Project Summary Spontaneous intestinal perforation (SIP) is a gastrointestinal complication affecting 3-8% of extremely low birth weight infants, corresponding to approximately 4,000 prematurely born babies annually in the US. All affected infants require surgery and are at an increased risk of brain injury, infection, and death. Yet, the pathogenesis and etiology of SIP are poorly understood. Consequently, there are currently no biomarkers for early recognition or disease-specific therapy. The foundation for this proposal is preliminary and published data where we showed alterations in T cell and epithelial cell subsets in SIP-affected mucosa that was not present in non-SIP mucosal samples. Intestinal immune dysregulation in SIP was characterized by reduced tissue-resident memory T cells, increased naïve T cells that produce more IFNγ upon stimulation, and a decreased proportion of epithelial cells in SIP compared to fetal and neonatal controls. This suggests that the inability to generate tissue-resident memory T cells in utero and the proinflammatory effects of the naïve T cells that infiltrate the intestine in their place after birth could contribute to the development of SIP. We hypothesize that susceptibility to intestinal perforation in extremely premature infants is exacerbated by defects in memory T cell generation and concomitant epithelial cell damage. We will perform a comparative analysis of T cells’ transcriptome and spatial location in patients with SIP compared to samples from fetal and neonatal mucosa. Using a T cell co-culture organoid model, we will investigate the impact of aberrant T cell activation and IFNγ exposure on epithelial cells in healthy and affected mucosa. Completing these aims will provide data to inform a next-step large-scale study into the role of epithelial-immune dysfunction in SIP. The candidate is committed to a career in studying epithelial-immune cell interactions using spontaneous intestinal perforation as a model and is strongly supported by her mentors and her department at the Yale School of Medicine. The proposal builds upon the candidate’s prior research and clinical experience in immune dysregulation in neonatal intestinal disease. It integrates two new domains of expertise in epithelial-immune interactions using organoids and integrative network analysis in a comprehensive training and didactic plan. This proposal is supported by dedicated and experienced mentors with expertise in mucosal immunology, immunobiology, T-cell signaling in immune disorders, neonatal nutrition, and biostatistics. The proposed experiments and didactics will provide the candidate with interdisciplinary skills that will foster her transition to independence as a physician-scientist in studying epithelial-immune cell interactions in intestinal disorders in children.