Suppressive Function Of Induced Regulatory T Cells In Allograft Rejection

BackgroundReconstructive vascularized composite allotransplantation shows promising results inreconstruction of serious injury, while rejection remains however a major hurdle that itconfronts. T cells play an important role in transplant rejection. Regulatory T cells, whichsuppress the function of effective T cells, have been widely studied. Tregs consist ofnatural Treg and induced Treg subsets. Na ve CD4+T cells can be converted into iTregs invitro, which can reverse the conversion from Tregs to Th17cells. iTregs show a bettercharacter in therapy for self-immune diseases than nTregs, while its function in graftrejection has not been fully studied.ObjectiveThis experiment investigated the suppressive function of iTregs both in vitro and in vivo.iTregs are developed from na ve CD4+T cells in vitro and determined by the decrease ofTeffs proliferation rate. To monitor the migration of iTregs and confirm the journey afteriTregs infusion. To observe if injection of iTregs can prolong the survival time of allograftskin. All of these can verify the suppressive function of iTreg in allograft rejection.Methods Na ve CD4+T cells were isolated and cultured with IL-2and TGF-β. The percentage ofiTreg was tested with fluorescence-conjugated anti-mouse CD4, CD25and Foxp3antibody through FACS. iTregs and CD4+CD25-Teffs were mixed in different ratios inorder to test in vitro suppressive fuction of iTregs. Fully MHC mismatched C57BL/6andBalb/c mice were used as donors and recipients respectively in skin allograft model.CFSE-labeled iTregs were injected into mice to observe the migration of iTregs in theblood. Mice were divided into experiment group and control group. Experiment groupwere injected with iTregs and control group were injected with PBS. Survival time ofgrafted skin in the two groups was compared and immune cells infiltration rate in rejectionskin was compared through HE staining.ResultsThe percentages of na ve CD4+T cells in isolated cells were higher than90％. Theaverage percentage of CD4+CD25+Foxp3+cells in induced cells is44.2％. iTregsshowed suppressive function in proliferation of Teffs with a dose-dependent pattern. Afterinjection of CFSE-labeled iTregs, CFSE could be detected in thymus and lymph node inthe third day. Six days later, no signal of CFSE could be found in any organ tested.Average skin grafts survival time was higher in iTregs group than control group(P＜0.05).HE staining also showed a lower immune cells rate infiltration in the rejection skin ofiTregs group than control group.ConclusionMethod used in this article could help us develop enough Tregs to suppress theproliferation of Teffs in vitro. iTregs migrated to thymus and lymph node after injectionand prolonged survival time of allograft skin. iTregs showed suppressive function inallograft rejection, but iTregs alone cannot establish immune tolerance.