Fig. 5. IFN-γ is the major factor responsible for CD56⁺ T supernatant (SN)-mediated anti-HCV activity.

(A) Anti-IFN-γ antibody blocks CD56⁺ T SN-mediated anti-HCV activity. HCV JFH-1-infected Huh7.5.1 cells were cultured in the presence or absence of CD56⁺ T SN and/or antibody against IFN-γ (10μg/mL). For the cultures in the presence of CD56⁺ T SN and the antibody against IFN-γ, the CD56⁺ T SN was preincubated with the antibody against IFN-γ for 30 min prior to the addition to Huh7.5.1 cultures. IFN-γ (200U/mL) alone was added to the cell cultures as a positive control to determine the neutralization ability of the antibody to IFN-γ. Mouse IgG2A was used as a control antibody to determine the specificity of the antibody to IFN-γ. (B) Antibodies to IFN-γR1 and IFN-γR2 block anti-HCV activity of CD56⁺ T SN. HCV JFH-1-infected Huh7.5.1 cells were incubated with or without antibodies to IFN-γR1 (10μg/mL) and/or IFN-γR2 (10μg/mL) for 1 h prior to the addition of the CD56⁺ T SN. Goat IgG was used as a control IgG to determine the specificity of the antibodies to IFN-γR. HCV RNA levels in Huh7.5.1 cells were determined at 48 h post exposure to CD56⁺ T SN. Total cellular RNA extracted from Huh7.5.1 cells was subjected to the real-time RT-PCR for HCV and GAPDH RNA quantification. The data are expressed as HCV RNA levels relative (%) to control cultures (no antibody treatment and no CD56⁺ T SN added, which is defined as 100). The results shown are mean ±SD of triplicate cultures, representative of three separate experiments (* p<0.05, ** p<0.01).