Fig. 7

miR-455-5p overexpression decreases T cell apoptosis. A Representative flow cytometry histogram plot (upper panel) and quantification (lower panel) of PD-1 MFI in Jurkat cells stimulated either with ± CD3/CD28/CD2 soluble antibody complexes for 72 h. Results are shown as fold change of MFI relative to not active cells. Data are mean ± SEM (N = 4). P value was calculated by one-sample t test. (B and C) NCI-H1975 cells transfected with the indicated oligos were exposed to IFN-γ for 8 h and then co-cultured for 72 h with Jurkat cells in the presence of T cell activator. B Representative flow cytometry histogram plots (left) and quantification (right) of PD-L1 MFI at the indicated experimental conditions. Results are shown as fold change of MFI relative to control conditions. Data are mean ± SEM (N = 4). P values were calculated by one sample t test. C Analysis of Jurkat apoptosis rate co-cultured with the indicated cell lines by Annexin V/7-AAD staining. Right panels: Representative flow cytometric plots (left) and quantification (right) of apoptotic dead Jurkat cells (Annexin V+, 7-AAD+; highlighted in red). Results are shown as fold change of apoptotic dead cells relative to matched control conditions. Data are mean ± SEM (N = 4). P values were calculated by one sample t test. D–E–F Distribution of the percentage of CD8+ cells and miR-455-5p expression, expressed as z-score, in NSCLC primary tumors from CD8-CIMA-CUN (D), CD8-CSS Cohort (E) and after pooling together the two cohorts (F). Tumors were stratified in high and low CD8 tumors based on the median value of CD8 + z-score. G Left: correlation analysis of miR-455-5p levels with PD-L1 mRNA, gene signature for exhausted CD8 + T cell (GET), IFN-γ and IFN-α response in tumors from TGCA-LUAD and TGCA-LUSC cohorts. Bubble plots reported the correlation coefficients for miR-455-5p expression with the indicated variables. The size of the bubbles indicates statistical significance calculated by the Spearman correlation analysis. Right: Bar plot reporting the Thorsson immune subtype of TGCA-LUAD and TCGA-LUSC tumors according to miR-455-5p expression. P value was calculated using the t test for equality of proportions (High vs Low). ****P < 0.001 (referred to C3). H Schematic model of the effects of miR-455-5p-dependent PD-L1 regulation in NSCLC