Chow diet promotes microbiota-independent epithelial adaptation and cytotoxic transcriptional programming of intestinal CD4 ⁺ T cells. (A–C) 16S rRNA sequencing of small intestine (SI) or cecum contents of 8-wk-old SPF mice fed AA or standard chow diet represented by detrended correspondence analysis (DCA; A), relative SI phyla abundance (B), and SI Chao1 alpha diversity with mean ± SD and unpaired t test, ****P < 0.0001 (C). Data is from four independent experiments using 11–15 mice per condition. (D) Flow cytometry from the IE of GF or Oligo-MM¹² mice fed AA or standard chow diet measuring frequency of the indicated cell subsets. Dashed lines show mean value from SPF Chow (red) or SPF AA (blue). Mean + SD from three to five independent experiments with 7–16 mice per condition. Two-way ANOVA P values beneath each plot, and Holm-Šidák multiple comparison test between diets within each colonization within each plot, *P < 0.05, **P < 0.01 ****P < 0.0001. (E–J) scRNAseq of 12,139 IE and LP CD4⁺ T cells from GF or Oligo-MM¹² mice fed AA, AA + OVA, or standard chow diet with two to four mice per condition. (E) UMAP visualization of sequenced cells positioned by gene expression similarity and colored by gene expression cluster. (F) Frequency of cells within each cluster from the IE (top) or LP (bottom). (G and H) Expression (Pearson residuals) of IE signature genes within the three IE mature clusters (G) or within all IE CD4⁺ T cells (H). For H, Wilcoxon rank sum test with Bonferroni correction for multiple comparison, P-adj < 1e−5 were considered statistically significant. Groups labeled with asterisk (*) are significantly higher than AA diet mice within the same colonization group. Groups labeled with a circle (∘) are significantly higher than GF mice from the same dietary group. (I) IE gene signature score grouped by condition. Each data point contributing to the violin plots represents a single sequenced cell. Wilcoxon rank sum test within each colonization group, *P-adj < 1e−5. (J) Three-way volcano plot showing differential gene expression between diets in all sequenced IE CD4⁺ T cells. Colored genes are differentially expressed (P-adj < 0.05 from FDR-corrected Kruskal–Wallis Test and log₂ fold change > 0.5), colored by the diet(s) in which they are upregulated. Select genes of interest are labeled on each plot.