Differential IRF8 Transcription Factor Requirement Defines Two Pathways of Dendritic Cell Development in Humans

The formation of mammalian dendritic cells (DCs) is controlled by multiple hematopoietic transcription factors, including IRF8. Loss of IRF8 exerts a differential effect on DC subsets, including plasmacytoid DCs (pDCs) and the classical DC lineages cDC1 and cDC2. In humans, cDC2-related subsets have been described including AXL⁺SIGLEC6⁺ pre-DC, DC2 and DC3. The origin of this heterogeneity is unknown. Using high-dimensional analysis, in vitro differentiation, and an allelic series of human IRF8 deficiency, we demonstrated that cDC2 (CD1c⁺DC) heterogeneity originates from two distinct pathways of development. The lymphoid-primed IRF8^hi pathway, marked by CD123 and BTLA, carried pDC, cDC1, and DC2 trajectories, while the common myeloid IRF8^lo pathway, expressing SIRPA, formed DC3s and monocytes. We traced distinct trajectories through the granulocyte-macrophage progenitor (GMP) compartment showing that AXL⁺SIGLEC6⁺ pre-DCs mapped exclusively to the DC2 pathway. In keeping with their lower requirement for IRF8, DC3s expand to replace DC2s in human partial IRF8 deficiency.