CD39介导GSCs抵抗辐射诱导ICD的初步探究

Preliminary study of CD39-mediated GSCs resistance to radiation-induced ICD

脑胶质瘤干细胞(glioma stem cells,GSCs)的辐射抗性造成放疗后脑胶质瘤复发率高,CD39介导GSCs抵抗辐射诱导的免疫原性细胞死亡(immunogenic cell death,ICD)可能是放疗耐受的重要原因。目的:深入阐释CD39介导GSCs抵抗辐射诱导ICD的分子机制。方法:免疫荧光染色法和流式细胞术检测GSCs干性标记物、ICD标记物、抑制CD39后胞外ATP变化、ATP对DCs炎症小体激活以及吞噬功能的影响。利用Western blot和流式细胞术检测HIF-1α与CD39相关性。利用流式细胞术检测抑制CD39后激活T淋巴细胞的增殖分化和杀伤效应。结果:GSCs的ICD标记物分子普遍低表达,通过膜表面CD39水解胞外ATP,维持免疫抑制微环境。抑制CD39则阻断ATP水解,显著增强“吃我”信号,吸引树突状细胞(dendritic cells,DCs)聚集。ATP激活DCs的P2X7受体,从而激活NF-κB p65-NLRP3-Caspase-1 p20-IL-1β炎症小体信号通路,促进pro-IL-1β转向成熟的IL-1β释放,引发炎症反应。DCs也向T细胞递呈抗原增加,MHC分子表达水平上调,最终激活淋巴细胞杀伤效应。结论:抑制CD39表达能明显增强辐射诱导的肿瘤细胞ICD,通过激活NF-κB p65-NLRP3-Caspase-1 p20-IL-1β信号通路,促进淋巴细胞免疫反应。

Radiation resistance of glioma stem cells(GSCs)causes high recurrence rate of glioma after radiotherapy.CD39 mediated GSCs resistance to radiation-induced immunogenic cell death(ICD)may be an important cause of radiotherapy tolerance.Objective:It is important to further elucidate the CD39 mediated GSCs resistance to radiation-induced ICD.Methods:Immunofluorescence staining and flow cytometry were used to detect stemness markers in GSCs,ICD markers,extracellular ATP changes after CD39 inhibition,the effect of ATP on inflammatory vesicle activation in DCs,and phagocytosis.The correlation between HIF-1αand CD39 was examined using Western blot and flow cytometry.The proliferative differentiation and killing effects of activated T lymphocytes after CD39 inhibition were examined by using flow cytometry.Results:ICD marker molecules of GSCs were generally under-expressed and the immunosuppressive microenvironment was maintained by hydrolyzing extracellular ATP through CD39 on the membrane surface.Inhibition of CD39 could block ATP hydrolysis,which significantly enhanced“find me”signal and attracted dendritic cells(DCs)to aggregate.ATP activated the P2X7 receptor of DCs,which could further activate the NF-κB p65-NLRP3-Caspase-1 p20-IL-1βinflammasome signaling pathway,promote the release of pro-IL-1βto mature IL-1β,and trigger an inflammatory response.DCs also increased antigen delivery to T cells,the expression level of MHC molecules was upregulated,and ultimately the lymphocytic killing effect was activated.Conclusion:Inhibition of CD39 expression can significantly enhance radiation-induced tumor cell ICD,and promote lymphocyte immune response by activating NF-κB p65-NLRP3-Caspase-1 p20-IL-1βsignaling pathway.