Human ILC methylomes uncover stable lineage codes and disease-linked ILC2 regulators in allergy

Abstract

The transcriptional programs of human ILCs are increasingly defined, but the DNA-methylation landscapes that stabilize their identity and function remain poorly understood. Here, we generated genome-wide DNA methylomes of human NK cells, ILC1, ILC2, and ILC3 from blood and lymphoid tissues. Subset-specific differentially methylated regions distinguished all populations and mapped to canonical regulators, including TBX21, GATA3, and RORC, as well as genes not previously linked to ILC biology, such as ERN1, DDX47, JAML, BTLA, and NRROS. Because ILC2 showed a particularly distinct methylation landscape and contribute to allergic inflammation, we tested whether selected ILC2-specific regions were functionally relevant. ILC2 marker regions were largely stable across tissues and during cytokine-driven expansion. CRISPR/Cas9-mediated deletion of the HPGDS or NRROS DMR revealed that these elements act as cis-regulatory sites controlling HPGDS/NRROS expression and promoting production of the type 2 cytokines IL-4, IL-5 and IL-13. Finally, methylome profiling of ILC2 from healthy, atopic, and asthmatic children identified disease-associated DMRs linked to PTGS2, QKI, and GIMAP4. These findings define stable epigenetic signatures of human ILC identity and uncover regulatory elements connecting ILC2 methylation to allergic disease.