乙型肝炎相关肝癌患者乙型肝炎病毒不同抗原肽特异性CD3+CD8+人白细胞抗原-A2+细胞表达

摘要

目的 探讨乙型肝炎相关肝细胞癌(HCC)患者外周血单个核细胞(PBMC)中HBV不同抗原肽特异性CD3+ CD8+人白细胞抗原( HLA)-A2+细胞的表达.方法 从4例HLA-A2阳性乙型肝炎相关HCC患者外周血中分离PBMC,分别与HBV抗原肽sAg( FLLTRILTI、GLSPTVWLSV、WLSLLVPFV),HBV eore(FLPSDFFPSV)和HBV pol(FLLSLGIHL)及抗-CD3-pacific blue、抗-CD8-异硫氰酸荧光素共育,流式细胞仪分析HBV/HLA-A2-CD3-CD8阳性细胞,克隆培养,筛选出克隆培养的抗HBV T淋巴细胞;再与含有HBV的肝癌细胞株HepG2( HLA-A2+)共育,ELISA法检测其分泌IFN-γ水平.结果 4例患者体内受GLSPTVWLSV肽诱导的特异性抗HBV T淋巴细胞占所有CD8+细胞的1.44％±0.04％,高于FLLTRILTI肽的0.68％±0.08％、FLPSDFFPSV肽的1.06％±0.09％、FLLSLGIHL肽的0.56％±0.04％和WLSLLVPFV肽的0.46％±0.08％(t=0.001,P＜0.05).将GLSPTVWLSV/HLA-A2获得的HBV/HLA-A2 PentamerCD3-CD8阳性细胞克隆,获得2株抗HBV CD8 T淋巴细胞,与负荷GLSPTVWLSV肽段的HepG2 (HLA-A2+)细胞共育,CD8 T淋巴细胞分泌较高水平的IFN-γ.结论 乙型肝炎相关HCC患者外周血中存在针对不同HBV抗原肽的特异性抗HBV CD8 T淋巴细胞,其表达量与抗原肽段有关.%Objective To explore the expression of CD3+ CD8+ human leukocyte antigen (HLA)-A2+T lymphocytes with specificity to the different hepatitis B virus (HBV) peptides in the peripheral blood mononuclear cells (PBMC) from the patients with hepatitis B associated hepatocellular carcinoma (HCC).Methods The HLA-A2+ PBMC from four patients with hepatitis B associated HCC were incubated with five HBV/HLA-A2 pentamers respectively,which were HBV sAg (FLLTRILTI),HBV sAg (GLSPTVWLSV),HBV sAg (WLSLLVPFV),HBV core (FLPSDFFPSV),and HBV pol (FLLSLGIHL),as well as anti-CD3-pacific blue and anti-CD8-fluorescein isothiocyanate (FITC).Then,HBV/HLA-A2-CD3-CD8 positive cells were detected by flow cytometry. The monoclonal HBV/HLA-A2-CD3-CD8+ cells were acquired by fluorescenceactivated cell sorter,and cultured and identified by flow cytometry.The anti-HBV specific T lymphocytes were then cultured with HepG2 (HLA-A2+ ) cells and the release of interferon γ (IFN-γ)were determined by enzyme-linked immunosorbent assay (ELISA),Res(a)lts The percentage of antiHBV T lymphoeytes with specificity to GLSPTVWLSV in total CD8+ T lymphoeytes from four patients with hepatitis B associated HCC was 1.44％±0.04％,which was higher than those to other four HBV antigen peptides (0.68％±0.08％ of FLLTRILTI,1.06％±0.09％ of FLPSDFFPSV,0.56％ ±0.04％ of FLLSLGIHL,and 0.46％ ±0.08％ of WLSLLVPFV) (t=0.001,P＜0.05).The two lines of monoclonal cell with specificity to GLSPTVWLSV both exhibited high level of IFN-γ expression after incubated with hepatic carcinoma cell line HepG2 (HLA-A2+) with HBV GLSPTVWLSV peptide.Conclusions CD3+ CD8+ HLA-A2+ cells with specificity to the different HBV peptides exist in PBMC of patients with hepatitis B associated HCC.The expression level depends on HBV antigen peptide sequences and genomic sites.