膀胱癌来源的外泌体通过促进髓系抑制性细胞的扩增介导毒性T细胞的免疫抑制

Exosomes derived from bladder cancer mediate the immunosuppression of cytotoxic T lymphocyte by promoting the amplification of myeloid inhibitory cells

目的:探究膀胱癌细胞系MB49来源的外泌体(MB49-Exo)对髓系来源的抑制性细胞(MDSCs)的影响及后者对MB49抗原特异性细胞毒性T淋巴细胞(CTL)免疫功能的调控。方法:试剂盒分离MB49-Exo,并应用透射电子显微镜(TEM)观察外泌体形态,纳米粒径颗粒跟踪分析仪(NTA)和Western blot进行鉴定;构建Balb/c小鼠MB49-Exo荷瘤模型,设置PBS作为阴性对照,MB49荷瘤小鼠为阳性对照,使用流式细胞术检测小鼠骨髓与脾脏中MDSCs的表达及分型和脾脏中CD8^(+)T淋巴细胞数量;分离培养小鼠原代骨髓细胞,流式细胞术检测PBS与MB49-Exo对其向MDSCs分化的影响,并将两组不同处理的MDSCs记为PBS-MDSCs组与MB49-Exo-MDSCs组;使用MB49特异性抗原诱导小鼠成熟树突状细胞(DCs),并利用其制备膀胱癌抗原特异性CTL,再使用PBS-MDSCs与MB49-Exo-MDSCs组的MDSCs进行刺激,ELISA检测不同处理条件下CTL对白介素-1(IL-2)和干扰素-γ(IFN-γ)的表达水平,羟基荧光素二醋酸盐琥珀酰亚胺脂染色结合流式细胞术检测CTL的增殖水平,乳酸脱氢酶法检测CTL对MB49的杀伤活性,最后通过Western blot检测两组MDSCs中信号转导因子和转录激活因子3(STAT3)的磷酸化水平(p-STAT3/STAT3)。结果:成功分离获得MB49-Exo;与对照组相比,MB49-Exo不仅能够明显促进荷瘤小鼠骨髓与脾脏中以粒细胞型MDSCs类型为主MDSCs的形成(P<0.001),还能显著减少脾脏中CD8^(+)T淋巴细胞数量(P<0.001);在体外研究中,较PBS-MDSCs组比较,MB49-Exo-MDSCs组中MDSCs的扩增水平明显升高(P<0.001),且其处理的CTL对IL-2和IFN-γ的分泌水平,细胞增殖能力和对MB49的杀伤活性均明显降低(P<0.001);但MB49-Exo-MDSCs组中STAT3的磷酸化水平较PBS-MDSCs组明显升高(P<0.001)。结论:本研究发现来源于膀胱癌细胞的外泌体能通过促进MDSCs的形成来抑制肿瘤特异性CTL的免疫功能,从而协助肿瘤的免疫逃逸,而外泌体的上述作用可能通过促进MDSCs中激活STAT3信号通路发挥作用。

Objective:To investigate the effect of bladder cancer cell line MB49-derived exosomes(MB49-ExO)on myeloid derived suppressor cells(MDSCs),and the regulation of the MDSCs on the immune function of MB49 antigen-specific cytotoxic T lymphocytes(CTLs).Methods:MB49-Exo was isolated by the exosome isolation kit,and the exosomes morphology was examined by transmission electron microscope(TEM),and identified by nanoparticle size analyzer(NTA)and Western blot,MB49-Exo tumor-bearing model was established in Balb/c mice.PBS was used as negative control,and MB49 tumor-bearing mice as positive control the espression and typing of MDSCs in bone marrow and spleen and the number of CD8^(+)T.Lymphocytes in spleen were determined by drain cell assay.Primary bone marrow cells of mice were isolated and cultured,and the effects of PBS and MB49-Exo on their differentiation to MDSCs were detected by flow cytometry,and the two groups of MDSCs treated differently were recorded as PBS-MDSCs group and MB49-Exo-MDSCs group.Mice mature dendritic cells(DCs)were induced by MB49-specific antigen and used to prepare bladder cancer antigen specific CTL,which was then stimulated by PBS-MDSCs and MDSCs of the MB49-Exo-MDSCs group.ELISA was used to detect the expression levels of interleukin-1(IL-2)and interferon-γ(IFN-γ)of CTL under different treatment conditions.CFSE staining combined with flow cytometry was used to detect the proliferation levels of CTL.LDH was used to detect the cytotoxic activity of CTL against MB49.Finally,the phosphorylation levels of signal transduction factor and transcriptional activator 3(STAT3)in MDSCs of the two groups(p-STAT3/STAT3)were detected by Western blot.Results:MB49-Exo was obtained by successful separation.Compared with the control group,MB49-Exo not only significantly promoted the formation of MDSCs in bone marrow and spleen of tumor-bearing mice(P<0.001),but also significantly reduced the number of CD8^(+)T lymphocytes in spleen(P<0.001).In vitro studies,compared with PBS-MDSCs group,MDSCs amplification level was significantly increased in MB49-Exo-MDSCs group(P<0.001),and the secretion of IL-2 and IFN-γ,cell proliferation ability and cytotoxicity against MB49 of CTL treated by MB49-Exo-MDSCs group were significantly decreased(P<0.001).However,the phosphorylation level of STAT3 in MB49-Exo-MDSCs group was significantly higher than that in PBS-MDSCs group(P<0.001).Conclusion:Exosomes from bladder cancer cells could inhibit the immune function of tumor specific CTLs by promoting the formation of MDSCs,thus assisting the immune escape of tumors.The above effects of exosomes may play a role by promoting the activation of STAT3 signaling pathway in MDSCs.