Efficient and Treg-specific deletion of <i>Dgcr8</i> and miR-150.

<p>Conventional CD4⁺GFP⁻ (Tc) and CD4⁺GFP⁺ regulatory (Tr) T cells were FACS sorted from FoxP3-GFP reporter mice (a) and FoxP3-GFP-hCre:DGCR8^wt/lox (het) or FoxP3-GFP-hCre:DGCR8^lox/lox (KO) mice (b-d). qPCR analysis of Dgcr8 mRNA in Tc and Tr (a,b) and miR-150 (c,d). Pooled data from 3 independent experiments with 3 mice total (a,b) and representative data from 2 independent experiments with 2 mice total (c,d).</p

Mice with Treg lacking canonical miRNAs develop scurfy-like disease.

<p>Macroscopic (a), flow cytometric (b,c) and histologic (d) analysis of the disease spontaneously occurring in FoxP3-GFP-hCre:DGCR8^lox/lox (KO) mice. FoxP3-GFP-hCre:DGCR8^wt/lox (het) served as control mice. (a) Splenomegaly (top) and lymphadenopathy (bottom) in het and KO mice. (b) Lymph nodes (LN) were harvested from ≥3 week old het and KO mice. Single cell suspensions stained with anti-CD44 and anti-CD62L to determine activation of CD4⁺ Tconv (gated on CD4⁺GFP⁻) as an indirect readout of Treg function were analyzed by flow cytometry (FACS). The increased frequency of CD44^hiCD62L^lo cells among CD4⁺GFP⁻ lymphocytes represents spontaneous activation of Tconv in lymph node cells. Representative FACS plots from >10 independent experiments. (c) Quantification of frequency of activated CD44^hiCD62L^lo among CD4⁺GFP⁻ Tconv in peripheral blood of 3–4 week old mice. n = 35 (het), n = 27 (KO). p<0.0001 (Two-tailed Mann-Whitney Test). (d) Representative paraffin-embedded sections of liver and lung tissues stained with Hematoxylin & Eosin of het and KO mice. Pictures were taken with 100×optical magnification. 4/4 KO livers and 4/4 KO lungs had infiltrates, 0/4 het livers or lungs were infiltrated.</p

Early lethality in mice with a Treg-specific <i>Dgcr8</i> deficiency.

<p>Survival curve of pooled female and male FoxP3-GFP-hCre:DGCR8^wt/lox (het) or FoxP3-GFP-hCre:DGCR8^lox/lox (KO) mice. Mice found dead or required to be euthanized due to severe body condition as per institutional requirements were collectively flagged as “dead” for the analysis. P<0.0001 (Log-rank (Mantel-Cox) Test.</p

Canonical miRNAs stabilize FoxP3 expression.

<p>Flow cytometric analysis of FoxP3 stability in lymphocytes isolated from FoxP3-GFP-hCre:DGCR8^wt/lox (het) and FoxP3-GFP-hCre:DGCR8^lox/lox (KO) mice. (a) Representative FACS histograms of FoxP3 intracellular staining of CD4⁺GFP⁻ Tconv and CD4⁺GFP⁺ Treg with heterozygous (het) or homozygous deletion of Dgcr8 (KO) isolated from lymph nodes (LN). (b) Quantification of FoxP3 median fluorescence intensity (MFI) in het and KO Treg cells isolated from LN. The MFI was normalized due to inter-experimental variability of the relative FoxP3 MFI. For normalization the MFI of the control Treg was set to 1 and the relative reduction of MFI was calculated for the ko. In experiments with more than one control the mean MFI of the het controls was set to 1. In each individual experiment the het control Treg displayed a higher FoxP3 MFI than ko Treg. Statistical analysis was performed on pooled normalized data from four independent experiments. p = 0.0075 (Two-tailed Mann-Whitney Test). (c) FACS sorted CD4⁺GFP⁺ Treg were cultured with beads coated with anti-CD3 and anti-CD28 antibodies and 2000 U/ml IL-2 and live cells were counted. (d) Treg were purified and cultured under expansion conditions as described for panel c. At 41 h and after 4 days samples were stained for intracellular FoxP3 as in panel a. The bar indicates the gate used to define the cutoff for FoxP3 staining. Numbers indicate the % of FoxP3 expressing cells. Representative growth curve (c) and representative FACS plots (d) from 2 independent experiments.</p

Generation and Characterization of miR-155-5p sensor transgenic mice.

<p>(A) Schematic illustration of the BAC transgenic constructs. Left panel-R26-DTR-BAC lacking miR-155-5p target sequence. Right panel-R26-DTR-155T-BAC containing 4×miR-155-5p target sequence in the 3’-UTR of DTR-BFP fusion gene. The fusion cassette gene was then placed downstream of a STOP element flanked by LoxP sites driven by Rosa26 promoter. (B) Schematic illustration of the determination of miR-155-5p activity in single living cell by flow cytometry. The relative activity of miR-155-5p will be inversely proportional to the expression level of DTR or BFP. (C) Phenotype of R26-DTR-155T mice. Figures in the upper panel show FACS profiling of DTR expression in conventional (conv) CD4-gated cells and Treg-gated cells of different mice as shown. Two founders, 47 and 84, of the R26-DTR-155T mice and a R26-DTR mouse were crossed with CMV-Cre mice; lymph node (LN) cells of the littermates were stained with anti-DTR-Biotin and then conjugated with Streptavidin-PE along with anti-CD4 and anti-Foxp3 (intracellular staining) and analyzed through FACS Calibur. Figures in the lower panel represent DTR MFI of indicated cells (shown in the figures of upper panel). Significance was calculated by Student’s <i>t</i>-test using GraphPad Prism 5 software. N = 3, ns: not significant; *P<0.05; **P<0.01.</p

Inhibition of miR-155-5p with antagomir-155 rapidly releases DTR expression.

