Figure 5: CD8+ T cells are required for the efficacy of miR-424(322) treatment in ID8 tumours.

(a) The luciferase vectors that contained the mouse wild-type (WT) and mutant (MUT) PD-L1 3′-UTR regions were co-transfected into ID8 cells with miR-424(322) or scramble miRNA precursors (miR-Scr). The relative luciferase/Renilla activities were analysed in the cells 48 h after the transfection. t-test, **P≤0.01. The results represent the mean±s.e.m. from three independent experiments. (b) ID8 cells with stable overexpression of miR-424(322) were treated with anti-PD-L1 or IgG control for 24–48 h. PD-L1 protein levels were determined by western blotting. (c–f) ID8 cells (5 × 10⁶) with stable overexpression of miR-424(322) were injected into the syngeneic C57BL/7 mice followed by CD8+ blocking antibody (anti-CD8) treatment. (c) Kaplan–Meier survival analysis of tumour-bearing mice in different treatment groups. t-test, **P≤0.01. (d) The number of tumours was determined in different treatment groups. t-test, *P≤0.05, **P≤0.01. Bar graphs are shown as the mean±s.e.m. (n=12 mice per group). (e,f) At the time of necropsy, CD8 and PD-L1 expression was detected by flow cytometry. (e) FACS analysis of CD8 expression in ID8 tumours. CD8 expression was decreased in the anti-CD8-treated versus untreated mice. (f) FACS analysis of cell-surface PD-L1 expression in ID8 tumours. PD-L1 expression was decreased in the ID8 tumours with stable overexpression of miR-424(322).