Breast milk-derived signals regulate epithelial cells maturation and γδ-IEL colonization to shape early life intestinal homeostasis 2258727

Abstract Introduction The intestinal epithelium is a specialized barrier that balances tolerance and defense against luminal antigens and microbes. Its maturation during early life is shaped by intraepithelial lymphocytes (IELs), the microbiota, and maternal factors. Natural CD8αα+ αβ and Vγ7+ γδ IELs are critical for epithelial homeostasis, repair, and microbial regulation, yet the mechanisms controlling their postnatal maintenance remain poorly understood. Methods Jackson (Jax) and Taconic (Tac) mice were evaluated by flow cytometry after cross-foster, co-house, and adoptive transfer experiments. Bulk RNA sequencing, and milk proteomic analysis were also performed. Results Tac mice showed fewer natural CD8αα+ and Vγ7+ γδ IELs throughout the small intestine (SI), while colonic frequencies remained comparable to Jax mice, indicating a compartmentalized defect. CD8αα+ IEL loss was microbiota-dependent, as co-housing with Jax mice restored their abundance, whereas Vγ7+ IEL loss was microbiota-independent and emerged at weaning despite normal thymic output. Cross-fostering Tac pups onto Jax dams fully rescued Vγ7+ IELs, implicating milk-derived factors. Proteomic profiling of Tac versus Jax milk revealed broad protein reductions, notably in IGF transport and uptake pathways, with decreased IGF-1 and osteopontin. Adoptive transfers showed a tissue-intrinsic defect, as Tac IELs engrafted in Jax RAG⁻/⁻ hosts but not vice versa. SI epithelial transcriptomics revealed downregulation of c-Myc targets after day 28, coinciding with Vγ7+ IEL loss and defective epithelial maturation. Conclusion Together, these findings reveal that postnatal maintenance of natural IELs depends on distinct environmental cues: while CD8αα+ IELs are shaped by the microbiota, Vγ7+ γδ IELs require maternal milk—derived factors that support epithelial maturation and c-Myc—dependent pathways. This identifies a critical early-life window in which deficient milk signals can impair intestinal immune—epithelial development and long-term mucosal homeostasis. Funding Source n/a Topic Categories Mucosal and Regional Immunology (MUC)