成熟树突状细胞的分子特征：对树突状细胞疗法质量检测的影响。

BACKGROUND: Dendritic cells (DCs) are often produced by granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) stimulation of monocytes. To improve the effectiveness of DC adoptive immune cancer therapy, many different agents have been used to mature DCs. We analyzed the kinetics of DC maturation by lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) induction in order to characterize the usefulness of mature DCs (mDCs) for immune therapy and to identify biomarkers for assessing the quality of mDCs. METHODS: Peripheral blood mononuclear cells were collected from 6 healthy subjects by apheresis, monocytes were isolated by elutriation, and immature DCs (iDCs) were produced by 3 days of culture with GM-CSF and IL-4. The iDCs were sampled after 4, 8 and 24 hours in culture with LPS and IFN-gamma and were then assessed by flow cytometry, ELISA, and global gene and microRNA (miRNA) expression analysis. RESULTS: After 24 hours of LPS and IFN-gamma stimulation, DC surface expression of CD80, CD83, CD86, and HLA Class II antigens were up-regulated. Th1 attractant genes such as CXCL9, CXCL10, CXCL11 and CCL5 were up-regulated during maturation but not Treg attractants such as CCL22 and CXCL12. The expression of classical mDC biomarker genes CD83, CCR7, CCL5, CCL8, SOD2, MT2A, OASL, GBP1 and HES4 were up-regulated throughout maturation while MTIB, MTIE, MTIG, MTIH, GADD45A and LAMP3 were only up-regulated late in maturation. The expression of miR-155 was up-regulated 8-fold in mDCs. CONCLUSION: DCs, matured with LPS and IFN-gamma, were characterized by increased levels of Th1 attractants as opposed to Treg attractants and may be particularly effective for adoptive immune cancer therapy.

背景：树突状细胞（DCs）通常通过粒细胞-巨噬细胞集落刺激因子（GM-CSF）和白细胞介素-4（IL-4）刺激单核细胞产生。为了提高 DC 过继免疫癌症治疗的效果，已经使用了许多不同的药物来使 DC 成熟。我们通过脂多糖（LPS）和干扰素-γ（IFN-γ）诱导分析了 DC 成熟的动力学，以便对成熟 DC（mDC）在免疫治疗中的有用性进行特征描述，并确定用于评估 mDC 质量的生物标志物。

方法：通过离心分离从 6 名健康供体采集外周血单个核细胞，分离单核细胞，并通过 3 天 GM-CSF 和 IL-4 培养生成未成熟 DC（iDCs）。将 iDCs 在 LPS 和 IFN-γ培养 4、8 和 24 小时后取样，然后通过流式细胞术、ELISA 和全基因和 microRNA（miRNA）表达分析进行评估。

结果：在 LPS 和 IFN-γ刺激 24 小时后，DC 表面 CD80、CD83、CD86 和 HLA Ⅱ类抗原的表达上调。在成熟过程中上调了 Th1 趋化因子基因，如 CXCL9、CXCL10、CXCL11 和 CCL5，但未上调 Treg 趋化因子，如 CCL22 和 CXCL12。经典 mDC 生物标志物基因 CD83、CCR7、CCL5、CCL8、SOD2、MT2A、OASL、GBP1 和 HES4 的表达在整个成熟过程中均上调，而 MTIB、MTIE、MTIG、MTIH、GADD45A 和 LAMP3 仅在成熟后期上调。miR-155 在 mDCs 中的表达上调了 8 倍。

结论：用 LPS 和 IFN-γ成熟的 DC 具有增加的 Th1 趋化因子水平而不是 Treg 趋化因子，对于过继免疫癌症治疗可能特别有效。