Supplementary Figure 4: TGF-β regulates the transition of c-Kit⁻ ILC2s into RORγt⁺ ILC2s.

(a) Principle component analysis of global gene expression of c-Kit⁺ ILC2 and c-Kit⁻ ILC2 populations isolated from 3 healthy donors, stimulated with indicated cytokines. c-Kit⁺ ILC2s (grey) and c-Kit⁻ ILC2s (dotted circle) incubated under different cytokine combination (dot shown in different colors as indicated) are displayed in PC1 vs PC2 (top) and in PC1 vs PC3 (bottom) are shown. (b) Venn diagrams comparing differently expressed genes of c-Kit⁺ ILC2s and c-Kit⁻ ILC2s stimulated with indicated cytokines from global gene expression data in (a). Red circle represents gene-expression differences of c-Kit⁺ ILC2s stimulated with IL-1β vs IL-1β IL-23. The blue circle represents differences of c-Kit⁻ ILC2 stimulated with IL-1β vs IL-1β plus IL-23. Differently expressed genes of c-Kit⁻ ILC2 stimulated with IL-1β vs IL-23 or IL-1β, IL-23 and TGF-β are shown in the green circle. Numbers in the Venn diagram represent numbers of differently expressed genes. (c) Expression of GATA3, IL17RB, IL1RL1 and CRLF2 determined by RT-qPCR in c-Kit⁺ ILC2s (n = 8) and c-Kit− ILC2s (n = 6, all independent donors) stimulated with IL-1β or IL-1β TGF-β for 7 days. ns, not significant. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P< 0.0001. 1-way ANOVA followed by Tukey’s test. Error bars represent SEM. (d) Flow cytometry analysis of RORγt and GATA-3 expression in c-Kit⁺ ILC2s, c-Kit⁻ ILC2s, CD56^bright NK and CRTH2⁻c-Kit⁺ ILCs stimulated with IL-1β or IL-1β TGF-β for 7 days. Data are representative of 3 independent experiments with individual donors. Numbers adjacent to outlined areas indicate percent of each cell.