Allograft Tolerance with Fas/FasL-Mediated Apoptosis

带血管同种异体移植是治疗终末期器官衰竭的有效方法。然而，在基因不同的个体之间进行器官移植，受到我们控制受者对移植物的免疫排斥反应的能力的限制。免疫抑制药物可能会降低排斥反应的严重程度，但不能产生对移植物的永久特异性耐受状态。在此，我们提出了一种免疫调节方法，使用具有强大的凋亡活性的改良形式的FasL来消除同种异体反应性T细胞，以防止同种异体心脏移植排斥反应和诱导耐受。FasL诱导的细胞凋亡是激活诱导细胞死亡的主要机制，与免疫稳态和自身耐受有关。使用野生型(Wt)FasL预防同种异体移植排斥反应一直存在争议。FasL最初是作为膜分子合成的，当阻止细胞表面时，它会诱导细胞凋亡。WtFasL也在表达后几分钟内被金属蛋白酶从细胞表面脱落，其可溶性形式对中性粒细胞具有抗凋亡和趋化作用。我们假设，如果FasL的凋亡活性与抗凋亡和趋化功能分离，那么它可以作为一种免疫调节分子。我们获得了具有很强的凋亡活性的修饰形式的FasL，并开发了一种新的方法，在1小时内在抗原提呈细胞(APC)和血管内皮细胞的表面蛋白水平表达FasL。在初步实验中，我们证明了在供体APC上传递FasL可以阻止幼年和预敏动物的同种异体反应，并防止胰岛同种异体移植排斥反应。在适应临床环境的条件下，血管内皮细胞上FasL蛋白的表达延长了移植心脏的存活时间。在这项计划中，大鼠和小鼠将在移植前和移植后的不同时间用表达FasL的同种异体APC免疫。FasL也将表达于心脏内皮细胞表面，用于免疫逃避和预防排斥反应。几种突变和转基因动物将被用来测试FasL诱导的细胞凋亡是否是观察到的免疫无应答的主要机制。这种基于蛋白质的方法可以很容易地应用于临床，并可能提供一个显著的优势，因为它的安全性和简单性，与基于DNA的表达的免疫调节方法相比。

Transplantation of vascularized allografts is an effective therapeutic alternative for patients with end-stage organ failure. Transplantation of organs between genetically different individuals, however, is limited by our ability to control the immunological rejection of the graft by the recipient. Immunosuppressive drugs may reduce the severity of rejection, but fail to create a state of permanent specific tolerance to the graft. We herein propose an immunomodulatory approach using a modified form of FasL with potent apoptotic activity to eliminate alloreactive T cells for the prevention of cardiac allograft rejection and induction of tolerance. FasL-induced apoptosis is the main mechanism of activation-induced cell death that is responsible for immune homeostasis and self-tolerance. The use of wild type (wt)FasL to prevent allograft rejection has been controversial. FasL is initially synthesized as a membranous molecule that induces apoptosis when prevention the cell surface. wtFasL is also shed from the cell surface by metalloproteinases within minutes of expression and the soluble form is antiapoptotic and chemotactic for neutrophils. We hypothesize that FasL can be used as an immunomodulatory molecule if its apoptotic activity is separated from anti-apoptotic and chemotactic functions. We generated modified forms of FasL with potent apoptotic activity and developed a novel approach to express FasL at the protein level on the surface of antigen- presenting cells (APCs) and vascular endothelium within 1 hour. In preliminary experiments, we demonstrated that delivery of FasL on donor APCs blocked alloreactive responses in naive and presensitized animals and prevented islet allograft rejection. Expression of FasL protein on vascular endothelium under conditions adapted from clinical settings prolonged survival of cardiac allografts. In this proposal, rats and mice will be immunized with allogeneic APCs expressing FasL at various times pre-and post- transplantation. FasL will also be expressed on the surface of heart endothelium for immune evasion and prevention of rejection. Several mutant and transgenic animals will be used to test whether apoptosis induced by FasL is the main mechanism of the observed immune nonresponsiveness. This protein-based approach may be readily applied to the clinic and may provide a significant advantage because of its safety and simplicity as compared with immunomodulatory approaches using DNA-based expression.