A Study Of Immunotherapy And Immunomodulation Mechanism In The Treatment Of Experimental Autoimmune Myasthenia Gravis By RelB-Silenced DC

Part 1Objective:To construct lentiviral vectors targeting mouse RelB gene by RNA interference and to observe its effect on RelB expression in bone marrow-derived dendritic cells and its effect on cell maturation/activation.Methods:According to the Genbank information of RelB,four RNA interfering sequences and a negative sequence were designed and inserted into the lentiviral vector pGCSIL and 5 kinds of plasmids were packaged:LV-SiRelB1,LV-SiRelB2,LV-SiRelB3,LV-SiRelB4 and LV-SiNC;and they were used to transfect NIH3T3 cells.Real-time PCR was used to investigate the interfering efficiency.The plasmid with high interfering efficiency was packaged.Recombinant lentivirus that produced RelB siRNA and control lentivirus were prepared and were used to infect BMDCs.The infected BMDCs were stimulated with LPS, the resulting cells were designated as RelB-Silenced DC or Control DC, respectively.The mRNA and protein expression of RelB were examined by Real-time PCR and Western-blot analysis.Cell surface markers of DC (CD86,CD80 and MHCâ…¡) were evaluated by Flow cytometry,IL-6, IL-12 and IL-23 in supernatant were detected by ELISA.Results:DNA sequencing showed that the sequences of 5 recombinant lentivirus plasmids were correct.Lentivirus packaging was successfully done.Real-time PCR analysis confirmed that LV-SiRelB3 had the highest interfering efficiency(82%).Real-time PCR and Western-blot analysis confirmed that Lv-SiRelB3 infection result in about 78.8%reduced RelB mRNA and 75.2%reduced RelB protein.Compared with Control DC,RelB-Silenced DC expressed a significantly lower level of CD86,CD80 and MHC classâ…¡on their surface,produced a significantly lower levels of IL-6,IL-12 and IL-23,as demonstrated by Flow cytometry and ELISA respectively.Conclusion:We have successfully constructed RelB RNAi recombination plasmid LV-SiRelB3,which can effectively inhibit RelB expression in BMDC and cell maturation/activation.This approach provides a basis for further study of the therapeutic potential of RelB-Silenced DC in autoimmune diseases. Part 2Objective:To observe the therapeutic effect of RelB-Silenced Dendritic cells(DCs) in experimental autoimmune myasthenia gravis (EAMG),further more to investigate the mechanism of this immune therapeutic.Methods:â‘ RelB-silenced DCs/Control DCs were co-cultured with AChR-specific CD4⁺T cells,and stimulated with T-AChR,Tα_146-162 peptide,OVA or Medium.The proliferation of CD4⁺T was estimated by 3H-TdR incorporation method.â‘¡RelB-silenced DCs/Control DCs were co-cultured with AChR-specific CD4⁺T cells,and stimulated with T-AChR or Tα_146-162 peptide.The supernatant were collected to observe the IL-17,IFN-γ,IL-4,IL-10 production by ELISA.â‘¢To inducing EAMG,C57BL/6 mice were immunized with T-AChR in CFA at day 0, 30,60.EAMG mice with 1 to 2 clinical score were collected and enrolled randomly into RelB-silenced DCs group and Control DCs group. RelB-Silenced DCs or equal Control DCs pulsed with Tα_146-162 were injected iv at day 0,7,14 after enrollment.At 20 days after allotment, serum from individual mice was collected to detect serum concentrations of anti-mouse AChR IgG,IgG1,IgG2b and IgG2c by ELISA.At 21 days after allotment,the splenocytes were isolated for analysis of lymphocyte proliferative responses,cytokines(IL-17,IFN-γ,IL-4,IL-10) production and protein levels of RORγt,T-bet,GATA-3,and FoxP3(special transcription factors of Th17,Th1,Th2,Treg respectively).Results:â‘ Low proliferation responses were observed in cocultured cells with no Ag or with control Ag OVA,while high proliferation responses were observed in cocultured cells with T-AChR, Tα_146-162 peptide.T-AChR-reactive CD4⁺ T cells primed by RelB-Silenced DC exhibited significantly lower proliferation response as compared with those primed by Control DC.â‘¡Significantly reduced production of IFN-γ,and IL-17,as well as higher levels of IL-4 and IL-10,were found in cocultures of T cells and RelB-Silenced DCs,as compared with supernatants of co-cultures of T cells and Control DC.â‘¢Treatment with RelB-Silenced DCs effectively reduced myasthenic symptoms in mice with ongoing EAMG,and levels of serum anti-acetylcholine receptor autoantibody as well.Th17-related markers(RORγt,IL-17) and Th1-related markers(T-bet,IFN-γ,) were down-regulated,whereas Th2 markers(IL-4 and GATA3) and Treg marker(FoxP3) were up-regulated.Conclusion:RelB-Silenced DCs effectively reduced myasthenic symptoms in mice with ongoing EAMG,and levels of serum anti-acetylcholine receptor autoantibody as well.The protective effect was associated with reduced AChR-specific T cell proliferation and a shift of a T Helper Th17/Th1 to a Th2/FoxP3⁺ Regulatory T Cell Profile differentiation.