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Development of endocytosis, degradative activity, and antigen processing capacity during GM-CSF driven differentiation of murine bone marrow.

ABSTRACT: Dendritic cells (DC) are sentinels of the immune system, alerting and enlisting T cells to clear pathogenic threats. As such, numerous studies have demonstrated their effective uptake and proteolytic activities coupled with antigen processing and presentation functions. Yet, less is known about how these cellular mechanisms change and develop as myeloid cells progress from progenitor cells to more differentiated cell types such as DC. Thus, our study comparatively examined these functions at different stages of myeloid cell development driven by the GM-CSF. To measure these activities at different stages of development, GM-CSF driven bone marrow cells were sorted based on expression of Ly6C, CD115, and CD11c. This strategy enables isolation of cells representing five distinct myeloid cell types: Common Myeloid Progenitor (CMP), Granulocyte/Macrophage Progenitor (GMP), monocytes, monocyte-derived Macrophage/monocyte-derived Dendritic cell Precursors (moMac/moDP), and monocyte-derived DC (moDC). We observed significant differences in the uptake capacity, proteolysis, and antigen processing and presentation functions between these myeloid cell populations. CMP showed minimal uptake capacity with no detectable antigen processing and presenting function. The GMP population showed higher uptake capacity, modest proteolytic activity, and little T cell stimulatory function. In the monocyte population, the uptake capacity reached its peak, yet this cell type had minimal antigen processing and presentation function. Finally, moMac/moDP and moDC had a modestly decreased uptake capacity, high degradative capacity and strong antigen processing and presentation functions. These insights into when antigen processing and presentation function develop in myeloid cells during GM-CSF driven differentiation are crucial to the development of vaccines, allowing targeting of the most qualified cells as an ideal vaccine vehicles.

SUBMITTER: Olatunde AC

PROVIDER: S-EPMC5944997 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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.

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Development of endocytosis, degradative activity, and antigen processing capacity during GM-CSF driven differentiation of murine bone marrow.

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