Neonatal RTEs produce higher levels of IL-2 and Th1/Th2 cytokines than adult RTEs in response to PBαCD3 and αCD28 stimulation. (A) GFP^hi RTEs and GFP⁻ resident cells were sorted from purified neonatal and adult LN CD4⁺ cells, as described in “Preparation of cells from neonatal and adult RAG2p-GFP^+/− mice.” Neonatal (left) and adult (right) RTEs and resident cells were activated for 48 hours with 0.5 μg/well PBαCD3 and 0.5 μg/mL soluble αCD28 (lower concentrations of PBαCD3, 0.005 or 0.05 μg/well, elicited only low-level cytokine production). Cytokine production and the number of cytokine-secreting cells were then determined. Graphs depict pooled data from 8 to 11 ELISA experiments and 3 ELISPOT experiments and are shown as the mean ± SEM; n = 35 to 60 neonates and 4 to 6 adults per experiment. (B) The data from panel A were regraphed to show a direct comparison of neonatal and adult RTEs. (C) Neonatal and adult RTEs were stimulated with PBαCD3 and αCD28 for 68 hours. Cytokine production was determined by ELISA. Graphs depict pooled data from 4 experiments; n = 18 to 35 neonates and 2 to 17 adults per experiment. (D) Sorted RTEs and resident cells from neonates and adults were stimulated with PBαCD3 and soluble αCD28 as described in panel A for 24 and 48 hours. Supernatants were harvested at each time point and assayed for IL-2 by ELISA. Graphs depict pooled data from 4 or 5 experiments; n = 18 to 25 neonates and 2 adults per experiment. (E) The data from panel D were regraphed to show a comparison of neonatal and adult RTEs.