Identification of dendritic cell precursor from the CD11c+ cells expressing high levels of MHC class II molecules in the culture of bone marrow with FLT3 ligand.

Dendritic cells (DCs) are readily generated from the culture of mouse bone marrow (BM) treated with either granulocyte macrophage-colony stimulating factor (GM-CSF) or FMS-like tyrosine kinase 3 ligand (FLT3L). CD11c⁺MHCII⁺ or CD11c⁺MHCII^hicells are routinely isolated from those BM cultures and generally used as in vitro-generated DCs for a variety of experiments and therapies. Here, we examined CD11c⁺ cells in the BM culture with GM-CSF or FLT3L by staining with a monoclonal antibody 2A1 that is known to recognize mature or activated DCs. Most of the cells within the CD11c⁺MHCII^hi DC gate were 2A1⁺ in the BM culture with GM-CSF (GM-BM culture). In the BM culture with FLT3L (FL-BM culture), almost of all the CD11c⁺MHCII^hi cells were within the classical DC2 (cDC2) gate. The analysis of FL-BM culture revealed that a majority of cDC2-gated CD11c⁺MHCII^hi cells exhibited a 2A1^- CD83^- CD115⁺CX₃CR1⁺ phenotype, and the others consisted of 2A1⁺CD83⁺CD115^- CX3CR1^- and 2A1^- CD83^- CD115^- CX₃CR1^- cells. According to the antigen uptake and presentation, morphologies, and gene expression profiles, 2A1^- CD83^- CD115^- CX₃CR1^- cells were immature cDC2s and 2A1⁺CD83⁺CD115^- CX₃CR1^- cells were mature cDC2s. Unexpectedly, however, 2A1^- CD83^- CD115⁺CX₃CR1⁺ cells, the most abundant cDC2-gated MHCII^hi cell subset in FL-BM culture, were non-DCs. Adoptive cell transfer experiments in the FL-BM culture confirmed that the cDC2-gated MHCII^hi non-DCs were precursors to cDC2s, i.e., MHCII^hi pre-cDC2s. MHCII^hi pre-cDC2s also expressed the higher [ABSTRACT FROM AUTHOR]