Abstract
CD4(+) regulatory T cells (Tregs) are essential for controlling immune responses and preventing autoimmunity. Their development requires regulation of gene expression by microRNAs (miRNAs). To understand miRNA function in Treg development, we searched for important miRNAs and their relevant target genes. Of the more abundantly expressed miRNAs in Tregs, only miR-15b/16, miR-24, and miR-29a impacted the production of in vitro-induced Tregs (iTregs) in overexpression and blocking experiments. miRNA mimics for these significantly enhanced the induction of iTregs in Dicer(-/-) CD4(+) T cells. Furthermore, the overexpression of miR-15b/16 in conventional CD4(+) T cells adoptively transferred into Rag2(-/-) mice increased the in vivo development of peripheral Tregs and diminished the severity of autoimmune colitis. In searching for targets of miR-15b/16, we observed that the mammalian target of rapamycin (mTOR) signaling pathway was enhanced in Dicer(-/-) CD4(+) T cells, and its pharmacological inhibition restored induction of iTregs. Suppression of mTOR signaling is essential for induction of iTregs from naive CD4(+) T cells, and the mTORC2 component, Rictor, contained a functional target site for miR-15b/16. Rictor was more abundantly expressed in Dicer(-/-) T cells as was mTOR, and their expression was downregulated by the overexpression of miR-15b/16. This led to a reduction in mTOR signaling, as measured by phosphorylation of the downstream target, ribosomal protein S6. Finally, knockdown of Rictor by small interfering RNAs enhanced Treg induction in Dicer(-/-) CD4(+) T cells. Therefore, an important mechanism of miRNA regulation of Treg development is through regulation of the mTOR signaling pathway.