Cross-presentation of extracellular Ags to CD8⁺ T cells. Human CD1c⁺ mDCs, CD16⁺ mDCs, BDCA3⁺ mDCs, and pDCs were incubated with 10μM irrelevant peptide (tyrosinase_369-376), 10μM gp100 short peptide (gp100_280-288), 25μM gp100 long peptide (gp100_272-300) (A-B), or 50 μg/mL necrotic BLM cells expressing gp100 or tyrosinase (C-D). 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 (mDCs) or 4 μg/mL R848 only (pDC). Ag-specific T-cell activation was assessed by analysis of CD69 expression (A-C) and IFN-γ production (B-D). IFNγ production is shown relative to irrelevant peptide. For panels A and B the graphs show the mean ± SEM of CD1c⁺ mDCs (n = 5), CD16⁺ mDCs (n = 4), BDCA3⁺ mDCs (n = 6), pDCs (n = 12), and for panels C and D the graphs show CD1c⁺ mDCs (n = 2), CD16⁺ mDCs (n = 2), BDCA3⁺ mDCs (n = 2), and pDCs (n = 9) experiments with different donors. (A-B) Significance was determined by ANOVA and Newman-Keuls testing (*P < .05; **P < .01; ***P < .001) compared with irrelevant peptide. (D) Significance was determined by a paired Student t test.