调节性T细胞和共刺激通路阻断剂抑制大鼠肝移植急性排斥反应的研究

目的探讨CD4^＋CD25^＋调节性T细胞、共刺激通路阻断剂CD154单抗及两者联合应用在抑制大鼠肝移植急性排斥反应中的作用。方法48例原位肝移植大鼠（DA→Lewis）分为A组：对照组;B组：术前7d回输经DA大鼠脾细胞体外激活的Lewis大鼠CD4^＋CD25^＋细胞;C组1术后第1、2d腹腔注射CD154单抗（15m/kg）;D组：联合应用CD4^＋CD25^＋细胞和CD154单抗。术后7d各组处死6只受体,观察移植肝病理变化,检测移植肝内T细胞亚群和细胞因子白细胞介素之（IL-2）、IL4、IL-10和转化生长因子B1（TGFβ1）表达情况;分离脾淋巴细胞与供体行单向混合淋巴细胞反应观察刺激指数。余大鼠观察生存情况。结果D组平均存活时间（52．00±10．64）d明显长于其他各组（P〈0．01）;移植肝内淋巴细胞浸润数量（2．47±0．61）×10^6和CD8^＋细胞百分比（14．2±3．0）％明显低于B、C组（P〈0．05、P〈0．01）,而CD4^＋CD25^＋细胞比例（16．4±4．3）％高于B、C组（P〈0．05,P〈0．01）。移植物内IL-2mRNA表达A组最高,D组最弱;IL4mRNA各组均弱表达;IL-10mRNAB、D组高表达,A、C组未表达;TGFβ1mRNAB、D组表达明显强于A、C组。单向混合淋巴细胞反应D组刺激指数最低（P〈0．05）。结论CD4^＋CD25^＋调节性T细胞和共刺激通路阻断剂CD154单抗均能抑制大鼠肝移植急性排斥反应;联合应用CD154单抗明显增强CD4^＋CD25^＋调节性T细胞对急性排斥反应的抑制作用。

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.