肾细胞癌源性exosomes体外诱导单核细胞分化为PD-L1髓源性抑制细胞

目的研究肾细胞癌源性exosomes诱导人外周血单核细胞分化为髓源性抑制细胞及该细胞PD-L1分子表达情况的效应。方法超滤和蔗糖重水密度梯度离心相结合的方法分离纯化肾细胞癌源性exosomes。Ficoll密度梯度离心法分离外周血单个核细胞,贴壁法获取外周血单核细胞。倒置显微镜观察获得的单核细胞形态。流式细胞仪检测单核细胞比例及活性、经exosomes诱导的单核细胞活性情况、髓源性抑制细胞(MDSCs)及其上PD-L1分子的表达情况。结果贴壁的单核细胞形状为圆形或类圆形具少许短小伪足状突起;贴壁法获得的单核细胞比例达83.5%,活细胞比例约90.0%;髓源性抑制细胞的生成较对照组明显增加[(8.91±0.21)%vs(3.19±0.91)%,P<0.05],且该髓源性抑制细胞表达PD-L1较对照组明显增高[(88.93±8.57)%vs(44.53±5.35)%,P<0.05];体外经exosomes诱导刺激的单核细胞损伤率较对照组明显升高[(39.93±16.51)%vs(12.87±2.43)%,P<0.05]。结论肾细胞癌源性exosomes在体外能够促进单核细胞损伤,诱导单核细胞分化为髓源性抑制细胞,且该髓源性抑制细胞表达PD-L1增高,可能是其抑制T细胞免疫功能的负性调节机制之一。

Renal cell carcinoma-derived exosomes induce monocytes to differentiate into PD-L1 myeloid derived suppressor cells in vitro

Objective To assess the process of renal cell carcinoma-derived exosomes(RCC-exo) inducing human peripheral blood monocytes to differentiate into myeloid-derived suppressor cells(MDSCs) and determine the expression of programmed death ligand 1(PD-L1) in the differentiated cells.Methods RCC-exo were isolated and purified by ultrafiltration and sucrose gradient centrifugation,and characterized by transmission electron microscopy.Ficoll density gradient centrifugation was used to isolate peripheral blood mononuclear cells(PBMC) from healthy donor,and monocytes were amplified and purified from PBMC,which characterized by inverted microscopy.The obtained monocytes were induced by RCC-exo for 48 h,and those without treatment served as control.Flow cytometry were used to analyze the impairment of monocytes,the presence of MDSCs and the expression of PD-L1.Results Attached monocytes were round or round-like in shape,with a few pseudopod processes.The yield rate was 83.5%,and 90.0% obtained cells were alive.Compared with control group,RCC-exo obviously impaired monocytes [(39.93±16.51)% vs(12.87±2.43)%,P<0.05)],and promoted myeloid cells differentiation into MDSCs [(8.91±0.21)% vs(3.19±0.91)%,P<0.05].Among the obtained cells,the percentage of PD-L1 MDSCs was(88.93±8.57)%,significantly higher than that of control group [(44.53±5.35)%,P<0.05].Conclusion RCC-exo induces monocytes impairment,and the cells differentiate into MDSCs with significantly increased PD-L1.PD-L1 and PD-1 interaction may be one of the pathways that MDSCs suppress T cells immune response.