Endosomal preprocessing of antigen associated with apoptotic or necrotic cells involves cathepsin D. Immature HLA A^*0201⁺ DCs were preincubated with increasing doses of pepstatin A (A), leupeptin (B), or cathepsin D (C). Pretreated DCs were cocultured about 12 hours with apoptotic or necrotic monocytes expressing MP or with MP peptide and the same doses of the inhibitors along with a maturation stimulus. All DC groups were fixed in glutaraldehyde and cultured with Flu16. Responses were assessed by IFN-γ ELISPOT. (D) IDCs were preincubated with cathepsin D (1 μM) alone, or combined with either pepstatin A (200 μM) or NH₄Cl (50 μM). The pretreated DCs were cocultured with apoptotic or necrotic monocytes together with same doses of cathepsin D alone or combined with the inhibitors and exposed to maturation stimuli for about 12 hours (D). All groups of DCs were fixed by glutaraldehyde and assessed with Flu16 in IFN-γ ELISPOT assay. In panels A-D, the left axis shows cross-presentation; the right, peptide presentation. The ratio of dead cells to DCs was 2:1 and the concentration of MP peptide was 100 nM. One representative set of experiments of 4 is shown.