Research Of The Immune Regulatory Activity Of The Dendritic Cell Modified By RNA Interference

Background:Allogeneic transplantation rejection is mainly because of immune response mediated by T lymphocyte.Dendritic cells(DC) are the most potent APC,and CD80/CD86 are defined as stimulatory molecules on DC.When the CD28 binds to CD80/CD86,naive T lymphocyte will be transformed to activated T lymphocyte or memory T lymphocyte,leading to their proliferation and the role of immune rejection.RNA interference is,an specific and efficient approach to knock down some gene expression,by which the expression of co-stimulatory molecules on DC is inhibited and co-stimulatory pathways is intermitted,so that we could induce immune tolerance.Objective:To evaluate the effects of lentiviral vectors targeting mouse CD86 gene by RNA interference on mouse bone marrow-derived dendritic cells in vitro;to examine the immune regulatory activity of the dendritic cells(DC) that were knocked down the expression of CD86 by RNA interference(RNAi) approach,and study the related mechanisms.Methods:DC propagated from C3H(H-2~k) mouse bone marrow of femurs and tibias in the presence of GM-CSF and IL-4 were infected by the recombinant lentiviruses targeting mouse CD86 gene by RNA interference the second day of cell culture according to manufacturer's protocol.The infection efficiency was assessed by fluorescence microscope.DC were collected after LPS was added to the culture to allow for maturation,and the expression of molecules on DC were determined by flow analysis.DC allo-stimulatory activity was examined in vitro by mixed lymphocyte reaction(MLR) culture in which irradiated DC were cultured with C57BL/6(B6,H-2~b) spleen T cells at different ratio for 3 days.The proliferation of T cells were evaluated by[~3 H]-TdR incorporation,and the apoptotic T lymphocytes were identified by annexin V and CD3 staining. Results:The infection efficiency of lentiviral vectors was 79.78%when MOI was 20. Infection with the lentiviruses targeting CD86 gene by RNAi markedly inhibited expression of CD86 on dendritic cells compared with either blank control group or negative control group(P＜0.05),but did not affect the expression of CD80 and MHC-Ⅱ,indicating the effectiveness and specificity of the RNAi.In both blank control group and negative control group,DC stimulated profound proliferative responses in allogeneic T cells,whereas DC infected with lentiviruses targeting CD86 gene by RNAi induced significantly less allogeneic T cells proliferation(P＜0.05).The incidence of apoptotic T lymphocytes cultured with DC infected with lentiviruses targeting CD86 gene by RNAi was significantly higher than either blank control group or negative control group(P＜0.05).Conclusions:RNA interference is an efficient and specific approach to knock down expression of CD86 on DC.CD861ow DC could inhibit the proliferation of T lymphocytes and show tolerogenic activity induction of apoptosis in T lymphocytes in vitro.This approach could enhance the potential clinical application of tolerance induction by DC vaccination.