Chow diet promotes microbiota-independent epithelial adaptation and cytotoxic transcriptional programming of intestinal CD4 ⁺ T cells. Additional data supporting Figs. 2 and 3. (A–E and G–J) CD4⁺ T cells were sorted from the IE or LP of 8-wk-old GF or Oligo-MM¹² mice weaned onto AA, AA + OVA, or standard chow diet and scRNAseq was performed using the 10X Genomics platform, pooling two to four mice per diet/colonization group. The data shown is for all sequenced CD4⁺ T cells (A–E) or subclustered Tregs (G–J). (A) Number of cells sequenced per indicated sample, colored by sequencing batch (left), and violin plots showing number of detected RNA molecules, number of sequenced genes, or percentage of mitochondrial DNA per cell per sequencing batch (right). (B–G) Top five differentially expressed genes (ranked by fold change) in each UMAP gene expression cluster from total CD4⁺ T cells (B) or Tregs (G). Wilcoxon rank sum test (P < 0.01). (C–J) Frequency of mature clusters (IE1, IE2, IE3, and Th1 combined; C) or Il10 high Tregs (J). (D–I) IE signature score of total CD4⁺ T cell (E) or Treg (I) gene expression clusters. (E) Frequency of IE2 or IE3 out of total IE CD4⁺ T cells. (F) Flow cytometry from the IE or LP of 8-wk-old GF or Oligo-MM¹² mice weaned onto AA or standard chow diet measuring frequency or absolute count of the indicated cell subsets. Dashed lines show mean value from SPF Chow (red) or SPF AA (blue). Bar plots show mean + SD representative of two to three independent experiments using three to nine mice per group. (H) Frequency of IE subclusters in IE or LP. (K) Flow cytometry from the IE of 8-wk-old SPF mice weaned onto standard chow diet measuring frequency of Granzyme B out of the indicated cell subsets. Mean + SEM representative of three independent experiments using eight mice per group. (C, E, F, J, and K) Two-way ANOVA with P values beneath each plot and Holm-Šidák multiple comparison test between diets within each colonization within each plot (C, F, and J) or one-way ANOVA with Tukey’s multiple comparison test (E–K), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.