Our overall goal is to define the development, trafficking, and functional properties of a novel gut-homing dendritic cell (DC). These mucosal ?DC reside in the intestinal lamina propria and Peyer's patches (PP), and in the bone marrow (BM). Preliminary studies have provided insights leading to the following general hypotheses: a) ?DC are distinct from conventional DC subsets in phenotype, microenvironmental localization, and development. b) They use novel trafficking cascades to home to the gut from their origin in the bone marrow. c) Gut-homing ?DC are key progenitors of intestinal DC populations including CD103+ cDC; and they can also give rise to CCR9+ plasmacytoid DC (pDC). d) ?DC may have specialized roles in mucosal immunity as evidenced by unique TLR responses and effector activities. Thus we hypothesize that ?DC are both specialized progenitors of intestinal DC, and participants in intestinal immune responses. Aims include: Aim 1. To define the precursors and progeny of ?DC: bone marrow development and intestinal DC homeostasis. ?DC, and CCR9+ pDC are generated in Flt3L-stimulated BM cultures. To identify their origin, common DC progenitors (CDP) and other DC progenitors will be sorted from BM, and their development into DC subsets will be monitored by immunophenotyping after in vitro culture. Sorted ?DC will also be studied to evaluate their progenitor and renewal potential in vitro and in vivo. The effects of regulatory factors including retinoic acid on ?DC generation will be assessed. Aim 2. To determine the homing properties of ?DC vs. CCR9+ pDC, and to define trafficking mechanisms involved. Short term homing assays will determine the ability of ?DC to migrate via the blood into the intestines vs. other sites, monitoring localization by flow cytometry of recovered cells from recipient tissues. Immunohistochemistry will be used to ask whether ?DC home to specific target microenvironments in the gut wall. CCR9+ pDC will be studied in parallel for comparison. Neutralizing antibodies, and/or DC from gene-targeted mice, will be used to define novel trafficking mechanisms/cascades involved. Aim 3. To define the effects of activating (TLR ligands) and gut regulatory factors (e.g. retinoic acid) on the progenitor and immunologic activities of ?DC. In addition to their progenitor activities, ?DC stimulate T cells and display unique patterns of cytokine expression. ?DC will be assayed for maturation and altered progenitor ability in response to TLR ligands; production of immunostimulatory vs. modulatory cytokines; and education or imprinting of T cells responding to presented antigen. Regulation of their progenitor vs. immunologic activities by gut-associated factors including retinoic acid and wnts will also be assessed. Elucidating the origin, function and regulation of intestine-associated DC subsets, as proposed here, will help us understand mucosal immune homeostasis and responses to infection; and has the potential to lead to novel approaches to enhance mucosal vaccination (e.g. for HIV or rotavirus) and to control pathologic inflammation (e.g. in inflammatory bowel diseases or celiac disease). PUBLIC HEALTH RELEVANCE: Dendritic cells (DC) are specialized white blood cells that control the body's balance between good (immunity to infection) and bad (for example autoimmune diseases like diabetes or inflammatory bowel disease) immune responses. We have discovered a new kind of mucosa (intestine)- associated DC that acts as a progenitor for other intestinal DC, and that may help control immune responses in the intestines. Our goal is to understand how this mucosal DC develops and gets to the intestines, what controls its differentiation into other DC types, how it responds to infection-related stimuli, and what factors control its contribution to desirable vs. damaging immune responses. In the future our findings may lead to new approaches to enhancing vaccination for AIDS or diarrheal disease, or to treating inflammatory bowel diseases.