调节性DC分泌的外体诱导免疫耐受功能的体外研究

为了探讨树突状细胞(DC)分泌的外体(Dex)在诱导T细胞免疫耐受中的作用,体外研究采用供体Dex降低同种异体移植排斥的可能性。从正常人外周血单个核细胞中诱导培养未成熟DC(imDC),用TGF-β1联合IL-10诱导调节性DC,LPS诱导DC成熟。采用流式细胞术方法观察TGF-β1和IL-10对DC表型、吞噬功能的影响;采用超速离心和超滤的方法提纯Dex;Western blot方法检测imDC分泌的Dex(imDex)与调节性DC分泌的Dex(rDex)表达的相关分子;通过CCK-8法分析异源iDex和mDex在混合淋巴细胞反应(MLR)中的生物学功能,并比较rDex与iDex诱导免疫耐受的能力。结果显示,TGF-β1和IL-10可下调DC表面的共刺激分子CD80、CD83、CD86的表达,并诱导调节性DC分泌更多的rDex;异源的mDC分泌的Dex(mDex)在mDC存在时增强MLR,而异源的imDex在imDC存在时一定程度上抑制MLR,rDex诱导的抑制T细胞增殖作用显著强于iDex;rDex表达更多的FasL,提示TGF-β1和IL-10诱导的调节性DC分泌的rDex在免疫耐受中发挥重要作用,有望应用于同种异体移植抗免疫排斥。

In vitro induction of immune tolerance by exosomes secreted from regulatory dendritic cells

To investigate the role of the exosomes(Dex) secreted from dendritic cells(DC) to induce the immune tolerance of T lymphocytes and the feasibility of reducing allo-transplantation rejection by donor Dex in vitro,the immature DC(imDC)were induced from peripheral blood mononuclear cells and the regulatory DC(rDC)were induced by treating the imDC with cytokines TGF-β1 and IL-10. Meanwhile,the mature DC(mDC)were treated with LPS,and then the phenotypes of regulatory and normal DC as well as their capacity in phagocytosis were assayed by flow cytometry. The Dex from rDC and normal DC were isolated by ultracentrifugation and ultrafiltration and the Dex secreted by imDC(imDex) and rDC(rDex)function associated molecules were assayed by Western blotting. In addition,the biological function of allogeneic imDex and mDex in allogeneic mixed lymphocyte reaction (MLR)and the effect of immune tolerance induced by rDex and imDex were analyzed by cell counting kiy-8. It was demonstrated that TGF-β1 and IL-10 could down-regulate the expressions of the costimulatory molecules,including CD80,CD83 and CD86 and induced rDC to secrete more rDex than imDC. In the presence of mDC,the allogeneic mDex could stimulate the proliferation of the allogenic T lymphocytes,however,the imDex suppressed the proliferation of allogeneic T lymphocytes,when imDex was presented in MLR. Moreover,the suppression of rDex was significantly stronger than that of imDex,and the rDex expressed more FasL,leading to a more potent suppression on MLR. From these observations,it is evident that rDex secreted by DC induced with TGF-β1 and IL-10 plays an important role in immune tolerance and it might has the potential application to be used in the prevention of allo-transplantation rejection in the future.