Targeting CD40 results in superior MHC class II presentation than targeting DEC205. (A) NY-ESO–extended peptide sequence. The amino acid change from cysteine → alanine is indicated in bold. (B) DPB1*0401⁺ BDCA1⁺ DCs were incubated with indicated doses of irrelevant HIV reverse transcriptase peptide (HIV RTp), WT NY-ESO peptide (WT NYESOp), or C → A mutated NY-ESO peptide (mut NYESOp) for 1.5 hours, followed by washing and coculture with an NY-ESO peptide–specific CD4⁺ T-cell clone. Percentage of CD4⁺ T cells with intracellular IFNγ results are shown as 1 representative of 3 independent DC donors. (C-E) Antibody-NY-ESO peptide conjugates (0.1-10 μg/mL) were fed to BDCA1⁻ DCs for 1.5 hours, followed by washing and coculture with an NY-ESO–specific CD4⁺ T-cell clone at a ratio of DCs to T cells of 1:1, and subsequent staining for intracellular cytokines. The percentage of CD4⁺ T cells that are positive for (C) IFNγ, (D) IL-2, and (E) TNFα are depicted. One representative of 3 independent DC donors is shown. (F) As in panels C through E, 1 μg/mL antibody–NY-ESO conjugates were used with varying ratios of DCs to T cells. The percentage of CD4⁺ T cells that are positive for IFNγ is depicted. One representative of 3 independent DC donors is shown.