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

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

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。

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 CDS0, 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 ceils 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 development of full maturity of these cells loaded with Tk should combine with the exogenous maturing signal, such as provided by anti-CD40 monoclonal antibody 5Cll, 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 alone, 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 Th2 cell polarization in vitro.