Notch信号通路对甲型H1N1流感病毒感染者特异性CD8+T细胞的调控作用  

Regulatory effects of Notch signaling pathway on viral specific CD8 T cells in patients with influenza A H1N1 virus infection

作　　者：李吉梅[1] 许玲芬[1] LI Jimei;XU Lingfen(Department of General Medicine,Qinghai Provincial People’s Hospital,Xining 810007,China)

机构地区：[1]青海省人民医院全科医学科,西宁810007

出　　处：《免疫学杂志》2021年第6期528-534,540,共8页Immunological Journal

摘　　要：目的检测Notch受体和配体在甲型H1N1流感患者中的表达,观察Notch信号通路对甲型H1N1流感病毒特异性CD8+T细胞的调控作用。方法本研究入组轻症甲型H1N1流感患者27例、重症甲型H1N1流感患者11例和健康对照者18例。实时定量PCR法检测外周血单个核细胞(PBMC)中Notch受体和配体mRNA的相对表达量。PBMC与Notch信号通路抑制剂DAPT刺激培养24 h后,流式细胞术检测病毒特异性CD8+T细胞分泌肿瘤坏死因子-α(TNF-α)和干扰素-γ(IFN-γ)的变化,酶联免疫斑点试验(ELISPOT)检测病毒特异性CD8+T细胞分泌穿孔素和颗粒酶B的变化。纯化HLA-A2限制性患者CD8+T细胞,DAPT刺激培养24 h后建立与甲型H1N1病毒株感染的NCI-H345细胞的直接接触和间接接触培养系统,通过检测乳酸脱氢酶的释放计算CD8+T细胞诱导靶细胞死亡比例,酶联免疫吸附试验(ELISA)检测培养上清TNF-α和IFN-γ水平,评估CD8+T细胞非细胞杀伤功能。结果轻症和重症甲型H1N1流感患者PBMC中Notch受体(Notch1、Notch2、Notch3、Notch4)和Notch配体(DLL1、DLL4、Jagged1、Jagged2)mRNA相对表达量均高于对照组(P<0.05)。重症甲型H1N1流感患者病毒特异性CD8+T细胞分泌IFN-γ的细胞比例、分泌穿孔素和颗粒酶B的细胞数量均高于轻症患者(P<0.05)。DPAT刺激抑制甲型H1N1流感患者病毒特异性CD8+T细胞分泌TNF-α和IFN-γ的细胞比例以及分泌穿孔素和颗粒酶B的细胞数量(P<0.05)。直接培养系统中,DAPT刺激均可降低病毒特异性CD8+T细胞诱导的靶细胞死亡比例(P<0.05),培养上清中TNF-α和IFN-γ水平均亦降低(P<0.01)。间接接触培养系统中,虽然DAPT刺激后培养上清TNF-α和IFN-γ水平降低(P<0.01),但CD8+T细胞诱导的靶细胞死亡比例的差异在经DAPT刺激和无DAPT刺激之间的差异无统计学意义(P=0.149)。结论甲型H1N1流感患者中升高表达的Notch受体和配体可增强病毒特异性CD8+T细胞的直接细胞杀伤功能,促进疾病进�This study was performed to investigate the expression of Notch receptors and ligands in influenza A H1N1 patients,and to assess the modulatory activity of Notch signaling pathway to influenza A virus specific CD8+T cells.Total of 27 mild influenza A H1N1 patients,11 severe influenza A H1N1 patients,and 18 healthy controls were enrolled in this study.Real-time PCR was used to semi-quantify the relative levels of Notch receptors and ligands m RNAs in peripheral blood mononuclear cells (PBMC).PBMC was stimulated with Notch signaling inhibitor DAPT for 24 hours,then flow cytometry was used to investigate the secretion of tumor necrosis factor-α(TNF-α)and interferon-γ(IFN-γ) by viral specific CD8+T cells,while ELISA was used to assess the secretion of perforin and granzyme B by viral specific CD8+T cells.CD8+T cells were purified from HLA-A2 restricted patients,and stimulated with DAPT for 24 hours to set up direct contact and indirect contact culture systems between CD8+T cells and influenza A H1N1 virus strain infected NCI-H345 cells.Target cell death,which induced by CD8+T cells,was cultured supernatants using ELISA.Data showed that the mRNA relative levels of Notch receptors (Notch1,Notch2,Notch3,and Notch4) and Notch ligands (DLL1,DLL4,Jagged1,and Jagged2) were elevated in PBMC from mild and severe influenza A H1N1 patients compared with controls (P<0.05);the percentage of IFN-γ-secreting viral specific CD8+T cells as well as cell counts of perforin and granzyme B-secreting viral specific CD8+T cells were increased in severe influenza A H1N1 patients compared with mild patients (P<0.05),while DAPT stimulation could suppress this increase (P<0.05).In direct contact culture system,DAPT stimulation down-regulated the percentage of target cell death induced by viral specific CD8+T cells (P<0.05),and the levels of TNF-αand IFN-γin the cultured supernatants were also reduced (P<0.01).In indirect contact culture system,although TNF-αand IFN-γproduction were reduced in response to DAPT stimulation (P<0.01),ther

关 键 词：甲型流感病毒 NOTCH信号通路 CD8+T细胞 免疫调控 

分 类 号：R512.6[医药卫生—内科学]