IL-7Rα is down-regulated more rapidly on neonatal RTEs than on adult RTEs after exposure to IL-7 and is associated with higher levels of pSTAT5. (A) Purified CD4⁺ lymph node cells from RAG2p-GFP^+/− neonates and adults were either stained directly ex vivo with anti–IL-7Rα (left panel) or were cultured overnight in complete media before staining (right panel). IL-7Rα expression on RTEs was determined by gating on CD4⁺GFP^hi cells. (B) Purified CD4⁺ LN cells from neonatal and adult RAG2p-GFP^+/− mice were either stained directly ex vivo (0 minutes) or cultured with 10 ng/mL IL-7 for 30 minutes, 60 minutes, or 6 hours, and then stained with anti–IL-7Rα. IL-7Rα expression on RTEs was determined by gating on CD4⁺GFP^hi cells. IL-7Rα mean fluorescence intensity (MFI) for neonatal (in black) and adult (in gray) RTEs are shown in each histogram. Histograms represent data from 2 independent experiments; n = 2 adults and 15 neonates per experiment. In panels A and B, curves represent 800 to 2000 gated CD4⁺GFP^hi RTEs for adults and 7000 to 13 000 gated CD4⁺GFP^hi RTEs for neonates. (C) Sorted neonatal and adult RTEs were cultured overnight in complete media. The cells were then stimulated with IL-7 (10 ng/mL) for 5 to 60 minutes, fixed and permeabilized, and stained intracellularly for pSTAT5. The specificity of the staining was confirmed by competition with an excess of unlabeled pSTAT5 antibody (data not shown). Points represent pSTAT5 MFI data from 2 independent experiments; n = 40 to 49 neonates and 4 adults per experiment. ΔMFI was calculated by subtracting the MFI of pSTAT5 in the absence of IL-7 from the pSTAT5 MFI at each time point.