Type I interferons drive the maturation of human DC3s with a distinct costimulatory profile characterized by high GITRL

Human mononuclear phagocytes comprise specialized subsets of dendritic cells (DCs) and monocytes, but how these subsets individually regulate expression of the molecular signals involved in T cell costimulation is incompletely understood. Here, we used multiparameter flow cytometry and CITE-sequencing to investigate the cell type-specific responses of human peripheral blood DC and monocyte subsets to type I interferons (IFN-I), focusing on differential regulation of costimulatory molecules. We report that IFN-β drives the maturation of the recently identified human CD1c⁺ CD5^- DC3 subset into cells with higher GITRL and lower CD86 expression compared with other conventional DC subsets. Transcriptomic analysis confirmed that DC3s have an intermediate phenotype between that of CD1c⁺ CD5⁺ DC2s and CD14⁺ monocytes, characterized by high expression of MHCII, Fc receptors, and components of the phagocyte NADPH oxidase. IFN-β induced a shared core response in human DC and monocyte subsets as well as subset-specific responses, including differential expression of costimulatory molecules. Gene regulatory network analysis suggests that upon IFN-β stimulation NFKB1 drives DC3s to acquire a maturation program shared with DC2s. Accordingly, inhibition of NF-κB activation prevented the acquisition of a mature phenotype by DC3s upon IFN-β exposure. Collectively, this study provides insight into the cell type-specific response of human DC and monocyte subsets to IFN-I and highlights the distinct costimulatory potential of DC3s.