Fig. 7: CD45⁺lineage^–CRTH2⁺CD127⁺GATA3⁺ST2^–KLRG1⁺IL-17RB⁺ ILC2s in peripheral blood of asymptomatic asthmatic patients are identical to mice ml-ILC2s.

Flow cytometry was performed to analyze CD45⁺lineage^–CRTH2⁺CD127⁺GATA3⁺ST2^–KLRG1⁺IL-17RB⁺ ILC2s in PBMCs purified from asthmatic patients during remission phase (all rested for 50 days or longer) and healthy donors. A Gating strategy, B statistical plots, C IL-13⁺ percentage and MFI and D IL-5⁺ percentage and MFI of CD45⁺lineage^–CRTH2⁺CD127⁺GATA3⁺ST2^–KLRG1⁺IL-17RB⁺ ILC2s. E KLRG1^–, KLRG1⁺IL-17RB^– and KLRG1⁺IL-17RB⁺ subsets were purified from the total CD45⁺lineage^–CRTH2⁺CD127⁺ST2^– ILC2s in PBMC of 6 asymptomatic asthmatic patients (marked as A-KLRG1^–, A-KLRG1⁺IL-17RB^– and A-KLRG1⁺IL-17RB⁺). KLRG1⁺IL-17RB⁺ subsets were sorted from total ILC2s in PBMC of 3 healthy donors (marked as H-KLRG1⁺IL-17RB⁺). Cells were then cultured (5 × 10³/well) with 10 ng/mL IL-2 and 20 ng/mL IL-7, and treated with (alarmins+) or without (alarmins−) 200 ng/mL recombinant human IL-17E/IL-25 and 200 ng/mL recombinant human IL-33 every other day for 21 days. Subsequently, cells were collected for further analysis. F Ki-67⁺ percentage, G absolute number and H fold changes of expansion of cells were determined. Representative plots and statistical results (bar graph) showing the percentages of I IL-13⁺ and J IL-5⁺ of three ILC2 subsets after stimulation with PMA, ionomycin, brefeldin A and monensin. KLRG1^–, KLRG1⁺IL-17RB^– and KLRG1⁺IL-17RB⁺ subsets in total CD45⁺lineage^–CRTH2⁺CD127⁺ST2^– ILC2s in PBMC of asymptomatic asthmatic patients (marked as A-KLRG1^–, A-KLRG1⁺IL-17RB^– and A-KLRG1⁺IL-17RB⁺) and KLRG1⁺IL-17RB⁺ subsets in total ILC2s in PBMC of healthy donors (marked as H-KLRG1⁺IL-17RB⁺) were sorted (4.5 ~ 6.8 × 10⁵ for every subset, purity > 98%). K, L Total RNA was isolated, and qRT-PCR was performed. K Heat map of mRNA expression values of selected genes. L Bar plots of mRNA expression values (relative to GAPDH) for genes significantly changed. Data was shown as Means ± SD. n = 8 H and 15 A for (A–D). n = 11 (H) and 18 A for E–J. n = 6 (A-KLRG1^–, A-KLRG1⁺IL-17RB^– and A-KLRG1⁺IL-17RB⁺) and 3 (H-KLRG1⁺IL-17RB⁺) for (K, L). Statistical comparisons were performed using unpaired, two-sided Welch’s t test for (B–D), and unpaired one-way ANOVA with Dunnett’s test for (L). p values are shown on the graphs. Source data are provided as a Source Data file. Fig. 7E Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en.