Donor cells persist in the neonate for at least 6 days after injection. (A) Neonates (top panel) and adults (bottom panel) were injected with allogeneic GFP⁺ cells, as described in Figure 2. Two to 9 days later, spleen cells were harvested for DNA isolation. DNA (0.1 μg) from individual animals was used in a quantitative GFP-specific real-time PCR assay to test for the presence of donor GFP⁺ cells, as described in Figure 1. The limit of sensitivity (dotted line) for this assay was 0.001%. Data are pooled from 2 to 3 independent experiments. (B) Cytokine-secreting memory CD4⁺ spleen cells were sorted, and the frequency of N-region addition in the CDR3 region of the TCR was determined as described in “Determination of N-region addition in the CDR3 region of the TCR.” Data represent 19 clones from IFNγ-secreting cells and 24 clones from IL-4–secreting cells.