Th144 - Single Cell Analysis Reveals Altered Regulatory T Cells in Neonates with Necrotizing Enterocolitis

Preterm infants are susceptible to necrotizing enterocolitis (NEC). This devastating disease remains poorly understood and treatment is non-specific. There is an urgent need to identify biomarkers and to develop targeted therapies. Single-cell analysis with spatial resolution of NEC-affected mucosa can identify altered signaling and cellular interactions that could serve as potential therapeutic targets. Small intestinal tissue from infants with NEC (n=20, gestational age (GA) 23-39 weeks) and in neonates with non-immune congenital anomalies (neonatal n=13, GA 34-40 weeks) were compared. Single cell RNA sequencing (scRNAseq), deconvolution of bulk sequencing data, and imaging mass cytometry (IMC) was performed.

 scRNAseq identified 7 distinct populations of lymphocytes. Analysis of differentially expressed genes showed that genes required for appropriate T reg suppressive function (FOXP3, CTLA4) were decreased, and SELL (CD62L), CCR7, SOCS1  were increased in NEC compared to neonates. This is consistent with the upregulation of SOCS1, a downstream target of STAT3 signaling and IMC analysis showed an increase in pSTAT3 in NEC associated Tregs. Interactions between T regs and macrophages were altered in NEC signaled with increased ligand-receptor interactions of IL1R1/IL1B, CAV1-TGFB1, and ABCA1-CALM1. Furthermore, IMC highlighted altered interactions in NEC Tregs. Specifically, decreased interaction with other T cells (p< 0.01), and increased interaction with inflammatory macrophages. In summary, we characterize altered cellular interactions and transcriptional signature in NEC-associated regulatory T cells that contribute to disease pathogenesis and could serve as potential targets for future therapies.