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. 2020 Nov 27;8(2):e001521. doi: 10.1136/jitc-2020-001521

Figure 3.

Figure 3

Effect of PD-1/PD-L1 triggering on transcriptional regulation and secretion of IL-2. (A, B) PD-1 expressing TPR (TPR-PD-1) cells were stimulated using TCS-CD86 or CD86/PD-L1 bearing TCS (TCS-CD86/PD-L1) cells for 24 hours. NFAT-GFP (left), NFκB-CFP (middle) or AP-1-Cherry (right) expressing cell percentage (A) or geometric mean fluorescence intensity (gMFI) (B) was analyzed. Transcription factor activity was calculated by normalizing data to the maximal activation. (C) Luciferase activity of an IL-2 reporter construct was determined in TPR-PD-1 cells stimulated with TCS-control, TCS-CD86 or TCS-CD86/PD-L1 cells for 24 hours. Luciferase activity was corrected using an internal renilla luciferase control. (D) IL-2 secretion to medium was assessed in supernatants of TPR-PD-1 cells stimulated with TCS-control, TCS-CD86 or TCS-CD86/PD-L1 cells for 24 hours. When indicated, a humanized anti-PD-1 antibody (nivolumab (nivo)) was added. IL-2 secretion was calculated by normalizing data to the maximal activation. Data were analyzed using parametric unpaired t-test (A, B) or one-way ANOVA and Bonferroni post-test (C, D); NS ***p<0.001, ****p<0.0001. Results are representative of at least three independent experiments with similar results. ANOVA, analysis of variance; IL-2, interleukin 2; NFAT, nuclear factor of activated T cells; NFκB, nuclear factor κ B cells; ns, not significant; PD-1/PD-L1, programmed cell death-1/ligand 1; TCS, T cell stimulator; TPR, triple parameter reporter.