小鼠肝脏非实质细胞在诱导Foxp3+CD4+CD25+调节性T细胞及肝移植免疫耐受中起重要作用
刘宏宇1，关连越1，辛敏刚2，王展鹏1，李卓男1，李巍1,3，Perkins JD3，Reyes  J3
基金项目：美国华盛顿大学校长基金（李巍）；中国国家自然科学基金（李巍81170416；81273264）；中国教育部博士点基金（李巍20100061110069）；吉林省科技厅国际合作基金（李巍2011742）；天普研究基金（李巍01201046)；吉林省自然科学基金（李巍201015178） 作者简介：刘宏宇（1980年生），男，主治医师，器官移植与免疫耐受 通信联系人：李巍（1963年生），女，教授，器官移植与免疫耐受.

（1. 吉林大学中日联谊医院肝胆胰外科，长春，130033； 2. 吉林大学中日联谊医院麻醉科，长春，130033； 3. 华盛顿大学器官移植外科，西雅图，美国）

摘要：我们最近的研究表明Foxp3+CD25+CD4+调节性T (Treg) 细胞在肝移植自发耐受诱导过程中起重要作用，但Treg是如何被诱导的、Treg与其他免疫细胞之间的关系及相互作用仍然不十分清楚。在本研究中, 我们使用供体B6小鼠肝脏非实质细胞（NPC）和树突状细胞（DC）在体外与同种异体C3H CD4 T细胞共同培养，以扩增Treg，然后将扩增的Treg输注给心脏移植受体，通过心脏移植物的存活时间以检验Treg的免疫调节功能。通过应用Flt3L-/-小鼠，研究肝脏DC在诱导Treg和肝移植免疫耐受过程中的作用。结果：与脾细胞或脾脏DC相比，肝脏NPC，尤其是DC，在体外与同种异体CD4 T细胞共同培养能够诱导产生较多的Foxp3+CD25+CD4+Treg。过继转输这些 Treg给同种异体鼠后，能显著延长与其同品系鼠的心脏移植物存活时间。然而，来自PD-L1-/-鼠的DC却不能诱导Treg。Flt3L-/- 和 PD-L1-/-小鼠的肝脏移植给C3H会诱导受体抗移植物急性排斥反应。免疫组化染色发现Flt3L-/- 和PD-L1-/-作为供体的肝移植物及受体脾内Foxp3+ Treg细胞明显减少。结论：肝脏NPC，尤其是肝脏DC在诱导Foxp3+CD25+CD4+Treg和肝移植耐受过程中起重要作用，其机理可能是依赖其表面PD-L1信号传导通路。
关键词：肝移植耐受；小鼠；Foxp3+CD4+CD25+ 调节性T细胞；肝脏非实质细胞
中图分类号：请查阅《中国图书馆分类法》
Liver nonparenchymal cells play a significant role in the induction of Foxp3+CD4+CD25+Treg and liver transplantation tolerance in mice
LIU Hongyu1, GUAN Lianyue1, XIN Mingang2, WANG Zhanpeng1, LI Zhuonan1, LI  Wei1,3, Perkins JD3, Reyes J3
(1. Dept of Hepatobiliary-pancreatic Surgery,China-Japan Union Hospital of Jilin University, 126 Xiantai Street, Changchun,130033; 2. Anesthesiology,China-Japan Union Hospital of Jilin University, 126 Xiantai Street, Changchun,130033;3. transplant surgery，Washington university, Seattle, USA)
Abstract: Our recent studies have demonstrated that Foxp3+CD25+CD4+ regulatory T cells (Treg) contribute significantly to liver transplant tolerance induction. It is largely unclear how Treg are induced and how these cells interplay in the regulation of liver transplant tolerance. Here, we attempted to expand the number of Treg in vitro by coculture of liver nonparenchymal cells (NPCs) or dendritic cells (DCs) with allogeneic CD4 T cells, and assessed their function by adoptive transfer to heart allograft recipients. We used Flt3 ligand (FL) mutation mice, which have severe reduction in all types of DCs, to examine the role of liver DCs in liver transplant tolerance in vivo after liver transplantation. Our results showed that liver NPCs, in particular DCs, induced a greater number of Foxp3+ Treg than did spleen cells (SCs) and spleen DCs. Adoptive transfer of the CD25+CD4+ T cells generated from liver NPCs or DCs prolonged heart allograft survival at a significantly higher level than the cells expanded by SCs or spleen DCs. The DCs which isolated from PD-L1-/- mice could not generate Treg in vitro. Moreover, the liver grafts from FL-/- or PD-L1-/- mice were rejected acutely in the C3H recipients and associated with reduction of Foxp3+ cells in the liver grafts and recipient spleens from FL-/- donors. Thus, liver NPCs, in particular DCs, play a critical role in the induction of Treg, which underpin spontaneous acceptance of MHC-mismatched liver allografts in mice.
Key words: liver transplantation tolerance；mice ；Foxp3+CD4+CD25+Treg；Liver nonparenchymal cells