CD4⁺ RTEs are defective in their commitment to the iTreg and Th17 lineages. (A) CD4⁺ RTEs and MN T cells were differentiated under iTreg conditions for 3 days and then stained for intracellular Foxp3. Left panels are representative plots, and right panel shows compiled data presented as mean ± SEM for 3 independent experiments with cells pooled from 2-3 mice per experiment. CFSE-labeled CD4⁺ RTEs and MN T cells were differentiated under Th17 conditions for (B) 5 days or (C) 3 days, stained for intracellular IL-17A and IFN-γ, and gated as CD4⁺. (B) Left panels are representative flow cytometry plots. Numbers in quadrants represent the percentage of cells that were cytokine-positive. MFI values are for IL-17A⁺ populations and are significantly different (*P < .05). In the right panel, compiled data are presented as mean ± SEM for 5 independent experiments with cells pooled from 3-4 mice per experiment and a range of anti-CD3 concentrations from 30-500 ng/mL. **Significant differences between populations (P < .005). (C) Numbers in left panels represent the percentage of cytokine producers among cells that had undergone ≥ 3 divisions. Histograms in right panel represent CFSE dilution by RTEs (shaded) and MN T cells (open), and numbers denote the percentage of cells that had undergone ≥ 3 divisions. Data are representative of 2 independent experiments with cells pooled from 3-4 mice per experiment and 30 ng/mL anti-CD3.