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

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

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

miR-10a expression in Treg inversely correlates with susceptibility to autoimmune disease.

<p>qRT-PCR for miR-10a expression by FACS-purified CD4⁺CD25⁺CD62L^hi Treg. a) Amplification plots for miR-10a on Treg cDNA from B6 and NOD mice. The sno202 signal for B6 and NOD completely overlapped. The signal for Tconv is comparable to miR-10a in NOD Treg (data not shown). b) Relative miR-10a expression in Treg from B6, 129X1/SvJ, 129S6/SvEvTac, DBA/2J, BALB/c and NOD/ShiLtJ mice. Bars represent means of pooled data from 3 (B6), one (129X1/SvJ), one (129S6/SvEvTac), 2 (DBA/2J), one (BALB/c) and 3 (NOD/ShiLtJ) biologic replicates. Error bars: SEM. All samples were normalized to miR-10a expression in BALB/c mice.</p

All-trans retinoic acid but not TGF-ß induces miR-10a in CD4⁺ T cells.

<p>FACS-purified CD4⁺CD62L^hiGFP^- cells from FoxP3-GFP reporter mice were cultured with plate-bound anti-CD3 and anti-CD28 antibodies +/− TGF-ß and/or retinoic acid. After 72 h the CD4⁺GFP^- and CD4⁺GFP⁺ cells were purified by flow cytometry for RNA extraction. miRNA levels were assessed by qPCR in technical triplicates. Shown is a representative experiment of two independent experiments. Error bars: SD of triplicates.</p

miR-10a is dispensable for TGFβ and retinoic acid-mediated FoxP3 induction.

<p>Naïve CD4⁺CD25^-CD62L^hi Tconv were activated with anti-CD3 and anti-CD28 in the presence of RA, TGFβ or a combination of the two. Cells were from wildtype (“control”) or littermate miR-10a-deficient (“ko”) mice. Representative of 4 independent experiments.</p

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

Identification of MiR-205 As a MicroRNA That Is Highly Expressed in Medullary Thymic Epithelial Cells

<div><p>Thymic epithelial cells (TECs) support T cell development in the thymus. Cortical thymic epithelial cells (cTECs) facilitate positive selection of developing thymocytes whereas medullary thymic epithelial cells (mTECs) facilitate the deletion of self-reactive thymocytes in order to prevent autoimmunity. The mTEC compartment is highly dynamic with continuous maturation and turnover, but the genetic regulation of these processes remains poorly understood. MicroRNAs (miRNAs) are important regulators of TEC genetic programs since miRNA-deficient TECs are severely defective. However, the individual miRNAs important for TEC maintenance and function and their mechanisms of action remain unknown. Here, we demonstrate that miR-205 is highly and preferentially expressed in mTECs during both thymic ontogeny and in the postnatal thymus. This distinct expression is suggestive of functional importance for TEC biology. Genetic ablation of miR-205 in TECs, however, neither revealed a role for miR-205 in TEC function during homeostatic conditions nor during recovery from thymic stress conditions. Thus, despite its distinct expression, miR-205 on its own is largely dispensable for mTEC biology.</p></div