Objective: Lentiviral-mediated shRNA against RelB was used to produce tolerogenic dendritic cells from murine bone marrow derived dendritic cells (BMDCs).
Method: RelB expression in the BMDCs was silenced by lentivirus carrying RelB shRNA. The apoptosis rate and surface markers of DCs were assessed by flow cytometry. IL-12,IL-10,TGF-β1 secreted by DCs and DNA binding capacity of NF-κB subunits in the nucleus were measured by ELISA, independently. MLR was used to analyze the capacity of DCs to inhibit immune response.
Results: RelB expression was significantly inhibited in DCs following lentiviral mediated delivery of RelB specific shRNA. The RelB shRNA-DC produced lower IL-12 and higher IL-10 than mature dendritic cells (mDCs) and silencing control DCs. There was no difference in the apoptosis rate between shRNA RelB-DCs and mDCs. The expression levels of co-stimulatory molecules (CD80, CD86 and CD83) and MHC-II class molecule were lower in the RelB shRNA-DCs than in the mDCs and silencing control DCs. In addition, RelB shRNA also inhibited the RelB DNA binding capacity but had no effect on other NF-κB subunits. The shRNA RelB-DCs can significantly inhibit mixed lymphocyte reaction (MLR) and down-regulate Th1 cytokines and prompt the production of Th2 cytokines.
Conclusion: Our results indicate RelB shRNA transfection of DCs can induce the immature status, and produce tolerogenic DCs.
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