Influence of IL-12 on PD-1 expression in HBV- and CMV-specific T cells.

<p>a) Frequency of PD-1+ HBV- or CMV-specific T cells (defined as CD8+ IFN-γ+) and (b) PD-1 expression levels as measured by MFI after short term culture in the presence or absence of IL-12, example FACS plots and summary data are gated on CD8+ IFN-γ+ T cells (n = 16 for overnight HBV, n = 27 for HBV- and n = 9 for CMV-specific 10 days). c) PD-1 MFI was compared on IFN-γ+ CD8+ T cells after stimulation with HBV-derived peptide in the presence or absence of IL-12. Samples were then divided according to whether PD-1 MFI decreased or not upon IL-12 stimulation. The graph shows the change in the frequency of IFN-γ producing cells upon IL-12 addition compared to peptide stimulation alone. d) PBMC were cultured with HBV derived peptides in the presence of IL-12; IFN-γ positive cells were divided into T-bet positive and negative cells according to an isotype staining control performed on IL-12 treated cells. PD-1 expression (mean fluorescence intensity) was compared in both groups. e) IFN-γ positive T cells were divided into IFN-γ low and IFN-γ high positive cells, defined by background IFN-γ production by CD8+ T cells in unstimulated samples. T-bet expression in IFN-γ low and high producing T cells was compared in samples tested after stimulation with HBV peptide and IL-12. f) Frequency of T-bet positivity in T cells producing either IFN-γ or TNF-α alone or co-producing both cytokines.