急性乙肝患者HBV特异性CD8T细胞受体基因的克隆分析

Cloning analysis of HBV-specific CD8 T cell receptor gene in patients with acute hepatitis B

目的探讨急性乙肝患者HBV特异性CD8 T细胞受体（TCR）参与免疫应答的分子机制。方法分离HLA-A2阳性急性乙型肝炎患者外周血单个核细胞（PBMCs）,用荧光标记的CD3、CD8抗体和五聚体染色,流式细胞仪检测HBsAg183-191和HB-sAg335-343特异性CD8 T细胞频率。取6例患者PBMCs,用HBsAg335-343表位多肽诱导培养34周,用磁珠分选出CD8 T细胞后再用流式细胞术分选HBV特异性CD8 T细胞,经IL-2/CD3抗体/CD28抗体扩增后提取细胞mRNA,用cDNA5′末端快速扩增技术获取TCR分子α链和β链全长可变区基因片段并进行克隆和测序,每个样本的克隆测序数≥50个。确定TCRα、β链基因家族并比较CDR3区序列特点。结果 6例患者样本600多个TCRα、β链克隆基因序列分析结果表明,HBV特异性CD8 T细胞均呈现TCR分子α、β链基因家族优势表达特点,每例患者样本以24种α链和14种β链家族为主,其中α2、α14、α15、β3、β13和23链家族同时出现在1例以上患者,有1例患者所有克隆分析的β链基因均为β13家族。同一患者同一家族α、β链CDR3区序列趋于一致,而不同患者同一家族α、β链CDR3区序列相差较大。结论经HBV抗原表位多肽诱导增殖的特异性CD8 T细胞具有明显的TCRα、β链取用优势特点,这种TCR链的优势表达特点可能是影响抗HBV感染特异性细胞免疫力的重要分子基础。

Objective To investigate the molecular mechanism of T cell receptor（TCR） in CD8 T cell-mediated immune response to HBV in patients with acute hepatitis B（AHB）.Methods Peripheral blood mononuclear cells（PBMCs） were collected from HLA-A2-positive AHB patients.To determine HBsAg183-191 and HBsAg335-343-specific CD8 T cell frequencies,the PBMCs were stained by fluorescence-labeled anti-CD3,anti-CD8 and pentamers,and analyzed by flow cytometry.PBMCs from 6 patients were stimulated with epitopic peptide HBsAg335-343 in vitro for 3 to 4 weeks.HBV-specific CD8 T cells were isolated by magnetic activated cell sorting followed by flow florescence activated cell sorting.The mRNA of sorted cells was extracted after expanding by IL-2,anti-CD3 and anti-CD8.The full-length gene fragments of variable region of TCR α and β chains were gained by 5’-RACE,and then cloned and sequenced（≥50 clones for single chain of each sample）.The gene families of TCR α and β chains were identified and the sequence characters of CDR3 were compared.Results Analysis of more than 600 cloned gene sequences of TCR α and β chains showed that the proliferated HBV-specific CD8 T cells from 6 AHB patients presented a predominant expression in TCR α and chains,with 2-4 α chain families and 1-4 chain families in each case.The α2,α14,α15,β3,β13 and 23 families were detected in more than one case.The chain genes were all 13 for all tested clones in one case.For the same α chain or-chain family,CDR3 sequences tended to be identical in one case but different among cases.Conclusions HBV-specific CD8 T cells with antigenic peptide-induced proliferation present predominance in the usage of TCR α and β chains.This property might be one of the important molecular factors influencing anti-HBV immunity.