Dreg1 deletion results in a specific reduction in peripheral ILC2 cells.

(A) Deletion of Dreg1 in mice using CRISPR-Cas9. (B-E) Representative FACS plots and % of CD45+ of NK and ILC populations from the visceral adipose tissue (VAT), small intestinal lamina propria (SI LP), and lung from wild type (WT) or Dreg1-/- mice. Shown is one representative experiment of two with n=4 mice/group. Mean and SEM together with individual data points are shown. Data were statistically analyzed by Student t test.

Dreg1 deletion results in a bottleneck in ILC development and a reduction in ILC2P.

(A) Boxplots showing expression of Gata3 and Dreg1 from RNA-Seq (GSE77695). (B-G) Quantification of ILC progenitor subsets with percentages (upper) and total numbers (below). (H) Mean fluorescence intensity (MFI) of Gata3 measured on the CHILP, ILCP and ILC2P. Shown is one representative experiment of two with n=3-4 mice/group. Mean and SEM together with individual data points are shown. Data were statistically analyzed by Student t test.

Dreg1 locus is dynamically regulated in a Tcf1-dependent manner during ILC2 development.

(A) Chromatin accessibility around Dreg1 in ILC progenitors. (B) Motifs enriched in accessible regions around Dreg1. (C) Tcf1 binding around Dreg1 locus in EILP. (D) Histone marks around Dreg1 at different stages of ILC development and in Tcf7-/- EILP. (E) Gata3 and Dreg1 expression quantified from RNA-Seq data in ILC progenitors and Tcf7-/- EILP.

A syntenic region in humans represents a GATA3 enhancer element with a transcriptional profile akin to murine Dreg1.

(A) GATA3 and DREG1.1 and DREG1.2 expression quantified from RNA-Seq data in ILC and T helper populations from isolated from healthy human blood. (B) Alignment of Functional Sequence 23 (FS23) that overlaps the syntenic region revealed as regulating GATA3 expression in a CRISPR deletion screen of human TH2 cells (26). (C) Chromatin accessibility around FS23 guides from the CRISPR deletion screen (26).

Dreg1 deletion in does not affect T or NK cells.

(A-C) Representative FACS plots with quantification (right) showing expression of (A) CD8α and CD4 in splenic T cells. (B) CD44 and CD62L in splenic CD8+ T cells (C) CD44 and CD62L in splenic CD4+ T cells. (D) Quantification of splenic γδ T cells and NK cells. Shown is one representative experiment of two with n=3 mice/group. Mean and SEM together with individual data points are shown. Data were statistically analyzed by Student t test.

Gating of ILC populations in peripheral tissues and bone marrow.

(A) Gating scheme for bone marrow ILC progenitors corresponding to Figure 2.