Tumor antigens delivered via CLEC9A to BDCA3⁺ mDCs are cross-presented to CD8⁺ T cells. Gp100 long peptide was targeted to CLEC9A via PLGA nanoparticles encapsulating gp100:272-300 and Atto488, coated with rabbit polyclonal αCLEC9A antibodies or rabbit IgG as control. (A-C) Uptake of nanoparticles by BDCA3⁺ mDCs after overnight incubation analyzed by flow cytometry. (A) Example of nanoparticle uptake by control BDCA3⁺ mDCs (no particles added; left), mDCs incubated with isotype antibody-coated particles (middle), or mDCs incubated with αCLEC9A-coated nanoparticles (right). Numbers indicate mean fluorescence intensity. (B) Percentage of Atto488-positive BDCA3⁺ mDCs. The graph shows mean ± SEM of 4 experiments with different donors performed in duplicate. (C) Relative fluorescence intensity of BDCA3⁺ mDCs. Data are presented as percentage of BDCA3⁺ mDCs incubated with RbIgG-coated nanoparticles. The graph shows mean ± SEM of 4 experiments performed in duplicate. (D-E) Freshly isolated BDCA3⁺ mDCs were incubated for 1-2 hours with 10μM irrelevant peptide (tyrosinase:369-376), 10μM gp100 short peptide (gp100:280-288), 10μM gp100 long peptide (gp100:272-300), or 50 μg/mL αCLEC9A- or RbIgG-coated PLGA nanoparticles encapsulating gp100:272-300 long peptide (corresponds to 0.5μM gp100:272-300) and Atto488. Next, DCs were cocultured overnight with allogeneic CD8⁺ T cells expressing gp100:280-288-specific TCR in the presence of 4 μg/mL R848 and 2 μg/mL poly I:C. T-cell activation was assessed by analysis of CD69 expression (D) and IFN-γ production (D). (D) Mean ± SD of percentage of CD8⁺ T cells expressing CD69 and is a representative example of 3 experiments with different donors performed in duplicate. (E) Mean ± SD IFN-γ production and is a representative example of 3 experiments with different donors performed in duplicate. **P < .01; ***P < .001; NS, not significant.