ABSTRACT Plasmacytoid dendritic cells (pDCs) are a unique subset of innate immune cells capable of multiple functions essential for antiviral responses, including type I interferon production, antigen-presentation and T cell activation. The mechanisms that govern these distinct pDC functions remain poorly defined; however, they could be mediated by distinct subpopulations. Using high-dimensional single-cell proteomic and transcriptomic approaches, we and others recently discovered a novel human dendritic cell (DC) population that is captured within traditional pDC definitions. These cells harbor phenotypic features of both pDCs and conventional DC subsets (cDCs); thus, we called them transitional DCs or tDCs. We have now performed an integrated multidimensional comparison that resulted in the identification of the mouse homolog of human tDCs. The discovery that tDCs occur in both human and mouse suggests they have an evolutionarily conserved role during immune responses. However, tDC function has never been investigated. Similarly, the developmental origin of tDCs has not yet been analyzed. This represents a fundamental gap in our understanding of the cellular components that mediate innate immune responses against viruses and poses an impediment to the development of therapeutics. Based on our preliminary data generated in mouse, we hypothesize that tDCs and pDCs form a distinct developmental lineage that cooperates at the site of viral infection to modulate immune responses. In three specific aims, we propose to query tDC origin, function and relationship with pDCs. To achieve these aims, we will take advantage of high-dimensional approaches already established in our lab, in vitro and in vivo differentiation assays, and novel lineage tracing and cell-specific depletion mouse models. We anticipate that findings from this proposal will enhance our current understanding of innate cellular pathways that result in the positive outcome of viral infection. Importantly, our integrated approach will incorporate analyses of both mouse and human tDCs; thus, it has the potential to reveal features of the innate immune compartment that are conserved between species. This proposal has the potential to impact the rational design of future therapeutic strategies.