Antigens delivered to BDCA3⁺ DCs via CLEC9A are presented to CD4⁺ T cells. Freshly isolated BDCA3⁺ mDCs were loaded with KLH via a 3-step labeling procedure, consisting of mouse polyclonal anti–human CLEC9A antibodies, followed by biotinylated goat anti–mouse IgG and subsequently KLH coupled to antibiotin molecules. As negative controls, mouse IgG or nonbiotinylated goat anti–mouse IgG were used. BDCA3⁺ mDCs cultured in the continuous presence of 10 μg/mL KLH served as a positive control. A total of 1 × 10⁴ KLH-loaded BDCA3⁺ mDCs were cocultured with 1 × 10⁵ autologous purified CD4⁺ T lymphocytes in the presence of 2 μg/mL poly I:C and 4 μg/mL R848. (A) After 4 days, proliferation of CD4 T lymphocytes was analyzed by 3H thymidine incorporation. (B) IFN-γ production in the supernatant was measured by ELISA. The graphs show mean ± SEM T-cell proliferation or IFN-γ production relative to the isotype control (mouse IgG) of 3 experiments with different donors performed in duplicate. *P < .05; **P < .01; ***P < .001.