Allograft Tolerance with Fas/FasL-Mediated Apoptosis

血管化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，受到我们控制受体对移植物的免疫排斥的能力的限制。 免疫抑制药物可以降低排斥反应的严重程度，但不能建立对移植物的永久特异性耐受状态。我们在此提出了一种免疫调节方法，使用具有强效凋亡活性的改良形式的FasL来消除同种异体反应性T细胞，以预防心脏同种异体移植物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，负责免疫稳态和自身耐受。 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最初是作为膜分子合成的，当阻止细胞表面时诱导凋亡。wtFasL也在表达的几分钟内通过金属蛋白酶从细胞表面脱落，并且可溶形式对中性粒细胞具有抗凋亡和趋化性。 我们假设，FasL可以被用作免疫调节分子，如果其凋亡活性是从抗凋亡和趋化功能分离。 我们产生了具有有效凋亡活性的FasL的修饰形式，并开发了一种在1小时内在抗原呈递细胞（APC）和血管内皮表面上以蛋白水平表达FasL的新方法。在初步实验中，我们证明了在供体APC上递送FasL阻断了幼稚和致敏动物的同种异体反应性，并防止了胰岛移植排斥反应。FasL蛋白在血管内皮细胞上的表达延长了心脏移植物的存活。在该提议中，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也会在心脏内皮细胞表面表达，用于免疫逃避和预防排斥反应。将使用几种突变体和转基因动物来测试由FasL诱导的细胞凋亡是否是所观察到的免疫无反应性的主要机制。 这种基于蛋白质的方法可以容易地应用于临床，并且可以提供显著的优势，因为与使用基于DNA的表达的免疫调节方法相比，其安全性和简单性。

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.