Huh 7.5.1 cells were seeded at 3 × 106 cells in T75 plate for 24 hours. They were then infected with 4 × 104 focus-forming unit (FFU) (multiplicity of infection [MOI] 0.01) of HCV strain JFH-1, and infected cells were cultured for 10 days in DMEM/10% FCS media. Cells were expanded 2 days following infection. Infection was confirmed by immunofluorescence. Hepatocytes

were stained with monoclonal antibodies to HCV core (clone C7-50, Thermo Scientific, Rockford, IL) and subsequently stained with Alexa Fluor 488-conjugated donkey antimouse antibodies (Invitrogen). Nuclei were visualized using DAPI (Invitrogen). To isolate JFH-1, centrifugation using CP-690550 price an Amicon Ultra-15 (100,000 MWCO) centrifugal filter unit was used. Briefly, 10 mL of JFH-1 infected culture media was concentrated to 1 mL. Next, peripheral blood mononuclear cells (PBMCs) were treated with indicated amounts of the concentrated virus for 7 days. Human PBMCs were isolated from healthy blood donors (Virginia Blood Services, Richmond, VA) by lympholyte gradient centrifugation (Cedarlane Laboratories, Buparlisib Burlington, NC). Infected hepatocytes

were plated at 0.1 × 106 cells/mL in a T25 cm2 flask and cultured overnight. PBMCs were then thawed and 10 × 106 cells were cocultured with the hepatocytes for 7 days in complete media (RPMI 1640 supplemented with 10% [vol/vol] FBS) (Hy-Clone, Logan, UT), penicillin/streptomycin (100 μg/mL), and L-glutamine (2 mM). Following 7 days of coculture, CD33+ cells were selected using magnetic beads (Miltenyi Biotec) according to the manufacturer’s instructions. CD33+ cells were cocultured

with autologous magnetic bead selected (MACS) CD4 and CD8 T cells at a ratio of 1:2 (250,000 CD33+ cells to 500,000 T cells) for 3 days in the presence of 5 μg/mL anti-CD3 (OKT3; eBioscience, San Diego, CA) and 10 μg/mL anti-CD28 (CD28.6; eBioscience). Human PBMCs were cultured in complete media at 1 × 106 cells/mL for 7 days in the presence of 1 μg/mL recombinant HCV core protein (Virogen, Watertown, MA) or recombinant protein control, β-galactosidase (Virogen). CD33+ cells were then selected using magnetic beads and cocultured with autologous CD4 and CD8 T cells as described above. T cells and CD33+ cells were cocultured in transwell plates (Corning, Corning, NY) containing 0.4 μm pores in indicated experiments L-gulonolactone oxidase (Fig. 3). Prior to coculture of CD4 and CD8 T cells with CD33+ cells, cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) according to the manufacturer’s instructions (Invitrogen). The cells were then washed in media and cocultured with CD33+ cells. Following 3 days of coculture in the presence of plate-bound anti-CD3/anti-CD28, cells were stained with APC-conjugated anti-CD4 (Leu-3a; eBiosciences) or APC-conjugated anti-CD8 (RPA-T8; eBiosciences), fixed, and collected on a FACSCanto (BD Bioscience, San Diego, CA).