Inhibition of miR-24 reconstitutes ICOSL expression and restores tumor immunogenicity in a MLR assay. (A) ICOSL MFI after anti–miR-24 expression in (Ai) U2932 MPA vector transfected with control inhibitor and anti–miR-24. The middle panel shows ICOSL MFI in 3 independent flow cytometry experiments as whiskers. The right panel shows fold change in ICOSL mRNA expression by RT-qPCR after anti–miR-24 transfection. GAPDH was used as a housekeeping gene. (Aii) An increase in ICOSL after inhibition of miR-24 expression in U2932 EBNA2. Three independent experiments by flow cytometry show statistically significant increase of ICOSL (middle panel), also confirmed by mRNA RT-qPCR (right panel). P values were calculated through 2-tailed unpaired t test; ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001. (B) MLR to evaluate T-cell activation as result of ICOSL reconstitution after miR-24 downregulation. T cells were activated for 72 hours in plates coated with anti-CD3/anti-CD28 antibodies. Irradiated U2932 MPA vector and U2932 EBNA2 were cocultivated with activated T cells (effector). The effector-to-target ratio was 1:10. The target cells were transfected with control inhibitor or anti–miR-24 molecules before cocultivation with the effector cells. The coculture was carried out for 48 hours, and the cells were stained for CD4/CD8 and IFN-γ and processed for flow cytometry. All experiments were repeated at least 3 times and with peripheral blood mononuclear cells (PBMCs) from 3 healthy donors. Statistical analysis was performed with two-way ANOVA and Tukey multiple comparisons test; ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001.