51Cr-release assays using a CTL line generated from an HLA-A2– and CMV-positive donor. (A) 51Cr-release assays using a pp65-peptide–specific CTL line generated from an HLA-A2– and CMV-positive donor. Autologous MDCs pulsed with the pp65-peptide or incubated with HLA-A2–negative HFFs that were either infected with CMV (CMV+) or left untreated (CMV−) served as target cells. MDCs incubated with an irrelevant HIV peptide or supernatant (SN) from infected HFFs were applied as controls. Inhibition of HLA class I or class II was performed by incubating MDCs prior to the assay with anti (α)–HLA class I or II antibodies. MDCs that were cocultured with HFFs transfected with pp65-IVT as well as MDCs that were cocultured with HFFs transfected with an irrelevant EGFP-IVT as a control were included. (B) Cross-presentation of pp65-derived HLA-A*02–binding T-cell epitopes is sensitive to proteasome inhibitor lactacystin or the blocker of the MHC class I processing pathway brefeldin A but not to the lysosomotropic agent chloroquine. Data represent means (± SD) of quadruplicates. One representative experiment is shown.
