Combination of CD4~+ CD25~+ regulatory T cell and costimulatory pathway blockade inhibits acute rejection after liver transplantation:experiment with rats

Objective To investigate the effect of CD4~+ CD25~+ regulatory T cell(Treg)combined with anti-CD154 mAb,a costimulatory pathway inhibitor,on acute rejection after liver transplantation. Methods CD4~+T cells were isolated from the spleen of a Lewis rat and labeled with CD25-PE antibody. Anti-PE microbeads were added to collect CD4~+ CD25~+T cells.Splenocytes were isolated form DA rat, treated with mitomycin C,and then co-cultured with the CD4~+CD25~+ T cells of Lewis rat for 5 days for ex vivo activation.Forty-eight Lewis rats received orthotopic transplantation of the livers of DA rats,and then were randomly divided into 4 equal groups:Group A,used as control group,Group B,undergoing intravenous injection of the CD4~+CD25~+Treg activated ex vivo by splenocytes of DA rat 7 days before the liver transplantation,Group C,undergoing intraperitoneal injection of anti-CD154 mAb twice 1 and 2 days after the liver transplantation,and Group D,undergoing injection of both CD4~+CD25~+Treg and anti- CD154.Seven days after the transplantation 6 Lewis rats in each group were harvested to observe the pathological changes of the hvers and to detect the infiltrating lymphocytes,and CD4~+,CD8~+,and CD4~+ CD25~+T cells.RT-PCR was used to detect the mRNA expression of IL-2,IL-4,IL-10,and TGFβ1 in the liver.Mixed lymphocyte reaction(MLR)was performed to evaluate the tolerance status.The remaining rats were used to observe the survival status.Results The mean survival time of Group D was 52.00±10.64 days,significantly longer than those of the other three groups(P0.05 or P0.01).Seven days after transplantation,the number of infiltrating lymphocytes in liver of Group D was significantly lower than those in the other 3 groups(P0.05 or P0.01),whereas the proportion of CD4~+ CD25~+ T cells was significantly higher than those of the other 3 groups(P0.01 or P0.05).The mRNA expression levels of IL-10 and TGFβ1 of Group D were both the highest,however,the mRNA level of IL-2 of Group D was the lowest.The MLR assay with the splenocytes of DA rat as stimulatory cells demonstrated that the SI of Group D was,significantly lower than those of other 3 groups(all P0.05),however,the MLR assay with the splenocytes of Wistar rat as stimulatory cells demonstrated that there were not significant differences among the SI levels of the 4 groups(all P0.05).Conclusion Acute rejection after liver transplantation can be inhibited by CD4~+ CD25~+ Treg and costimulatory pathway inhibitor,especially the combination of both. Anti-CD154 mAb significantly enhances the effect of CD4~+ CD25~+ Treg on inhibition of.