应用HLA-A2.1限制性T细胞表位库探索1型糖尿病特异性多靶点细胞免疫治疗的研究

Insulin treatment for type 1 diabetics can not delay progressive loss of islet mass and avoid the occurrence of fragile diabetes during the course of the disease. The research on the immunotherapy based on the immunology mechanism of type 1 diabetes become the hot spot recently years. Current reported protocols of clinical immunotherapy for autoimmune diabetes can delay or decrease the progress of the loss of islet mass, but autoimmunity injury to pancreatic islets can not be stopped completely due to non-antigen speicificity and single autoantigen targeted in the reported studies. Therefore, based on our previous research on clinical immunotherapy with anti-CD20 agent for type 1 diabetes and mapping epitopes of islet antigen specific CD8+ T cell, we design to expand islet autoreactive CD8+ T cell from peripheral blood mononuclear cells of type 1 diabetic patients with HLA-2.1 restricted epitopes pool of islet antigen, such as IGRP, ZnT8, GAD, INS and IA-2. Then the islet antigen epitopic specific T cell and human isolated islets were transplanted under renal capsule membrane of HLA-2.1 transgenic mice, as the evaluation model of immunological injury by autoreactive T cell in vivo. The priming spleenocytes of transgenic mice with epitope peptides pool were transfer to human islets plus autoreactive T cell transplated mice, as multiantigen specific cell immunotherpay. The inhibitory effect of immunological injury were evaluated and compared with different epitope peptides pool. The purpose of the study is to explore the protocol of islet multiantigen specific cell immunotherapy for type 1 diabetes.

胰岛素治疗挽救不了1型糖尿病不断丧失的胰岛数量并避免脆性糖尿病的出现，近年针对1型糖尿病发病机制的免疫治疗研究成为热点，目前报道的1型糖尿病免疫治疗方案可一定程度延缓胰岛功能衰竭速度，但尚不能完全终止免疫损伤进程，其原因主要是阻断的特异性不够和针对的胰岛自身抗原较为单一。为此本研究小组在1型糖尿病临床免疫治疗和致病性T细胞表位前期研究基础上，拟应用IGRP、ZnT8、GAD、INS和IA-2等HLA-2.1限制性胰岛自身抗原表位多肽库扩增1型糖尿病病人外周血中致病性CD8+ T细胞，与人胰岛联合移植至HLA-2.1转基因鼠肾包膜下，建立致病性T细胞致人胰岛免疫损伤的体内评估模型；再以表位多肽库诱导免疫耐受后的转基因鼠淋巴细胞为特异性细胞免疫干预手段，摸索不同表位多肽库对相应抗原特异性致病性T细胞致人胰岛免疫损伤的阻效作用，探讨特异性多靶点细胞免疫治疗方案，为1型糖尿病临床免疫治疗探索新路。

致病性CD8+T细胞在1型糖尿病胰岛细胞的破坏中发挥关键作用，致病性CD8+T细胞的表达谱的筛选有助于早期诊断及作为免疫干预治疗的终点指标。本研究旨在探讨建立合适高效的致病性CD8+T细胞检测模型从而早期，准确诊断及治疗1型糖尿病。我们对携带HLA-A*0201基因型的109例受试者分别采用ELISPOT方法检测致病性CD8+T细胞并用放射免疫配体法进行四种胰岛自身抗体（GADA、IA-2A、ZnT8A和IAA）检测，并将多肽与纳米微粒连接制成ZnT8-PSB，处理多肽免疫的HHD鼠。我们筛选出7种胰岛自身抗原的28种表位，建立精选表位报道库。与健康对照相比，28个表位中的16个在初发抗体阳性的经典1型糖尿病中能检测出阳性反应。采用ROC曲线分析，确定2个表位阳性作为致病性CD8+T细胞表位库检测的最佳切点（敏感性75%，特异性94.4%），该致病性CD8+T细胞表位库检测在儿童中临床应用价值更大（敏感性93.3%，特异性94.4%）。进一步优化表位库，建立包含5个表位的优化表位诊断库，仍然能高效诊断1型糖尿病，并不明显损失敏感性和特异性（敏感性71.4%，特异性94.4%）。值得注意的是，77.8%的初发抗体阴性及25%的长病程抗体阴性的1型糖尿病患者其致病性CD8+T细胞检测阳性。联合致病性CD8+T细胞及胰岛自身抗体检测，敏感性可达91.1%。进一步，我们将HLA-A2.1转基因鼠体内免疫相应多肽后用ZnT8-PSB处理，流式细胞术分析结果表明，ZnT8-PSB处理后可以显著降低CD8+T细胞以及Th1、Th17等CD4+T细胞亚群的数量；同时上调Treg的水平。因此，采用包含5个表位的优化表位库检测致病性CD8+T细胞能够用于1型糖尿病免疫学诊断及病因学分类，具有极大的临床应用价值, 结合纳米微粒治疗，也为T1DM免疫治疗提供新的思路。