致凋亡蛋白体外工程化异体胰岛移植治疗NOD小鼠1型糖尿病的机制研究

The objective of this grant proposal is to use a novel form of Fas ligand (SA-FasL) molecule having potent immunoregulatory activities and a unique method, ProtEx™, to rapidly and effectively display SA-FasL protein on allogeneic pancreatic islets as an alternative to gene therapy to regulate both auto and alloreactive immunity for the treatment of type 1 diabetes (T1D) using NOD as a mouse model of the human disease. .FasL-induced apoptosis plays an important role in immune homeostasis and tolerance to self antigens. These aspects of FasL led to intense studies for its use to induce tolerance to self and alloantigens for the treatment of autoimmunity and allograft rejection. However, ectopic expression of FasL in cells and tissues of interest using gene therapy is a technically challenging approach and has safety concerns. Therefore, we recently developed the ProtEx™ technology that involves i) the generation of chimeric proteins consisting of core streptavidin (SA) and extracellular portion of immunological ligands, and ii) the display of such molecules on biological membranes derivatized with biotin . Chimeric proteins exist as stable tetramers and oligomers, owing to the tetramer-forming properties of streptavidin, and as such have improved function by effectively crosslinking their counter receptors on immune cells for potent signal transduction. Inasmuch as receptor/ligand interactions on the surface of immune cells are critical to immune responses and these interactions need not to be extensive in duration, transient display of exogenous immunological ligands on the cell surface provides a powerful approach for immunomodulation. .A chimeric form of FasL (SA-FasL) protein was generated and shown to have potent apoptotic activity as a soluble molecule or when displayed on biological membranes. In this application, we propose to display SA-FasL on allogeneic pancreatic islets and use these engineered islets for immunomodulation to treat T1D in NOD mice. Rapamycin (Rapa), a clinically approved immunomodulatory agent, will be used transiently to further augment the tolerogenic effects of SA-FasL as well as establish a clinically applicable tolerance protocol. We hypothesize that immunomodulation with SA-FasL-islet grafts.under transient cover of Rapa will induce apoptosis in activated auto and alloreactive pathogenic T effector (Teff) cells expressing high levels of Fas receptor, initiating a cascade of immunomodulatory events resulting in the induction/generation of CD4+CD25+FoxP3+ T regulatory (Treg) cells that maintain tolerance to auto and alloantigens. Control of allo and autoimmunity is expected to result in the treatment/cure of T1D by allogeneic islet Tx and regeneration of endogenous β-cells.

I 型糖尿病(Type 1 Diabetes，T1D)是一种自身免疫性疾病，诱导同种胰岛移植免疫耐受是治疗T1D的重要方向，而Fas／FasL介导的细胞凋亡在调节免疫应答方面起到决定性作用。本课题组研发了新型的体外细胞工程化修饰技术—ProtEx，可使膜型FasL 快速高效的表达在小鼠胰岛表面，并以此为基础成功治疗初发型T1D。进一步计划在NOD小鼠完全型T1D模型中实现免疫调节，保护移植胰岛长期存活，并诱导内源性β细胞再生，从而实现可临床应用的免疫调节策略，克服胰岛短缺的现状，彻底改变T1D的治疗模式。建立SA-FasL工程化修饰移植胰岛诱导免疫耐受的理论体系，揭示该过程是否诱导内源性β细胞再生，阐明Teff细胞凋亡激活的免疫调节如何引起级联反应获得免疫耐受，Treg细胞的大量产生是否源于吞噬细胞吞噬Teff细胞凋亡，而后诱导免疫调节因子释放所致，揭示移植免疫新的机制。

I型糖尿病(Type 1 Diabetes，T1D)是一种自身免疫性疾病，诱导同种胰岛移植免疫耐受 是治疗T1D的重要方向，而Fas／FasL介导的细胞凋亡在调节免疫应答方面起到决定性作用。本课题组研发了新型的体外细胞工程化修饰技术—ProtEx，可使膜型FasL 快速高效的表达在小鼠胰岛表面，并以此为基础成功治疗初发型T1D。进一步计划在NOD小鼠完全型T1D模型中实现 免疫调节，保护移植胰岛长期存活，并诱导内源性β细胞再生，从而实现可临床应用的免疫调 节策略，克服胰岛短缺的现状，彻底改变T1D的治疗模式。建立SA-FasL工程化修饰移植胰岛诱导免疫耐受的理论体系，揭示该过程是否诱导内源性β细胞再生，阐明Teff细胞凋亡激活的免 疫调节如何引起级联反应获得免疫耐受，Treg细胞的大量产生是否源于吞噬细胞吞噬Teff细胞 凋亡，而后诱导免疫调节因子释放所致，揭示移植免疫新的机制。. 应用ProtExTM技术制备新型SA-FasL蛋白，在体外快速有效地工程化修饰于同种异基因胰岛细胞表面，在NOD小鼠完全型T1D模型中实现免疫调节，.保护移植胰岛长期存活，从而实现可临床应用的免疫调节策略。建立SA-FasL工程化修饰移植胰岛诱导细胞免疫耐受的理论体系，揭示该过程是否诱导内源性β细胞再生，Treg细胞是否其关键，阐明Teff细胞凋亡激活的免疫调节如何引起级联反应获得免疫耐受，从而发现在SA-FasL诱导移植免疫耐受过程中，Teff细胞凋亡、吞噬细胞、免疫调节因子以及Treg细胞产生存在的顺序关系。. 1.课题组建立了稳定的小鼠肾包膜下胰岛移植模型，建立稳定的NOD小鼠初发型及完 全性T1D模型。 . 2. 课题组采用ProtEx 技术体外修饰供体胰岛细胞，使其快速表达SA-FasL。. 3. 课题组在NOD小鼠初发型T1D模型中优化SA-FasL工程化修饰胰岛方案。. 可明确iTregs和吞噬细胞在FasL 发挥移植免疫调节中的作用，为FasL 作用机制提供新的科学解释和补充。了解以上机制，将有助于推进该技术向大动物和灵长类动物模型的转化，也可应用于异种胰岛移植，从而克服胰岛短缺的现状，彻底改变T1D的治疗模式。