Study On The Mechanism Of Inhibitory Dendritic Cells Induced By Multiple Routes And Its Mechanism Of Immunosuppression

PARTILentiviral vectors targeting CD80and CD86genes by RNA interference induce tolerogenic dendritic cells and their effects in immune suppressionObjective:To study the mechanism of immune suppression induced by recipient tolerogenic dendritic cells (DCs) modified by RNA interference in indirect recognition pathway.Methods:In the culture process of DCs from recipient C3H mice bone marrow, donor antigens from splenic lymphocyte of C57BL/6mice were added, these DCs were infected with lentiviral vector targeting mouse CD80/CD86gene by RNA interference. Flow cytometry was used to evaluate expression of CD80and CD86on the antigen-pulsed recipient DCs. The stimulating capacity to recipient T-cell proliferative response was testified in mixed lymphocyte reaction (MLR), in which irradiated DCs were cultured with C3H spleen T cells. IL-2, IL-4, IL-10and Interferon-y (INF-y) levels in MLR culture supematants were measured using enzyme-linked immunosorbent assay (ELISA). The apoptosis of T cells in indirect recognition pathway was detected by Annexin V/CD3staining.Results:Compared with the control group and blank group, there was a significant inhibition of CD80and CD86expression on DCs in recombinant lentivirus pretreated group (P<0.05), indicating the specificity of RNA interference. Recombinant lentivirus pretreated recipient DCs effectively inhibited the proliferation of recipient T cells in MLR. A remarkable reduction of Thl cytokines (IL-2and INF-γ) and a significant increase of Th2cytokines (IL-10) were found in MLR culture supematants (P<0.05). There was no significant difference in IL-4levels between the groups (P>0.05). We also found a higher percentage of apoptotic T cells in indirect recognition pathway (P<0.05).Conclusions:Lentivirus mediated RNA interference can effectively and specifically knock down CD80/CD86target genes in DCs. The tolerogenic DCs with low expression of CD80and CD86may show their immunosuppression activity by inducing apoptosis of T cells.PART ⅡHepatic stellate cells induce tolerogenic dendritic cells and their effects in immune suppressionObjective:To explore the signal pathways of B7-H1expression on hepatic stellate cells (HSCs) induced by INF-y. To further study the mechanism of immune suppression caused by HSCs induced tolerogenic DCs.Methods:In activation of HSCs isolated from C57BL/6mice by IFN-y, HSCs were exposed to graded concentrations of IFN-y in vitro, and these HSCs were also treated with constant concentration of IFN-γ for0.5to48hrs. In addition, HSCs isolated from IFN-yRl gene knockout mice or Statl gene knockout mice were exposed to IFN-y. During this process, protein synthesis inhibitor (CHX), RNA synthesis inhibitor (ActD), signal transduction inhibitors such as U0126, SP600125, and LY294002were added. The B7-H1mRNA expression and protein synthesis in HSCs were analyzed by RT-PCR and flow cytometry. ERK1/2phosphorylation in HSCs was measured using Western blot. In the culture process of DCs from C57BL/6mice bone marrow, activated HSCs of C57BL/6mice were added. Flow cytometry was used to evaluate the expression level of CD80and CD86on these DCs. The stimulating capacity to recipient T-cell proliferative response was testified in MLR, in which these DCs were cultured with BALB/c spleen T cells. The apoptosis of T cells in the MLR was detected by Annexin V/CD3staining.Results:B7-H1was markedly up-regulated following exposure to IFN-y, which was dependent on the IFN-y concentration and activating time. B7-H1on HSCs isolated from IFN-y R1gene knockout or Statl gene knockout mice showed no response to IFN-y. ActD completely blocked B7-H1mRNA synthesis. However, CHX had no effect on B7-H1mRNA synthesis. Blocking of PI3K with LY294002did not reduce B7-H1expression. However, blocking MEK1/2with U0126dramatically reduced B7-H1expression. A slight reduction of B7-H1expression was also observed after blocking JNK with SP600125. ERKl/2phosphorylation was found in HSCs activated by IFN-y. A significant inhibition of CD80and CD86expression on DCs was observed when DCs were incubated with activated HSCs. Activated HSCs-pretreated DCs effectively inhibited the proliferation of T cells in MLR. We also found a higher percentage of apoptotic T cells in MLR.Conclusions:The expression level of B7-H1on HSCs was up-regulated by IFN-y via MEK/ERK signaling pathway. The tolerogenic DCs induced by IFN-y activated HSCs may show immunosuppression activity via induction of T cell apoptosis.