MicroRNA 10a marks regulatory T cells

Abstract

MicroRNAs (miRNAs) are crucial for regulatory T cell (Treg) stability and function. We report that microRNA-10a (miR-10a) is expressed in Tregs but not in other T cells including individual thymocyte subsets. Expression profiling in inbred mouse strains demonstrated that non-obese diabetic (NOD) mice with a genetic susceptibility for autoimmune diabetes have lower Treg-specific miR-10a expression than C57BL/6J autoimmune resistant mice. Inhibition of miR-10a expression in vitro leads to reduced FoxP3 expression levels and miR-10a expression is lower in unstable "exFoxP3" T cells. Unstable in vitro TGF-ß-induced, iTregs do not express miR-10a unless cultured in the presence of retinoic acid (RA) which has been associated with increased stability of iTreg, suggesting that miR-10a might play a role in stabilizing Treg. However, genetic ablation of miR-10a neither affected the number and phenotype of natural Treg nor the capacity of conventional T cells to induce FoxP3 in response to TGFβ, RA, or a combination of the two. Thus, miR-10a is selectively expressed in Treg but inhibition by antagomiRs or genetic ablation resulted in discordant effects on FoxP3.

Figure 1. Treg miRNA expression signature.

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.

Figure 2. miR-10a marks Treg cells.

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.

Figure 3. All-trans retinoic acid but not TGF-ß induces miR-10a in CD4⁺ T cells.

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.

Figure 4. Treg-specific miR-10a modulates FoxP3 stability in vitro.

FACS-purified CD4⁺CD62L^hiGFP⁺ cells from FoxP3-GFP reporter mice were cultured with anti-CD3 and anti-CD28 beads and 2000U IL-2/ml for 48 h. Time course of antagomiR-DY547 transfection in cultured Treg (a). FoxP3 staining 48 h after culture in the presence of 50 µg/ml antagomiR-155 or antagomiR-10a (b). Grey shaded histograms represent nontransfected cells, black lines represent cells treated with antagomiR. Shown is one experiment (a) and one representative experiment from three (b) independent experiments.

Figure 5. miR-10a is dispensable for TGFβ and retinoic acid-mediated FoxP3 induction.

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.

Figure 6. miR-10a expression in Treg inversely correlates with susceptibility to autoimmune disease.

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.