Objectives: The main objective of this project is to elucidate the epigenetic mechanisms that control transcription in type 2 innate lymphoid cells (ILC2) from healthy donors and allergic and asthmatic subjects. To achieve this, we want to identify differentially methylated regions (DMRs) specific to human ILC2 cells to understand their role for the functionality of these cells. Further, we want to investigate which types of external inflammatory stimuli can activate the DMR-associated gene expression and determine pathway-specific trigger to understand the functions of DMRs linked to ILC2 biology.

Methods: We performed a genome-wide DNA methylation analysis of human ILC subsets and identified DMRs specific to each subset. Next, human ILC2s, sorted from PBMCs as Lin-CD45+CD127+CD161+CRTH2+ cells, were cultured in the presence of several cytokines for up to two weeks to achieve a 100-fold homeostatic expansion. The stability of the DNA methylation pattern in the expanded ILC2s was assessed by pyrosequencing. DMRs from IL26, IL10RA and HPGDS were cloned into luciferase reporter vectors and preliminary luciferase assays in expanded ILC2s demonstrated their transcriptional activity. Further, CRISPR technology in expanded ILC2s was established to study the impact of a loss of DMRs and/or associated genes on the functionality of the ILC2s.

Results: We have identified DMRs in genes like GATA3, MAF, CCR2, IL26 or IL10RA among others that are specifically demethylated in ILC2. During the in vitro culture, no marked deviation in the expression of ILC2 surface markers nor changes in the DNA methylation pattern of the identified DMRs were observed, suggesting that expanded ILC2s were able to sustain their phenotype and epigenetic profiles.

Conclusions: In summary, our study will provide novel approaches to identify the critical genetic elements controlling the differentiation and function of ILC2s, which will further improve our knowledge about the biology of these innate immune cells.