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

Cytotoxicity Assay. JY target cells were incubated with synthetic peptides (10 g/ml) in FCS-medium overnight. Target cells (peptide-pulsed JY cells or AHH-1 TK ϩրϪ -derived cells) were labeled with 100 Ci of Na 2 [ 51 Cr]O 4 (Amersham Pharmacia Biotech) for 1 h and washed four times with PBS containing 10% FCS-medium. Cytolytic activity was determined in a standard 4-h 51 Cr-release assay using U-bottomed 96-well plates containing 2,500 targets per well. Where indicated in the figure legends, 2,500 K562 cells per well were added to reduce unspecific lysis. Percentage of cytotoxicity was determined from the formula: 100 ϫ [(experimental release Ϫ spontaneous release)/ (maximum release Ϫ spontaneous release)]. Maximum release was determined by lysis of targets with HCl. Spontaneous release was Ͻ 25% of maximal release in all assays. Specific lysis was calculated as difference between lysis of targets with peptide (or plasmid) and targets without peptide (or plasmid). In peptide titration experiments, JY target cells were incubated with various peptide concentrations for 90 min after 51 Cr labeling, washed once with PBS, and used as described above. In functional MHC binding assays, JY target cells were pulsed with synthetic peptide for 90 min at indicated times before the assay, then washed twice with PBS containing 10% FCS-medium, incubated in FCS-medium until 51 Cr labeling, and used as described above. CD8 dependency of target recognition was tested by addition of 10 g/ml anti-CD8 antibody OKT8 (Ortho Diagnostic Systems Inc.) during the cytotoxicity assay.