Dendritic cells (DCs) are critical antigen presenting cells for initiating adaptive immune responses. Their function depends on being positioned in sentinel locations at pathogen-exposed surfaces, and being present in tissues at appropriate densities. The major antigen-presenting DCs are called classical DCs (cDCs) types 1 and 2. cDC2s are particularly important for supporting T follicular helper (Tfh) cell and Th2 cell generation and are a focus of our efforts to broadly understand requirements for mounting antibody responses. In this application we will study requirements for cDC2 positioning, homeostasis and function in spleen and lung. Very recently, we found that splenic cDC2 homeostasis was dependent on the G13-containing heterotrimeric G-protein. Using CRISPR/Cas9-based screening, we found that the adhesion GPCR Adgre5 (CD97) functions upstream of G13 to promote splenic cDC2 positioning, homeostasis and function. Importantly, we found that the presence of CD55, a CD97 ligand, on red blood cells (RBCs) was sufficient to promote splenic cDC2 homeostasis. As an adhesion GPCR family member, CD97 is predicted to be mechanosensitive. We hypothesize that the CD97-G13 signaling pathway is activated in splenic cDC2s following encounter with CD55-expressing RBCs under conditions of shear stress. We speculate that this pathway critically informs cDC2s of their positioning in blood-exposed regions of the spleen, sites that allow encounter with systemic pathogens. In Aim 1 of this application we will characterize the domain requirements of CD97 to support cDC2 homeostasis. Mechanosensing by CD97 will be studied and the role of key transcription factors, including Mrtf and Irf4, in the CD97 pathway will be examined. Finally, the mechanism for defective responses to TLR ligands in CD97-pathway mutant cDC2s will be characterized. In Aim 2 intravital two photon microscopy of the spleen will be used to define how CD97 promotes cDC2 positioning and to study the dynamics of cDC2 interaction with self and foreign RBCs. This work will define a new pathway acting in cDC2s to support their antigen capture and presentation function during the initiation of antibody responses to blood-borne antigens. In earlier work we discovered that EBI2 (GPR183) is a critical chemoattractant receptor in splenic cDC2s, and loss of EBI2 leads to defective T cell-dependent antibody responses to blood borne antigens. We also defined the cell types producing EBI2’s oxysterol ligands in lymphoid tissues. In new work we have discovered that EBI2-deficiency leads to a marked reduction in cDC2s in the lung. In Aim 3 we will define the role of EBI2 in positioning cDC2s within the lung and determine the cell types that serve as a source of oxysterol ligands. The impact of EBI2 deficiency on the lung allergic response will be examined. Definition of a chemoattractant receptor controlling lung cDC2 homeostasis and influencing lung immune responses may provide novel avenues for therapeut...