*2.10. T-Cell Proliferation and ELISpot Assays*

T-cell proliferation was assessed by the incorporation of [3H]-thymidine into the DNA of dividing cells. The spleens of 10 mice of each group were pooled, a suspension of splenocytes was seeded in U-bottomed 96-well microculture plates at a density of 5 <sup>×</sup> 105 cells/well, and specific stimulants (pools of the recombinant HCV NS3, NS4, NS5A, and NS5B proteins at a final concentration of 1 μg/mL) were added. As negative controls, we used medium alone (spontaneous proliferation) and recombinant HCV core protein. Mitogen concanavalin A (ConA, 5 μg/mL, Sigma, USA) was used as an unspecific positive control. All samples were set in at least three replicates. The cells were cultured in a RPMI-1640 medium containing 20% FCS (Invitrogen, USA), 4.5 mg/mL glucose, 2 mM glutamine, 0.2 u/mL insulin, and 50 μg/mL gentamicin at 37 ◦C in a 5% CO2 atmosphere. Four days later, aliquots of cell culture fluids were withdrawn and frozen at −20 ◦C. The remaining cells were labeled with 1 μCi/well [3H]-thymidine (TdR, Amersham-Pharmacia-Biotech) and 18 h later harvested onto the glass-fiber filters. The radioactivity was measured using a MicroBeta2 β-counter (PerkinElmer, Waltham, MA, USA). Results were expressed as stimulation indexes (SI), determined by dividing the mean radioactive 3H incorporation as counts per minute (c.p.m.) in the presence of antigens by means of 3H incorporation in the wells containing medium alone and control antigen.

The number of IFN-γ synthesizing cells was determined using the ELISPOT mouse IFN-γ Kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer's instructions. The results were expressed as the number of spot forming cells (SFC) per 106 cells.
