Allograft Tolerance with Fas/FasL-Mediated Apoptosis

带血管的同种异体移植是终末期器官衰竭患者的有效治疗选择。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体对移植物的免疫排斥的能力的限制。免疫抑制药物可以减轻排斥反应的严重程度，但不能对移植物产生永久性的特异性耐受。我们在此提出了一种免疫调节方法，使用具有强凋亡活性的FasL修饰形式来消除同种异体反应性T细胞，以防止心脏移植排斥和诱导耐受。fasl诱导的细胞凋亡是激活诱导的细胞死亡的主要机制，与免疫稳态和自我耐受有关。使用野生型（wt）FasL来预防同种异体移植排斥反应一直存在争议。FasL最初是作为一种膜性分子合成的，在防止细胞表面时诱导细胞凋亡。wtFasL也在表达后几分钟内被金属蛋白酶从细胞表面脱落，其可溶性形式具有抗凋亡和中性粒细胞趋化作用。我们推测，如果FasL的凋亡活性与抗凋亡和趋化功能分离开来，它可以作为一种免疫调节分子。我们产生了具有强凋亡活性的FasL修饰形式，并开发了一种新的方法，在抗原呈递细胞（APCs）表面和血管内皮1小时内蛋白水平表达FasL。在初步实验中，我们证明了FasL在供体apc上的递送阻断了幼稚和现敏化动物的同种异体反应，并防止了胰岛移植排斥反应。在临床条件下FasL蛋白在血管内皮上的表达延长了同种异体心脏移植的生存期。本研究将在移植前后不同时间用表达FasL的异体apc免疫大鼠和小鼠。FasL也可在心脏内皮细胞表面表达，用于免疫逃避和预防排斥反应。一些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反应的主要机制。与使用dna表达的免疫调节方法相比，这种基于蛋白质的方法可能很容易应用于临床，并且可能提供显著的优势，因为它的安全性和简单性。

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.