纳米微粒修饰致病性T细胞表位多肽诱导免疫耐受治疗1型糖尿病的研究

The induction of long-lasting T cell tolerance is a primary goal of therapies for type 1 diabetes (T1D). Many strategies have attempted to achieve this goal. However, they have had limited success and have been associated with adverse effects. Recent study showed that epitope-coupled particles(500nm) can safely induce tolerance in experimental autoimmune encephalomyelitis (EAE) and could have broad utility in a range of immune disorders. Our previous research found three major pathogenic CD8+ T cell epitopes derived from ZnT8. Therefore, based on our study, we will design to observe if nanoparticles bearing ZnT8 or IGRP epitopes could safely induce long-lasting T cell tolerance and attenuate disease in T1D. As evaluation of epitope-coulpled particles involved in induction of T cell tolerance, antigen specific T cell responses will be detected from immunized mice in vitro or from mice with epitope-coupled particles intravenous injection and explore the possible mechanism. Then the antigen-specific CTL will be evaluated by cocultured with T cell clones and human isolated islets after treated with ZnT8 or IGRP epitope-coulpled particles. Furthermore, antigen-specific T cells in T1D patients will be analyze after treated with epitope-coulpled particles in vitro. The purpose of the study is to demonstrate antigen attached to nanoparticles could induce tolerance in T1D. It will explore new ideas of clinical immunotherapy for T1D.

诱导长期T细胞免疫耐受是1型糖尿病(T1D)免疫治疗的主要目标。目前大多数的免疫治疗方案因疗效不确定或存在相关的副反应而未能达到预期目标。最近研究表明纳米微粒修饰的表位能够有效的诱导实验性自身免疫脑脊髓膜炎（EAE）小鼠产生长期免疫耐受，预防和治疗EAE的发生和进展。因此申请人在前期1型糖尿病致病性T细胞表位研究的基础上，观察了纳米微粒修饰的ZnT8或IGRP的混合表位在HLA-A2.1转基因鼠体外和体内能否减轻或阻止抗原表位特异性T细胞反应及其可能的机制。同时应用纳米微粒修饰的表位预处理人的ZnT8或IGRP的CD8+T细胞克隆与人胰岛体外共培养，进一步分析处理过后的CD8+T细胞对人胰岛的攻击能力。最后筛选相应表位阳性的T1D患者，进一步证明纳米微粒修饰致病性T细胞表位对T1D患者外周血中致病性T细胞反应的影响。本研究将纳米技术引入免疫耐受的治疗方案，为T1D免疫治疗探索新的思路。

目的：诱导抗原特异性的免疫耐受在NOD鼠模型中提供引人注目的免疫治疗方式，然而这一方式转化到1型糖尿病病人中则仍未取得显著的效果。我们的研究应用500nm聚苯乙烯纳米微球共价交联混合的在1型糖尿病病人中筛选出的免疫显性的HLA- A*02:01限制性的表位多肽（peptides-PSB），证明其是否可以促进免疫耐受，并探讨peptides-PSB促进T细胞耐受的机制。.方法：混合的HLA-A*02:01限制性的表位与PSB交联后尾静脉注射给人源化的HHDII鼠，观察其诱导耐受的现象及其机制。通过观察NOD.β2mnull HHD鼠发病率、胰岛炎及胰腺中调节性T细胞的表达，证明混合peptides-PSB是否可以有效延缓该人源化小鼠的发病过程。在HLA-A*02:01限制性1型糖尿病病人的外周血中通过ELIspot实验证明是否可以有效抑制抗原特异性T细胞反应。.结果：我们证明peptides-PSB在转基因鼠体内可以诱导抗原特异性的免疫耐受，其机制是通过抑制T细胞活化，介导T细胞无能以及发挥CD4+CD25+调节性T细胞功能。peptides-PSB在脾脏中可以快速的使CD169+巨噬细胞产生CCL22，使调节性T细胞和CD11C+DC细胞活化和聚集。更进一步，peptides-PSB可以阻止和延缓人源化 NOD.β2mnull HHD鼠进展为自身免疫糖尿病的进程。同时，在1型糖尿病病人的外周血中可以抑制外周血抗原特异性T细胞的反应。.结论：我们第一次证明通过混合多肽-PSB可以诱导T细胞耐受并阻止自身免疫糖尿病的进程。这一结果为今后的1型糖尿病抗原特异性免疫治疗提供新的思路，并证明先天免疫机制在早期通过细胞与细胞间相互作用参与了多肽-PSB诱导的免疫耐受。