Dendritic cells (DCs) in lymphoid tissue arise from precursors that also produce monocytes and plasmacytoid DCs (pDCs). Where DC and monocyte lineage commitment occurs and the nature of the DC precursor that migrates from the bone marrow to peripheral lymphoid organs is unknown. We show that DC development progresses from the macrophage and DC precursor (MDP), to common DC precursors (CDPs) that give rise to pDCs and classical spleen DCs (cDCs), but not monocytes, and finally to committed precursors of cDCs (pre-cDCs). Pre-cDCs enter lymph nodes through and migrate along HEVs and later disperse and integrate into the DC network. Further cDC development involves cell division, controlled in part by regulatory T cells (Treg) and fms-related tyrosine kinase-3 (Flt3).Dendritic cells (DCs) are immune cells specialized to capture, process and present antigens to T lymphocytes to induce immunity or tolerance (1). Where commitment to DC development takes place, at what stage the monocyte lineage diverges from DCs, and the precise nature of the migrating DC precursor that moves from the bone marrow to the peripheral lymphoid organs is not known. These questions have been difficult to resolve in part because DC subsets are functionally and phenotypically diverse (2). For example, classical spleen DCs (cDCs) include two major functionally distinct subsets distinguished by the expression of a variety of C-type lectins and CD8 (2-4). Spleen and other tissues also contain plasmacytoid DCs (pDCs) that primarily initiate immune responses to nucleic acids (5,6).Lymphoid tissue cDCs, pDCs, and monocytes share a common progenitor called the macrophage and DC precursor (MDP) identified by its surface phenotype (Lin − cKit hi CD115 + CX 3 CR1 + Flt3 + ) (7,8), whereas a distinct progenitor called the common DC precursor (CDP) (Lin − cKit lo CD115 + Flt3 + ) is restricted to producing cDCs and pDCs (9,10). Although monocytes can develop many of the phenotypic features of DCs under inflammatory conditions (11-13), the cDC, pDC and monocyte lineages separate by the time they reach tissues and neither monocytes nor pDCs develop into cDCs under steady state conditions (8,14). Unlike monocytes and pDCs, cDCs in lymphoid tissues are thought to emerge from the bone marrow as immature cells that must further differentiate and divide in lymphoid organs (15,16 We searched for MDPs and CDPs in the blood and spleen by flow cytometry but could only detect them in the bone marrow ( Fig. 1A and Fig. S1). Although pre-cDCs can be identified in the spleen by combining density centrifugation and flow cytometry (18), we speculated that these cells could be identified directly by expression of Flt3 and the chemokine receptor, CX 3 CR1, which are expressed on other DC progenitors and also on mature cDCs (7,10,19). Indeed, we found a small but distinct population of lineage negative CD11c + MHC class II − SIRP-α int Flt3 + cells (pre-cDCs) in the bone marrow (0.2%), blood (0.03%), spleen (0.05%) and lymph nodes (LN) (0.03%) (Fig. 1B).To determine ...

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