A New Understanding Of The Role Of Tacrolimus On Immunosupressionand Immune-regulation In Allogeneic Skin Transplantation In Mice

Objective:Tacrolimus（FK506）is one of the most widely used immunosuppressant in clinical transplantation.The main mechanism of action is inhibiting calcineurin activity of nuclear factor of activated T cells（NFAT）pathway,impacting the transcription of cytokines such as interleukin（IL）-2,thereby blocking the activation and proliferation of T cells.The calcineurin signal pathway of NFAT is widely present in innate immune cells.In transplant recipients,the effects of FK506 on innate immune system are still insufficient.This study aims to establish a model of allogeneic skin transplantation in mice,and evaluate the influence of FK506,by flow cytometry,on acquired immune cells（T cells）,as well as innate immune cells（bone marrow derived suppressor cells,MDSCs）.Methods:We use BALB/c mice as donors and C57BL/6 mice as receptor,then establish allogeneic mice tail to back skin transplant model.Began with the surgery date,FK506 was dosed in 2mg/kg/days（group FK506）or equal volume of PBS（group PBS）by intraperitoneal injection.Skin allograft rejections were observed and the survival rate were calculated.After ninth days from the transplantation,mice skin allografts were harvested for pathological examination,peripheral blood,spleen and skin graft draining lymph node（dLN）were prepared for flow cytometry.The proportion of CD44^highCD62L^low effector memory T cells,the expression of IFN-gamma and IL-4,and the CD25⁺Foxp3⁺regulatory T cell proportion were observed between two groups.Bone marrow-derived CD11b⁺Gr1⁺MDSCs proportion,TNF-alpha,IL-10 expression from Gr1⁺ bone marrow cells of and F4/80 macrophage phagocytosis function were also compared.Results:The median survival period of skin allograft in FK506 group（n=10）was 19 days,was significantly prolonged comparing with PBS（n=10）group mice for 8 days（P<0.0001）;The proportion of CD4⁺CD44^highCD62L^low T cells in peripheral blood in FK506（n=5）group was 4.740±0.261%,decreased significantly comparing with that in PBS group（n=5）,5.46±0.476%,p=0.023.The difference were also observed in the spleen and dLN.CD4⁺IFN-γ⁺T cell proportion was 3.860±1.953%in the FK506 group in spleen,which was significantly lower than that of PBS group,13.64±2.105%,P<0.0001.The proportion of CD4⁺IL-4⁺T cells in spleen in FK506 group was significantly higher than that of PBS group,5.600±0.570%versus 4.580±0.421%,p=0.0123.The proportions of CD25⁺Foxp3⁺Treg in spleen and dLN in FK506 group were 8.640±1.295%and 9.780±1.108%,significantly lower than that in PBS group were 14.48±1.521%and 12.60±1.799%,P were 0.0002 and 0.0175,respectively.The proportion of CD11b⁺Gr1⁺MDSCs in peripheral blood in FK506 group was 8.240±0.868%,which were significantly lower than that in PBS group,11.160±1.167%,p=0.002.The expression of TNF-α and IL-10 in the Grl⁺cell in FK506 group and PBS group ratio were comparable both in spleen and dLN.There was no difference observed in Escherichia coli phagocytosis rate in macrophages between FK506 and PBS group.Conclusion:The protective effect of FK506 from skin transplant rejection is specifically by suppressing antigen specific effect/memory T cells,leading to differentiation deviation from TH1 to TH2 T cells.Both Treg and MDSCs are not involved in the immunosuppression effect of FK506 in the allogeneic skin transplant model.