Figure S4.

Intestinal CD4⁺ T cell responses during OVA feeding, tolerance, or allergy. Additional data supporting Figs. 6 and 7. iSell^Tomato mice were analyzed on day 26 after treatment with tamoxifen to permanently label naïve T cells and then exposure to OVA in the context of feeding, tolerance, or allergy. (A) Representative H&E histology images with 200 μm scale bar (left) and pathology scores based on one image per tissue per mouse, where 40 is the maximum score (right). Mean + SD representative of two independent experiments using four to five mice per group. (B–D) Flow cytometry measuring frequency of Tomato⁺ CD4⁺ T cells in the large intestine (B) or of the indicated CD4⁺ T cell subsets out of Tomato⁺ or Tomato⁻ CD4⁺ T cells in the IE (C) or LP (D). Data are representative of two independent experiments with five to eight mice per group. (E–I) scRNAseq of 11,217 Tomato⁺ and Tomato⁻ CD4⁺ T cells from the IE and LP using four to five mice per condition pooled across two independent experiments and sequencing runs. (E) Captured cells per sample in 10X sequencing experiment with Tomato⁺ or Tomato^– assignments. (F) Violin plots showing number of detected RNA molecules, number of sequenced genes, or percent mitochondrial DNA per cell per sequencing run. (G) Top five differentially expressed genes (ranked by fold change) in each UMAP gene expression cluster from total CD4⁺ T cells. Wilcoxon rank sum test (P < 0.01). (H) UMAP visualization of sequenced cells positioned by gene expression similarity and colored by tissue (top left) or treatment group (top right) or Tomato assignment (bottom left). (A–D) One-way ANOVA with Tukey’s multiple comparison test, *P < 0.05.