Fig. 4: Aiolos supports IL-5 expression by suppressing PD-1.

a–c, i–q ILC2s were purified from the large intestine of wild-type (a–c) or Pdcd1^–/– mouse (o–q) and cultured in vitro in the presence of IL-7 and IL-33 for 5 days. ILC2s were infected with control retrovirus or retrovirus expressing Ikzf3-shRNA#1 or Ikzf3-shRNA#2 (all have GFP as reporter) for two rounds. a Expression of indicated genes in purified virus-infected ILC2s (GFP⁺ cells) was analyzed by real-time RT-PCR. b, c, i–q GFP⁺ virus-infected cells were purified and cultured starting with equal cell numbers for another 48 h before analysis (in the presence of hamster IgG or α-PD1 antibody for l–n). Expression of PD-1 (b and c), IL-5 (b, c, l, o), Ki67 (i, m, p) gated on GFP⁺ cells was analyzed by flow cytometry. b Isotype control was used to set up gates for IL-5⁺ or PD-1⁺ cells. j, n, q Absolute numbers of ILC2s were shown. k Concentration of IL-5 in culture supernatant was analyzed by ELISA. d–f ILC2s infected with control virus expressing GFP or virus expressing Ikzf3-shRNA#2 were transferred to Rag2^–/–Il2rg^–/– mice. d, e Four weeks after transfer, LI LPLs were isolated and analyzed. Expressions of IL-5 and PD-1 gated on Lin^–GFP⁺ cells from host mice were analyzed by flow cytometry. FACS plots and statistics are shown. d Gating control for IL-5 and PD-1 were gated on Lin⁺ cells and Lin^–GFP^– cells, respectively. f Mice were injected with IL-33 for 4 days and were then sacrificed for analysis. Percentages of eosinophils (CD45⁺CD11b⁺Siglec-F⁺) gated on live leukocytes (CD45⁺ cells) are shown. g, h ILC2s from human PBMC were purified and cultured in the presence with IL-2, IL-7 and IL-33 for 3 days. Cells were treated with control siRNA or siRNA targeting IKZF3 for 48 h before harvested for analysis. g IKZF3 and IL5 mRNA expression were analyzed by real-time RT-PCR with GAPDH as reference and normalized to the expression of control siRNA group as 1. h Brefeldin A was added 2 h before cells were harvested for analysis. Histogram shows expression of Aiolos and IL-5 gated on ILC2s analyzed by flow cytometry. a–q Data are representative of at least three independent experiments. Error bars are mean + SEM. c, i, j, k Connected lines indicate a data pair using cells from same mouse.