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

Abstract

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. 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 livers 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 TGFbeta1 in the liver. Mixed lymphocyte reaction (MLR) was performed to evaluate the tolerance status. The remaining rats were used to observe the survival status. The mean survival time of Group D was 52.00 +/- 10.64 days, significantly longer than those of the other three groups (P < 0.05 or P < 0.01). Seven days after transplantation, the number of infiltrating lymphocytes in liver of Group D was significantly lower than those in the other 3 groups (P < 0.05 or P < 0.01), whereas the proportion of CD4+ CD25+ T cells was significantly higher than those of the other 3 groups (P < 0.01 or P < 0.05). The mRNA expression levels of IL-10 and TGFbeta1 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 P < 0.05), 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 P > 0.05). 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.