负载灭活口蹄疫病毒树突状细胞对CD8^+T细胞的旁位活化效应

Bystander activation of CD8^+T lymphocytes by inactivated foot-and-mouth disease virus pulsed-dendritic cells

目的:探讨在体外条件下负载灭活口蹄疫病毒(FMDV)树突状细胞活化CD8+T细胞的机制。方法:用重组粒细胞-巨噬细胞集落刺激因子(rmGM-CSF)和白介素-4(rmIL-4)将小鼠外周血单核细胞诱导分化成树突状细胞(MoDCs),以信号阻断法从淋巴结T细胞制备CD8+T细胞,利用负载灭活FMDV的MoDCs与CD8+T细胞共培养,以FMDV+MoDCs+淋巴结T细胞作为对照,用ELISA检测不同时间培养上清液中IFN-γ的含量。用蛋白酶体抑制剂和溶酶体抑制剂处理MoDCs,2小时后再负载灭活FMDV,并与CD8+T细胞共培养,对照组则用FMDV+MoDCs+CD8+T细胞和FMDV+MoDCs,用ELISA检测不同时间培养上清液中IFN-γ的含量。结果:在共培养9小时,CD8+T细胞的IFN-γ释放量约为淋巴结T细胞IFN-γ释放量的1/6,随后,淋巴结T细胞和CD8+T细胞均产生少量IFN-γ。用灭活FMDV负载被lactacystin和氯喹处理的DCs与CD8+T细胞共培养后9～12小时,CD8+T细胞产生IFN-γ显著减少,在共培养24小时,IFN-γ释放显著增多,并在36小时达到峰值,与对照组相比,差异有统计学意义。结论:负载灭活FMDV的MoDCs可以活化淋巴结T细胞,而且,还可通过非MHCⅠ和非MHCⅡ类分子依赖途径激活CD8+T细胞,此即旁位活化效应。

Objective：To study the mechanism of CD8 ^ ＋ T cell activation by dendritic cells loaded with inactivated FMDV in vitro. Methods： Monocyte-derived dendritic cells（MoDCs） were generated from peripheral blood monoytes by adding to rmGM-CSF and rmIL-4 in the medium. CD8^＋ T cells were produced by depletion of CD4^ ＋ T cells with appropriate antibody in the lymph node T cells. MoDCs pulsed with inactivated FMDV were co-cultured with CD8^＋ T cells. Inactivated FMDV pulsed-MoDCs were cocultured with lymph node T cells as control. The production of the IFN-γ was determined at different time points with ELISA. The MoDCs were incubated with proteasome inhibitor and lysosome inhibitor for 2 h and then pulsed with inactivated FMDV and co-cultured with CD8^＋ T cells. Inactivated FMDV pulsed-MoDCs alone in culture was utilized as control.The production of the IFN-γ was determined at different time points by using ELISA. Results：At 9h post-co cuhuration, the level of IFN-γ in supematant of lymph node cell culture was approximately six fold higher than that of CD8 ^＋ T cells in culture medium. Subsequently, small amount of IFN-γ produced by the 1）anph node T cells and CD8^ ＋ T cells was determined, respectively. The inhibitor group＇s level of IFN-γ was remarkably reduced from 9 h through 12 h. By contrast, the quantity of IFN-γ produced was significantly higher in inhibitor group compared to the control groups at 24 h post-co cuhuration. And the level of IFN-γ in inhibitor group reached peak at 36 h post-co culturation in comparison with the control groups. Conclusion： MoDCs loaded with inactivated FMDV have the capability of initiating T cell response. Furthermore, inactivated FMDV pulsed MoDCs can activated naive CD8^＋ T cells independently of both MHC Ⅰ and MHC Ⅱ molecule pathways, which is known as bystander activation.