LN-TRCs trans-present IL-2 to CD4 ⁺ T cells through CD25. (A) STAT5 phosphorylation in naive CD4⁺ T cells cocultured with WT or CD25-deficient TRCs was assessed by Western blot analysis after 15-min treatment with rIL-2 (20 ng/ml). Total STAT5 and β-actin are shown as controls. (B) Representative flow cytometric histograms of STAT5 phosphorylation (P-Stat5) in naive CD4⁺ T cells cocultured with WT or CD25-deficient TRCs after treatment with rIL-2 (20 ng/ml) for the indicated time points. Mean fluorescence intensity is shown as a number. (C) STAT5 phosphorylation in naive CD4⁺ T cells cocultured with WT or CD25-deficient TRCs detected by immunocytochemical analysis after 30-min treatment with rIL-2 (20 ng/ml). Scale bar = 10 μm. Data are representative of three independent experiments.