Acquisition and data analysis were performed by FACS. To test whether HCV core

protein could have any effect on NK cells as previously observed with T cells [19, 20], the YTS NK cell line was transduced with a lentivirus construct expressing HCV core protein and GFP (coreGFP+ YTS NK cells). Initially coreGFP+ YTS NK cells were set up to study the effect of core in the proliferation rate and apoptosis Alvelestat of the cells. The expression of annexin-V was evaluated as an early marker of programmed cell death (Fig. 1 and data not shown). Annexin-V staining was performed every 24 h for a total of 7 days, starting 24 h after transduction. Untransduced cells were also set up in parallel as an additional control. After 24 h, HCV core induced a significant increase in the percentage of apoptotic cells (65 ± 5% annexin-V-positive YTS cells) compared with GFP-expressing YTS cells (41 ± 6%). Those cells expressing the highest level of core were more susceptible, suggesting that high levels of core protein induced the apoptosis in the NK cell line. After the first 24 h, there were no significant differences in the percentage of annexin-V-positive cells between core- and GFP-transduced YTS cells at any time point. The

level of apoptotic cells in untransduced YTS cell cultures ranged between 8% and 15% during the experiment (data not shown). Previous studies examining NK cells from patients infected with HCV have noted alterations in the NK cell phenotypes [21, 22]. While YTS cell line does not express inhibitory receptors, we examined the expression of activating receptors in PF-01367338 research buy the coreGFP+ YTS NK cells at 24 and 120 h

post-transduction (Fig. 2). After 24 h of core protein expression in YTS, only NKp46 showed a significant decrease in expression compared with GFP+ YTS control cells (MFI 16 ± 1 in coreGFP+ YTS NK cells compared with Cyclooxygenase (COX) 20 ± 2 in GFP+ YTS NK cells). At 120 h, coreGFP+ YTS NK cells continued to show altered NKp46 receptor expression (25 ± 3 in coreGFP+ YTS NK cells compared with 35 ± 3 in GFP+ YTS NK cells). None of the other receptors examined seemed to be altered in their expression (Fig. 2). Natural killer cells from HCV-infected patients have been observed to have reduced cytotoxic capabilities [6, 8, 23]. Natural cytotoxic activity of coreGFP+ YTS NK cells was measured against the K562 cell line in a standard 51Cr release assay, at 24 and 120 h after transduction (Fig. 3). At 24 h, no significant differences were found between coreGFP+ YTS NK cells and GFP+ YTS NK cells. However, at 120 h, coreGFP+ YTS cells exhibited a significant decrease in the cytotoxic ability (28.9 ± 6.1% by coreGFP+ YTS NK cells compared with 40.4 ± 2.1% lysis by GFP+ YTS NK cells at 30:1 effector/target ratio; 20.5 ± 3.4% lysis by coreGFP+ YTS NK cells compared with 29.7 ± 1.5% by GFP+ YTS NK cells at 10:1 ratio).