Figure 2. Processed Antigen from the Dying Cell Is Required for MHC I Presentation in TAP^−/− DCs.

To generate apoptotic cells lacking processed antigen, lactacystin pretreatment of influenza-infected H-2^d 3T3 cells was performed. Expression of influenza antigen was evaluated by intracellular FACS analysis using influenza NP mAbs followed by PE-conjugated goat anti-mouse mAb (A). Expression of MHC I/pep complexes in the lactacystin-treated 3T3 cells was evaluated by monitoring the activation of H-2^d-restricted influenza hemagglutanin-reactive T cells. The K^d-restricted immunodominant peptide (HA_210–219) derived from hemagglutanin was pulsed onto 3T3 cells and served as a positive control (B). The influenza-infected H-2^d 3T3 cells were then induced to undergo apoptosis, and co-cultures were generated using C57BL/6 WT DCs (C) or TAP^−/− DCs (D). To evaluate T cell activation and expansion, DCs cross-presenting antigen were cultured with CD8⁺ T cells in the presence of IL-12 for 7–8 d. T cells were then harvested and tested for influenza reactivity in a 20-h IFN-γ ELISPOT. H-2^b EL4 cells with or without influenza infection served as the stimulators in the ELISPOT assay as above. Data are representative of two experiments. Values are averages of triplicate wells with error bars indicating standard deviation.