人锌转运蛋白8的HLA-A~*0201限制性CTL表位的预测及初步验证

目的预测和初步鉴定1型糖尿病(T1DM)主要自身抗原锌转运蛋白8(ZnT8)的HLA-A*0201限制性细胞毒性T淋巴细胞(cytotoxic T lymphocytes,CTL)表位,为基于ZnT8抗原表位的特异性免疫治疗奠定基础。方法选取BIMAS预测工具预测该抗原HLA-A*0201限制性结合肽,人工合成待测表位肽,利用T2细胞株测定各肽与HLA-A*0201分子的结合力。利用酶联免疫斑点检测(enzyme-linked immunospotassay,ELISPOT)方法检测候选肽刺激T1DM患者外周血单个核细胞分泌IFN-γ和IL-2的能力,利用标准51Cr释放试验检测特异性CTL诱导活性。结果在所筛选的5个候选CTL表位中,ZnT8(107-115)、ZnT8(115-123)及ZnT8(145-153)与HLA-A*0201分子具有较高的结合荧光强度,可在体外有效诱导抗原特异性CTL的产生,刺激T1DM患者PBMC分泌IFN-γ和IL-2,并对抗原肽负载的T2细胞具有明显的杀伤效应。结论 ZnT8(107-115)、ZnT8(115-123)及ZnT8(145-153)可能是HLA-A*0201限制性CTL表位,为基于人ZnT8抗原表位的特异性免疫治疗奠定理论基础。

Identification of HLA-A~*0201-restricted CTL epitopes of human ZnT8

This study is aimed to identify HLA-A*0201-restricted CTL epitopes from human ZnT8,which may lay a foundation for specific immunotherapy based on epitope.We predicted HLA-A*0201 restricted cytotoxic T lymphocyte(CTL) epitope from BIMAS.Then the predicted peptides were synthesized and purified.Furthermore,T2 cell line was used to determine binding activity of the peptide to HLA-A*0201 molecule.Finally,five candidate peptides were utilized to elicited CTL response which detected by ELISPOT assay and 51Cr release assays.We found that ZnT8(107-115),ZnT8(115-123),and ZnT8(145-153) out of the 5 candidate peptides could bind to HLA-A*0201 molecule with high affinity.And these peptides could induce specific CTLs in vitro,stimulate peripheral blood mononuclear cell(PBMC) to secrete IFN-γ and IL-2,as well as lyse target cells.All these results suggest that ZnT8(107-115),ZnT8(115-123),and ZnT8(145-153) might be the HLA-A*0201 restricted epitopes of ZnT8,and the results provide a foundation for immunotherapy of ZnT8.