Molecular Mimicry of Human Cytochrome P450 by Hepatitis C Virus at the Level of Cytotoxic T Cell Recognition

Kammer, Andreas R.; van der Burg, Sjoerd H.; Grabscheid, Benno; Hunziker, Isabelle P.; Kwappenberg, Kitty M.C.; Reichen, Jürg; Melief, Cornelis J.M.; Cerny, Andreas

Measurement of MHC-Peptide Complex Stability. JY cells at a concentration of 1-2 ϫ 10 6 cells/ml were incubated with 10 Ϫ 4 M emetine (Sigma Chemical Co.) for 1 h at 37 Њ C to stop protein synthesis and the subsequent emergence of de novo synthesized class I molecules at the cell surface. Cells were washed twice with PBS and peptide stripped (see above). 10 6 cells were added to 200 g of peptide in 1 ml and incubated for 90 min at room temperature. Cells were washed twice with ice-cold IMDM and resuspended in 1 ml IMDM. Subsequently, the cells were incubated for 0, 2, 4, and 6 h at 37 Њ C and thereafter stained with BB7.2, an HLA-A2 confirmation-specific mAb (27) , and goat anti-mouse FITC. Thereafter, the cells were fixed by resuspension in PBA1% containing 0.5% paraformaldehyde and analyzed by FACScan ® . The fluorescence index (FI) was calculated as FI ϭ (mean fluorescence sample Ϫ mean fluorescence background) / mean fluorescence background without peptide. Samples were tested in duplicate and the variation between both samples was always Ͻ 10%.