Colocalization of IL-2 and CD4 ⁺ T cells on TRCs and complex of IL-2 and CD25. Purified, naive CD4 T cells (1 × 10⁶) from dsRed mice were cocultured with WT or CD25-deficient TRCs (1 × 10⁵). rIL-2 (20 ng/ml) was treated to each well. (A) After 1-h incubation and light wash, images were visualized by confocal microscopy. Arrowheads indicate colocalization of rIL-2 and CD4⁺ T cell on TRCs. Immunofluorescence staining of nuclear DAPI (blue), podoplanin (green) for TRCs, RFP-expressing CD4⁺ T cells isolated from dsRed mice (red), and rIL-2 (yellow). Scale bar = 50 μm. (B) Quantification of rIL-2 and colocalized CD4⁺ T cells are depicted. **, P < 0.01 compared with WT. Error bars indicate the mean ± SEM (n = 4 per each group). (C) Purified, naive CD4 T cells (1 × 10⁵) were cocultured with WT or CD25-deficient TRCs (2 × 10⁴). rIL-2 (20 ng/ml) was added to each well. After 1-h incubation, IL-2–CD25 complexes on TRC were visualized by PLA. Nuclear DAPI staining (blue); CD25-IL-2 complex (red). Arrows indicate CD4⁺ T cells. Scale bar = 50 μm. (D) TRCs form immune synapses with CD4⁺ T cells through their CD25 complexed with IL-2. Data are representative of three independent experiments.