LMP2混合肽负载树突状细胞与EB病毒感染患者外周血单个核细胞共培养产生识别EB病毒抗原的T细胞

T Cells Recognizing EBV-epitopes Arose in Co-culture of Peripheral Blood Mononuclear Cells from EB-infected Patients and Dendritic Cells Loaded with LMP2-mixed Peptides

LMP2是EB病毒感染细胞后表达的一种蛋白。本研究探讨应用适于不同HLA分型的EB病毒-LMP2抗原混合肽(MIX-LMP2)体外刺激EB病毒感染患者外周血单个核细胞,诱导产生EB病毒抗原特异性的细胞毒性T淋巴细胞(CTL)。取EB病毒相关噬血细胞综合症患者的外周血,分离并诱导培养树突状细胞,在培养过程中用EB病毒MIX-LMP2混合肽刺激,促成熟后在体外激活自体T淋巴细胞,每周刺激1次,共刺激2次;同时对部分分离的淋巴细胞在培养过程中不予以刺激作为对照。用基因扫描T细胞受体(TCR)β基因图谱的方法观察培养前后的T细胞克隆分布变化;用流式细胞术检测T淋巴细胞的表型变化;将培养的细胞和靶细胞共培养检测IFN-γ的分泌以研究CTL细胞抗原特异性的细胞毒作用。研究结果表明,基因扫描TCRβ基因结果显示体外培养改变了患者的TCRβ基因图谱,培养前为寡克隆的TCRβ基因家族在培养后出现与正常人相似的多峰分布,提示淋巴细胞亚群分布恢复正常;流式细胞分析显示培养前的淋巴细胞CD3+、CD3+CD8+、CD3+CD45RA-CD45RO+比例分别为70.73%、42.99%、27.56%,用负载EB病毒LMP2肽2次刺激体外培养后,上述的淋巴细胞表型分别升高为95.17%、52.54%、81.41%。NK细胞(CD3-CD56+)、调节性T细胞(CD4+CD25+FOXP3+)比例变化不大,分别从培养前的2.12%,0.03%变化为2.35%,0.02%。CD3+CD45RA-CD45RO+细胞增长比例较大,表明2次刺激后大部分的初始型T淋巴细胞被激活。IFN-γ分泌检测结果显示,当负载LMP2肽的DC细胞作为靶细胞时,刺激1次的细胞分泌IFN-γ量和刺激2次的细胞分泌IFN-γ量明显高于同期未刺激细胞分泌IFN-γ量(p<0.05)。而对于未负载LMP2的DC细胞作为靶细胞时,IFN-γ检测结果则显示无统计学意义(p>0.05)。结论:用负载EB病毒MIX-LMP2肽的树突状细胞(DC)刺激T淋巴细胞的培养方法,可以改变患者T淋巴细胞克隆分布,产生识别EB病毒的特异性T淋巴细胞。

The latent membrane protein 2 （LMP2） is a kind of protein expressed by EBV-infected cells. This study was aimed to investigate whether the stimulation of peripheral blood mononuclear cells with peptides induces EBV- specific cytotoxic T lymphocytes （CTL） . The peptides were mixture of LMP2 protein and available for people with different HLA types. Peripheral blood sample was collected from a patient with EBV-associated hemophagocytic syndrome. The mononuclear cells were isolated and cultured to obtain dendritic cells （DCs）. Immature DCs were pulsed with MIX-LMP2 and added with different maturation-promoting factors. The auto-T lymphocytes were stimulated weekly with the harvested mature DCs loaded with MIX-LMP2, and totally for two times. Part of isolated lymphocytes was cultured without any stimulation as control. T-cell receptor （TCR） 13 spectratyping was used to analyze the distribution of different T cell subgroups before and after culture. The phenotype of T lymphocytes was determined by flow cytometry. The IFN-γ assay was used to estimate specific cytoxic activity of the cultured T cells. The results showed that the distribution of TCRβ was changed according to analysis of TCR spectratypes. From the distribution of gene families of TCRβ,the T lymphocytes were oligoclonal before culture, but shifted to a polyclonal after culture in vitro like the normalization of TCR diversity, suggesting the subgroups of lymphocyte could return to normal. The percentage of CD3 ^＋ , CD3 ＋CD8 ^＋ CD3 ^＋ CD45RA^- CD45 RO^＋ on T lymphocytes from freshly isolated mononuclear cells were 70.73%, 42.99%, 27.56% respectively. After being stimulated twice with DC loaded with MIX-LMP2, they further increased to 95.17%, 52.54% and 81.41%. The percentages of CD3^- CD56 ^＋ NK cells and CD4 ^＋ CD35 ^＋ FOXP3 ^＋ regulation T cells seldom changed, from 2. 12%, 0.03% to 2. 35%, 0.02% respectively. The increase of CD3 ^＋ CD45RA^- CD45RO ^＋ cells obviously indicated that most naive T cells could be activated. ELISA for IFN-γ showed that when DCs loaded with LMP2 peptide were used as target cells, IFN-γ level secreted by the T cells stimulated with LMP2 peptide- pulsed DCs was 805 ± 16 pg/ml and 1729 ± 49 pg/ml, the IFN-γ level secreted by T cells stimulated twicely with LMP2 peptide-pulsed DCs was 956± 23 pg/ml and 2325 ± 58 pg/ml respectively at effector-target ratios of 10： 1 and 10： 2. They were both significantly higher than that secreted by T cells without any stimulation （441 ±27 pg/m and 557 ± 19 pg/ml） （p 〈0.05）. But DCs unpulsed with LMP2 peptide were used as target cells, there were no significant differences between the T cells stimulated with LMP2 peptide-pulsed DCs and the T cells without stimulation （p 〉 0.05 ）. It is concluded that the antigen specific T cells recognizing EBV epitopes can be obtained by using DCs pulsed with MIX-LMP2 peptide in vitro, meanwhile the distribution of T cell subgroups can be changed and normalized.