天花粉蛋白联合CD40信号激发的人MoDC介导Th2细胞分化的研究

探讨天花粉蛋白(Tk)联合CD40信号诱导人单核细胞来源DC(MoDC)的成熟活化及其介导Th2细胞分化的作用和机制。采用GM-CSF和IL-4联合方案体外诱导人MoDC,并利用鼠抗人CD40激发型单抗(5C11)刺激负载了Tk的DC制备成熟DC。采用免疫荧光标记和流式细胞术分析DC表型(CD80、CD83、CD86、CD14、PDL1)及其摄取FITC-Dextran的能力;ELISA法测定DC上清中IL-12p70的含量;胞内染色和流式细胞术检测经成熟DC活化的T细胞中CD4~+IFN-γ~+T和CD4~+IL-4~+T的比例。实验结果显示单独Tk不能刺激DC完全成熟,用Tk负载DC后必须联合外源性成熟刺激信号(如5C11),才能有效促进DC成熟,表现在CD80、CD83、CD86的上调表达,CD14下调表达,DC摄取FITC-Dextran的能力降低。与CD40信号单独诱导组相比,Tk联合CD40信号诱导的成熟DC表面PDL1分子呈下调表达,分泌IL-12的能力显著降低,明显提高T细胞中CD4~+IL-4~+T的比例。由此表明负载了Tk的成熟MoDC体外能有效介导Th2细胞分化。

The human monocyte-derived dendritic cells stimulated by trichosanthin combined with CD40 signaling mediate the Th2 cell polarization

To investigate the role and mechanism on the maturation and activation of the human monocyte-derived dendritic cells(MoDC)stimulated by trichosanthin(Tk)combined with CD40 signaling and its mediated differentiation of Th2 cells,the human MoDC were induced in vitro using GM-CSF and IL-4,and the Tk-pulsed DC were induced for further maturation by ag- onistic anti-CD40 monoclonal antibody 5C11 for 48 hus.The expression of the surface molecules including CD80,CD83, CD86,CD14,PDL1 on DC and the capacity to take up FITC-Dextran were determined by flow cytometry(FCM).Mean- while,the concentration of IL-12p70 in the supernatants of DC cultures was detected by ELISA.Intracellular staining and FCM were used to detect the percentages of the CD4~+ IFN-γ~+ Tcells and CD4~+ IL-4~+T cells in T cell populations activated by mature DC.The experimental results showed that Tk alone was unable to induce the full maturity of MoDC.and the develop- ment of full maturity of these cells loaded with Tk should combine with the exogenous maturing signal,such as provided by an- ti-CD40 monoclonal antibody 5C11,which would make DC to up-regulate the expressions of CD80,CD83 and CD86,but to down-regulate that of CD14 and to decrease the uptake of FITC-Dextran by DC.Comparing to those with CD40 signaling a- lone,the Tk-combined CD40 signaling down-regulated the expression of PDL1 and obviously decreased the secretion of IL- 12,as well as remarkably increased the proportion of CD4~+ IL-4~- T cells in T cell population.Taken together,it is apparent that the mature human monocyte-derived dendritic cells loaded with trichosanthin are potent to mediate the Tb2 cell polarization in vitro.