Figure 1.
MiR-21 expression is associated with memory T-cell phenotype and is induced upon αCD3/CD28 activation of naive and memory T-cells.
A Baseline miR-21 expression was analyzed by qRT-PCR in naive (CD4+ CD45RO-) and memory (CD4+ CD45RO+) T-cells isolated from PBMC of healthy volunteers. Relative expression normalized to the RNU48 reference gene is shown. Each dot represents a separate donor. Lines represent median values (n=7, Wilcoxon signed ranked test). B MiR-21 expression was analyzed by qRT-PCR in naive (CD4+ CD45RO-) and memory (CD4+ CD45RO+) T-cells before (day 0), and at indicated time points after activation with plate-bound-anti-CD3/soluble-anti-CD28 mAbs. Relative expression normalized to the RNU48 reference gene is shown. Data are expressed as median with interquartile range (n=6 donors, statistical analysis inside the T-cell subset: Friedman test, with a Dunn’s Multiple Comparison test). *p<0.05, **p<0.01, ***p<0.0001.
Figure 2.
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.
Figure 3.
MiR-21 inhibition leads to increased CCR7 expression in activated T-cells.
A Representative FACS staining plot depicting CCR7 expression on resting naive (CD3+ CD8-CD45RO-CD25-, Day 0), and on PHA/IL-2 activated, GFP+ cells harboring miR-21 or control inhibitor (scrambled hairpin sequence) at day six post lentiviral transduction. The isotype staining control is depicted. B Quantification of CCR7 staining of GFP+ cells from A, depicted as MFI (geometric mean fluorescent intensity). Each line represents a separate donor (n=8, Wilcoxon signed ranked test). C Percentage of CCR7 positive, GFP+ cells from A. Each line represents a separate donor (n=8, Wilcoxon signed ranked test). D GFP+, PHA/IL-2 activated naive T-cells harboring miR-21 or control inhibitor were FACS isolated at day six post-lentiviral transduction and CCR7 transcript expression was determined by qRT-PCR. Each line represents a separate donor (n=3, paired t-test, ns). Relative expression normalized to the U6 reference gene is shown. E Representative FACS staining plot depicting CCR7 expression on resting memory (CD3+ CD8-CD45RO+CD25-, Day 0), and on PHA/IL-2 activated, GFP+ cells harboring miR-21 or control inhibitor (scrambled hairpin sequence) at day six post lentiviral transduction. The isotype staining control is also depicted. F Quantification of CCR7 staining of GFP+ cells from E, depicted as MFI (geometric mean fluorescent intensity). Each line represents a separate donor (n=7, Wilcoxon signed ranked test). G Percentage of CCR7 positive, GFP+ cells from E. Each line represents a separate donor (n=7, Wilcoxon signed ranked test). D GFP+, PHA/IL-2 activated memory T-cells harboring miR-21 or control inhibitor were FACS isolated at day six post-lentiviral transduction and CCR7 transcript expression was determined by qRT-PCR. Each line represents a separate donor (n=3, paired- t-test, ns). Relative expression normalized to the U6 reference gene is shown. *p<0.05, **p<0.01.
Figure 4.
CCR7 is a direct target of miR-21 and correlates inversely with miR-21 expression in resting and activated T-cells.
A Ratio of Renilla luciferase (RL) to firefly luciferase (FL) signal, determined in lysates of Cos-7 cells co-transfected with psiCHECK-2 construct harboring the CCR7 3’ UTR and a synthetic miR-21 or control precursor. Median values with range of data normalized to control precursor are depicted (n=4 independent experiments, paired t-test). B MiR-21 and CCR7 expression levels were assessed by qRT-PCR in freshly isolated naive (CD4+ CD45RO-) and memory (CD4+ CD45RO+) T-cells. Expression levels are shown relative to the RNU48 and U6 reference genes respectively. Each line represents a separate donor (n= 8, Wilcoxon signed ranked test). C Representative FACS staining plot depicting CCR7 expression on isolated naive (CD4CD45RO-) T-cells before (day 0), and after seven days of activation with plate-bound-anti-CD3/soluble-anti-CD28 mAbs. Isotype staining is depicted. D Quantification of CCR7 staining from C, depicted as MFI (geometric mean fluorescent intensity). Each line represents a separate donor (n=3, paired t-test, ns). E Percentage of CCR7 positive, GFP+ cells from C. Each line represents a separate donor (n=3, paired t-test). F CCR7 transcript expression was determined by qRT-PCR in naive (CD4+ CD45RO-) T-cells before (day 0), and after seven days of activation with plate-bound-anti-CD3/soluble-anti-CD28 mAbs. Each line represents a separate donor (n=5, paired t-test). Expression levels shown in the graph are normalized to the U6 reference gene. *p<0.05, **p<0.01.