原发性胆汁性肝硬化患者中自身抗原丙酮酸脱氢酶特异性CD8+CTL表位预测及鉴定

Prediction and Identification of Autoepitopes of PDC-E2 SpecificCD8+CTL in Primary Biliary Cirrhosis Patients

目的鉴定原发性胆汁性肝硬化(PBC)患者中自身抗原丙酮酸脱氢酶(PDC-E2)上的HLA-A觹0201限制性CD8+CTL表位,为临床探索特异性免疫治疗奠定基础。方法应用数据库SYFPEITHI预测PDC-E2上两段涵盖B细胞表位和CD4+T细胞表位的氨基酸序列(163～184aa和36～49aa)附近可能存在的HLA-A觹0201限制性T细胞表位,再通过T2细胞株、细胞增殖试验和细胞毒性检测分别分析各抗原肽与HLA-A觹0201的结合力、诱导患者外周血单个核细胞(PBMCs)增殖能力及抗原肽诱导的抗原特异性T细胞杀伤毒性,逐步鉴定HLA-A觹0201限制性CD8+CTL细胞表位。结果数据库初步预测到5个可能性较大的HLA-A觹0201限制性抗原肽,其中两个抗原肽(159～167aa和165～174aa)显示与T2细胞上HLA-A觹0201分子有较高的亲和力,这两个抗原肽能刺激大部分HLA-A觹0201阳性PBC患者PBMC增殖,并且由其诱导产生的CTL具有特异杀伤活性。结论位于PDC-E2内酯酰区上的KLSEGDLLA穴159～167aa)和LLAEIETDKA穴165～174aa雪是PBC患者体内HLA-A觹0201限制性的CD8+CTL表位。

Objective To identify autoepitopes of E2 subunit of pyruvate dehydrogenase complex?穴PDC-E2?雪specific CD8+CTL in primary biliary cirrhosis?穴PBC?雪patients. Methods An online database SYFPEITHI was applied to predict HLA-A?觹0201 restricted epitopes which located in PDC-E2 30 - 50 aa and 150 -190 aa where B-cell epitopes clustered with CD4+ T-cell epitopes. T2 cell line reconstitution and stabilization assay?熏 induction of specific CTL lines from peripheral blood mononuclear cells?穴PBMCs?雪of patients with PBC and cytotoxicity of peptides-induced CTL were performed to screen the epitopes from those candidates. Results Five potential epitopes were predicted by database. Of the 5 candidates?熏 two peptides 159-167 aa and 165 - 174 aa?熏 with highly binding activity to HLA-A?觹0201 molecules?熏 could stimulate PBMCs from most HLA-A?觹0201 positive PBC patients to proliferate and peptide-induced CTL lines showed specific cytotoxicity. Conclusion Peptides of KLSEGDLLA(159 - 167 aa)and LLAEIETDKA(165 - 174 aa)in the inner lipoyl domain of PDC-E2 are HLA-A?觹0201 restricted CD8+ CTL immunodominant epitopes in PBC.