CD4⁺ RTEs exhibit striking functional defects under Th0 conditions in vitro. CFSE-labeled CD4⁺ RTEs and MN T cells were stimulated under Th0 conditions for (A) 24 and 48 hours and (B-C) 3 days, stained for intracellular cytokines, and gated as CD4⁺. (A) Representative data for cells pooled from 3 mice. Numbers in the top right of the gate represent percentage of total IL-2⁺ cells, whereas numbers in the top left (for the 48-hour plots) represent the percentage of IL-2 producers among those cells that had divided at least once. MFI indicates mean fluorescence intensity. (B) Left panels are representative data; the numbers in quadrants represent the percentage of cytokine-positive cells. In right panels, 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. (C) Histograms (left panel) represent CFSE dilution by RTEs (shaded) and MN T cells (open), and numbers denote the percentage of cells that had undergone ≥ 3 divisions. Numbers in right panels represent the percentage of cytokine producers among those 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. Probability values were calculated with either a paired or unpaired 2-tailed Student t test: *P < .05, **P < .005.