Allograft Tolerance with Fas/FasL-Mediated Apoptosis

Fas/FasL 介导的细胞凋亡的同种异体移植耐受

• 批准号: 6621531
• 负责人: Haval Shirwan
• 金额: $ 28.6万
• 依托单位: UNIVERSITY OF LOUISVILLE
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-12-15 至 2005-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6621531
• 关键词: T cell receptor; antigen presenting cell; apoptosis; chimeric proteins; cytotoxic T lymphocyte; disease /disorder prevention /control; enzyme linked immunosorbent assay; flow cytometry; gene induction /repression; genetically modified animals; heart function; heart transplantation; helper T lymphocyte; homeostasis; homologous transplantation; immune tolerance /unresponsiveness; immunocytochemistry; immunoprecipitation; isoantigen; laboratory mouse; laboratory rat; membrane proteins; nucleic acid sequence; transplant rejection; vascular endothelium; western blottings

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.

血管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，基因不同的个体之间的器官移植受到我们控制受者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，但无法对移植物产生永久的特异性耐受状态。我们在此提出了一种免疫调节方法，使用具有有效凋亡活性的修饰形式的 FasL 来消除同种异体反应性 T 细胞，从而预防心脏同种异体移植排斥和诱导耐受。 FasL 诱导的细胞凋亡是激活诱导细胞死亡的主要机制，负责免疫稳态和自我耐受。 使用野生型 (wt)FasL 预防同种异体移植排斥一直存在争议。 FasL 最初被合成为膜分子，当阻止细胞表面时诱导细胞凋亡。 wtFasL 也会在表达后几分钟内被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作用。 我们假设，如果 FasL 的凋亡活性与抗凋亡和趋化功能分开，它可以用作免疫调节分子。 我们生成了具有强效凋亡活性的 FasL 修饰形式，并开发了一种新方法，可在 1 小时内在抗原呈递细胞 (APC) 和血管内皮表面的蛋白质水平上表达 FasL。在初步实验中，我们证明在供体 APC 上传递 FasL 可以阻断初始动物和预致敏动物的同种反应反应，并防止胰岛同种异体移植物排斥。在适应临床环境的条件下，FasL 蛋白在血管内皮上的表达延长了同种异体心脏移植物的存活率。在此提议中，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 进行免疫。 FasL也会在心脏内皮表面表达，用于免疫逃避和预防排斥反应。将使用几种突变和转基因动物来测试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。 这种基于蛋白质的方法可以很容易地应用于临床，并且与使用基于 DNA 的表达的免疫调节方法相比，由于其安全性和简单性，可以提供显着的优势。