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 (FIt3-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 F1t3-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/Jak3(null) (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 F1t3-L and GM-CSF. Following IVT, F1t3-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(hi) and CD45RA-Foxp3In T cells. Whereas CD45RA-Foxp3(hi) T cells were increased only after treatment with GM-CSF alone, CD45RA-Foxp3(lo)) T cells were increased only after treatment with both F1t3-L and GM-CSF. Treatment with F1t3-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 F1t3-L and GM-CSF on human DCs and regulatory T cells.