Inhibitory Mechanism Of Tolerogenic Dendritic Cells In Vitro Induced By RMT1-10 On High Risk Corneal Allograft Rejection In Mice

Objective:To investigate the inhibitory effect of tolerogenic dendritic cells in vitro induced by RMT1-10 on high risk corneal allograft rejection in mice.Methods:Immature dendritic cells(im DCs)derived from the donor bone marrow were obtained and divided into four groups according to intervention:im DCs group(no intervention);m DCs group(lipopolysaccharide was added);RMT1-10 group(RMT1-10 and lipopolysaccharide were added);Ig G isotype control group(Ig G isotype antibody and lipopolysaccharide were added).The DCs phenotypes were detected by flow cytometry including CD11C,CD80,CD86,MHC-II,TIM-4 and CD103.The concentrations of IL-10 and TGF-βin the cell cultured supernatants were determined by enzyme-linked immunosorbent assay.A mixed lymphocytes culture system was established,CCK-8 method was used to detect the effect of DCs on the proliferation of CD4~+T cells.First,corneal neovascularization was induced by corneal stromal suture in BALB/c mice,and the mice with four quadrants neovascularization growing into the middle and peripheral area of cornea were used as recipients.80 recipient mice were randomly divided into 4groups with 20 mice in each group.DCs obtained above were injected into the tail vein of recipient mice(1×10~6cells/100μL).7 days later,C57BL/6mice were used as donors and penetrating keratoplasty was performed.The rejection signs of corneal graft were observed under slit lamp every day after operation,including opacity,edema and neovascularization,and then scored to 30 days after operation.At 21 days after operation,5 recipients were selected from each group and the delayed hypersensitivity(DTH)response were detected.Results:Compared with im DCs group,there was no significant difference in expression of CD80,CD86 and MHC-II on DCs of RMT1-10 group(all at P>0.05),while the expression of Tim-4 was significantly decreased and the expression of CD103 was significantly increased(all at P<0.0001).The concentrations of IL-10 and TGF-βin the cell culture supernatant of RMT1-10 group were significantly higher than those of other groups(all at P<0.0001).The stimulating index of DCs in RMT1-10 group were significantly lower than that in other groups(all at P<0.05).The survival rate of the RMT1-10 group was significantly higher than that of the other groups(all at P<0.0001),and the DTH responses in RMT1-10 group were significantly weaker than that in the other groups(all at P<0.05).Conclusions:RMT1-10 can inhibit the rejection of high-risk corneal transplantation in mice,whose mechanism may be attribute to induce the expansion of Tol-DCs in vitro and promote antigen-specific immune tolerance by secreting TGF-βand IL-10,hereby prolonging the survival of corneal grafts.