Figure 1 miR-128, miR-27 and miR-340 are overexpressed in multiple sclerosis CD4⁺ T cells. (A) miR-128 (left) and miR-27a or miR-27b (right) expression in purified naïve CD4⁺ T cells from healthy donors (HD; n = 16) and multiple sclerosis (MS; n = 22, including five primary progressive, 12 relapsing–remitting and five secondary progressive) by Taqman real-time polymerase chain reaction array. (B) Distribution of miR-128 and miR-27b expression by Taqman array in primary progressive (PP) multiple sclerosis, relapsing–remitting (RR) multiple sclerosis and secondary progressive (SP) multiple sclerosis subtypes. (C) miR-340 expression counts in purified memory CD4⁺ T cells from healthy donors (n = 17), all multiple sclerosis (n = 19), primary progressive multiple sclerosis (n = 4), relapsing–remitting multiple sclerosis (n = 11) or secondary progressive multiple sclerosis (n = 4) by Nanostring nCounter detection. miRNA fold-changes were calculated relative to healthy donors C_t value geometric mean and geometric mean values for healthy donors and patients with multiple sclerosis are shown by the lines (A and B). Statminer's Limma test P-values for the healthy donors to multiple sclerosis groups comparisons shown in A–C. From: Micro-RNA dysregulation in multiple sclerosis favours pro-inflammatory T-cell-mediated autoimmunity Brain. 2011;134(12):3578-3589. doi:10.1093/brain/awr262 Brain | © The Author (2011). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Figure 2 Multiple sclerosis-associated miRNAs target Th2 pathway genes Figure 2 Multiple sclerosis-associated miRNAs target Th2 pathway genes. (A) Relative luciferase units (RLU) from a luciferase vector carrying the predicted multiple sclerosis-associated miRNA wild-type (WT) or mutant binding sites (mut) from the hBMI1 gene 3′-UTR (indicated above bars) in cos-7 cells transfected with multiple sclerosis-associated miRNAs. Mean relative luciferase units ± SEM are shown as percentage of the control NS miRNA. Results from three independent experiments are shown. The miR-128 wild-type data have been previously reported (Godlewski et al., 2008) and are shown here for confirmation and comparison of the relative effects of each miRNA. The miR-128 mutant data have also been published (Godlewski et al., 2008). (B) Non-linear regression Pearson's correlation analysis of the relationship between BMI1 and miR-128 expression in naïve CD4⁺ T cells from healthy donors and patients with multiple sclerosis. Pearson P (indicating statistical significance) and r (indicating negative correlation) values shown. (C) Luciferase assay with a hGATA3 3′-UTR luciferase vector after transfection of cos-7 cells with multiple sclerosis-associated miRNAs predicted to bind GATA3 messenger RNA. Mean relative luciferase units ± SEM results from multiple replicates of three independent experiments shown. (D) Luciferase assay with a hIL-4 3′-UTR luciferase vector after transfection with miR-340. Mean relative luciferase units ± SEM results from multiple replicates of two independent experiments shown. (E) IL-4 (left) and IL-5 (right) expression determined by ELISA in 24-h supernatants from a myelin basic protein Ac1-11–specific Th2 cell line transfected overnight with NS miRNA or miR-340 prior to stimulation with myelin basic protein Ac1-11 peptide. Mean ± SEM results shown are intra-experimental replicates, representative of two independent experiments. Dunnett's post hoc (A and C–E) or t (E) test P-value: **P ≤ 0.005. RU = relative expression units. From: Micro-RNA dysregulation in multiple sclerosis favours pro-inflammatory T-cell-mediated autoimmunity Brain. 2011;134(12):3578-3589. doi:10.1093/brain/awr262 Brain | © The Author (2011). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Figure 3 Model of the effects of multiple sclerosis-associated miRNAs on Th2 pathway genes. Th2 cell commitment is mediated by IL-4 receptor engagement during T-cell activation, resulting in STAT6 phosphorylation and transactivation of the GATA3 gene. In turn, GATA3 initiates transcription of the IL-4, IL-5 and IL-13 cytokine genes. BMI1 stabilizes GATA3, and BMI1 repression by increased multiple sclerosis-associated miR-27, and miR-128 expression in naïve CD4⁺ T cells would result in enhanced GATA3 degradation and reduced Th2 differentiation. In addition, increased miR-340 in memory CD4⁺ T cells of patients with multiple sclerosis would directly target the IL-4 gene at the effector cell level. From: Micro-RNA dysregulation in multiple sclerosis favours pro-inflammatory T-cell-mediated autoimmunity Brain. 2011;134(12):3578-3589. doi:10.1093/brain/awr262 Brain | © The Author (2011). