[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 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.

Results: 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).

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.