a, A positive control showing immunofluorescence staining of IL-2 in situ, and the comparison of IL-2 detection sensitivity between flow cytometry and histo-cytometry. OVA_323-339 peptide-loaded splenic dendritic cells (DC, cyan) were stained with CMTMR (5-(and-6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine) and injected into the footpad, and Rag2^−/− TCR transgenic OT-II CD4⁺ T cells (green) stained with CMFDA (5-chloromethylfluorescein diacetate) were transferred 18 h post-transfer of dendritic cells. For each recipient, 16 h after T cell transfer, one draining popliteal lymph node was isolated for IL-2 (red) immunofluorescence staining, while the contralateral lymph node was isolated and dissociated into single cells for intracellular IL-2 staining ex vivo. Each dot in the middle panel of the upper row indicates a lymph node (n = 3, 3 lymph nodes total from 3 mice for each analysis). *P < 0.05 as calculated by two-tailed Student’s t-test. b, Immunofluorescence staining of IL-2 and pSTAT5 in lymph nodes from FOXP3–eGFP reporter mice. Upper row indicates an IL-2⁺ cell associated with clustered T_reg cells among a group of pSTAT5⁺ T_reg cells. Lower row indicates an unassociated IL-2⁺ cell surrounded by a group of pSTAT5⁺ T_reg cells. c, Quantification of IL-2⁺ cells associated or unassociated with T_reg clusters. Data are pooled from seven lymph nodes.