Renca细胞exosomes所诱导的CD8^+T细胞的特异性杀伤作用

Specific cytotoxicity of CD8^+ T cells induced by Renca cell-derived exosomes

目的检测Renca细胞exosomes对CD8^+T细胞的激活效应以及exosomes负载的树突状细胞(DCs)所诱导的CD8^+T细胞对Renca细胞特异性杀伤作用。方法采用超速离心法从Renca细胞无血清上清液中提取出exosomes并鉴定,体内通过流式和ELISA;体外通过CFSE荧光染色和ELISA检测exosomes对CD8^+T细胞的激活效应。小鼠骨髓分离出DCs并进行体外培养,用exosomes冲击成熟的DCs然后活化CD8^+T细胞,测定其对Renca、4T1、CT26细胞的杀伤率。结果透射电镜下观察exosomes是直径在30~100 nm的盘状囊泡,Western blot检测exosomes高表达MHC-1和HSP70。Exosomes能在体内和体外促进CD8^+T细胞的增殖和激活效应。在体外杀伤实验中,exosomes冲击的DCs所诱导的CD8^+T细胞相对于4T1和CT26细胞,其对Renca细胞具有明显更强的杀伤作用。结论 Renca细胞exosomes对CD8^+T细胞具有明显的激活作用且exosomes负载的DCs所诱导的CD8^+T细胞对自体肿瘤细胞具有强烈的特异性杀伤能力。

Tumor exosome is a kind of intercellular communication material that can induce anti-tumor immune effect. This study was performed to detect the activation effect of exosomes on CD8^+T cells and the specific cytotoxicity of CD8^+T cells induced by exosomes-loaded DCs against Renca cells. Exosomes derived from Renca cells were separated and purified by traditional ultracentrifugation. The activation effect of exosomes on CD8^+T cells was determined by fluorescence staining with carboxyfluorescein diacetate succinimidylamino ester(CFSE), flow cytometry and ELISA. Then, DCs were isolated from mouse bone marrow and cultured in vitro to induce CD8^+T cells. The specific cytotoxicity of CD8^+T cells induced by exosomes-loaded DCs was investigated by LHD release assays. Data showed that exosomes were isolated as expected and visualized as spherical membrane-bound vesicles with a diameter of 30-100 nm. In addition, the expression of molecular markers MHC-1, HSP-70 were increased in exosomes compared with Renca cells. Exosomes can promote the proliferation and activation of CD8^+T cells in vivo and in vitro. Furthermore, immunization with exosomes facilitated the stimulation of a stronger specific cytotoxic CD8^+T cell response against Renca cells than against 4 T1 and CT26 cells in vitro. In conclusion, exosomes from Renca cells have a strong activation effect on CD8^+T cells, and the CD8^+T cells induced by exosomes-loaded DCs can trigger strong specific cytotoxicity against autologous tumor cells.