人端粒酶逆转录酶HLA-A^* 0201限制性CTL表位的预测和鉴定

Prediction and identification of HLA-A~* 0201-restricted CTL epitopes from hTERT

目的人端粒酶逆转录酶(human telomerase reverse transcriptase,hTERT)在正常组织中不表达而在肿瘤组织中广泛表达,且与肿瘤生长密切相关,是肿瘤免疫治疗的理想靶位。HLA-A*0201是中国人群中最为常见的HLA-I类分子的型别,阳性率达30%。筛选hTERT HLA-A*0201限制性细胞毒性T淋巴细胞(cytotoxic lymphocyte,CTL)表位,对于有hTERT表达的人群的肿瘤免疫治疗有重要临床意义。文中采用生物信息学和免疫学方法,预测和鉴定hTERT的HLA-A*0201限制性CTL表位。方法联合利用表位预测数据库SYFPEITHI、BIMAS和NetCTL和分子模拟技术对hTERT的HLA-A*0201限制性CTL表位进行预测。用T2细胞株测定各肽与HLA-A*0201分子的亲和力和稳定性。酶联免疫吸附斑点法(enzyme linkedimmunospot assay,ELISPOT)检测HLA-A*0201+健康人对肽的T淋巴细胞反应。结果预测得出hTERT抗原的6个CTL表位,即ILAKFLHWL(540～548)、LMSVYVVEL(548～556)、ILSTLLCSL(836～844)、CLKELVARV(76～84)、LLGASVLGL(675～683)、FLDLQVNSL(923～931)。分子模拟的计算结果提示ILAKFLHWL(540～548)和LLGASVLGL(675～683)肽与HLA-A*0201分子之间的结合较其他多肽稳固。亲和力实验结果表明上述2个肽与HLA-A*0201的亲合力较强,荧光系数FI分别为3.20和2.98,肽-HLA-A*0201复合物半数解离时间DC50均大于8 h。ELISPOT显示ILLGASVLGL(675～683)与其他5个肽相比,可诱导较强的T淋巴细胞反应,T细胞频率为(534±57)个斑点形成细胞(spot-forming cell,SFC)/106个外周血单个核细胞(peripheral blood mononeuclear cell,PBMC)。结论 ILLGASVLGL(675～683)有可能是人端粒酶逆转录酶HLA-A*0201的限制性CTL表位。

Objective Human telomerase reverse transcriptase(hTERT),widely present in human cancers,generally absent in normal tissues,and required for tumor cell survival,is essentially identified as an almost ideal 'universal' tumor associated antigen.As HLA-A*0201-expressing individuals constitute more than 30% of the Chinese population,to identify peptides derived from hTERT HLA-A*0201-restricted CTL epitopes is of great clinical significance for specific immunotherapy against tumor.The present study was to predict and identify hTERT specific HLA-A*0201 restricted epitopes using bioinformatic methods and immunological assay.Methods The online databases SYFPEITHI,BIMAS and NetCTL and molecular modeling were applied to predict the potential HLA-A*0201 restricted epitopes from hTERT.T2 cells were used to determine the peptide binding affinity and HLA-A*0201 peptide complex stability.IFN-γ ELISPOT was employed to monitor the magnitude of specific T lymphocytes to candidate peptides.Results We obtained 6 epitopes with high immunogenicity scores,i.e.,ILAKFLHWL(540-548),LMSVYVVEL(548-556),ILSTLLCSL(836-844),CLKELVARV(76-84),LLGASVLGL(675-683),and FLDLQVNSL(923-931),of which,2 satisfied the criteria of HLA-A*0201-restricted CTL epitopes in molecular modeling analysis.ILAKFLHWL(540-548) and ILLGASVLGL(675-683) could bind to HLA-A*0201 with high affinity and flurorescene indexes(FI) of 3.20 and 2.98,respectively,and the dissociation time of 50% HLA-A*0201 peptide complex was > 8 h.ELISPOT analysis showed that ILLGASVLGL(675-683) could induce T lymphocyte responses.The frequency of peptide-specific T cells was 534±57 SFC/106 PBMCs.Conclusion ILLGASVLGL(675-683) might be a new HLA-A*0201 restricted CTL epitope.