In vitro-generated murine monocyte-derived dendritic cells habor a unique identity compared to other mononuclear phagocytes, largely conserved with their human and putative in vivo counterparts.

Whether the cells differentiating in vitro from total murine bone marrow (BM) cultures with GM-CSF +/- IL-4 are homologous to the human cells derived in vitro from purified circulating monocytes (Mo) cultured with GM-CSF and IL-4, mostly correspond to monocyte-derived dendritic cells (moDCs) or rather to macrophages (MFs), and have in vivo counterparts, is debated. It has been proposed to define cell types based on their ontogeny, leading to the proposal of excluding from the dendritic cell (DC) family the cells that do not arise from the common DC progenitors (CDP). However, recent discoveries support defining cell types rather by functions, or, as a surrogate, their instructive molecular networks. Indeed, Langerhans cells, Rorgt+ DCs, some cDC2As and human in vitro-derived moDCs do not arise from the CDP but exert hallmark DC functions instructed by specific transcriptomic modules shared with cDCs. Actually, the study of both LCs and moDCs has been key for dissecting DC biology. Yet, the existence of moDCs is put into questions. The functions once attributed to moDCs are now proposed to be exerted by type 2B conventional DCs (cDC2Bs) or type 3 DCs (DC3s) that have been characterized recently, harbor strongly overlapping phenotypes with moDCs and a similar mixed expression of genes from both the cDC and Mo/MF lineages, but a different ontogeny. To clarify these issues, we generated a transcriptomic atlas of in vitro-derived mononuclear phagocytes (MNPs). We show that moDCs are the only DC type arising in BM cultures in the presence of GM-CSF and IL-4. They express a unique transcriptomic and phenotypic signature, different from those of cDC2Bs and DC3s that are produced in FLT3-L cultures and appear to correspond to the same DC type differentiating though two different but converging ontogenetic paths. By harnessing the phenotypic and transcriptomic signatures identified from the analysis of our in vitro MNP atlas, we are able to identify in vivo a population of putative DCs differentiated from monocytes derived from the common monocyte-granulocyte progenitor (GMP), in a respiratory allergy model, specifically in the lungs of mice sensitized to house dust mite (HDM). Using the model of thioglycolate-induced peritonitis, we also characterized the CD226+ Mo-derived cells that have been reported to be moDCs or small cavity MFs (SCM) depending on studies. We showed that these cells are mainly differentiated from monocytes derived from the MDP, and split into two populations, one resembling MFs (SCMs) and the other one closer to lung moDCs but expressing distinct genes suggesting a different functional potential. Collectively, we show that the generation and characterization of specific MNP types in vitro can be harnessed to rigorously define their in vivo counterparts based on phenotypic analysis and gene module assessment, as we demonstrate for moDCs.