Tsuyuki Shigeru, Kono Mari, Bloom Eda T
Laboratory of Immunology and Virology, Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892, USA.
Xenotransplantation. 2002 Nov;9(6):410-21. doi: 10.1034/j.1399-3089.2002.01114.x.
Endothelial cells (EC) are primary targets of the recipient's immune response to transplanted organs and constitutively express Fas (CD95) ligand (FasL) on their surface. We investigated the role of porcine FasL in the generation of the human anti-pig response in vitro. Porcine aortic endothelial cells (PAEC) lysed a Fas+ human T-cell line, Jurkat. Anti-human Fas monoclonal antibody (mAb) specifically inhibited this killing in a dose-dependent manner, suggesting that porcine FasL recognizes and binds human Fas to induce apoptosis of human Fas+ cells. We next cloned porcine FasL, identifying an open reading frame of 849 base pairs predicting a protein of 282 amino acids. The predicted amino acid sequence was 85, 76, and 75% homologous to the predicted amino acid sequences of human, mouse, and rat, respectively, and found that PAEC expressed both FasL mRNA and protein. Transient transfection was used to increase or induce porcine FasL expression in PAEC or COS-7 cells. Transfection of PAEC with a plasmid encoding porcine FasL increased their ability to induce apoptosis in Jurkat cells, fresh human T cells activated with IL-2 and anti-CD3, and fresh IL-2-activated human (natural killer) NK cells. Moreover, porcine Fas L-transfected COS-7 cells induced significant apoptosis in Jurkat cells compared with that induced by mock-transfected COS-7 cells. Finally, the overexpression of porcine FasL in PAEC reduced their susceptibility as target cells to lysis by activated human NK or T cells. These findings suggest that porcine FasL overexpression in EC of vascularized xenografts may provide protection from cellular xenograft rejection.
内皮细胞（EC）是受体对移植器官免疫反应的主要靶细胞，且在其表面组成性表达Fas（CD95）配体（FasL）。我们在体外研究了猪FasL在人类抗猪反应产生过程中的作用。猪主动脉内皮细胞（PAEC）裂解了Fas+人T细胞系Jurkat。抗人Fas单克隆抗体（mAb）以剂量依赖性方式特异性抑制这种杀伤作用，表明猪FasL识别并结合人Fas以诱导人Fas+细胞凋亡。接下来，我们克隆了猪FasL，鉴定出一个849个碱基对的开放阅读框，预测其编码一个282个氨基酸的蛋白质。预测的氨基酸序列分别与人、小鼠和大鼠预测的氨基酸序列具有85%、76%和75%的同源性，并发现PAEC表达FasL mRNA和蛋白质。利用瞬时转染来增加或诱导PAEC或COS-7细胞中猪FasL的表达。用编码猪FasL的质粒转染PAEC，增强了它们诱导Jurkat细胞、用IL-2和抗CD3激活的新鲜人T细胞以及新鲜IL-2激活的人（自然杀伤）NK细胞凋亡的能力。此外，与mock转染的COS-7细胞相比，猪FasL转染的COS-7细胞诱导Jurkat细胞发生显著凋亡。最后，PAEC中猪FasL的过表达降低了它们作为靶细胞被激活的人NK或T细胞裂解的敏感性。这些发现表明，在血管化异种移植物的内皮细胞中过表达猪FasL可能提供针对细胞异种移植排斥的保护作用。
