725 - T cells derived from spontaneous intestinal perforation affected mucosa are detrimental to epithelial cell growth

Publication Number: 725.6927

Oluwabunmi Olaloye, Yale School of Medicine, New Haven, CT, United States; Chino Eke, The Warren Alpert Medical School of Brown University, Providence, RI, United States; Alisha Mason, Yale School of Medicine, Frederick, MD, United States; Long Phan, Yale School of Medicine, New Haven, CT, United States; Liza Konnikova, Yale School of Medicine, North Haven, CT, United States

Background: Spontaneous intestinal perforation (SIP) is a gastrointestinal complication with high morbidity and mortality in preterm infants. Little is known about the underlying mechanisms driving the disease. Recent data from our group shows that T cells in SIP mucosa exhibit increased IFNgamma expression after activation suggesting a proinflammatory role in the disease.

Objective: Investigate the impact of T cells derived from SIP-affected mucosa on immature and SIP epithelium using an organoid model.

Design/Methods: Intestinal organoids were derived from crypts isolated from viably cryopreserved small intestine from elective terminations (fetal n = 2, Gestational Age (GA) 23 weeks) and resected tissue from infants who had surgery for SIP (n = 2, GA 25 - 27 weeks, Fig 1A). After organoid lines were established fetal and SIP organoids were co-cultured with and without Fetal/SIP T cells for 7 days in organoid growth media (Fig 1B, OGM, Stemcell). Organoids were imaged on Days 1, 3, 5, and 7 on an Echo Revolve Microscope at 10X. Organoid growth and generation were determined from the number and area of organoids quantified in Image J. OGM was saved and stored for cytokine analysis on Days 3, 5, and 7 of culture. On day 7, single-cell RNA sequencing was performed on the organoids.

Results: Co-culture with fetal T cells resulted in the generation of more organoids on Day 7 in fetal and SIP organoids. However, organoid size and generation from SIP were significantly limited compared to fetal small intestine (Fig 1C-D). scRNAseq analysis revealed downregulation of SERPINA1, APOA4/A1/B genes that regulate intestinal inflammation, modulate immune responses as well as OLMF4 a marker of intestinal stem cells in in SIP compared to fetal organoids (Fig 1E, F) suggesting a defect in stem cells in SIP. After 7 days of co-culture with SIP T cells, size and generation of fetal organoids was significantly limited (Fig 2A,B). Meanwhile, exposing SIP organoids to SIP T cells limited organoid size but not generation (Fig 2C, D). IL22 was abundant in collected media after co-culture with SIP T cells in both SIP and fetal organoids (Fig 2E) and IL29, IL34 and IFNB were also present in SIP organoids.

Conclusion(s): Both T cells and epithelium are profoundly impaired after neonatal spontaneous intestinal perforation. A pro-inflammatory signature in T cells and defect in epithelial stem cells likely contribute to the pathogenesis of SIP. Further analysis of epithelial-T cell interactions in SIP can elucidate targets for future therapies.

Figure 1: Organoids derived from SIP affected mucosa exhibit decreased growth and reduced expression of the stem cell marker OLMF4.
Fig 1 SIP organoids_PAS 2025.pdf

Figure 2: Co-culture of SIP T cells impairs growth of fetal and SIP organoids.
Fig 2 SIP organoids_PAS 2025.pdf