PROJECT SUMMARY/ABSTRACT CD1d-restricted, invariant natural killer T cells (iNKT) cells play a critical role in regulating the commensal micro- biota and resistance to mucosal pathogens. Conversely, iNKT cell levels are suppressed by microbiota in early (pre-weaned), but not later (post-weaned), life. If critical microbial signals are not provided during early-life, co- lonic iNKT cell levels remain elevated leading to increased susceptibility to colitis in later-life. Recently, we dis- covered that colon iNKT cells are resident cells that establish themselves in early-life in a pathway controlled locally by macrophages of fetal origin and a novel stromal cell population that is marked by Wnt4 which are in turn regulated by the commensal microbiota. The current research proposal addresses the unanswered question about how this newly identified stromal cell population regulates intestinal iNKT cells in early-life. Our long-term goals are to parse out the mechanisms that microbes and the host use to establish an iNKT cell niche that influences mucosal disease such as infection and colitis in later-life. The objective of this research is to better understand the pathways that regulate iNKT cells in early-life as a paradigm for imprinting cellular residency during a critical period of development. Our central hypothesis is that a unique early-life colonic stromal cell population is part of a local tissue niche that directly imprints iNKT cells through bone morphogenic protein (BMP) signals and determines the host’s susceptibility to inflammatory bowel disease (IBD) and enteropathogens in later-life. Specifically, it is proposed that embryonic macrophages and microbes regulate a unique colonic stromal cell population that is marked by Wnt4 and determines the tissue receptivity to early-life engraftment of thymic iNKT cell emigrants through stromal cell-derived cognate (CD1d) and non-cognate (BMP2) signals. The rationale for our proposed research is that there is very little known about early-life colonic stromal cells and their interac- tions with immune cells. Our central hypothesis will be tested with four specific aims: 1) determine whether colonic Wnt4+ stromal cells are an early-life specific population that forms a tissue niche with embryonic macro- phages to support local iNKT cells; 2) Identify the specific molecules generated from colonic Wnt4+ stromal cells that could directly support colonic iNKT proliferation during early-life and specifically the role of BMP2; 3) deter- mine whether Wnt4+ stromal cells specifically imprint colonic iNKT cells during early-life which results in durable effects that influence later-life disease, and; 4) determine how microbial signals regulate the cell abundance and transcriptome of colonic Wnt4+ stromal cells during early-life. Specifically, we expect we will show that this dis- tinctive type of stromal cell forms a tissue niche that supports iNKT cells in early-life (Aim 1) and operates through BMP2 and CD...