MHC II expression on IECs is enriched in the distal small intestine, constituting a favorable niche for differentiation of DP IELs but not other IEL subtypes. (A) Significantly enriched GO terms of DEGs among IECs sorted from duodenum (d), jejunum (j), and ileum (i). DEG analysis was performed using the PANTHER classification system. (B–D) Heatmap of MHC II–related genes (B), cytokines and DEGs annotated in oxidation–reduction process (GO:0055114; C), and DEGs annotated in response to IFN-γ (GO:0034341; D). The same numbers below d, j, and i correspond to the replicates from the same mouse. (E) Expression of Ifngr1 and Ifngr2 from RNA-seq analysis of IECs sorted from d, j, and i (mean ± SEM). (F) Gating scheme of DP IELs. (G) MHC II expression (mean fluorescence intensity [MFI]; mean ± SEM) on IECs in d, j, and i of MHC II^fl/fl and MHC II^△IEC mice (n = 8). The data shown are pooled from three independent experiments. (H) Frequencies of IEL subsets in MHC II^fl/fl and MHC II^△IEC mice (n = 5; mean ± SEM). The data shown are pooled from two independent experiments. IEL populations were gated as TCRβ⁺CD4⁻CD8α⁺CD8β⁺ (TCRβCD8αβ), TCRβ⁺CD4⁺CD8α⁻ (TCRβCD4⁺CD8α⁻; SP IEL), TCRβ⁺CD4⁻CD8α⁺CD8β⁻ (TCRβCD8αα), or TCRγδ⁺CD8α⁺ (TCRγδ). *, P < 0.05; ***, P < 0.001, ns, not significant. One-way ANOVA with Tukey’s post hoc test (E) or unpaired Student’s t test (G and H). FDR, false discovery rate; FSC-A, forward scatter area; FSC-H, forward scatter height; SSC-A, side scatter area; SSC-H, side scatter height.