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.
Highlights
We hypothesized that important miRNAs would be abundant and more highly expressed in induced Tregs (iTregs) compared with conventionally activated CD4+ T cells or those polarized to other Th subsets
These miRNAs were felt to be the most likely candidates to be involved in the induction of iTregs, so their relative level was determined by quantitative real-time PCR (qPCR) in iTregs and compared with other Th subsets
These included resting naive CD4+ T cells and those activated under nonpolarizing or polarizing conditions toward Th1, Th2, or Th17 subsets. miRNAs that were more abundantly expressed in ex vivo regulatory T cell (Treg) were the most abundant in iTregs compared with T cells cultured in other conditions; those that were not abundantly expressed in ex vivo Tregs were not abundantly expressed in iTregs (Fig. 1A)
Summary
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. We found three miRNAs (miR15b/16, miR-24, and miR-29a) that regulated the induction of Tregs from naive CD4+ T cells, with miR-15b/16 having the greatest effect in overexpression and blocking experiments. The miRNAs that had the greatest effect in these experiments were miR-15b and miR-16, which are encoded within the same primary transcript and are closely related, such that they target the same sequences in mRNAs. Overexpression of miR15b/16 significantly increased the induction of iTregs compared with cells transduced with a control retrovirus lacking any miRNA sequences (Fig. 1B).
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