Abstract
Stem cell factor (SCF), the ligand of c-kit, is a key cytokine for hematopoiesis. Hematopoietic precursors express c-kit, whereas differentiated cells of hematopoietic lineage are negative for this receptor, with the exception of NK cells, mast cells, and a few others. While it has long been recognized that dendritic cells (DCs) can express c-kit, several questions remain concerning the SCF/c-kit axis in DCs. This is particularly relevant for DCs found in those organs wherein SCF is highly expressed, including the bone marrow (BM). We characterized c-kit expression by conventional DCs (cDCs) from BM and demonstrated a higher proportion of c-kit+ cells among type 1 cDC subsets (cDC1s) than type 2 cDC subsets (cDC2s) in both humans and mice, whereas similar levels of c-kit expression were observed in cDC1s and cDC2s from mouse spleen. To further study c-kit regulation, DCs were generated with granulocyte-macrophage colony-stimulating factor (GM-CSF) from mouse BM, a widely used protocol. CD11c+ cells were purified from pooled non-adherent and slightly adherent cells collected after 7 days of culture, thus obtaining highly purified BM-derived DCs (BMdDCs). BMdDCs contained a small fraction of c-kit+ cells, and by replating them for 2 days with GM-CSF, we obtained a homogeneous population of c-kit+ CD40hi MHCIIhi cells. Not only did BMdDCs express c-kit but they also produced SCF, and both were striking upregulated if GM-CSF was omitted after replating. Furthermore, a small but significant reduction in BMdDC survival was observed upon SCF silencing. Incubation of BMdDCs with SCF did not modulate antigen presentation ability of these cells, nor it did regulate their membrane expression of the chemokine receptor CXCR4. We conclude that the SCF/c-kit-mediated prosurvival circuit may have been overlooked because of the prominent use of GM-CSF in DC cultures in vitro, including those human DC cultures destined for the clinics. We speculate that DCs more prominently rely on SCF in vivo in some microenvironments, with potential implications for graft-versus-host disease and antitumor immunity.
Highlights
Dendritic cells (DCs) are cells of hematopoietic origin that develop in the bone marrow (BM) and play a key role in immunity and tolerance
Since granulocyte-macrophage colony-stimulating factor (GM-CSF) can inhibit c-kit expression by mast cells [49, 50], we evaluated the effects of GM-CSF in our BM-derived DCs (BMdDCs) cultures, by comparing c-kit membrane expression by BMdDCs cultured in complete Opti-MEM medium either with or without GM-CSF at 20 ng/ml
We found that BMdDCs produced cell-associated Stem cell factor (SCF) and its levels remained stable after 2 days of culture in complete Opti-MEM medium with GM-CSF
Summary
Dendritic cells (DCs) are cells of hematopoietic origin that develop in the bone marrow (BM) and play a key role in immunity and tolerance. DCs mostly derive from common myeloid progenitors (CMPs) in the BM, with some contribution by common lymphoid precursors [2] Both FMS-like tyrosine kinase 3 ligand (Flt3-L) and granulocytemacrophage colony-stimulating factor (GM-CSF) regulate DC development and maintenance of normal differentiated DC numbers [20,21,22]. Some residual cDCs were found in the spleen and BM of Flt3-L ko mice, and they were still present in Flt3-L/GM-CSF double ko mice [21] These results suggest that some unknown mechanisms might contribute to differentiated DC survival in lymphoid organs [21]. Binding of SCF to its membrane receptor c-kit ( called CD117) activates intracellular signaling molecules including PI-3 kinase, PLCγ, src-family kinases, and regulating cell survival, proliferation, adhesion [32,33,34]. We evaluated c-kit expression by DC subsets in mouse and human BM and studied SCF/c-kit axis in DCs generated in vitro from mouse BM
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