Figure 2. Retinoic acid dynamically regulates developmental balance between ILC subsets.

SiLP cells from WT Ctrl and VAI or VAI Rag1^−/− mice treated with all-trans retinoic acid every three days for 12 days (VAI + RA). A) GATA3 and RORγt expression in Lin⁻, Thy1.2⁺ cells (upper panel). Intracellular IL-13 and IL-22 expression in Lin⁻ Thy1.2⁺ cells following stimulation with PMA and ionomycin (lower panel). B) Total numbers of ILC3 and ILC2 cells in SiLP and C) total numbers of IL-22 and IL-13 producing ILC in the SiLP. D) Frequencies of Ki67 expression in ILC3 and ILC2 isolated from SiLp of Ctrl, VAI or VAI +RA Rag1^−/− mice. E) SiLP cells from Rag1^−/− mice treated with RAi or Veh for 8 days stained for intracellular Ki67 in ILC3 (upper panel) cells and ILC2 (lower panel). F) 100,000 CD45.1⁺ common lymphoid progenitors (CLP) were transferred into congenic CD45.2⁺ Rag2^−/−/gc^−/− mice and treated either with vehicle (Veh), RA or RAi. 14 Days after transfer, SiLP cells were isolated, stained for GATA3 and RORγt and gated on CD45.1⁺, Lin⁻ and Thy1.2⁺ cells. G) Quantification of relative proportion of ILC3 and ILC2 in recipient mice and H) frequencies of ILC2 and ILC3 from RARa^fl/fl mice. Transferred cells were gated on GFP⁺ cells I) ILC2 progenitors (ILC2P) were sort purified from bone marrow and cultured in vitro with IL-7 and SCF in the presence of Veh, RA and RAi.for 7 days. Total numbers of Thy1.2⁺ GATA3⁺ cells (left panel) and IL-13 production in the culture supernatant (right panel). J) Ki67 expression in small intestinal ILC2 from mice treated with RAi or Veh and anti-IL7Ra. K) Total number of Ki67⁺ ILC2 and L) total number of ILC2. Data are representative of three (A–E, G–H) or two (I–L) independent experiments with 3–4 mice in each experimental group or at least two independent experiments with 3 experimental groups of cells isolated from 2 mice each (F). (G) displays pooled data from three independent experiments. All graphs display means ±SEM.