Plasmacytoid dendritic cells (pDCs) rapidly secrete type I interferon (interferon α/β, IFN) in response to virus- derived nucleic acids, facilitating both innate and adaptive antiviral responses. Conversely, aberrant IFN production by pDCs is associated with autoimmune diseases, establishing pDCs as important emerging therapeutic targets. The overall goal of this project is to elucidate the molecular control of pDCs lineage, including its development, homeostasis and function in various immune responses. In the previous award cycles, we have identified E protein transcription factor TCF4 (E2-2) and several other factors as important regulators of pDC development. The proposed work will build upon these findings to address major unanswered questions in pDC biology. In Aim 1, we will use high-dimensional single-cell analysis methods to characterize the stage and regulation of pDC lineage specification, as well as the functional heterogeneity of mature pDCs. In Aim 2, we will use genetic approaches to dissect transcriptional control and regulation of the unique IFN-producing capacity of pDCs. In Aim 3, we will explore the mechanism of virus recognition and IFN production by pDCs in vivo. Collectively, the proposed studies should yield a comprehensive molecular view of pDC development and function, paving the way for therapeutic approaches focused on this cell type. RELEVANCE (See instructions): Plasmacytoid dendritic cells (pDCs) play an important role in immune responses to infection and in autoimmunity, and therefore represent attractive targets for novel immunoterapies. The proposed work aims to build a comprehensive molecular view of pDC development and function, paving the way for pDCfocused translational approaches in human diseases.