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Figure 4 Multiple sclerosis-associated miRNAs repress BMI1 and GATA3 in CD4⁺ T cells and exacerbate experimental autoimmune encephalomyelitis (EAE). (A) BMI1 versus forward scatter (FSC) (top), GATA3⁺ versus FSC (middle) and BMI1 versus GATA3 (lower) intranuclear staining of CD4⁺ T cells from myelin basic protein-specific TcR-tg cells transfected with NS, miR-27, miR-128, miR-340 or a combination of miR-27, miR-128 and miR-340 (miR mix) prior to stimulation with MBPAc1-11 peptide. Results are representative of three independent experiments, which are pooled in the bar graphs in B (mean ± SEM, *t-test P ≤ 0.05). (C) Histogram of BMI1 and GATA3 expression in cells from A to B, showing the mean fluorescence intensity in CD4⁺ T cells transfected with control miRNA or miR mix. The plots show individual results representative of three independent experiments. (D) Myelin basic protein Ac1-11-specific TcR-tg splenocytes were transfected with control miRNA or miR mix before stimulation with myelin basic protein Ac1-11 in Th neutral conditions for 72 h, transferred to B10.PL mice and monitored for signs of experimental autoimmune encephalomyelitis. Four out of seven mice receiving control miRNA-transfected cells and six out of seven mice receiving multiple sclerosis miRNA-transfected cells developed experimental autoimmune encephalomyelitis. Mean ± SEM experimental autoimmune encephalomyelitis score is shown. The plots show one experiment and are representative of two independent experiments. Significance was calculated using Mann–Whitney's t-test. From: Micro-RNA dysregulation in multiple sclerosis favours pro-inflammatory T-cell-mediated autoimmunity Brain. 2011;134(12):3578-3589. doi:10.1093/brain/awr262 Brain | © The Author (2011). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Figure 5 Multiple sclerosis-associated miRNAs suppress Th2 and favour Th1 cytokine production. (A) Per cent change (over NS) in IFN-γ secretion detected by ELISA in 72 h supernatants of purified CD4⁺ T cells from Th1-prone B6 mice after stimulation with plate-bound anti-CD3/CD28 in Th neutral conditions following overnight transfection with miRNAs (top) or miRNA inhibitors (bottom). Mean ± SEM data from multiple replicates of two independent experiments. (B) IFN-γ (top), IL-4 (middle) and IL-5 (bottom) % change (over NS) detected by ELISA in supernatants 72 h after restimulation of DO11.10 TcR-tg Balb/c mouse T cells with OVA 323–339 peptides. Initial stimulation was performed 7–10 days earlier in Th neutral conditions, following overnight transfection with miRNAs or miRNA inhibitors. Mean ± SEM data from multiple replicates of two independent experiments. After significant ANOVA analysis, Dunnett's post hoc test P-values were calculated for the multiple sclerosis miRNA to control miRNA comparison and are shown above each group (**P ≤ 0.001, *P ≤ 0.05). From: Micro-RNA dysregulation in multiple sclerosis favours pro-inflammatory T-cell-mediated autoimmunity Brain. 2011;134(12):3578-3589. doi:10.1093/brain/awr262 Brain | © The Author (2011). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Figure 6 Multiple sclerosis-associated miRNAs suppress BMI1 and GATA3 in human CD4⁺ T cells and Th2 cytokine production. (A) BMI1 versus forward scatter (FSC) (top), GATA3⁺ versus FSC (middle) and BMI1 versus GATA3 (lower) intranuclear staining of CD4⁺ T cells from peripheral blood mononuclear cells of healthy donors transfected with NS, miR-27, miR-128, miR-340 or a combination of miR-27, miR-128 and miR-340 (miR mix), stimulated with anti-CD3/CD28 in Th neutral conditions, and analysed for intranuclear staining by flow cytometry. The panels in B show the mean ± SEM pooled results from three independent experiments (*t-test P ≤ 0.05). (C) Histogram of BMI1 and GATA3 expression in cells from A to B, showing the mean fluorescence intensity in CD4⁺ T cells transfected with control miRNA or a combination of multiple sclerosis-associated miRNAs. The plots show individual results representative of three independent experiments. (D) Peripheral blood mononuclear cells from healthy donors (HD) were transfected with multiple sclerosis-associated or control miRNAs, and peripheral blood mononuclear cells from patients with multiple sclerosis were transfected with miRNA inhibitors prior to primary stimulation with anti-CD3/CD28 in Th neutral conditions. Cells were rested and restimulated with anti-CD3/CD28 and IL-5 was measured by ELISA. Shows mean ± SEM of individual patient replicates, representative of two independent experiments. Dunnett's post hoc test P-values for the multiple sclerosis miRNA to control miRNA comparison are shown above each group (**P ≤ 0.01, *P ≤ 0.05). (E) Association between endogenous miRNA expression and Th2 pathway differentiation. Healthy donors (black) or multiple sclerosis (grey) purified naïve CD4⁺CD45RA⁺ T cells with low or high, respectively, miR-27+ miR-128 expression (shown on right axis) were stimulated with anti-CD3/CD28 in Th neutral conditions and, after differentiation, the percentage of IL-4 + Th2 cells assessed by flow cytometry (shown on the left axis). From: Micro-RNA dysregulation in multiple sclerosis favours pro-inflammatory T-cell-mediated autoimmunity Brain. 2011;134(12):3578-3589. doi:10.1093/brain/awr262 Brain | © The Author (2011). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com