四聚体技术检测原发性胆汁性肝硬化患者血循环中抗原特异性T细胞

Quantitation of antigen specific T lymphocytes in peripheral blood of patients with primary biliary cirrhosis using tetramer technology

目的：定量检测原发性胆汁性肝硬化（PBC）患者体内抗原特异性T淋巴细胞含量，探讨其在PBC发病机制中的作用。方法：采用四聚体技术检测15例HLA-A＊0201阳性（A2^＋）PBC患者外周血单个核细胞（PBMC）经抗原肽诱导生长的细胞毒性T淋巴细胞（CTL）中PDC-E2 159～167aa与PDC—E2 165～174aa特异性CD8^＋T细胞频率，以A＊0201阴性（A2^-）PBC患者与A2^＋的其他慢性肝病和健康自愿者为对照组：结果：在A2^＋PBC患者PDC-E2 159～167aa与PDC-E2165～174aa诱导的CTL中可检测到其相应的四聚体／CD8^＋细胞，平均频率分别为0．42%±0．24％（0．17％～1．08％）、0．27%±0．17%（0．05％～0．56％），各对照组的四聚体阳性细胞频率均低于0．1％，差异非常显著（P〈0．001）；A2^＋PBC组中PDC-E2 159～167aa特异性的CTL频率与PDC—E2 165～174aa特异性的CTL无显著性差异（P〉0．05）：处于临床Ⅰ、Ⅱ期的A2^＋PBC患者中CD8^＋特异性CTL频率均较Ⅲ期的要高（P〈0．05）：PDC—E2159～167aa特异性CTL与PDC—E2165～174aa特异性CTL频率在抗．PDC阳性PBC组和抗．PDC阴性组之间均无显著性差异（P〉0．05）：结论：HLA—A＊0201限制性的PDC-E2159～167aa和PDC-E2165～174aa特异性CD8^＋CTL在PBC疾病进展中起重要作用，抗线粒体抗体阴性或抗-PDC阴性PBC患者与阳性患者可能有着相似的T细胞介导的免疫发病机制。

AIM： To quantitate antigen specific T lymphocytes in peripheral blood from patients with primary biliary cirrhosis （PBC） and study the role of antigen specific T lymphocytes in the development of PBC. METHODS： Using tetramers and CD8 monoclonal antibody staining, PDCE2 159 - 167aa and PDC-E2 165 - 174aa specific CD8^＋ T lymphocytes were determined respectively in the peptide-induced cytotoxic T cell lines prepared from peripheral blood mononuclear cells （PBMC） of 15 PBC patients. The frequencies of these two kinds of antigen specific T lymphocytes in HLA-A ＊ 0201 positive （ A2 ^＋ ） PBC were compared with those in A2- PBC patients, patients with other A2 ^＋ chronic liver diseases and healthy controls. RESULTS： PDC-E2 159 - 167aa/HLA-A ＊ 0201 and PDC-E2 165 - 174aa/HLA-A, 0201 tetramer positive CD8 ^＋ T lymphocytes were detected in all of A2 ^＋ PBC patients with average percentages of 0.42% ±0.24% （0. 17% ± 1.08%） and 0.27% ±0.17% （0.05%±0.56% ）, respectively. The frequencies of the two kinds of antigen specific CD8^ ＋ T lymphocytes from peripheral blood were significantly high in earlier stages I and II of PBC as compared with stage Ⅲ （ P 〈 0. 001 ）, while no difference was found between PDC-E2 159 - 167aa and PDC-E2 165 - 174aa specific CD8＋ T lymphocytes at the same stages. In addition, there existed no statistical difference between frequencies of antigen specific T lymphocytes in AMA or anti-PDC positive and negative PBC patients（ P 〉0.05 ）. CONCLUSION： This study suggests that HLA-A ＊ 0201 restricted PDC-E2 165 - 174aa and PDC-E2 159 - 167aa specific CTL play important roles in the development of PBC, and there might be a similar mechanism of T cell-mediated damage between AMA or anti-PDC positive and negative PBC patients.