TLR ligands enhance cross-presentation of extracellular Ags. Human CD1c⁺ mDCs, CD16⁺ mDCs, BDCA3⁺ mDCs, and pDCs were incubated with 0.01 to 10μM gp100 short peptide (gp100_280-288; n and ▾), or 1 to 25μM gp100 long peptide (gp100_272-300; ▴ and ♦). Next, DCs were cocultured overnight with allogeneic CD8⁺ T cells expressing gp100_280-288–specific TCR in the absence (n and ▴) or presence (▾ and ♦) 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) and IFN-γ production (B). IFNγ production is shown relative to irrelevant peptide. The graphs show mean ± SEM CD69 expression or relative IFNγ production of 2 independent experiments performed in duplicate with different donors.