摘要目的 探讨阻断PD-1/PD-L1信号通路后,负载HBsAg的树突细胞(dendritic cells,DC)诱导HBV转基因小鼠免疫应答的作用特点.方法 体外诱导扩增BALB/c小鼠骨髓来源的DC,尾静脉免疫HBV转基因小鼠(其中在DC免疫前1 d腹腔注射PD-L1抗体,共3次).实验分5组进行:DC/HBsAg+PD-L1抗体组、DC/HBsAg组、Dc组、mlgG组和磷酸盐缓冲液(PBS)组,分别以流式细胞术、乳酸脱氢酶(LDH)释放法、酶联免疫吸附试验(ELISA)和荧光定量聚合酶链反应(PCR)等检测HBV转基因小鼠脾脏淋巴细胞增殖及细胞内干扰素γ(IFNγ)、特异性细胞毒性T淋巴细胞(CTL)活性及血清HBsAg、HBV DNA和丙氨酸转氨酶(ALT)水平.采用SPSS 13.0统计软件进行分析,组间比较采用ANOVA方差分析和q检验.结果免疫后第3天和第6天,DC/HBsAg+PD-L1抗体组与其他各组比较在诱导CD_3~+CD_8~+T淋巴细胞增殖及IFNγ分泌方面差异有统计学意义(细胞增殖:F值分别为25.22和39.01,P值均<0.01;IFNγ分泌:F值分别为32.35和36.98,P值均<0.01);而DC/HBsAg组与Dc组淋巴细胞增殖能力比较仅在第3天有统计学意义(t=6.79,P=0.012).特异性CTL的活性在DC/HBsAg+PD-L1抗体组明显升高.各组血清HBsAg在第6天差异有统计学意义(F=6.12,P<0.05).DC/HBsAg+PD-L1抗体组血清ALT水平与DC/HBsAg组比较在第3天无差异,与其他各组比较在第3和第6天差异有统计学意义(F值分别为19.22和14.30,P值均<0.05).各组对HBV DNA的清除能力没有差别,但DC/HBsAg+PD-L1抗体组有1只小鼠病毒载量下降1个数量级.结论封闭PD-1/PD-L1信号通路能通过提高特异性CD_3~+CD_8~+T淋巴细胞增殖及其分泌IFNγ能力,进而促进负载HBsAg的DC诱导转基因小鼠抗HBV免疫能力.

abstractsObjective To investigate the effect of programmed death(PD)-1/PD-L1 interaction on immunc response of HBsAg-pulsed dendritic cells(DCs)vaccine.Methods DCs were genermed from bone marrow cells from BALB/c mice and pulsed with HBsAg protein.HBV transgenic mice were injected i.D with PD-L1 blocking monoclonal antibodies(Abs)1 d before DC/HBsAg transfer with an interval of 3 d for 3 times,and then divided into 5 groups:DC/HBsAg+PD-L1 Abs group,DC/HBsAg group,DC group,mlgG group and phosphate buffered saline(PBS)group.Extracellular molecule and intraceilular cytokine of splenic T cells were analyzed by flow cytometry,HBsAg-specific activity of splenic cytotoxic T lymphocytes (CTLs)was detected by LDH release assay,HBsAg and HBV DNA titers in sera were monitored by ELISA and fluorescent quantitative PCR,and hepatic injury Was monitored by measuring serum ALT at 3th and 6th after immunization,respectively.SPSS 13.0 software Was used for data processing,and ANOVA(analysis of variance)as well as q test were performed to compare the measurement data among the groups.Results At 3th and 6th after immunization,PD-L1 blockade stimulated splenic CD_3~+CD_8~+T lymphocytes proliferation more efficiently than that with no PD-L1 blockade(mice with DC/HBsAg treatment alone)(F=25.22 and 39.01,P<0.01),and the secretion of IFNγ WaS hisher(F=32.35 and 36.98.P<0.01);meanwhile the differences between mice with DC/HBsAg treatment and those with DC alone were only observed at 3th(t=6.79,P=0.012).The activity of HBsAg-specific CTL and serum HBsAg levels at 6th were significantly higher in mice with anti-PD-L1 Abs treatment compared with those in other groups (F=6.12,P<0.05).Serum ALT levels in mice with anti-PD-L1 Abs treatment were the same with DC/HBsAg group at 3th,but were higher than those in other groups at both 3th and 6th(F=19.22 and 14.30,P<0.05).All groups were similar in the clearance of HBV DNA.but HBV DNA hvel decreased one log copy in one mouse with anti-PD-L1 Abs treatment.Conclusion Blocking PD-1/PD-L1 interaction can enhance specific CD_3~+CD_8~+ T lymphocyte proliferation and the capability of secreting IFNγ,which improves the inhibition of HBV in transgenic mice induced by HBsAg-pulsed DC vaccine.