Comparing the transcriptome of small intestinal epithelial cells in organoids after co-culture with NKT cells

Intestinal homeostasis is maintained through the combined functions of epithelial and immune cells that collaborate to preserve the integrity of the intestinal barrier. However, the mechanisms by which immune cell populations regulate intestinal epithelial cell (IEC) homeostasis remain unclear. Here, we use a multi-omics approach to study the immune-epithelial crosstalk and identify CD1d-restricted Natural Killer T (NKT) cells as regulators of IEC biology. We find that NKT cells are abundant in the proximal small intestine and show hallmarks of activation at steady state. Subsequently, NKT cells regulate the survival and the transcriptional and cellular composition landscapes of IECs in intestinal organoids, through mechanisms dependent on IFN-? and IL-4 secretion by NKT cells but independent of the expression of CD1d on IECs. In vivo, lack of NKT cells results in an increase in IEC turnover, while NKT cell activation leads to IFN-?-dependent epithelial apoptosis. Our findings propose NKT cells as potent producers of cytokines that contribute to the regulation of IEC homeostasis. Overall design: 8 samples were sequenced; replicates of four samples from WT organoids co-cultured with NKT cells or alone for 3 days were obtained from 2 individual experiments. After co-culture, media was removed and organoids were washed with 1mL pre-warmed ADEMEM+++ to remove NKT cells before proceeding to mRNA isolation. Samples were frozen at —80°C before RNA extraction.