Fig 2: Single-cell RNA sequencing analysis of PDOs from 4 non-EoE patients. PDOs from 4 non-EoE patients were treated with IL13 (10 ng/mL) or vehicle (phosphate-buffered saline) from day 7 to day 11, and harvested and dissociated for single-cell RNA sequencing analysis on day 11. (A) Seurat’s Uniform Manifold Approximation and Projection (UMAP) visualization of 4 classified cell groups, basal-1, basal-2, suprabasal, and superficial, within PDOs from non-EoE controls grown with or without IL13 (10 ng/mL). (B) Average normalized gene expression values of MKI67, CD73, basal markers (TP63, NGFR), and differentiated markers (IVL, KRT13, FLG) in each cell cluster. Circle size reflects the percentage of cells with a nonzero expression level for the indicated genes. Color intensity reflects the average expression level across all cells within each cluster. (C) Expression z-scores for the top 8 up-regulated genes in each cluster. (D) The proportions of each cell cluster were compared in PDOs (n = 4) with IL13 or vehicle. (E–G) The normalized expression of (E) IVL and FLG, (F) SOX2, and (G) CD73 in nontreated (NT) PDOs was compared with that in PDOs treated with IL13 for each cluster. ****P < .0001, Wilcoxon signed-rank test. (H) GO analysis of differentially expressed gene profiles of CD73+CD104+ cells, compared with CD73-CD104+ cells in NT PDOs.

Fig 3: Omeprazole treatment prevents Th2-mediated depletion of CD73+CD104+population. (A) EPC2-hTERT organoids were treated with omeprazole (50 µmol/L), IL4 (10 ng/mL), or IL13 (10 ng/mL). The frequencies of CD73+CD104+ cells in organoids are shown as means ± SD (n = 3). (B and C) The treated EPC2-hTERT organoids were harvested on day 11 and dissociated into single cells, followed by seeding in Matrigel to assess organoid formation capacity. (B) OFR was evaluated manually on day 11. (C) Representative phase-contrast images of organoids from corresponding groups. The data are shown as means ± SD (n = 6). Analysis of variance was performed with multiple comparisons. ****P < .0001. DMSO, dimethyl sulfoxide.

Fig 4: The therapeutic potential of STAT6 inhibition in EoE. (A) IHC staining for phosphorylated STAT6 was performed, with representative images shown. Scale bars: 100 µm. (B) The positive rate of phosphorylated STAT6 in esophageal epithelium are compared. The data are shown as means ± SD (n = 5). (C) Relative expression of CD73 gene in EPC2-hTERT organoids treated with STAT6 inhibitor (AS151749, 400 nmol/L), IL4 (10 ng/mL), or IL13 (10 ng/mL). (D) The frequencies of CD73+CD104+ cells in organoids treated with AS151749 or dimethyl sulfoxide (DMSO) under the presence of Th2 cytokines. STAT6 silencing efficiency was evaluated by (E) qRT-PCR and (F) Western blot. (G) CD73 gene expression in EPC2-hTERT organoids treated with STAT6 siRNA or nonsilencing (scramble) siRNA with or without IL13. The data are shown as means ± SD (n = 3). Analysis of variance was performed with multiple comparisons. **P < .01, ****P < .0001.

Fig 5: Th2 cytokines shift the basaloid population from CD73+to CD73-in human esophageal 3D organoids. PDOs from non-EoE controls (n = 6) or patients with active EoE (n = 6), or immortalized esophageal epithelial (EPC2-hTERT) organoids were treated with IL4 (10 ng/mL) or IL13 (10 ng/mL) from day 7 to day 11. Nontreated (NT) organoids were used as control. (A) Representative flow cytometry plots showing the percentage of CD73+CD104+ cells in PDOs from non-EoE controls or patients with active EoE, or EPC2-hTERT organoids, treated with IL4 or IL13. (B) The percentage of CD73+CD104+ cells in each PDO treated with IL4 or IL13, paired with NT control. The data are shown as means ± SD. ***P < .001, ****P < .0001, 2-tailed Student t test. (C) The representative images of H&E staining, and IHC for CD73 or CD104 in EPC2-hTERT organoids treated with IL4 or IL13. Scale bars: 100 µm. (D) CD73, CD104, SOX2, and IVL gene expression in EPC2-hTERT organoids treated with IL4 or IL13 were validated by qRT-PCR. The relative expression over NT is represented. The data are shown as means ± SD (n = 3). *P < .05, **P < .01, and ****P < .0001, analysis of variance.