CD4⁺ RTEs are defective in Th1 lineage commitment in vitro. CFSE-labeled CD4⁺ RTEs and MN T cells were differentiated under Th1 conditions for (A) 5 days and (B) 3 days, stained for intracellular IFN-γ and IL-17, and gated as CD4⁺. (A) Left panels are representative flow cytometry plots. Numbers in quadrants represent the percentage of cells that were cytokine positive. MFI values are for IFN-γ⁺ 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 difference (P < .05) between populations. (B) 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. Graph represents the percentage of cytokine-producing cells in each division (undiv indicates undivided). Data are representative of 2 independent experiments with cells pooled from 3-4 mice per experiment and 30 ng/mL anti-CD3.