Department of Immunology, National Institute of Infectious Diseases, Tokyo, Japan.
Department of Life Science and Medical Bioscience, Waseda University, Tokyo, Japan.
Front Immunol. 2018 May 28;9:1042. doi: 10.3389/fimmu.2018.01042. eCollection 2018.
Two cytokines, fms-related tyrosine kinase 3 ligand (Flt3-L) and granulocyte-macrophage colony-stimulating factor (GM-CSF) are considered to be the essential regulators of dendritic cell (DC) development . However, the combined effect of Flt3-L and GM-CSF on human DCs has not been evaluated . In this study, we, therefore, aimed at evaluating this using a humanized mouse model. Humanized non-obese diabetic/SCID/Jak3 (hNOJ) mice were constructed by transplanting hematopoietic stem cells from human umbilical cord blood into newborn NOJ mice, and transfection (IVT) was performed by hydrodynamic injection-mediated gene delivery using plasmids encoding human Flt3-L and GM-CSF. Following IVT, Flt3-L and GM-CSF were successfully induced in hNOJ mice. At 10 days post-IVT, we found, in the spleen, that treatment with both Flt3-L and GM-CSF enhanced the reconstitution of two myeloid DC subsets, CD14CD1c conventional DCs (cDCs) and CD14CD141 cDCs, in addition to CD14 monocyte-like cells expressing CD1c and/or CD141. GM-CSF alone had less effect on the reconstitution of these myeloid cell populations. By contrast, none of the cytokine treatments enhanced CD123 plasmacytoid DC (pDC) reconstitution. Regardless of the reconstitution levels, three cell populations (CD1c myeloid cells, CD141 myeloid cells, and pDCs) could be matured by treatment with cytokines, in terms of upregulation of CD40, CD80, CD86, and CD184/CXCR4 and downregulation of CD195/CCR5. In particular, GM-CSF contributed to upregulation of CD80 in all these cell populations. Interestingly, we further observed that Foxp3 cells within splenic CD4 T cells were significantly increased in the presence of GM-CSF. Foxp3 T cells could be subdivided into two subpopulations, CD45RAFoxp3 and CD45RAFoxp3 T cells. Whereas CD45RAFoxp3 T cells were increased only after treatment with GM-CSF alone, CD45RAFoxp3 T cells were increased only after treatment with both Flt3-L and GM-CSF. Treatment with Flt3-L alone had no effect on the number of Foxp3 T cells. The correlation analysis demonstrated that the development of these Foxp3 subpopulations was associated with the maturation status of DC(-like) cells. Taken together, this study provides a platform for studying the effect of Flt3-L and GM-CSF on human DCs and regulatory T cells.
两种细胞因子，fms 相关酪氨酸激酶 3 配体（Flt3-L）和粒细胞-巨噬细胞集落刺激因子（GM-CSF）被认为是树突状细胞（DC）发育的必需调节剂。然而，Flt3-L 和 GM-CSF 对人 DC 的联合作用尚未得到评估。因此，本研究旨在使用人源化小鼠模型对此进行评估。通过将人脐血造血干细胞移植到新生 NOJ 小鼠中构建非肥胖型糖尿病/SCID/Jak3（hNOJ）人源化小鼠，并通过质粒编码的人 Flt3-L 和 GM-CSF 进行水力喷射介导的基因传递进行转染（IVT）。IVT 后，hNOJ 小鼠中成功诱导了 Flt3-L 和 GM-CSF。在 IVT 后 10 天，我们在脾脏中发现，与单独使用 GM-CSF 相比，同时使用 Flt3-L 和 GM-CSF 可增强两种骨髓来源的 DC 亚群的重建，即 CD14CD1c 经典 DC（cDCs）和 CD14CD141 cDCs，以及表达 CD1c 和/或 CD141 的 CD14 单核样细胞。GM-CSF 单独对这些骨髓细胞群的重建影响较小。相比之下，细胞因子处理不会增强 CD123 浆细胞样 DC（pDC）的重建。无论重建水平如何，三种细胞群（CD1c 髓样细胞、CD141 髓样细胞和 pDC）均可通过细胞因子处理成熟，表型为上调 CD40、CD80、CD86 和 CD184/CXCR4，并下调 CD195/CCR5。特别地，GM-CSF 有助于所有这些细胞群中 CD80 的上调。有趣的是，我们还进一步观察到，GM-CSF 的存在使脾 CD4 T 细胞中的 Foxp3 细胞明显增加。Foxp3 T 细胞可分为两个亚群，CD45RAFoxp3 和 CD45RAFoxp3 T 细胞。虽然仅在用 GM-CSF 单独处理后才会增加 CD45RAFoxp3 T 细胞，但在用 Flt3-L 和 GM-CSF 两者处理后才会增加 CD45RAFoxp3 T 细胞。单独用 Flt3-L 处理对 Foxp3 T 细胞的数量没有影响。相关性分析表明，这些 Foxp3 亚群的发育与 DC（样）细胞的成熟状态相关。总之，这项研究为研究 Flt3-L 和 GM-CSF 对人 DC 和调节性 T 细胞的影响提供了一个平台。
