Judge T A, Desai N M, Yang Z, Rostami S, Alonso L, Zhang H, Chen Y, Markman J F, DeMateo R P, Barker C F, Naji A, Turka L A
Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104, USA.
Transplantation. 1998 Aug 27;66(4):426-34. doi: 10.1097/00007890-199808270-00003.
One of the best-defined mechanisms for the induction of apoptosis involves signaling via the cell surface molecule Fas, after binding of Fas ligand. Expression of Fas ligand is tightly regulated, being expressed primarily by T cells after activation, where it serves as a self-regulatory mechanism for immune responses. Fas ligand has also been found to be expressed constitutively at sites of immune privilege such as the testes and the anterior chamber of the eye. Recently, co-transplantation of Fas ligand-transfected myoblasts in association with islet cell allografts was shown to prolong islet allograft survival but only rarely led to indefinite graft survival. Graft rejection was associated with loss of Fas ligand on the myoblasts, suggesting that direct expression of the transgene on the islets might be more effective.
A replication-defective adenoviral construct containing murine Fas ligand (Ad/MFL) was prepared by homologous recombination. NIH 3T3 cells, rodent splenocytes, and murine islets were infected with Ad/MFL and examined in vitro for functional murine Fas ligand expression. Survival of Ad/MFL-infected islets was subsequently evaluated in vivo in both syngeneic and allogeneic islet transplantation models.
Cell lines and islet allografts transfected with Ad/MFL expressed a functional Fas ligand, capable of inducing apoptosis (confirmed by three distinct assays for DNA fragmentation) in Fas+ targets, but not in Fas- controls. Furthermore, Ad/MFL was able to modify allogeneic immune responses in vitro, as addition of this virus, but not a control adenovirus, significantly reduced proliferation in a mixed lymphocyte reaction. Surprisingly, however, transplantation of islet allografts transfected with Ad/MFL resulted in long-term allograft survival in only 1 of 30 recipients. Moreover, adenoviral-mediated Fas ligand gene transfer was complicated by transient, dose-dependent islet dysfunction, perhaps contributing to the lack of long-term engraftment.
These data suggest that adenoviral-mediated Fas ligand expression may impair normal islet function in vivo, and indicate that alternative strategies for Fas ligand transgene delivery may be required in this setting.
诱导细胞凋亡的最明确机制之一涉及在Fas配体结合后通过细胞表面分子Fas进行信号传导。Fas配体的表达受到严格调控，主要在激活后的T细胞中表达，在那里它作为免疫反应的一种自我调节机制。Fas配体也被发现在免疫赦免部位如睾丸和眼前房中组成性表达。最近，与胰岛细胞同种异体移植联合移植Fas配体转染的成肌细胞可延长胰岛同种异体移植的存活时间，但很少能导致移植长期存活。移植排斥与成肌细胞上Fas配体的丧失有关，这表明转基因在胰岛上的直接表达可能更有效。
通过同源重组制备含有鼠Fas配体的复制缺陷型腺病毒构建体（Ad/MFL）。用Ad/MFL感染NIH 3T3细胞、啮齿动物脾细胞和鼠胰岛，并在体外检测功能性鼠Fas配体的表达。随后在同基因和异基因胰岛移植模型中体内评估Ad/MFL感染的胰岛的存活情况。
用Ad/MFL转染的细胞系和胰岛同种异体移植表达功能性Fas配体，能够在Fas+靶细胞中诱导细胞凋亡（通过三种不同的DNA片段化检测方法证实），但在Fas-对照细胞中则不能。此外，Ad/MFL能够在体外改变同种异体免疫反应，因为添加这种病毒而不是对照腺病毒可显著降低混合淋巴细胞反应中的增殖。然而，令人惊讶的是，用Ad/MFL转染的胰岛同种异体移植在30名受者中只有1例实现了长期移植存活。此外，腺病毒介导的Fas配体基因转移因短暂的、剂量依赖性的胰岛功能障碍而变得复杂，这可能是导致缺乏长期植入的原因。
这些数据表明腺病毒介导的Fas配体表达可能会损害体内正常胰岛功能，并表明在这种情况下可能需要替代的Fas配体转基因递送策略。
