Abstract
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 in vivo. However, the combined effect of Flt3-L and GM-CSF on human DCs has not been evaluated in vivo. In this study, we, therefore, aimed at evaluating this using a humanized mouse model. Humanized non-obese diabetic/SCID/Jak3null (hNOJ) mice were constructed by transplanting hematopoietic stem cells from human umbilical cord blood into newborn NOJ mice, and in vivo 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, CD14−CD1c+ conventional DCs (cDCs) and CD14−CD141+ 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, CD45RA−Foxp3hi and CD45RA−Foxp3lo T cells. Whereas CD45RA−Foxp3hi T cells were increased only after treatment with GM-CSF alone, CD45RA−Foxp3lo 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 in vivo effect of Flt3-L and GM-CSF on human DCs and regulatory T cells.
Highlights
Dendritic cells (DCs) play a pivotal role in maintaining the immune responses [1, 2]
Fms-related tyrosine kinase 3 ligand (Flt3-L) and granulocyte-macrophage colony-stimulating factor (GM-CSF), are considered to be the essential regulators of DC development in vivo: fms-related tyrosine kinase 3 ligand (Flt3-L) supports the development of conventional DCs (cDCs) and plasmacytoid DCs (pDCs) derived from bone marrow (BM) progenitors, while GM-CSF contributes to the development of monocyte-derived DCs (MoDCs) as well as inflammation-induced myeloid DCs [3, 4, 10, 12, 13]
When humanized NOJ (hNOJ) mice were injected with the Flt3-L-expressing plasmid (Group F), the GM-CSF-expressing plasmid (Group G), or both plasmids (Group F + G), the corresponding cytokines could be induced within 3 days of IVT
Summary
Dendritic cells (DCs) play a pivotal role in maintaining the immune responses [1, 2]. One study using knock-out mice showed that combined deficiency of Flt3-L and GM-CSF, rather than a single deficiency of either cytokine, massively reduced DCs in the periphery and monocyte-macrophage DC progenitors and further downstream common DC progenitors in the BM, indicating the concerted action of Flt3-L and GM-CSF on DC homeostasis in vivo [13]. Cytokines, such as IL-3, IL-4, IL-15, TNF-α, and TGF-β are selectively responsible for the development and maturation of specific DC subsets, which affects the type of immune response that develops [6, 7]. In humans, the effect of Flt3-L and GM-CSF singly or in combination in the absence of any other cytokine on the development of DCs remains to be evaluated in vivo
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