Flt3 Receptor Expression Rescues Dendritic Cell Developmental Potential in Megakaryocyte/Erythrocyte Progenitor Cells.

Abstract

Flt3/Flt3-ligand has a non-redundant role in dendritic cell (DC) and interferon producing cell (IPC) development from hematopoietic progenitor cells. Flt3-ligand−/− mice display a severe reduction of DCs and IPCs, and both, Flt3-ligand injection and conditional over- expression of Flt3-ligand in mice leads to an increased pool size of both cellular subsets. Furthermore, DC and IPC developmental precursor activity segregates with Flt3+ progenitor populations in mouse bone marrow. We have reported that common myeloid progenitors (CMPs) and their progeny granulocyte/macrophage progenitors (GMPs) as well as common lymphoid progenitors (CLPs) and pro-T cells can give rise to DCs in vitro and in vivo. DC developmental potential was lost, however, once definitive megakaryocyte/erythrocyte or B cell commitment occurs, both steps that are accompanied by loss flt3 receptor expression. To test if DC/IPC developmental potential can be regained, we evaluated the effect of ecotropic expression of human Flt3 in lin−c-kit+Flt3−Thy1.1−IL-7Ra− cells (flt3− progenitors) and in megakaryocyte/erythrocyte progenitors (MEPs).hFlt3 was transduced into purified flt3− progenitors, MEPs, and GMPs using a retrovirus vector carrying both hFlt3 and GFP. The transduction efficiencies were 20~25%. hFlt3-transduced flt3− progenitors gave rise to functional DCs and IPCs, but not control-GFP-transduced flt3− progenitors in vitro. Similarly, hFlt3-transduced MEPs differentiated into functional DCs in Flt3L, SCF, GM-CSF, IL-4, and TNF-a supplemented cultures but not into TER119+ erythroid cells. DC developmental potential of hFlt3-transduced MEPs was comparable to GFP-transduced GMPs. Over expression of hFlt3 in GMPs resulted in augmentation of its DC developmental potential. Interestingly, both hFlt3-tarnsduced MEPs and GMPs preferentially differentiated into IPCs in Flt3L supplemented medium. RT-PCR analysis revealed that enforced Flt3 signaling in MEPs lead to up-regulation of GM- and/or DC-development affiliated genes such as G-CSF receptor, M-CSF receptor, and GM-CSF receptor, PU.1, C/EBPa and STAT3. Furthermore, hFlt3-transduced MEPs gave rise to myeloid colonies (CFU-GM/G/M) but not CFU-Mix colonies in GM-CSF, IL-3, SCF, Flt3L, Tpo, and Epo supplemented methylcellulose medium. These results show that artificial Flt3 signaling in otherwise Flt3− progenitors is sufficient to reestablish DC/IPC differentiation and GM-differentiation programs.