黑色素瘤B16细胞通过释放外泌体诱导naïve CD4^(+) T细胞分化为Treg  

B16 cells induce naïve CD4^(+)T cell differentiation into Tregs via releasing exosomes

作　　者：赵丽 顾洪照 张红玉[2] ZHAO Li;GU Hongzhao;ZHANG Hongyu(Department of Dermatology,901 Hospital of Joint Logistic Support Force of the People's Liberation Army,Hefei 230031,China;Department of Emergency,Second Affiliated Hospital of Chongqing Medical University,Chongqing 400000,China)

机构地区：[1]中国人民解放军联勤保障部队第九〇一医院皮肤科,合肥230031 [2]重庆医科大学附属第二医院急诊科,400000

出　　处：《免疫学杂志》2022年第6期524-529,共6页Immunological Journal

摘　　要：目的检测小鼠黑色素瘤细胞B16通过释放外泌体诱导naïve CD4^(+)T细胞分化为调节性T细胞(regulatory T cell,Treg)的能力。方法通过试剂盒和超速离心获得B16培养上清中的外泌体,用透射电镜观察外泌体形态,Western blot检测外泌体标记性蛋白的表达水平,通过Transwell共培养验证外泌体或B16细胞对naïve CD4^(+)T细胞诱导分化的能力,流式细胞术检测小鼠脾脏、肿瘤内Th17、Treg细胞的频率,qPCR检测T细胞亚群特异性转录因子、炎症因子的mRNA水平。结果B16细胞外泌体的直径为30~100 nm,呈圆形碟状结构,外泌体高表达Tsg101、HSP60和Alix,低表达GAPDH;在体外,B16细胞外泌体可直接诱导naïve CD4^(+)T细胞分化成Treg,但不能诱导其分化为Th17;小鼠在体实验证实,B16细胞外泌体能够诱导Treg细胞并聚集于小鼠脾脏中,同时能够增加B16瘤体内Treg细胞的数量,并活化使其表达更高水平的IL-10、TGF-β和Foxp3。结论B16细胞外泌体通过诱导naïve CD4^(+)T细胞分化为Treg,并将其募集至肿瘤组织,从而维持肿瘤的免疫抑制微环境。This study was designed to detect the ability of mouse melanoma cell B16 to induce naïve CD4^(+)T cells differentiation into regulatory T cells(Treg)by releasing exosomes.The exosomes in B16 culture supernatant were obtained by kit and ultracentrifugation.The morphology of exosomes was observed by transmission electron microscopy,and the level of exosome marker proteins were detected by Western blotting.Transwell co-culture was used to verify the differentiation of naïve CD4^(+)T cells induced by exosomes or B16 cells.The frequency of Th17 and Treg in spleen and tumor were detected by flow cytometry,and the expression levels of T cell subsets specific transcription factors and inflammatory factors were detected by q-PCR.Data showed that the diameter of B16 cell exosomes was 30~100 nm,with circular disc structure and high expression of Tsg101,HSP60 and Alix,but low expression of GAPDH.In vitro,B16 cell exosomes could directly induce naïve CD4^(+)T cells to differentiate into Treg,but not Th17;in vivo experiments in mice confirmed that B16 cell exosomes could induce more Treg cells to accumulate in the spleen of normal mice.At the same time,B16 cell exosomes also increased the number of Treg cells in B16 tumor and activated them to express higher levels of IL-10,TGF-βand Foxp3.In conclusion,B16 cell exosomes maintain the immunosuppressive microenvironment of tumor by activating naïve CD4^(+)T cells to differentiate into Treg and recruit to tumor.

关 键 词：外泌体 naïve CD4^(+)T细胞 TREG 黑色素瘤细胞B16 

分 类 号：R392.32[医药卫生—免疫学]