Project Summary Conventional dendritic cells (cDCs) are crucial for both innate and adaptive immunity. In general, cDCs can be categorized roughly into two functionally and developmentally distinct subsets, cDC1 and cDC2, which preferentially activate CD8T cells and CD4T cells, respectively. cDC2s are phenotypically and functionally more heterogeneous compared to cDC1s, but how different cDC2 subsets acquire distinct phenotype and function remains poorly understood. We previously showed that a mouse cDC2 subset expressing CD301b (Mgl2) is functionally distinct and plays critical role in type 2 immune responses to allergens and helminth parasites. CD301b+ DCs are present in nearly all peripheral organs and expand during type 2 inflammation, but their differentiation mechanism is unknown. Our long-term goal is to understand the mechanism of CD301b+ DC differentiation at the cellular and molecular levels and explore its potential as a therapeutic targets for diseases with type 2 inflammation such as allergies. In general, cDC2s are thought to originate from the pre- cDC, a cDC-committed circulating precursor, through the action of the transcription factor IRF4. However, it remains unclear whether the heterogeneity in cDC2s originates in their precursors, or is acquired later during terminal differentiation. Likewise, while IRF4 seems to play a significant role in cDC2 development in general, its role in diversification of cDC2 subsets remains elusive and additional factors may be responsible for their heterogeneity. We hypothesize that pre-cDCs and/or monocytes acquire the CD301b+ DCs phenotype in a context-dependent manner with context-dependent requirement of IRF4. Our specific aims in this proposal are (1) to identify the precursor cell populations that give rise to CD301b+ DCs in peripheral organs, and (2) to elucidate the role of IRF4 and CD301b+ DC-specific transcription factors in their differentiation, under both naive and infected conditions. Understanding the mechanism of cDC2 differentiation might give us clues to understand the etiology and to identify potential therapeutic targets in allergies and other diseases with type 2 inflammation.