1例急性乙型肝炎患者HBV特异性CD8 T细胞的快速诱导扩增

Rapid induction and expansion of HBV-specific CD8 T lymphocytes from a patient with acute hepatitis B

目的鉴定急性乙型肝炎患者HBV特异性CD8 T淋巴细胞识别的优势表位并建立对其进行快速诱导扩增的方法,为研究HBV抗原特异性CD8 T细胞的功能奠定基础。方法从1例HLA-A2阳性的急性乙型肝炎患者外周血分离外周血单个核细胞（PBMC）,使用针对HLA-A2限制性HBsAg表位（S181-181、S335-343）和HBcAg表位（C18-27）的3种HLA-A2/抗原肽荧光标记五聚体试剂（Pentamers）检测HBV特异性CD8 T细胞频率,筛选可能的优势表位。用细胞因子和相应的抗原多肽对PBMC进行1-2周刺激培养,用Pentamers检测特异CD8 T细胞,动态分析特异性CD8 T细胞的增高并用流式细胞仪对其进行分选。结果经过抗原多肽刺激并在合适条件下培养后,HBV特异性T细胞频率显著增高,培养2周时达到高峰,S183-191特异性T细胞从0.21%增高到15.17%,升高了73.8倍；S335-343特异性T细胞占总CD8 T细胞的高分比从0.44%增高到1.79%,升高了3.1倍；但C18-27特异性T细胞的频率很低且刺激培养后无明显升高。结论通过HBV多肽诱导在适当条件下可使抗原特异性CD8 T细胞在短期内获得明显诱导扩增,S-183-191特异性T细胞在控制急性HBV感染中可能具有重要作用。

Objective To develop a rapid expansion assay and determine the predominant epitope for HBV-specific CD8 T cells from peripheral blood mononuclear cells （PBMC） of patients with acute hepatitis B （AHB）. Methods PBMC were isolated from a patient with HLA-A2 positive AHB, and the frequency of HBV-specific CD8 T cells was determined using 3 kinds of HLA-A2 antigenic peptide flores-cence-labeled pentamer agents, i.e. S183-191 and S335-343 derived from HLA-A2 restricted HBsAg epitope, and C18-27 derived from HBcAg epitope. Cytokines and corresponding epitopic peptides were used for induction of positive CD8 T-cell expansion and enrichment in PBMC for 1-2 weeks. T epitope-specific CD8 T-cells were quantified and sorted by flow cytometry. Results After cultivation with antigenic peptide stimulation and proper cultural condition, the frequency increased significantly in the pentamer positive HBV-specific CD8 T cells in total CD8 T cells. The peak value was observed in 2 week cultivation. At the time the HBV-specific CD8 T cell frequencies increased from 0.21% to 15.17% for S183-191, and from 0. 44% to 1.79 % for S335-343, representing 73.8-folds and 3.1-folds increase for each epitope respectively. However, the frequency of C18-27 specific CD8 T cells was negative and without obvious proliferation after stimulation. Condusions Under stimulation of HBV epitopic peptide in proper culture condition, the specific CD8 T lymphocytes may be induced and expanded in quantity via sort-term conditioned culture. CD8 T cells specific for S183-191 may play an important role in the control of acute HBV infection.