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(hi) cells 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(+)CX(3)CR1(+) phenotype, and the others consisted of 2A1(+)CD83(+)CD115(-)CX(3)CR1(- )and 2A1(-)CD83(-)CD115(-)CX(3)CR1(-) cells. According to the antigen uptake and presentation, morphologies, and gene expression profiles, 2A1(-)CD83(-)CD115(-)CX(3)CR1(-) cells were immature cDC2s and 2A1(+)CD83(+)CD115(-)CX(3)CR1(-) cells were mature cDC2s. Unexpectedly, however, 2A1(-)CD83(-)CD115(+)CX(3)CR1(+) 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 MHCIIhi non-DCs were precursors to cDC2s, i.e., MHCIIhi pre-cDC2s. MHCIIhi pre-cDC2s also expressed the higher level of DC-specific transcription factor Zbtb46 as similarly as immature cDC2s. Besides, MHCIIhi 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(hi )pre-cDC2s are close lineage to cDCs. All in all, our study identified and characterized a new cDC precursor, exhibiting a CD11c(+)MHCII(hi)CD115(+)CX(3)CR1(+) phenotype, in FL-BM culture.