Efficient cross-presentation of melanoma particulate antigens requires NOX2 activity. (A) Control HLA-A2–positive DCs (gray) and DPI-treated DCs (white) were pulsed with either MelanA/MART-1 long peptides coated to latex beads (top left panel) or MelanA/MART-1 short peptides (bottom panel). DCs were pretreated with 10 μM DPI 30 minutes before the pulse and washed before the coculture with the CTL clone. The percentage of phagocytosis of FITC/FluoProbes 647 latex beads by control and DPI-treated DCs was assessed by FACS (top right panel). Cross-presentation was evaluated by measuring the production of IFN-γ by the HLA-A2–restricted CTL clone LT12 after DC-CTL coculture. (B) HLA-A2–negative Mel888 melanoma cells were treated overnight with oxaliplatin to induce apoptosis. Apoptosis and necrosis were quantified using annexin V–FITC and propidum iodide, respectively, by FACS. (C) Expression of MelanA/MART-1 in whole-cell lysates from apoptotic and nontreated Mel888 cells, assessed by Western blot. (D) Control HLA-A2–positive DCs (black) and DPI-treated DCs (white) were pulsed with either MelanA/MART-1 short peptides (left panel) or different numbers of apoptotic Mel888 cells, as described in “Methods.” Cross-presentation was evaluated as indicated.