Recovery of neonatal IFN-γ response coincides with down-regulation of IL-13Rα1 expression. Newborn BALB/c mice were given 3 × 10⁵ purified neonatal CD4⁺ DO11.10 T cells and injected i.p. with 60 μg/g Ig-OVA in saline at day 1, 2, 4, 6, or 10 after birth, and 2 wk later, the splenic cells (10⁷ cells/ml) were stimulated for 10 h with 10 μM OVA peptide. (A) Subsequently, Th1 cells were isolated, and RNA was extracted and used for analysis of IL-13Rα1 gene expression as well as GAPDH control by spot-blot technology, as described in Materials and methods. (B) The intensity of radioactive spots was analyzed on a Molecular Imager FX using Quantity One software and presented as a ratio of IL-13Rα1 to GAPDH after deduction of the background intensity obtained with pUC19 DNA negative control. The bars represent the mean ± SD of two experiments. (C and D) Newborn BALB/c mice were given 3 × 10⁴ neonatal CD4⁺ DO11.10 T cells and injected i.p. with 60 μg/g Ig-OVA in saline at day 1 or 6 after birth, and 2 wk later, the splenic cells (10⁶ cells/ml) were stimulated for 10 h with 10 μM OVA peptide. In C, the cells were stained with KJ1-26 and rabbit anti–IL-13Rα1 antiserum (1:100 dilution), and Th1 cells were identified by staining for intracellular IFN-γ with anti–IFN-γ antibody. Expression of surface IL-13Rα1 was determined by flow cytometry on cells gated for KJ1-26 expression and intracellular IFN-γ production. In D, Th1 cells were purified as in A, lysed using NP-40 detergent, and run on a 10% acrylamide gel. Transfer was made onto a nitrocellulose membrane, and IL-13Rα1 protein was detected using rabbit anti–IL-13Rα1 antibodies (1:1,000 dilution). For control purposes, we used lysates from CTLL-2 cells transfected with a plasmid coding for cell-surface IL-13Rα1 (reference 17) or wild-type untransfected CTLL-2 cells. Band intensity was analyzed using the Molecular Imager FX, and the relative expression of IL-13Rα1 represents a percent ratio with 1 d as 100%. Each bar represents the mean ± SD of duplicate samples. (E) IL-13Rα1 mRNA expression measured by spot intensity from the experiments in A and B is illustrated together with the IFN-γ production presented in Fig. 1 C to correlate IL-13Rα1 down-regulation together with an increase in secondary IFN-γ production.