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). CD11cMHCII or CD11cMHCII cells are routinely isolated from those BM cultures and generally used as -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 CD11cMHCII 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 CD11cMHCII cells were within the classical DC2 (cDC2) gate. The analysis of FL-BM culture revealed that a majority of cDC2-gated CD11cMHCII cells exhibited a 2A1CD83CD115CXCR1 phenotype, and the others consisted of 2A1CD83CD115CXCR1 and 2A1CD83CD115CXCR1 cells. According to the antigen uptake and presentation, morphologies, and gene expression profiles, 2A1CD83CD115CXCR1 cells were immature cDC2s and 2A1CD83CD115CXCR1 cells were mature cDC2s. Unexpectedly, however, 2A1CD83CD115CXCR1 cells, the most abundant cDC2-gated MHCII cell subset in FL-BM culture, were non-DCs. Adoptive cell transfer experiments in the FL-BM culture confirmed that the cDC2-gated MHCII non-DCs were precursors to cDC2s, i.e., MHCII pre-cDC2s. MHCII pre-cDC2s also expressed the higher level of DC-specific transcription factor Zbtb46 as similarly as immature cDC2s. Besides, MHCII pre-cDC2s were generated only from pre-cDCs and common DC progenitor (CDP) cells but not from monocytes and common monocyte progenitor (cMoP) cells, verifying that MHCII pre-cDC2s are close lineage to cDCs. All in all, our study identified and characterized a new cDC precursor, exhibiting a CD11cMHCIICD115CXCR1 phenotype, in FL-BM culture.