<p>LN cells of CMV-Cre x R26-YFP × R26-DTR-155T mice founder 47 were stimulated with anti-CD3 for 48 hours. The activated cells were then transfected with 200 nM antagomir-155 or 200 nM antagomir-142 as negative control. FACS analysis was performed after 18 hours of transduction for detecting DTR expression. Left panel shows the dot plots of DTR expressing cells transduced with indicated antagomir. Middle panel shows the overlaid histograms of DTR expression between antagomir-155 and antagomir-142 transfected cells. Right panel shows DTR MFI of indicated cells. Significance was calculated by Student’s <i>t</i>-test using GraphPad Prism 5 software. N = 3, ****P<0.0001.</p

Validation of miR-155-5p-OFF system <i>in vitro</i>.

<p>(A) Validation of miR-155-5p target using luciferase assay, HEK293T cells were transfected with pmirGLO-4xmiR-155-5pT plus pEF-BOS-EX-miR-155, pEF-BOS-EX-miR-146a, and pEF-BOS-EX-empty vector (control), respectively, at a molar ratio of 1:1. (B) Validation of miR-155-5p target by flow cytometry, expression vector for miR-155 (pEF-BOS-EX-miR-155) or a control vector (pEF-BOS-EX) was co-transfected into HEK293T cells with an expression vector for miR-155-5p target reporter sequence (pDTR.BFP-155T) at various molar ratios. The expression of BFP reporter signal was analyzed through flow cytometry after 48 hours of transfection. (C and D) Sensitivity of miR-155-5p-OFF system. Fold change of mean fluorescent intensity of DTR expression in HEK293T cells transfected with pDTR.BFP-155T-N1 plasmid with increasing concentrations (0, 1.25, 2.5, 5, 10, 20, and 40 nM) of synthetic miR-155 or miR control. Significance was calculated by Student’s <i>t</i>-test using GraphPad Prism 5 software. N = 3, ns: not significant; *P<0.05; **P<0.01, ***P<0.001, ****P<0.0001.</p

A Novel Transgenic Mouse Line for Tracing MicroRNA-155-5p Activity <i>In Vivo</i>

<div><p>MicroRNA-155 (miR-155) plays significant role in various physiological processes involving both innate and adaptive immunity. miR-155 expression level changes dynamically during various immune responses. However, current approaches for miR-155 detection at the RNA level do not precisely reflect the real-time activity. Herein, we generated a transgenic mouse line (R26-DTR-155T) for determination of miR-155-5p activity <i>in vivo</i> by inserting miR-155-5p target sequence downstream of a reporter transgene comprising Diphtheria Toxin Receptor and TagBlue fluorescence protein. Using this approach, R26-DTR-155T mice were able to measure variation in levels of miR-155-5p activity in specific cell types of interest. The DTR expression levels were inversely correlated with the endogenous miR-155 expression pattern as detected by quantitative RT-PCR. Our data demonstrate a novel transgenic mouse line which could be useful for tracing miR-155-5p activity in specific cell types through measurement of miR-155-5p activity at single cell level.</p></div

miR-10a marks Treg cells.

<p>qPCR analysis of relative expression of miR-10a in purified T cells. a) Thymocytes: CD4^-CD8^- double negative (DN), CD4⁺CD8⁺ double positive (DP), CD4⁺CD8^- single positive (CD4SP), CD8⁺CD4^- single positive (CD8SP), CD4⁺CD8^-FoxP3-GFP^- (CD4SP GFP^-) and CD4⁺CD8^-FoxP3-GFP⁺ (CD4 SP GFP⁺). b) CD4 SP FoxP3-GFP^-R26YFP^- (GFP^-YFP^-), CD4 SP FoxP3-GFP⁺R26YFP^- (GFP⁺YFP^-) and CD4 SP FoxP3-GFP⁺R26YFP⁺ (GFP⁺YFP⁺) thymocytes. c) CD4⁺GFP^-YFP^- (Tconv), CD4⁺GFP⁺YFP⁺ (nTreg) and CD4⁺GFP^-YFP⁺ (exFoxP3) cells purified from pooled LN and spleen. Shown is one representative experiment from four (a) and two (b, c) independent experiments. Error bars: SD of triplicates.</p

Treg miRNA expression signature.

<p>a) miRNA microarray analysis of CD4⁺CD25^-GFP^- (Tconv) and CD4⁺CD25^hiGFP⁺ (Treg cells) purified from lymph nodes from female FoxP3-GFP-hCre reporter mice. Shown are 4 technical replicates from the same slide (one biologic replicate). b) qPCR of relative miR-10a expression by sorted Tconv (GFP^-) and Treg (GFP⁺). One representative example of >7 independent experiments from >7 independent biologic replicates. Error bars: SD of technical triplicates.</p