Cross-presentation of FcγR-targeted viral antigen requires antigen processing in both the endosomal pathway and by the proteasome. Human MoDCs were allowed to process 3 μg pp65-IC in absence (A-H) or presence of proteolysis inhibitors indicated (O/N, 37°C; B-G, I-N). To ascertain that inhibitors do not counteract HLA-A2–mediated presentation indiscriminately, DCs were loaded with 1−6 M NLVPMVATV peptide and T-cell activation assessed after 4 hours in the presence of Golgi-stop (A-G). Shown are representative plots of IFN-γ production by A2/NLVPMVATV-reactive T cells (n > 3 independent experiments, summarized in supplemental Figure 2) for peptide control experiments (A-G) and representative plots (top) and summarizing graphs for each inhibitor (H-N, n = 4-5). (B-I) MG132 proteasome inhibitor, 50μM. (C-J) Lactacysin proteasome inhibitor, 100μM. (D-K) Epoxomicin proteasome inhibitor, 10μM. (E-L) chloroquine endosomal acidification inhibitor, 50μM. (F-M) Leupeptin lysosomal cysteine protease inhibitor,15μM. (G-N) Primaquine recycling endosome inhibitor, 50μM.
