CpG ODN Stimulates Lymphocytes And Dendritic Cells In Human Pancreatic Cancer Cell Conditioned Medium

Objective：The morbidity of Pancreatic Cancer（PC） is increasing everyyear.Tumor vaccine based on the dendritic cell(DC) has been a hot spot. But DCvaccine is much more inefficient because of the Immune tolerance mediated by tumors.CD4+CD25+Treg s play a important role in tumors’ immune tolerance.It could inhibitthe function of DCs and effector T cells by secrete inhibitory factor such as TGF-β、IDO、 IL-10.This study is to investigate the effect of CpG ODN on human lymphocytesand dendritic cells in human pancreatic cancer cell conditioned medium in vitro and tolearn the mechanism of tolerance breaking lead by CpG ODN through the reduction ofTregs,so as to provide theoretical basis for Improving tihe efficiency of DC vaccine.Methods: T lymphocytes were purified by Nylon Fiber Column.Human peripheralblood mononuclear cells were isolated from healhy volunteers. Dendritic cell（DC）wasinduced by IL-4and GM-CSF.Mature DC was induced by TNF-α.Mediumconditioned by cell line BxPC-3(BxCM) was used for this study.ODN2006was used tostimulate lymphocyte in BxCM.TGF-β secretion after24and48hours was tested byELISA.Surpernatant of lymphocytes stimulated by ODN2006was added intoDCs.Flow cytometry was used to test CD80、CD83、CD86、MHC-II in DCs toinvestigate the maturity and function of DCs. IL-6secretion by DCs after24and48hours was tested by ELISA. CD4+CD25+Treg differentiation was tested by flowcytometry.After mixed lymphocyte culture,IFN-γ was tested by ELISA. The cytotoxict lymphocyte（CTL） response and tumor killing effects were estimated after tumorantigen loading on DC.Results:.1.After24and48hours, TGF-β in BxCM was obviously lower downwith ODN2006.2.CD83and CD86on DC with ODN2006were higher than controlgroup in BxCpG group while the expression of MHC-II and CD80had no significant difference.3.High expression of IL-6was detected after48h stimulation on DCs.4.After48hours，CD4+CD25+Tregs in BxCpG group were significantly reduced.The inhibitionof ODN2006on Tregs was weaker after the addion of anti-IL-6neotralization antibody.5.Results of Westernblot and Immunofluorescence demonstrated that the expression ofNF-kB in DCs in BxCpG group was significantly higher than it was in Bx group.Butafter the addion of anti-IL-6neotralization antibody,NF-kB was lower down.6.Mixedlymphocyte culture leads to a increased INF-γ secretion in BxCpG group. But thesecretion of IFN-γ was lower down after the addion of anti-IL-6neotralizationantibody.7.A stronger CTL response and tumor killing effects were found in BxCpGgroup while the killing ability in antiIL-6group was weaker.Conclusion:DCs were effetely stimulated by CpG ODN in BxCM.The secretion ofTGF-β in BxCM can be reduced by CpG ODN.CD4+CD25+Tregs which leads toimmune tolerance would be lower down with CpG ODN and IL-6may play a importantrole in this hidden mechanism. Moreover,CpG ODN could heighten IFN-γ secreted bylymphocytes and make a stronge CTL response and tumor killing effects.