SFB induce a switch of cognate CD4 T cells to granzyme - expressing IEL dependent on epithelial MHCII. (A–G) CD90.1/2 CD4 T cells carrying transgenically expressed TCR, specific for SFB antigen (SFBtg) were adoptively transferred to CD90.2/2 mice. (A) Schematic of the experimental setup. (B–D) scRNA-seq of TCRβ⁺ SI-LP and SI-IEL cells. (B) UMAP of annotated cell types. (C) UMAP showing the origin of cells from either donor (SFBtg) or host mice (polyclonal). (D) Heatmap of differentially expressed genes that define cell types among host cells, shown in all host cell types and SFBtg T cells from IEL cluster. Data are shown as z-score of expression. (E–G) FACS analysis of SFBtg T cell mLN, PP, SI-LP, and SI-IEL. (E) Representative FACS plots of CD8α and Gzmb and gating for Gzmb⁺ cells. (F) Plot showing the frequency of Gzmb⁺ cells among SFBtg T cells in mLN, PP, SI-LP, and SI-IEL. Two independent experiments, n = 3–4. (G) Plot showing the MFI of CD8α cells in SFBtg T cells in mLN, PP, SI-LP, and SI-IEL. Three independent experiments, n = 3–4. (H and I) BM from SFBtg (CD90.1/2) and WT (CD90.2/2) mice were mixed in 5:95 ratio and i.v. injected into lethally irradiated MHCII^ΔIEC mice and their Cre-negative littermates (MHCII-fl), which were both i.p. injected with 1 mg of tamoxifen in 50 μl of sunflower oil at 21 and 22 days of age. Mice were analyzed 6 wk after BM transplantation. (H) Schematics of the experimental setup. (I) FACS analysis of counts and frequencies of SFBtg T cells in SI-LP and SI-IEL. Plots on the left show representative gating. Plots on the right show overview of the results and statistical analysis. Three independent experiments, n = 7. Data in I were tested by Student’s t test. **, P < 0.001; ***, P < 0.0001; P values >0.05 are shown. Numbers beside gates represent frequencies from parent population. Horizontal lines show mean ± SEM.