SUMMARY: The GI tract recruits lymphocytes from the blood to establish an immune barrier to pathogens, while enforcing tolerance to self and, after birth, to food antigens and enteric bacteria. "Natural" (unconventional) thymus- derived T cells of the intraepithelial compartment (IEL) comprise the most abundant T cell pool in the adult and neonatal intestines, and yet how they are recruited to the colon and small intestines, why they populate the GI tract before birth, and their roles in epithelial homeostasis and pathogen resistance in the perinatal period remain incompletely understood. The objective of the current proposal is to define mechanisms of innate-like T cell trafficking to the GI tract in fetal and perinatal life, and to establish the role(s) of these receptors, and of the specialized IEL T cell subsets they control, in intestinal development and immunity. In Aim1 we will identify the major innate T cell subsets in the mouse fetal intestines, determine the chemoattractant receptors that recruit them into the SI and colon from the thymus, and define their function in the prenatal gut environment. We will manipulate fetal T cell recruitment in vivo and perform organoid co- culture ex vivo to test the hypothesis that specialized fetal T cells drive maturation of the intestinal epithelium and program epithelial defenses in preparation for birth. In Aim 2 we will establish the importance of these mechanisms and T cells in pathogen responses in models of neonatal viral and bacterial infection. In Aim 3 we will identify human innate T cell counterparts of the major mouse fetal colon T cell, as defined by common developmental trajectories; shared gene programs, homing receptor profiles and cytokine responses; and common functions in intestinal epithelial differentiation. We will employ innovative approaches and develop new protocols and tools to address these aims. Results from these studies will significantly advance our understanding of the cells, pathways and mechanisms that prepare the fetal GI tract for microbial assault at birth. These insights could lead to novel diagnostic, therapeutic or preventative strategies targeting neonatal immunity.