Fig. 5: FFAR3 signaling expands ILC2 numbers and reprograms ILC2s to an anti-inflammatory effector state in vitro.

A Schematic of 6-day ILC2 culture with IL-33 (10 ng/mL), TSLP (10 ng/mL), and IL-2 (100 U/mL). AR420626 concentration = 10 µM. All data shown are from Day 6 unless indicated. B Total ILC2 numbers after 6 days of culture (n = 5, paired biological replicates; representative of 3 independent experiments) ** p = 0.0029. C IL-5 (** p = 0.0014) and IL-13 (** p = 0.0084) in culture supernatant (n = 5, paired biological replicates; representative of 3 independent experiments). D Representative flow cytometry plots of IL-5 and IL-13 by intracellular cytokine staining. E Quantification of IL-5 (** p = 0.0015) and IL-13 (** p = 0.0034) expression by intracellular staining as MFI (mean fluorescence intensity) (n = 4, paired biological replicates; representative of 1 experiment for IL-5 and 2 for IL-13). F Representative flow cytometry of ST2 expression on cultured ILC2s (FMO = fluorescence minus one control). G Percentage of ST2⁺ ILC2s (n = 5, paired biological replicates; representative of 1 experiment) **** p < 0.0001. H Quantification of ST2 expression on ST2⁺ ILC2s by MFI (n = 5, paired biological replicates; representative of 1 experiment) ** p = 0.0012. I IL-10 in supernatant (n = 5, paired biological replicates; 3 samples in Vehicle group below detection limit; representative of 3 independent experiments) ** p = 0.0023. J Representative flow cytometry plot of Ki-67⁺ ILC2s. K Percentage of Ki-67⁺ ILC2s (n = 5, paired biological replicates; representative of 1 experiment) * = 0.044. (L) Representative CFSE dilution plot to assess proliferation after 48 h of culture. M Proliferation and division index quantification between groups (n = 4, paired biological replicates; representative of 2 independent experiments). N Representative flow cytometry of early/late apoptosis by Annexin V and propidium iodide (PI) staining after 6 days. Annexin V⁻/PI⁻ = healthy, Annexin V⁺/PI⁻ = early apoptotic, Annexin V⁺/PI⁺ = late apoptotic/dead. Numbers in quadrants = percentages. O Percentage of early apoptotic (Annexin V⁺/PI⁻) (* p = 0.038) and P late apoptotic/dead (Annexin V⁺/PI⁺) (*** p = 0.0002) cells (n = 5 per strain; representative of 1 experiment). B, C, E, G–I, K, M, O, P bar height = mean, error bars = SD, significance assessed by two-tailed paired Student’s t-test. ns not significant.