Panel A shows a volcano plot with transcriptomic comparison showing significant gene expression changes in Blimp-1-deficient intestinal ILC2s, with Irf4 notably reduced. This suggests Blimp-1 positively regulates Irf4 expression at steady state. Panel B shows Gene Ontology (GO) pathway analysis reveals altered immune and inflammatory pathways in Blimp-1–deficient ILC2s. Affected processes include cytokine signaling and leukocyte differentiation. Panel C shows heatmap analysis highlights distinct transcriptional profiles between control and cKO ILC2s. Several immune-related genes are downregulated in the absence of Blimp-1. Panel D shows quantitative gene expression confirms reduced Irf4 levels in Blimp-1-deficient ILC2s, while other transcription factors show minor or no significant change. Panel E shows flow cytometry histograms demonstrating decreased IRF4 protein levels in intestinal ILC2s lacking Blimp-1. Panel F shows protein quantification, which shows selective reduction of IRF4, whereas GATA3, cMaf, and Bcl6 remain largely unchanged, indicating specific regulation of IRF4. Panel G shows Irf4 expression increases with IL-33–containing conditions, suggesting cytokine-dependent regulation. Panel H shows flow cytometric analysis of IRF4 expression is reduced, indicating that IL-33 and TSLP signaling contribute to maintaining IRF4 levels.
IRF4 expression is dysregulated in Blimp-1–deficient ILC2s. (A) Volcano plot showing differentially regulated genes in sort-purified small intestinal ILC2s from naive Nmur1CrePrdm1fl/fl (cKO) vs. Prdm1fl/fl (Ctrl) mice. The genes Prdm1 and Irf4 are indicated in the plot. (B) GO pathway analysis of differentially regulated genes from bulk RNA-seq of small intestinal ILC2s comparing naive cKO and Ctrl mice. (C) Heatmap of the top differentially regulated genes from naive cKO and Ctrl mice. Genes of interest are indicated in red (A–C, n = 4 mice per group). (D) Relative expression of genes of interest in small intestinal ILC2s from naive cKO and Ctrl mice. (E) Histogram of flow cytometric analysis of IRF4 in intestinal ILC2s from naive cKO and Ctrl mice. (F) Quantification of E and of the transcription factors GATA3, cMaf and Bcl6 in intestinal ILC2s from naive cKO and Ctrl mice. (G) Relative expression of Irf4 in sort-purified small intestinal (Si) ILC2s after in vitro stimulation with IL-7 alone, or with IL-7 or TSLP, or in combination with IL-33 and IL-7, IL-33, or in combination with IL-25. (H) Flow cytometric analysis of IRF4-expressing small intestinal (Si) ILC2s from WT, Il33−/−, and Crlf2−/− mice. (D–H) Data are representative of two independent experiments; n = 3–5 mice per group. Mean ± SD; Student’s t test (D and F) or one-way ANOVA (G and H); ns, nonsignificant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.