Figure 4

HLA-A*0201+ BKV LTag transfected target cells are recognized by LTag _579–587 specific CTLs. HLA-A*0201+ HBL cells at 90–95% confluency were transfected by liposome-mediated gene transfer with plasmid DNA pTRE2LTag and pTet-Off (mixed at 1:1 ratio) and peaks of cell permissibility to plasmid were monitored by using a parallel pEGFP-N1 transfected cell culture as analysed by fluorescence microscopy at 2 different plasmid concentrations at 24 and 48 hours after transfection (panel A). Quantitative rt-PCR transient LTag gene expression showing means of Ct value of 31 for HBL transfected cell (2) and Ct value of 47 for untransfected cell (3), as compared to β-actin Ct values of 15 for either HBL transfected or untransfected culture (1) is also reported in panel B. Panel C reports fold increases of IFN-γ gene expression in five LTag 579 in vitro expanded CTL lines upon stimulation with transfected HBL (white bars) as compared with autologous peptide (LTag 579)-loaded APC (black bars). Results refer to IFN-γ relative gene expression (2^-ΔΔCt method) related to either untransfected HBL or non peptide-loaded APC. Panel D reports amounts of IFN-γ protein released by LTag 579 specific CTLs following culture in the presence of transfected (grey bars) or untransfected cells (black bars).