活化B淋巴细胞诱导健康人外周血HBcAg短肽特异性细胞毒性T淋巴细胞

Induction of HBcAg peptide specific cytolytic T lymphocyte on healthy human peripheral blood monocular cell by activated B lymphocytes

目的探讨负载乙型肝炎病毒核心抗原18-27(HBcAg 18-27)序列肽的活化B淋巴细胞诱导自体外周血单个核细胞(PBMC)产生HBcAg 18-27特异性细胞毒性T淋巴细胞(CTL)的能力。方法免疫磁珠法分选B淋巴细胞,与CpG寡核苷酸(CpG-ODN)(2μg/mL)和白细胞介素-4(IL-4)(2 ng/mL)共培养48 h,再加入人工合成的HBcAg 18-27肽(50μg/mL),继续温育12 h。将负载抗原肽的活化B淋巴细胞与自体PBMC进行混合淋巴细胞培养5 d,采用Pro5TMMHC Pentam ers技术检测HBcAg 18-27特异性CTL。结果免疫磁珠法分选B淋巴细胞纯度为89.60%。荧光显微镜观察到HBcAg 18-27抗原肽进入活化B细胞,流式细胞仪定量检测抗原负载率为41.3%。以此作为HBcAg 18-27特异性抗原递呈细胞(APC)能够从自体PBMC中诱导出HBcAg 18-27特异性CTL。对照组(不加APC)HBcAg 18-27特异性CTL为(0.20±0.10)%,实验组(加APC)为(0.50±0.19)%,2组差异有统计学意义(P<0.01)。结论负载了HBcAg 18-27肽的活化B淋巴细胞能够诱导自体PBMC产生HBcAg 18-27特异性CTL。

Objective To activate peripheral blood B lymphocytes by CpG-oligodeoxynucleotides （CpG-ODN） and load HBcAg 18-27 peptide to make them as antigen presenting cells （APC）, then induce hepatitis B virus （HBV）-specific cytolytic T lymphocytes （CTL） from peripheral blood monocular cells （PBMC） of healthy human. Methods B lymphocytes were isolated from PBMC with anti-human CD20 immunomagnetic beads. The B ceils were cultured in the presence of CpG-ODN and interleukin-4 （IL-4） for 48 h followed by a further incubation for 12 h after adding synthetic HBcAg 18-27 peptide （50 μg/mL）. The PBMC were co-cultured with HBcAg 18-27 peptide specific APC for 5 d. The HBcAg 18-27-specific CTL were detected by Pro5TM MHC Pentamers. Results According to the results of flow cytometry and fluorescence microscopy, the purity of B ceils was 89.60% , and 41.3% of the activated B cells were loaded with HBcAg 18-27 peptide. After co-culture with activated and the peptide-loaded B cells, （0.50± 0. 19）% of the PBMC were induced to be HBcAg 18-27-specific CTL, while such CTL were only （0.20 ±0. 10）% in the absence of these B cells. The induction of HBcAg 18-27-specific CTL was significant （ P 〈 0.01 ）. Conclusions Activated B cells with loaded HBcAg 18-27 peptide may induce HBcAg 18-27-specific CTL on healthy human PBMC.