自发免疫耐受大鼠移植肝内调节性T细胞的生物学特性

The biological features of intrahepatic CD4^＋ CD25^＋ T cells in the natural tolerance of rat liver transplantation

目的 研究大鼠肝移植后自发免疫耐受的形成与移植肝内CD4^＋CD25^＋调节性T细胞（Tr细胞）的关系。方法 按供、受者不同将实验分为3组。急性排斥组：DA大鼠为供者，LEW大鼠为受者；自发耐受组：LEW大鼠为供者，DA大鼠为受者；同基因组：供、受者均为DA大鼠。各组均建立大鼠原位肝移植模型。分别在肝移植术后4、7、14、30和90d时采用密度梯度离心法分离移植肝内淋巴细胞，免疫磁珠分离（MACS）法分选出CD4^＋CD25^＋Tr细胞；用流式细胞术（FCM）检测细胞纯度，同时分析CD4^＋CD25^＋Tr细胞比例的变化；体外细胞增殖试验研究CD4^＋CD25^＋Tr细胞对CD4^＋CD25^-T细胞的免疫抑制作用。结果 肝移植早期，急性排斥组和自发耐受组移植肝内CD4^＋CD25^＋Tr细胞比例均明显增加，其中急性排斥组增加更为明显；移植后4d左右，两组CD4^＋CD25^＋Tr细胞比例开始下降，急性排斥组的下降幅度较大；移植后30d，自发耐受组受者的移植肝内CD4^＋CD25^＋Tr细胞比例达到第2次高峰，约在移植后90d时下降至正常生理水平。移植后7d左右，急性排斥组受者均因发生排斥反应而死亡，而自发耐受组受者均存活。此外，CD4^＋CD25^＋Tr细胞能有效抑制CD4^＋CD25^-T细胞的增殖。结论 CD4^＋CD25^＋Tr细胞是一种具有特异免疫调节功能的T细胞亚群，其主动的免疫抑制功能可能是诱导大鼠肝移植自发免疫耐受的机制之一。

Objective To study the relationship between the spontaneous immune tolerance and the intrahepatic CD4^＋CD25^＋ Tr regulatory cells following rat liver transplantation. Methods The experiments were divided into three different groups according to different donors and recipients： tolerance group, LEW-to-DA; rejection group, DA-to-LEW; s3nageneic group （control group）, DA-to-DA. CD4^＋ CD25^＋ Tr cells were isolated by density gradient centrifugation on day 4,7, 14,30 and 90 after liver transplantation. The intrahepitc CD4^＋ CD25^＋ Tr cells were isolated by MACS and identified by flow cytometry （FCM）. The proportion of intrahepatic CD4^＋ CD25^＋ Tr cells from three groups was determined by FCM. And their suppression on the proliferation of CD4^＋ CD25^＋ T effector cells was analyzed by cell proliferation assay in vitro. Results In the early stage of transplantation, the ratio of Treg cells was increased over time in both syngeneic group but was significantly greater in rejection group. The quotient of Treg cells was declined after day 5, a reduction that was more dramatic in rejection group than in tolerance group. Animals in tolerance group showed a second increase in the quotient after day 14. Intrahepatic CD4^＋ CD25^＋ Tr cells isolated from spontaneous tolerance models inhibited the proliferation of mixed lymphocyte reaction. The purity of CD4^＋ CD25^＋ Tr cells sorted by MACS was 86%-93%. The CD4^＋ CD25^＋ Tr cells could specifically express the Foxp3 gene as compared with CD4^＋ CD25^- T cells. In vitro, the spleen cells from LEW rats could irritate the proliferation of CD4~ CD25~ T cells more obviously than the syngeneic spleen cells. CD4^＋CD25^＋ Tr cells could suppress the proliferation of CD4^＋ CD25^- T cells,but the inhibition was reversed by exogenous IL-2 （200 U/ml）. Conclusions The CD4^＋ CD25^＋ Tr regulatory cells, a subpopulation of T cells, have specific immuno regulation function,which may be one of the mechanisms which can induce spontaneous immune tolerance following rat liver transplantation.