Exogenous IL-12 inhibits developmental IL-13Rα1 up-regulation and restores secondary Th1 IFN-γ response in vivo. Newborn BALB/c mice were given 3 × 10⁴ neonatal DO11.10 T cells within 24 h after birth. 1 d later, the recipient mice were given 100 μg Ig-OVA and 50 ng rIL-12. The mice were given additional IL-12 on days 2 and 3 after T cell transfer. 7 wk later, the mice were challenged with 125 μg OVA peptide in CFA, and 10 d after the challenge, the splenic cells (10⁶ cells/well) were stimulated for 24 h with 10 μM OVA peptide. (A and B) Subsequently, IFN-γ was measured by ELISPOT (A) and apoptosis was evaluated using Annexin V staining of KJ1-26⁺/IFN-γ⁺ splenic cells (B). For Annexin V staining, 10 μg/ml BFA was added during the last 8 h of peptide stimulation. Each bar in A represents the mean ± SD of triplicate wells. (C and D) Mice that received neonatal DO11.10 T cells were given Ig-OVA and IL-12 as in A, and 2 wk later, Th1 cells were isolated and IL-13Rα1 expression was assessed by spot blot (C) and real-time PCR (D). The real-time PCR used 200 ng RNA and the Absolute QRT-PCR SYBR kit to determine IL-13Rα1 mRNA. For the spot blot in C, each bar represents the mean ± SD of duplicate spots. For the real-time PCR, the bars represent the comparative threshold cycle (C_T). The value of the sample from mice not receiving IL-12 was set as 1. (E and F) Newborn BALB/c mice given 3 × 10⁴ neonatal CD4⁺ DO11.10 T cells were exposed to Ig-OVA in the presence of IL-12 as in A and B, and 2 wk later, the splenic cells (10⁶ cells/ml) were stimulated for 10 h with 10 μM OVA peptide. The cells were then 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.