5A), although it led to increased expression of CD86 and CD40 by both subsets (Fig

5A), although it led to increased expression of CD86 and CD40 by both subsets (Fig. mesenteric lymph node (MLN) following systemic immunization with soluble flagellin (sFliC). In contrast, IRF8-dependent CD103+CD11b- (SP) are not required for these responses. The lack of this response correlated with a complete absence of sFliC-specific plasma cells in the MLN, small intestinal lamina propria and surprisingly also the BM. Many sFliC-specific plasma cells accumulating in the BM of immunized wildtype mice expressed 47+suggesting a mucosal Ufenamate origin. Collectively these results suggest that mucosal DP cDC, contribute to the generation of the sFliC-specific plasma cell pool in the Ufenamate BM and thus serve as a bridge linking the mucosal and systemic immune system. Introduction Flagellin is the filament protein component of bacterial flagella. Extracellular flagellin is usually recognised primarily through TLR5 and this can induce profound responses in innate and adaptive immune cells1. Immunization with purified, soluble flagellin (sFliC) protein from Typhimurium (STm) is sufficient to drive T and B cell responses against itself and co-immunized antigens in the absence of additional adjuvant 2C5. This autoadjuvant activity of flagellin has led to its use as a carrier protein in a number of vaccine strategies6C8, including an influenza fusion vaccine tested in humans9, 10. Additionally, immunization of sFliC in mice has been shown to enhance protection against viral infections 11 and radiation exposure12, promote antigen presentation through MHCII 13 and reduce Th1 differentiation after co-immunization with STm14. While such findings show that flagellin is an important modulator of the adaptive immune system, the cellular mechanism(s) underlying its mode-of-action remain unclear. Previously, it has been shown that systemic immunization with sFliC, given subcutaneously in the footpad or intraperitoneally, induces IgG responses in the spleen and concurrent IgG and IgA responses in the intestinal draining mesenteric lymph nodes (MLN)15. This unexpected induction of intestinal responses after systemic immunization was TLR5-dependent and associated with the quick and considerable recruitment of antigen-loaded CD103+ classical dendritic cells (cDC) into the MLN. This coincided with a decrease in the frequency of these cells in the small intestine lamina propria (SI-LP) and suggests that the autoadjuvant activity of sFliC may, in part, be mediated through the activation of mucosal CD103+ cDC. The intestinal mucosa contains three major subsets of cDC; CD103+CD11b+, CD103+CD11b- and CD103- cDCs16, 17 that require different transcription factors for their development and survival. Deletion of the transcription factors interferon regulatory factor (IRF) 8, BATF3 or ID2 results in a loss of intestinal and MLN CD103+CD11b- cDC18C20 while deletion of IRF4 and NOTCH-2 results RAB21 in a loss of intestinal derived CD103+CD11b+ cDC in the MLN 21, 22. We, as well as others, have recently exhibited that these subsets play important nonredundant functions in regulating intestinal immune homeostasis. For example, IRF4-dependent cDC play an important role in intestinal Th17 21, 23 and Th2 responses 24 and for driving post-operative ileitis25. In contrast, IRF8 dependent CD103+CD11b- cDC are required for the maintenance of T cells within the small intestinal epithelium and for the generation and maintenance of intestinal IFN- generating Th1 cells26, 27. In the current study, we assessed the role of mucosal cDC in the generation of sFliC-specific IgG and IgA responses in MLN following systemic immunization, and the impact of this response around the accumulation of plasma cells in the BM. We demonstrate that mucosal CD103+CD11b+ but not CD103+CD11b- cDC are essential for the generation of sFliC-specific responses in MLN, and that the absence of this response impacts on long-term systemic Ab response in the BM. Collectively these results suggest that mucosal CD103+CD11b+ cDC act as a bridge to link adaptive immune responses of the intestinal mucosa to serological memory and systemic protection. Results DP cDCs recruited to the MLN after direct activation by sFliC are functional It has been previously exhibited that i.p. or s.c. immunization with sFliC drives a TLR5-dependent accumulation of CD103+ cDC in intestinal draining MLN 15. To determine which CD103+ cDC subset in the MLN in response to sFliC, WT mice were immunized Ufenamate i.p. with sFliC and numbers of CD103+CD11b+ (DP), CD103+CD11b- (SP) and CD103- cDC in the MLN and SI-LP were assessed 24 h later by circulation cytometry (for gating strategy observe Fig. S1A). sFliC immunized mice experienced an increased frequency of CD11c+MHC-IIhi cDC in MLN (Fig. 1A). In the steady-state this populace has.