TLR7a-conjugate vaccination engages multiple DC subsets to cross-prime CD8⁺ T cells. WT mice were subcutaneously immunized with 50 μg of the OVA-TLR7a conjugate, and the dLNs were harvested 18 or 36 hours after immunization. DCs were isolated as described above, and the indicated DC subsets were sorted using the MoFlow cell sorter on the basis of surface marker expression as outlined in (A). Sorted cells were then cocultured either at the indicated titration of cell numbers (B,C) with 1.0 × 10⁴ purified and CFSE-labeled OTI T cells. After 3 days, OTI T cells were harvested from the culture and the dilution of CFSE was assessed by flow cytometry, gating on CD8+, CD3+, and B220- cells. The data shown are representative of 2 independent experiments.