Peptides derived from CHP/HER2-147 are presented on HLA-A2402 molecules through the conventional MHC class I pathway.

DCs (1 × 10⁶) were cultured with cytochalasin D (CCD, 30 μg/mL), amiloride (AML, 0.2 mM), chloroquine (CHQ, 100 μM), lactacystin (Lact, 100 μM), brefeldin A (BFA, 5 μg/mL), or cycloheximide (CHX, 10 μg/mL) for 1 hour in the serum-free AIM-V medium, followed by incubation with or without 20 μg/mL protein CHP/HER2-147 for 3 hours. DCs were then cultured in the absence of the inhibitors, CHP/HER2-147, or HER2p63 for 18 hours in 10% FCS RPMI 1640 supplemented with GM-CSF and IL-4. DCs not cultured with CHP/HER2-147 were incubated with 10 μM HER2p63 for 1 hour at room temperature and for an additional hour at 37°C. DCs (1 × 10⁶) that were not treated with the inhibitors were also cocultured with CHP/HER2-147 or HER2p63, as described in Figure 2B. For IFN-γ production assay, 1 × 10⁵ cells/well of these DCs were incubated with 5 × 10⁴ cells/well of the CTL clone Y.I.1a for 18 hours. IFN-γ production was measured in duplicate using an ELISA kit. Data are the average of duplicate measurements.