Figure 4

IK14004 increases the IL-12RΒ2: IL-12RΒ1 expression ratio irrespective of TCR activation in CD4+ /CD8+ T cells. Buffy coat samples were obtained from human volunteers following ethics approval. PBMC cultures were either not stimulated or stimulated with anti-CD3 antibody for either 24 or 72 h. Each tissue culture experiment was performed using triplicate wells (technical replicates) and cell-based flow cytometry experiments in the presence of IK14004 were repeated at least three or four times (n = experimental replicates) as indicated below each panel. ELISA studies were performed to detect IL-12p40/IL-12p70 and IFN-γ levels in supernatants from anti-CD3-stimulated PBMC cultures exposed to either anti-PD-1 mAb or isotype control IgG1 antibody. All error bars represent standard error of the mean (SEM). Flow cytometry data are shown as either mean fluorescence intensity (MFI) or percentage of receptor-expressing cells and dot plot/gating strategies are shown in Supplementary Figs. S7 and S8. (a) Percentage of Ki67-expressing CD4+ T cells within stimulated PBMC cultures. (b) Percentage of IL-12Rβ1-expressing CD4+ T cells within stimulated PBMC cultures. (c) Percentage of IL-12Rβ2-expressing CD4+ T cells within stimulated PBMC cultures. (d) Percentage of Ki67-expressing CD8+ T cells within stimulated PBMC cultures. (e) Percentage of IL-12Rβ1-expressing CD8+ T cells within stimulated PBMC cultures. (f) Percentage of IL-12Rβ2-expressing CD8+ T cells within stimulated PBMC cultures. (g) Expression of IL-12Rβ1 in CD4+ T cells within unstimulated PBMC cultures. (h) Expression of IL-12Rβ2 in CD4+ T cells within unstimulated PBMC cultures. (i) Expression of IL-12Rβ1 in CD8+ T cells within unstimulated PBMC cultures. (j) Expression of IL-12Rβ2 in CD8+ T cells within unstimulated PBMC cultures. (k) IL-12p70 levels in supernatant from stimulated PBMC cultures exposed to either anti-PD-1 mAb or isotype control IgG1 antibody. (l) IL-12p40 levels in supernatant from stimulated PBMC cultures exposed to either anti-PD-1 mAb or isotype control IgG1 antibody. (m) IFN-γ levels in supernatant from stimulated PBMC cultures exposed to either anti-PD-1 mAb or isotype control IgG1 antibody. Data were analysed by two-way ANOVA with Dunnett’s post-test for studies testing the effect of IK14004 and by paired t-test for anti-PD-1 experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.