鼠胰岛素B链HLA-A*0201限制性CTL表位拮抗型改造肽的设计与鉴定

Generation and identification of HLA-A*0201-restricted CTL antagonist altered peptide derived from murine insulin B chain

目的设计并鉴定具有负调致病细胞毒性T细胞(cytotoxic Tlymphocyte,CTL)免疫应答功能的改造肽配体(al-tered peptide ligand,APL),为基于胰岛素自身抗原的I型糖尿病特异性免疫治疗奠定基础。方法利用InSilico技术模拟构建TCR-pMHC(TCR-HLA-A*0201-mInsB5-14)复合物三维结构,根据结构选择改造位点P6;通过保守/不保守单个氨基酸虚拟替换得到若干候选APL;通过自由能变化计算、相对亲和力检测、细胞因子分泌水平检测等体外功能实验,初步鉴定具有拮抗效应的APL。结果筛选得到的APL mInsB5-14H6F(H→F)与天然肽mInsB5-14空间结构相似,能与HLA-A*0201分子结合,其HLA-A*0201分子的相对亲和力与mInsB5-14相近。该APL作用下mInsB5-14刺激CD8+T细胞分泌IFN-γ的水平明显低于天然肽单独刺激下特异性CTL分泌IFN-γ的水平(P<0.05)。结论基于天然自身抗原表位mInsB5-14改造得到的mInsB5-14H6F是HLA-A*0201限制性的APL,该APL通过降低致病CTL特异性分泌IFN-γ的水平,可能诱导HLA-A*0201转基因NOD小鼠的自身耐受,从而为I型糖尿病治疗性疫苗的研发奠定了基础。

Evidences have suggested that autoreactive CD8＋ T cells are the foremost and important contributors to insulin producing beta-cell destruction in the pathogenesis of type 1 diabetes.Administration of self-peptides or altered peptide ligands（APLs） for the induction of diabetogenic CD8＋ T cell tolerance provides a new strategy for diabetes immunotherapy.Murine insulin B chain（5-14） is an HLA-A＊0201 restricted immunodominant epitope that can induce diabetogenic CTL response in humanized NOD mice.In this study,we designed several altered peptide ligands of mInsB5-14 with single TCR contact residue substitution by in silico strategy.The antagonist APLs were identified by HLA-A2 binding and IFN-γ secretion inhibition assay.InsB5-14H6F,an APL with phenylalanine substitution at residues 6,was verified as a potential antagonist APL which could significantly downregulate the IFN-γ secretion of mInsB5-14-specific CTLs.The InsB5-14H6F could be used for treating a CD8＋ T cell-mediated diabetes model.