Summary The herpes virus entry mediator (HVEM) is a member of the TNF receptor super family. Our published findings indicated that HVEM expressed by mucosal epithelial cells is important for innate defense against pathogenic bacteria in the intestine and the lung. Our recent work suggests that HVEM also functions in intestine epithelial cells under steady state conditions. Mice with an epithelial cell-specific HVEM deletion have a decrease in the survival of intraepithelial lymphocytes (IEL) in the small intestine, and an increase in segmented filamentous bacteria (SFB), which causes an increase in Th17 cells. The experiments in this application are designed to explore the mechanistic bases for the effects of HVEM on immune and microbial homeostasis in the intestine, and the connection, if any, between decreased IEL populations and increased SFB. HVEM has three binding partners: CD160, BTLA, and LIGHT. In specific aim 1, we will use mice with germ line or conditional deficiency for the genes encoding each of these molecules to identify which one(s) is relevant and which cell type expresses this protein. In specific aim 2, we will investigate how epithelial HVEM regulates the size of the IEL population. Our preliminary data suggest an indirect mechanism, in which HVEM signals cause the epithelium to increase synthesis of basement membrane proteins that bind to β1-containing integrins expressed by the IEL. The integrin-basement membrane interaction leads to increased IEL survival. We will test this indirect mechanism, in part by analyzing IEL from β1 integrin deficient mice. As an alternative mechanism, we will investigate if HVEM acts as a ligand that carries out reverse signals that promote survival directly back to IEL through one of its binding partners. In specific aim 3, we will determine if the decrease in IEL and increase in SFB in the absence of epithelial HVEM are causally linked. We also will carry out in vitro and in vivo experiments to understand how epithelial HVEM affects the microbiome, in particular if there are effects on the IL-23-IL-22 axis and type 3 innate lymphoid cells (ILC3), and using organoid cultures, we will determine if signals through NF-κB and/or Stat3 are relevant. Overall, these experiments will provide a deeper understanding of the function of the numerous resident, innate-like lymphocytes in the intestine, the cross talk between resident lymphocytes and epithelial cells, and how these molecular and cellular interactions contribute to tissue homeostasis and the microbiome. The increase in Th17 cells in the intestine of mice lacking epithelial HVEM, and the association of HVEM polymorphisms with inflammatory diseases in the intestine, indicate that the fundamental issues addressed in this application are important for understanding the function of the mucosal immune system in providing protection while avoiding destructive inflammation.