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.