Tetrameric STAT5 regulates the formation of immune niche cells to protect stem cell regenerative repair against mucosal inflammation.

How intestinal stem cells (ISCs) are regulated during inflammation remains largely unexplored, leading to a lack of effective treatments for inflammatory bowel diseases (IBD). ISC-mediated intestinal epithelial regenerative repair can be regulated by intra-epithelial T lymphocytes, whose maturation is controlled by STAT5 dimeric or tetrameric activation. However, the mechanisms by which the T lymphocytes can protect ISCs are unclear. Here we hypothesize that tetrameric STAT5 regulates intra-crypt T cells to act as niche cells for ISC regeneration. Using IBD biospecimens, STAT5-hyperactive, tetramer-deficient mice and organoids, we found IBD-ulcerative colitis exhibited more crypt TCRγδ+STAT5+ T cells and less ISC pluripotency than healthy patients. Compared with wild-type mice, depleting tetrameric STAT5 in mice significantly increased ISC-mediated intestinal epithelial hyperplasia, TCR gene signatures, crypt TCRγδ+ T cells with elevated STAT5 tyrosine phosphorylation (pYSTAT5) and IL-17A levels, amplified both Lgr5hi and Lgr5low ISC proliferation and promoted de novo crypt regeneration with increased TCRγδ+ cell influx post irradiation or colitis. By contrast, depleting tetrameric STAT5 in organoids reduced ISC pluripotency and organoid growth post irradiation. Mechanistically, chromatin immunoprecipitation and single-cell RNA sequencing analyses with crypt cells revealed that depleting STAT5 tetramers decreased STAT5-binding on the Metallothionein 1 (Mt1) locus in crypt T cells and increased Mt1 expression, which leads to T cell migration into crypts and enhanced ISC regeneration. Together, the tetrameric STAT5 suppresses the formation of the crypt T cell niche. Interrupting STAT5 tetramers promotes the expansion of crypt TCRγδ cells, providing a target for promoting ISC regenerative repair during IBD-ulcerative colitis.