Neonates acquire the ability to develop secondary Th1 responses when the primary encounter with Ag occurs on or beyond day 6 after birth. Newborn BALB/c mice were given 3 × 10⁴ neonatal or adult DO11.10 CD4 T cells and injected with 100 μg Ig-OVA in saline, and 2 wk later, the splenic cells (10⁶ cells per well) were stimulated with 10 μM OVA peptide in vitro. (A) Production of IFN-γ was measured by ELISA. (B) Apoptosis of IFN-γ–producing KJ1-26⁺ Th1 cells was evaluated by Annexin V staining. In B, 10 μg/ml BFA was added after 6 h of stimulation with OVA peptide, and the culture was continued for another 6 h to facilitate intracellular accumulation of IFN-γ. The cells were then labeled with KJ1-26 and Annexin V, and stained for intracellular IFN-γ. Histogram shows gating on KJ1-26⁺/ IFN-γ⁺ cells. (C and D) Newborn BALB/c mice were given 3 × 10⁴ neonatal DO11.10 T cells within 24 h after birth and were injected with 60 μg/g Ig-OVA (to adjust for growth) on day 1, 2, 4, 6, 8, or 10 after T cell transfer. 2 mo later, the mice were challenged with 125 μg OVA peptide in CFA. 10 d later, the splenic (10⁶ cells/well) and lymph node (0.5 × 10⁶ cells/well) cells were stimulated with 10 μM OVA peptide for 24 h, and production of IFN-γ was determined by ELISPOT. Each bar represents the mean ± SD of triplicate wells. The data are representative of two experiments.