CD4⁺ RTEs are biased toward the Th2 effector lineage in vitro. (A) CD4⁺ RTEs and MN T cells were differentiated under Th2 conditions for 5 days and then stained for IL-4. Numbers in quadrants of representative flow cytometry plots show the percentage of IL-4⁺ cells. In the right panel, compiled data are presented as mean ± SEM for 8 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 between populations (P < .0005). (B-D) Supernatants from Th0- and Th2-differentiated CD4⁺ RTEs and MN T cells were assayed by ELISA for (B) IL-4, (C) IL-5, and (D) IL-13. Error bars represent SD for triplicate wells. Data are representative of ≥ 2 independent experiments with cells pooled from 2-4 mice per experiment. **P < .005, ***P < .0005. (E) CFSE-labeled CD4⁺ RTEs and MN T cells were differentiated under Th2 conditions for 3 days and stained for IL-4. Numbers represent the percentage of IL-4 producers among cells that had undergone ≥ 4 divisions. Histograms represent CFSE dilution by RTEs (shaded) and MN T cells (open), and numbers denote the percentage of cells that had undergone ≥ 4 divisions. (F) Graph represents the percentage of IL-4–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. (G) Purified CD4⁺ RTEs and MN T cells were stimulated directly ex vivo for 5 hours with or without PMA and ionomycin (P/I). Cells were stained for intracellular IL-4 and IFN-γ and gated as CD4⁺CD44^lo. Numbers in quadrants represent the percentages of cytokine-positive cells.