两种白血病细胞抗原负载DC的体外诱导特异性CTL应答的比较

The Comparison of inducing specific CTL by two methods of leukemia cell antigen loading of DCs and fusion of DCs with leukemia cells

目的: 比较树突状细胞(DC)与白血病细胞融合及白血病细胞冻融抗原负载DC两种白血病DC疫苗诱导抗原特异性CTL应答的能力.方法: 采用羟乙基淀粉-Ficoll两步法分离外周血单个核细胞(PBMC), 通过贴壁2 h获得黏附的单核细胞, 用GM-CSF加IL- 4诱导培养5 d收获细胞.将细胞分为4组: A组将K562细胞或原代慢性粒细胞白血病(CML)细胞在500 g/L PEG-100 mL/L DMSO诱导下与DC融合; B组加入相应细胞数量的白血病细胞冻融抗原; C组将K562细胞或CML细胞与DC共培养; D组: 单独DC培养组.融合前以红色荧光染料PKH26标记K562细胞, 采用流式细胞仪(FCM)检测PKH26/FITC-抗HLA-ABC抗体双标细胞并评估融合率.培养的第6天, 加入TNF-α诱导DC成熟, 然后分别与自体T细胞共培养, 用MTT比色法检测各组CTL对靶细胞的杀伤活性.结果: GM-CSF加IL- 4和TNF-α依次诱导成熟的DC具有经典的DC的形态和表型特征, 在PEG-DMSO的介导下, DC与白血病细胞的融合率为(17.33～29.94)%.两种抗原负载方案激活的CTL均对表达K562抗原的细胞具有特异性的细胞毒作用.在效靶比相同时, 融合组诱导CTL的杀伤活性强于冻融抗原致敏DC组.结论: 与冻融抗原致敏的DC相比, DC与白血病细胞融合细胞递呈抗原的效率更高, 体外诱导的CTL特异性杀伤靶细胞的作用更强, 有可能用于白血病的免疫治疗.

AIM: To compare the potency of the fusion of DCs with leukemia cells and freeze-thawed leukemia antigen-loading DCs in inducing antigen-specific CTLs. METHODS: Peripheral blood mononuclear cells(PBMC) isolated by HES-Ficoll two-step method were incubated 2 hours to select adherent monocytes. The adherent monocytes were then cultured in the presence of GM-CSF and IL-4 for 5 days and then divided into four groups: DCs were fused with K562 cells or CML cells from CML patients in the presence of 500 g/L PEG-100 mL/L DMSO (Group A); DCs were loaded with lysates from K562 cells or CML cells (group B); DCs were co-cultured with K562 cells or CML cells (group C) and DCs were cultured alone (group D). Before cell fusion, K562 cells were labeled using a red fluorescent dye, PKH26. After fusion, flow cytometry was used to detect hybrids labeled by both PKH26 and FITC-conjugated anti-HLA-ABC mAb to assess the efficiency of fusion. On day 6, TNF-α was added to induce the terminal maturation of DCs. Then each group DCs were co-cultured with autologous T cells respectively. The cytotoxicity of CTLs of each group against different target cells was measured using MTT coloremetry. RESULTS: DCs induced by GM-CSF+IL-4 and TNF-α had DC-classical phenotypic characteristics. The efficiency of cell fusion ranged from 17.33% to 29.94%. Both DC-K562 hybrids and DC loaded with leukemic freeze-thaw lysates could stimulate specific CTL responses against target cells. Furthermore, the cytotoxicity induced by the former DCs were much stronger than that induced by lysates-loaded DCs at the same E/T ratio. CONCLUSION: Compared with freeze-thaw lysates loaded DCs, DC-leukemic hybrids showed higher efficiency in antigen presenting and the stimulation of leukemia-specific CTLs.