抗原特异性ZnT8-CAR修饰Tregs调控1型糖尿病致病性T细胞的机制研究

Type 1 diabetes is a antigen-specific T cell mediated autoimmune disease. Natural regulatory T cells (Tregs) play a critical role in maintaining immune tolerance. Tregs suppress immune responses in a dominant and antigen-specific manner. Numerous examples exist that antigen-specific Tregs are more effective in several autoimmune syndromes when compared to polyclonal populations. Tregs can confer long-term protection against auto-inflammatory diseases in mouse models. A major challenge in antigen-specific Treg therapy in humans lies in the isolation of sufficient quantities of antigen-specific T cells - a challenge that is not easily overcome in humans where sampling is largely limited to peripheral blood. A preferred approach to endow Tregs with a desired specificity uses a chimeric antigen receptor (CAR) with antibody-type specificity. Therefore, based on our previous research on a clinically-relevant FACS-based isolation and in vitro expansion procedure for generating Tregs and the humanized scFv antibody library against type 1 diabetes, we are to initiate efforts with a fully mature polyclonal Trega population isolated from peripheral blood and redirect cell specificity by utilizing a lentiviral vector system to express CAR composed of an antibody variable region specific for β-cell-specific zinc transporter 8. Antigen-specific suppression will be evaluated in vitro in the mixed leukocyte reaction and human islet-leukocyte reaction and in vivo in NOD/SCID mice. We will examine the mechanisms how ZnT8-CAR modified Tregs regulate autoreactive T cells. Our findings could lead to the development of new therapies to protect beta cells and improve islet graft survival from the immune system in patients with type 1 diabetes.

1型糖尿病是一种抗原特异性T细胞介导的自身免疫疾病，阻断自身免疫损伤是保护和逆转胰岛细胞功能的希望。天然调节性T细胞(Tregs)在维持自身和移植耐受方面起着重要作用。TCR转基因动物模型研究证实Tregs具有抗原特异性。由于抗原特异性Tregs数量极少难以直接分离获得,目前对人胰岛抗原特异性Tregs在1型糖尿病发病机制知之甚少。为此申请人在与美国UCSF合作建立体外高效扩增Tregs技术和构建1型糖尿病scFv抗体库前期研究基础上,拟筛选胰岛自身抗原特异性ZnT8-scFv,构建ZnT8-CAR修饰 Tregs,再联合ELISOPT和FACS分选ZnT8致病性T细胞,与人胰岛共培养在多个水平探讨抗原特异性ZnT8-CAR Tregs调控致病性T细胞的机制和对人胰岛的可能保护作用。研究结果将阐明Tregs抗原特异调控方式,为1型糖尿病Tregs细胞免疫治疗和诱导胰岛移植免疫耐受探索新路。

1型糖尿病是一种抗原特异性T细胞介导的自身免疫疾病，阻断自身免疫损伤是保护和逆转胰岛细胞功能的希望。天然调节性T细胞(Tregs)在维持自身和移植耐受方面起着重要作用。TCR转基因动物模型研究证实Tregs具有抗原特异性。由于抗原特异性Tregs数量极少难以直接分离获得,目前对人胰岛抗原特异性Tregs在1型糖尿病发病机制知之甚少。构建胰岛抗原特异性Tregs是深入开展研究的关键。CAR修饰效应性T细胞在肿瘤治疗中展现出良好的效果和应用前景。本研究首先确定锌转运体8抗原表位在1型糖尿病免疫应答中抗原特异性，首次从新诊断1型糖尿病病人外周血基因中构建了一个库容达1×108的大容量scFv 全人源噬菌体抗体库，保证筛选结果的多样性，利用本研究构建人抗原特异性ZnT8（N/C）蛋白融合肽，最终筛选到的人源性抗1 型糖尿病ZnT8 特异性单链抗体scFv-C22/C27，其能够特异的识别人胰岛β细胞上的ZnT8蛋白，具有良好的生物学活性，申请专利2项。在以上基础之上，本研究成功构建ZnT8-CAR修饰的Tregs，其具有Tregs的表型和功能。研究结果为阐明Tregs抗原特异调控方式,为1型糖尿病Tregs细胞免疫治疗和诱导胰岛移植免疫耐受探索新路。