Figure 3

IK14004 enhances IL-2 production in human CD4+ T cells but not CD8+ T cells. Buffy coat samples were obtained from human volunteers following ethics approval. Non-TCR-stimulated PBMCs were cultured for 24–72 h. PBMCs were also exposed to IK14004 first for 48 h followed by either addition of anti-CD3 antibody for a further 48 h (unwashed cells; UC) or washed after the first 48 h to remove peptide followed by addition of anti-CD3 antibody for the remaining 48 h (washed cells; WC). Isolated CD4+ /CD8+ T cell cultures were exposed to peptide for the first 48 h followed by washing and then stimulated with anti-CD3/anti-CD28 antibodies for the next 48 h (washed T cells; WTC). Experiments to detect TCR α and β chain expression in CD4+ /CD8+ T cells within PBMC cultures were conducted with both unstimulated and anti-CD3-stimulated PBMCs over 24 h. Each tissue culture experiment was performed using triplicate wells (technical replicates) and cell-based flow cytometry/ELISA experiments were repeated at least three or four times (n = experimental replicates) as indicated below each panel. All error bars represent standard error of the mean (SEM). Flow cytometry data are shown as mean fluorescence intensity (MFI) and IL-2 production expressed as pg/mL. Dot plots and gating strategies are shown in Supplementary Figs. S5 and S6. (a) Expression of Ki67 in CD4+ T cells within unstimulated PBMC cultures. (b) IL-2 levels in supernatant from unstimulated PBMC cultures after 24 h. (c) IL-2 levels in supernatant from unstimulated PBMC cultures after 48 h. (d) IL-2 levels in supernatant from unstimulated PBMC cultures after 72 h. (e) Expression of CD25 in CD4+ T cells within unstimulated PBMC cultures after 72 h. (f) IL-2 levels in supernatant from unstimulated PBMC-UC cultures after 96 h. (g) IL-2 levels in supernatant from unstimulated PBMC-WC cultures after 96 h. (h) IL-2 levels in supernatant from isolated CD4+ T cell-WC cultures after 96 h. (i) IL-2 levels in supernatant from isolated CD8+ T cell-WC cultures after 96 h. (j) TCRα/β expression in CD4+ T cells within unstimulated versus TCR-stimulated PBMC cultures. (k) TCR α/β expression in CD8+ T cells within unstimulated versus TCR-stimulated PBMC cultures. Data were analysed by either two-way ANOVA with Dunnett’s post-test (a, e) or Sidak’s post-test (f, g, h, i), one-way ANOVA with Dunnett’s post-test (b, c, d) or two-way ANOVA with Holm-Sidak’s multiple comparisons post-test (j, k). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.