The Study On The Effect Of MDSC In Immune Tolerance Induced By HBeAg

Objective To research the immune regulation and mechanism of HBe Ag by a HBV replication mouse model.Methods 1.p BS-HBV1.3 contained 1.3 copies of the HBV genome(nt 1040–1986,Gen Bank AY220698)was constructed based on the p Bluescript II KS(+)vector.Then,HBe Ag(-)plasmid was constructed and the only difference between plasmid HBe Ag(-)and p BS-HBV1.3 is that 1896 th G mutation to A in the genome,whichcaused HBe Ag doesn't express.An acute replication mouse model was constructed by hydrodynamic injection(HI)of wild type plasmid p BS-HBV1.3 and mutant plasmid HBe Ag(-)into male,6-8 weeks C57BL/6 mice to explore the influence of HBe Ag on immune response.2.We bleed mice and monitored serum antigen and antibody by ELISA on days of 1th,4th,7th,14 th,21th,28 th after HI.In addition,on 1th,7th,14 th,21th and 28 th day after HI,serum HBV DNA was detected by realtime PCR.HBc Ag level were visualized by immunohistochemical on day of 28 th after HI.3.The amount and function of mononuclear cells from livers and spleens were assayed by FACS on every week after HI,total three weeks.4.MDSCs of spleens and Livers from different group mice were sorted out with magnetic bead separation and then co-incubated with T lymphocytes after 14 th days of HI.The secretion of IL-10 and IFN-? in the supernatant were detected by ELISA after 24 th hours of co-incubation.5.Total RNA of NPC from liver and T lymphocytes from spleen were extracted and performed RT-PCR to test relevant cytokines,such as IL-10 and Bcl-6.Results 1.p BS-HBV1.3-HBe Ag(-)was constructed successfully.2.In the HBV replication mouse model,serum HBs Ag and HBV DNA were higher in the group of H1.3 than group of HBe Ag(-)at the same time.The expression of HBc Ab appeared earlier in the group of HBe Ag(-).3.The results of FACS demonstrated that on day of 14 th after HI,MDSCs were more expanded and secreted lesser IL-6 in the H1.3 group.The number of T lymphocytes increased strikingly with the expression of CD44/CD62 L up-regulated but PD-1 down-regulated and the secretion of IFN??IL-2 and TNF? rising in the HBe Ag(-)group.Meanwhile,Treg cells had more augmenter and more PD-L1 expression,furthermore the IL-10 secretion of Tregs was up-regulated in H1.3 group compared to the HBe Ag(-)group.The results of 21 th were similar to 14 th.5.The results of co-incubation with MDSC and CD8+T cells suggests that the secretion of IL-10 from supernatant was increased significantly but the secretion of IFN-? was reduced markedly in the group of H1.3 compared to HBe Ag(-)group after 24 hours with co-incubation.6.The IL-10 was examined in each treatment various with time change by RT-PCR.On the 14 th and 21 th day post HI,IL-10 and Bcl-6 secretion in NPC and spleen from H1.3 were increased when compared to HBe Ag(-).Bcl-6 up-regulated in H1.3 imply that HBe Ag inducing tolerance may associated with TFH even B lymphocytes.Conclusions Our results highlight that HBe Ag plays a tolerance role in immune response in the HBV replication mouse model.According to the results in this study,the potential mechanisms as display: 1.MDSCs are recruited to liver and its function of IL-10 secretion is activated,however,the IL-6 secretion is suppressed by HBe Ag.2.HBe Ag could proliferat Treg cells in liver and enhances the expression of PD-L1 as well as the secretion of IL-10.This indicated that HBe Ag could activate PD-1-PD-L1 signal pathway by up-regulation PD-L1 of Treg cells or promote IL-10 secretion of Tregs to suppress the T cell response.3.MDSCs induced by HBe Ag could induce differentiation of Treg cells.Then Treg cells restrain the activation of T cells.