differential raft partitioning was epitope, cholesterol, and actin dependent but did not require Ab hyper-cross-linking. Fluorescence confocal imaging confirmed the spatiotemporal segregation of the CD4 complex inside rafts and concomitant Zap70 exclusion, which occurred within 10-30 s following rIgG(1) 13B8.2 ligation, reached a plateau at 1 min, and persisted until the end of the 1-h experiment. The differential spatiotemporal partitioning between the CD4 receptor and the Zap70-signaling kinase inside membrane rafts interrupts the proximal signal cross-talk leading to subsequent NF-kappaB nuclear translocation and explains how baculovirus-expressed CD4-CDR3-like-specific rIgG(1) 13B8.2 acts to induce its biological effects.
抗CD4 D1结构域上类CDR3环的重组免疫球蛋白G1（rIgG(1)）13B8.2的生物学效应部分归因于阻止核因子κB（NF-κB）核转位的信号，但确切的作用机制，尤其是在膜近端信号传导水平，仍不清楚。我们支持这样的假说，即rIgG(1) 13B8.2通过干扰膜筏内信号分子或受体分子的时空分布发挥作用。在Jurkat T淋巴细胞交联后，发现rIgG(1) 13B8.2在37℃时可诱导CD4分子在聚氧乙烯-20醚Brij 98抗去污剂膜内积聚/滞留，同时募集T细胞受体（TCR）、CD3ζ、p56 Lck、Lyn和Syk p70激酶、T细胞活化连接蛋白（LAT）以及与糖鞘脂衔接蛋白相关的Csk结合蛋白/磷蛋白和蛋白激酶Cθ，但排除了Zap70及其下游靶点含Src同源2结构域的76 kDa白细胞蛋白、磷脂酶Cγ1和p95(vav)。对关键上游事件如Zap70磷酸化的分析表明，酪氨酸（Tyr）292和Tyr319磷酸化的调节与13B8.2诱导的Zap70从膜筏中排除同时发生。13B8.2诱导的差异筏分区是表位、胆固醇和肌动蛋白依赖性的，但不需要抗体过度交联。荧光共聚焦成像证实了筏内CD4复合物的时空分离以及Zap70的同时排除，这在rIgG(1) 13B8.2连接后10 - 30秒内发生，1分钟时达到平台期，并持续到1小时实验结束。膜筏内CD4受体和Zap70信号激酶之间的差异时空分区中断了近端信号串扰，导致随后的NF-κB核转位，并解释了杆状病毒表达的CD4 - CDR3样特异性rIgG(1) 13B8.2如何发挥作用以诱导其生物学效应。
