Jagiellonian University Medical College, Faculty of Medicine, Department of Pharmacology, Ul. Grzegórzecka 16, 31-531 Kraków, Poland.
Jagiellonian University Medical College, Faculty of Medicine, Department of Pharmacology, Ul. Grzegórzecka 16, 31-531 Kraków, Poland.
Int Immunopharmacol. 2018 Apr;57:165-171. doi: 10.1016/j.intimp.2018.02.015. Epub 2018 Feb 28.
Dendritic cells (DCs) play a crucial role in the development of adaptive immune response. Monocyte-derived dendritic cells (MDDCs) are generated in vitro to study DC biology and for use in immunotherapy. However, procedures to generate MDDCs vary and an impact this may have on their final phenotype is insufficiently studied. Monocytes isolated from healthy blood donors were cultured for 7 days with granulocyte-macrophage colony stimulating factor (50 ng/mL) and low (500 IU/mL, L-IL4) or high (1000 IU/mL, H-IL4) interleukin 4 (IL4), to obtain immature DCs and for the following 2 days with addition of soluble CD40 ligand (500 ng/mL) and prostaglandin E (1 μg/mL) to obtain mature DCs. We measured mean fluorescence activity and percentage of cells, positive for CD14, HLA-DR, CD80, CD83, CD86, CCR7, and CD1a or CD209 markers after 7 and 9 days of culture, in both IL4 concentrations. Percentage of positively staining mature MDDCs was higher than among immature cells, for all studied markers. Interestingly, varying IL4 concentrations had negligible impact on staining of mature MDDCs. However, immature L-IL4 cultured MDDCs were less intensely stained for HLA-DR and CD209 than H-IL4 immature DCs. Flow cytometry revealed presence of 2 populations of cells (dominant P1 and less prevalent P2), when either L-IL4 or H-IL4 was used. Among mature MDDCs, population P1 had higher percentage of positively staining cells than P2, for all studied markers except CCR7. In conclusion, both concentrations of IL4 produce in vitro heterogeneous populations of mature MDDCs with similar staining for cell surface markers.
树突状细胞（DCs）在适应性免疫反应的发展中起着至关重要的作用。单核细胞衍生的树突状细胞（MDDCs）在体外生成，用于研究 DC 生物学和用于免疫治疗。然而，生成 MDDCs 的程序各不相同，其对最终表型的影响研究不足。从健康献血者中分离的单核细胞在含有粒细胞-巨噬细胞集落刺激因子（50ng/mL）和低（500IU/mL，L-IL4）或高（1000IU/mL，H-IL4）白细胞介素 4（IL4）的条件下培养 7 天，以获得未成熟的 DCs，并在接下来的 2 天中加入可溶性 CD40 配体（500ng/mL）和前列腺素 E（1μg/mL）以获得成熟的 DCs。我们测量了在两种 IL4 浓度下培养 7 天和 9 天后，细胞表面标记物 CD14、HLA-DR、CD80、CD83、CD86、CCR7、CD1a 或 CD209 阳性细胞的平均荧光强度和百分比。与幼稚细胞相比，成熟 MDDC 中所有研究标记物的阳性染色细胞百分比均较高。有趣的是，IL4 浓度的变化对成熟 MDDC 的染色几乎没有影响。然而，与 H-IL4 未成熟 DC 相比，L-IL4 培养的未成熟 MDDC 对 HLA-DR 和 CD209 的染色较弱。当使用 L-IL4 或 H-IL4 时，流式细胞术显示存在两种细胞群（占主导地位的 P1 和较少的 P2）。在成熟的 MDDC 中，除 CCR7 外，所有研究标记物的 P1 细胞群的阳性染色细胞百分比均高于 P2。总之，两种浓度的 IL4 均可产生体外异质性成熟 MDDC 群体，其细胞表面标记物的染色相似。
