A Study Of Antigen-specific Immunotherapy In The Treatment Of Experimental Autoimmune Myasthenia Gravis By Immature DC Derived Exosomes

Part1Objective:To prepare exosomes from antigen specific immature DCs by transfecting microRNA-146a mimics into DCs and to observe the phenotypes of those exosomes with electric microscope and flow cytometry oMethods:Firstly, to explore whether addition of Ta146-162altered maturation state of DC by detecting surface expressions of MHC II, CD80, CD86with flow cytometry and measuring supernatant levels of IL-6and IL-12with ELISA. Secondly, transfecting microRNA-146a mimics into DCs, and evaluate the ability of DC resistance to maturation stimuli of Ta146-162.Thirdly, select the immature DCs that can resist maturation stimuli of Ta146-162, and collect the supernatants. Extract exosomes (Dex) from the supernatants. Membrane markers of Dex (CD86, CD80and MHC Ⅱ) were evaluated by Flow cytometry, and morphology of Dex were observed under electrical microscope.Results:Surface markers (CD86、CD80and MHC Ⅱ) and supernatant levels (IL-6、IL-12) of mouse DCs were elevated after Ta146-162stimulation. DCs with microRNA-146a mimics transfection can resist maturation stimuli of Ta146-162, which showed decreased surface expression (CD86、CD80and MHC Ⅱ) both in percentage and mean flourscence indensity and reduced levels of cytokines(IL-6and IL-12)in supernatants significantly. Exosomes from DC with microRNA-146a mimics transfection and Ta146-162stimulation showed low levels of CD80and CD86expression.Conclusion:We have successfully obtained antigen-specific exosomes from immature DC induced by microRNA-146a mimics. Those exosomes showed low level of CD80and CD86in membrane. This approach provides a basis for further study of the therapeutic potential of antigen specific immature DC derived exosomes in autoimmune diseases.Part2Objective:To observe the therapeutic effect of antigen specific immature DC derived exosomes (imDex) in experimental autoimmune myasthenia gravis (EAMG), and further to investigate the mechanism of this immune therapeutic.Methods:①Bone marrow derived immature DCs (BM-imDCs) were co-cultured with AChR-specific CD4+T cells and stimulated with peptide Ta146-162-Ta146-162-imDex, Ta146-162-mDex, and PBS were added into the coculture system. The proliferation of CD4+T cells was estimated by CFSE method, and IL-17, IFN-y, IL-4, IL-10production were evaluated with ELISA.②Torpedo AchR (T-AchR) protein was extracted and mouse model of EAMG were induced with T-AchR.③To inducing EAMG, C57BL/6mice were immunized with T-AChR in CFA at day0,30,60. EAMG mice with1to2clinical score were collected and enrolled randomly into Ta146-162-imDex group and Ta146-162-mDex group. Ta146-162-imDex group was further divided into four subgroups according to their dosages (2ug,5ug, lOug). Ta146-162-imDex (2ug,5ug, lOug) and Ta146-162-mDex were injected IV at the day after enrollment. At20days after allotment, serum from individual mice was collected to detect serum concentrations of anti-mouse AChR IgG, IgGl, IgG2b and IgG2c by ELISA. At the meantime, serum concentrations of IL-17, IFN-y, IL-4, IL-10were measured with ELISA.④At21days after allotment, the splenocytes were isolated for analysis of lymphocyte proliferative responses, cytokines (IL-17, IFN-y, IL-4, IL-10) production and protein levels of RORyt, T-bet, GATA-3, and FoxP3(special transcription factors of Th17, Thl, Th2, Treg respectively).⑤At termination of the whole study, splenocytes of Ta146-162-imDex group(5ug) and Ta146-162-mDex group were cultured and stimulated with Ta146-162,T-AchR, PBS, and conA. The proliferation of CD4+T was estimated by CFSE.Results:①On day five, Low proliferation responses were observed in co-cultured cells with Ta146-162-imDex while high proliferation responses were observed in co-cultured cells with Ta146.162-mDex. T-AChR-reactive CD4+T cells primed by Ta146-162-imDex exhibited significantly lower proliferation response as compared with those primed by Ta146-162-mDex.②Significantly reduced production of IFN-γ and IL-17, as well as higher levels of IL-4and IL-10, were found in co-cultures of DC-T cells and Ta146-162-imDex as compared with co-cultures of DC-T cells and Ta146-162-mDex.③Treatment with Ta146-162-imDex effectively reduced myasthenic symptoms in mice with ongoing EAMG, and levels of serum anti-acetylcholine receptor autoantibody as well. Th17-related markers (RORyt, IL-17) and Thl-related markers (T-bet, IFN-y) were down-regulated, whereas Th2markers (GATA-3, IL-4) and Treg marker (FoxP3, IL-10) were up-regulated.④Low proliferation responses were observed in co-cultured cells with no Ag or with control Ag OVA, while high proliferation responses were observed in co-cultured cells with T-AChR, Ta146-162peptide. CD4+T cells of mouse splenocytes of Ta146-162-imDex group exhibited significantly lower proliferation response as compared with those of Ta146-162-mDex group.Conclusion:Ta146-162-imDex effectively reduced myasthenic symptoms in mice with ongoing EAMG, and levels of serum anti-acetylcholine receptor autoantibody as well. The therapeutic effect was antigen-specific and partly dose dependent. The protective effect was associated with reduced AChR-specific T cell proliferation and a shift of a T Helper Th17/Thl to a Th2/FoxP3+Regulatory T Cell Profile differentiation.