Introduction of Human Flt3-L and GM-CSF into Humanized Mice Enhances the Reconstitution and Maturation of Myeloid Dendritic Cells and the Development of Foxp3 + CD4 + T Cells

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, 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 Foxp3 and CD45RA Foxp3 T cells. Whereas CD45RA Foxp3 T cells were increased only after treatment with GM-CSF alone, CD45RA Foxp3 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.