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

LMF2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-LMF2抗原混合肽（MIX—LMF2）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—LMF2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）B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^＋、CD^＋ CD45RA^- CD45RO^＋比例分别为70．73％、42．99％、27．56％。用负载EB病毒LMF2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LMF2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—LMF2肽的树突状细胞（De）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。

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

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) beta 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-gamma assay was used to estimate specific cytoxic activity of the cultured T cells. The results showed that the distribution of TCRbeta was changed according to analysis of TCR spectratypes. From the distribution of gene families of TCRbeta, 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-gamma showed that when DCs loaded with LMP2 peptide were used as target cells, IFN-gamma level secreted by the T cells stimulated with LMP2 peptide-pulsed DCs was 805+/-16 pg/ml and 1729+/-49 pg/ml, the IFN-gamma level secreted by T cells stimulated twice 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.