(A) Identification of Foxp3⁺ Tregs among LN and SI IEL CD4⁺ T cells of Foxp3-GFP reporter mice. Foxp3⁺ Treg frequencies were assessed in 2 independent experiments with 5 Foxp3-GFP reporter mice. (B) Frequencies of CD25⁺Foxp3⁺ Tregs among CD4⁺ T cells in the LN, spleen, and IELs of Foxp3-GFP reporter mice. Contour plots (left) are representative, and the graph (right) provides a summary of 2 independent experiments with 5 Foxp3-GFP reporter mice. (C) Frequencies of CD25^– cells among Foxp3⁺CD4⁺ T cells in the LN and IELs of Foxp3-GFP reporter mice. The graph provides a summary of 2 independent experiments with 5 Foxp3-GFP reporter mice. (D) EpCAM versus CD45 expression in SI IELs upon Percoll enrichment (left) or EpCAM⁺ cell depletion (right). The results are representative of 9 independent experiments with a total of 13 WT mice. (E) Frequencies of CD45⁺ SI IELs after Percoll enrichment or EpCAM⁺ cell depletion. The graph shows a summary of 9 independent experiments with a total of 11 WT mice. (F) γδ and αβ T cell profile of CD45⁺TCRβ⁺ IELs that were isolated by either Percoll enrichment or EpCAM⁺ cell depletion (left). Graph shows the frequency and number of recovered γδ T cells and αβ T cells using the indicated methods (middle and right). The results show a summary of 2 independent experiments with a total of 4 WT mice. (G) CD4 and CD8 profiles of CD45⁺TCRβ⁺ IELs that were isolated by either Percoll enrichment or EpCAM⁺ depletion (left). Graphs show the frequency and number of recovered CD4⁺ and CD8⁺ IEL αβ T cells using the indicated methods (middle and right). The results show a summary of 9 independent experiments with a total of 11 WT mice. The data are represented as the mean ± SEM. P values were determined by paired Student’s t test (A, C, F, and G) or 1-way ANOVA with Tukey’s multiple comparison test (B and E). *P < 0.05, **P < 0.01, ***P < 0.001.