AUTHOR=In Hyunju , Park Ji Soo , Shin Hyun Soo , Ryu Seul Hye , Sohn Moah , Choi Wanho , Park Sejung , Hwang Soomin , Park Jeyun , Che Lihua , Kim Tae-Gyun , Chu Min Kyung , Na Hye Young , Park Chae Gyu TITLE=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 JOURNAL=Frontiers in Immunology VOLUME=14 YEAR=2023 URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2023.1179981 DOI=10.3389/fimmu.2023.1179981 ISSN=1664-3224 ABSTRACT=

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⁺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 level of DC-specific transcription factor Zbtb46 as similarly as immature cDC2s. Besides, MHCII^hi 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^hiCD115⁺CX₃CR1⁺ phenotype, in FL-BM culture.