MiR-21 mediates survival of activated memory CD4+ T-cells.

A Resting naive (CD3+ CD8-CD45RO-CD25-) T-cells were stimulated with 5µg/ml PHA and 100 U/mL IL-2, followed by transduction with lentiviral vectors harboring miR-21 or control inhibitor (scrambled hairpin sequence) and GFP. The percentage of GFP+ cells in culture over time was monitored by FACS. Data were normalized to the first measurement at day six. Each line represents a separate donor (n=5, two-way RM ANOVA with Bonferroni posttests, ns-not significant). B Data from A, depicted as median values with interquartile range. C Resting memory (CD3+ CD8-CD45RO-CD25-) T-cells were stimulated with 5µg/ml PHA and 100 U/mL IL-2, followed by transduction with lentiviral vectors harboring miR-21 or control inhibitor (scrambled hairpin sequence) and GFP. The percentage of GFP+ cells in culture over time was monitored by FACS. Data were normalized to the first measurement at day six. Each line represents a separate donor (n=5, two-way RM ANOVA with Bonferroni posttests). D Data from C, depicted as median with interquartile range. E Activated naive, and F activated memory GFP+ T-cells harboring miR-21 or control inhibitor were isolated by FACS from mixed cultures (A, and C respectively) at day six post lentiviral transduction. Isolated GFP+ cells, and activated not transduced cells (NT) were cultured in complete media supplemented with 100 U/mL of IL-2 for 48h (until day 8), and percentage of apoptotic cells was assessed by FACS-based measurement of mitochondrial trans-membrane potential loss, using DilC1(5). Each line represents a separate donor (naive: n=4, memory n=5, RM ANOVA with Bonferroni posttests). *p<0.05, **p<0.01, ***p<0.0001.