Immunogenic activation of dendritic cells (DCs) is essential for the initiation of adaptive immune responses following pathogen encounter or vaccination. While this transition involves profound transcriptional reprogramming, much of our current understanding derives from in vitro models that fail to capture DC heterogeneity. Defining the transcriptional regulation of activation across DC subtypes in vivo is critical to elucidating their contributions to immunity that will guide the rational design of DC-targeted therapeutics.

To this end, we integrated longitudinal transcriptomic (RNA-seq) and chromatin accessibility (ATAC-seq) profiling of mouse splenic DC subsets—including cDC1, ESAM+ cDC2, ESAM- cDC2, and DC3—across multiple timepoints following in vivo administration of CpG (a TLR9 ligand). This multi-omic, time-resolved approach enabled us to deconstruct the common and unique regulatory landscapes that define immunogenic activation across the spectrum of DC heterogeneity. Across subsets, stimulation induced a conserved core activation transcriptome; however, non-negative matrix factorization (NMF) successfully decoupled cell-type-specific and time-dependent gene expression modules. These modules allowed for the ontogenic identification of activated cDCs within mouse tumor scRNA-seq datasets, where activated states appeared transcriptionally convergent.  We observed that the kinetics and magnitude of both transcriptomic and epigenetic remodeling were lineage-specific. Specifically, cDC1s and DC3s exhibited a more rapid and robust activation, whereas ESAM+ and ESAM- cDC2s displayed delayed and attenuated responses.To identify the specific transcriptional regulators driving these divergent kinetics, we inferred activation gene regulatory networks (GRNs). This analysis recovered established master regulators of DC activation alongside previously uncharacterized, cell-type-specific TFs. The functional relevance of these candidates was further corroborated via in silico knockouts and experimental validation, identifying TFs with essential contribution for specific DC subsets.

Overall, our comprehensive multi-omic assessment of in vivo DC activation identified cell-type specific features and regulators, offering a novel blueprint for high-precision strategies to modulate specific DC subsets in immunotherapy and vaccination.