Wnt通路调控记忆性T细胞的增殖活化

The regulatory role of Wnt signaling pathway in the proliferation and activation of memory T cells

目的探讨Wnt通路在记忆性T细胞(TM)增殖活化中发挥的调控作用。方法建立同种异体(BALB/c-C57BL/6)小鼠皮肤移植模型,采用免疫磁珠方法分选培养C57BL/6小鼠未成熟树突状细胞(imDC)、成熟树突状细胞(mDC)和TM,流式细胞术鉴定。在96孔板或TranswellTM中用imDC和负载BALB/c小鼠抗原的mDC与TM进行混合淋巴细胞反应(MLR)。氚标记的胸腺嘧啶核苷(3H-TdR)检测TM的增殖情况。ELISA检测培养上清中白细胞介素2(IL-2)、IL-10、γ干扰素(IFN-γ)的水平。实时定量PCR检测imDC和mDC中Wnt基因的表达情况。双荧光素酶报告基因检测MLR中TM的β联蛋白/T细胞因子(β-catenin/TCF)信号通路转录活性。结果与对照组相比,在96孔板中负载BALB/c小鼠抗原的mDC显著诱导TM的增殖,上调IL-2和IFN-γ,下调IL-10的表达。mDC中Wnt3a和Wnt5a的mRNA表达水平显著高于imDC。mDC与TM共培养可显著增强TM的β-catenin/TCF信号通路转录活性。而在TranswellTM中培养的细胞基本未见细胞增殖。结论负载供者抗原的受者小鼠mDC可通过直接接触的方式有效激活受者效应型TM的增殖,Wnt通路的β-catenin/TCF转录激活可能是介导TM活化增殖的重要信号通路。

Objective To study the regulatory effect of Wnt signaling pathway on the activation and proliferation of memory T cells （TMs）. Methods We established the mouse model of skin allograft transplantation （BALB/c-C57BI_/6）, and then we sorted and cultured immature dendritic cells （imDCs）, mature dendritic cells （mDCs） and TMs from the C57BL/6 mice by immunomagnetic bead cell sorting. The imDCs or mDCs loading antigens from BALB/c mice were co-cultured with TMs in a 96-well plate or TranswellTM plate （MLR）. The 3H-TdR was used to detect the proliferation of TMs. In the co-culture supematants, IL-2, IL-10 and IFN- were detected by ELISA. The Writ mRNA expression levels in imDCs and mDCs were determined by real-time PCR. Dual-luciferase reporter assay was performed to detect the transcriptional activity of ^-catenin/ TCF signaling in TMs. Results Compared with the control group, mDCs loading antigens from BALB/c mice induced remarkably the proliferation of TMs, increased the expressions of IL-2 and IFN-y, and decreased the expression of IL-10. The expression levels of Wnt3a and Wnt.Sa were significantly higher in mDCs than in imDCs. The transcriptional activity of LS-catenin/TCF signaling pathway in TMs was obviously enhanced after co-cultured with mDCs. Conclusion The mDCs loading donor-specific antigens from the recipient mice could induce the proliferation of effector TMs of recipients through direct contact, and Wnt signaling pathway mediated β-catenin/TCF transcriptional activation may be one of the important signaling pathways to induce the activation and proliferation of TMs.