(A) Mice were immunized i.m. with graded doses of DNA, and 4 days later, 2 × 10⁶ CFSE-labeled CD8⁺ OT-I or 3 × 10⁶ CD4⁺ OT-II TCR transgenic T cells were injected i.v. At day 7, draining lymph node cells were harvested and cell division was evaluated by flow cytometry in CD4⁺ OT-II cells with Vα₂ T cell receptor (20.1) antibody and in CD8⁺ OT-I cells with Vβ_5.1/5.2⁺ T cell receptor (MR9-4) antibody. (A) Control mice received similar amounts of the scControl–OVA DNA vaccine. (B) As in A, but the experiments were performed with DEC^–/–, TAP^–/–, or WT B6 mice using 30 μg of each DNA vaccine. Representative results from 3 experiments are shown. (C) Longevity of in situ antigen presentation after vaccination with 30 μg DNA, then evaluation by antigen presentation to 2 × 10⁶ OT-I T cells injected at the indicated time points. (D) scDEC-OVA targets antigens to DCs after DNA vaccination. Mice were immunized with 30 μg of the indicated scFv DNA vaccines or backbone vector. 4 days later, CD11c⁺ and CD11c^– cells were isolated and cocultured with CFSE-labeled OT-I T cells at the indicated APC to T cell ratios to determine CFSE dilution in vitro at day 7.5 of the experiment. (E) CD11c-DTR mice were vaccinated with 30 μg scDEC-OVA or backbone vector and were depleted of CD11c⁺ DCs 4 days later by injection of 4 ng/g of DT (DT+); then CFSE-labeled OT-1 T cells were injected to monitor antigen presentation as in A. C–E are representative of 2 experiments.