目的：以造血干细胞移植后并发淋巴细胞增殖症（posttransplant lympholiferative disorder, PTLD)患者外周血单核细胞培养诱导DC，负载抗原肽后制备DCCIK（cytokineinduced killer cell），为探索新的PTLD治疗方法奠定基础。方法: 分离造血干细胞移植后EBV（EpsteinBarr virus)感染致PLTD患者外周血单个核细胞，贴壁细胞培养诱导DC，悬浮细胞诱导CIK；负载EBV抗原肽LMP2后建立DCCIK共培养体系。流式细胞仪分析共培养前后细胞的免疫表型，ELISA检测共培养前后细胞上清IFNγ的分泌水平，基因扫描仪分析T细胞受体(T cell receptor, TCR)β家族基因谱。结果: 成功制备负载EBV抗原肽的DCCIK，HLADR+CD86+DC细胞从诱导前的12.5%增加到91.17%；DCCIK共培养14 d后，两例患者的CIK数量分别增加了5.3和6.8倍；CD3+、CD8+、CD3+CD8+以及CD3+CD56+ 细胞比例在DCCIK共培养后均明显升高(均P<005)。抗原肽负载的DCCIK共培养体系中IFNγ的分泌水平明显高于未经抗原肽负载的DC组\[（1 332.6±92.38）pg/ml vs (693.42±62.41) pg/ml，P<0.01）\]。DCCIK培养后细胞的TCRβ家族基因在5.2家族出现单克隆表达峰。结论：EBV抗原肽负载后DC可诱导DCCIK共培养体系中CD3+CD8+以及CD3+CD56+ 细胞扩增，并分泌高水平IFNγ，为临床应用DCCIK对移植后EBV感染致PTLD患者进行过继性细胞免疫治疗提供实验基础。

Objective:To construct a DCCIK (cytokineinduced killer cell) coculture system using peripheral blood mononuclear cells (PBMCs) derivedDC from hematopoietic stem cell transplantation (HSCT) patients with posttransplant lympholiferative disorder (PTLD) after pulsed with EBVspecial peptides, so as to lay a foundation for new adoptive immunotherapy of patients with PLTD after HSCT. Methods: PBMCs were obtained from patients with PTLD after HSCT; DC was induced from adherent cells; and CIK was induced from suspension cells. DC was further pulsed with EBVspecial peptides and cocultured with CIK to establish the DCCIK coculture system; the immunophenotype of cells in DCCIK system before and after coculture were determined by FACS, IFNγ secretion was assayed by ELISA, and TCRβ genealogy was examined by genetic analyzer. Results: The ratio of HLADR+CD86+DC increased from 125% to 91.17% after cytokine stimulation. After coculture with DC for 14 d, the numbers of CIK in two patients with PTLD increased to 5.3 and 6.8 times,respectively. The ratios of CD3+, CD8+, CD3+CD8+, and CD3+CD56+ cells were significantly increased after DCCIK coculture. IFNγ level in peptidepulsed DCCIK group was significantly higher than that in peptideunpulsed DCCIK group（\[1 332.6±92.38\] pg/ml vs \[693.42±62.41\] pg/ml，P<0.05\]）; TCRβ genealogy assay found the clone expansion peak of 5.2 TCRβ subfamily in DCCIK coculture system. Conclusion: EBV peptidepulsed DC can induce CD3+CD8+ and CD3+CD56+ cell expansion in DCCIK coculture system with high level of IFNγ. DCCIK can be used as a new adoptive immunotherapy to HSCT patients with EBV infection and PTLD.

河北省自然科学基金资助项目（No. C2008001097）；国家高等学校博士学科点专项科研基金资助项目（No. 200800890011）；河北省卫生厅重点跟踪项目资助（No. GL200508）