Given the opposing roles of regulatory versus conventional helper CD4+
T cells, it is puzzling how few protein-coding genes are differentially expressed among these T cell subsets. Most differences are quantitative rather than qualitative 
, yet, the sum of differential gene expression is sufficient to imprint highly specialized functions in each subset. Likewise, the only miRNA specific for Treg cells was miR-10a. Interestingly, miR-10a expression seems to be specifically induced in Treg since the two flanking protein-coding genes are not detectable (HoxB5) or barely expressed (HoxB4) in naive, Th1, Th2, Th17, induced Treg (iTreg) or nTreg 
. These observations suggest that thymic Treg sense a signal to actively and specifically induce miR-10a.
The expression data suggests a correlation with more stable Treg and combined with the in vitro
data supports the possibility that this miRNA may be involved in long-term maintenance of Treg stability. Interestingly RA, secreted by a specialized subset of CD103+
dendritic cells, is important for inducing Treg in the gut 
. More importantly, in contrast to TGF-ß alone, the combination of TGF-ß and RA not only increases the frequency of FoxP3 expressing T cells in vitro
but the induced FoxP3+
cells are more stable, particularly in an inflammatory environment 
. Given the strong induction of miR-10a by RA, the added benefit of RA might partially be accomplished through miR-10a, which is embedded in the HoxB cluster, a genomic region with known RA responsiveness. In a pancreatic cancer cell line RA directly binds to a retinoic acid response element (RARE) in the HoxB cluster and induces miR-10a 
. Therefore, it seems likely that in T cells RA directly induces miR-10a through direct binding of RARE elements in the HoxB/miR-10a locus. Pharmacologic miR-10a inhibition did indeed inhibit the expression level of miR-10a. However, miR-10a-deficient mice are fertile and age without obvious phenotypic abnormalities (Stadthagen & Lund, unpublished results). Treg numbers and phenotype appear normal under homeostatic conditions (data not shown) and miR-10a was dispensable for FoxP3 induction in naïve T cells. Furthermore, miR-10a-deficient Treg cells suppress colitis in an adoptive transfer model (Jeker & Bluestone, unpublished results). Yet, the importance of this miRNA may only be apparent under certain pathogenic or other stress settings 
and deKouchkovsky et al., MS submitted. This notion is supported by the fact that NOD Treg express very low levels of miR-10a without NOD mice developing scurfy-like disease and miR-10a-deficient Treg are present in normal numbers and express normal levels of FoxP3 (data not shown).
How can the discrepancy between in vitro inhibition of miR-10a expression with antagomiRs versus genetic ablation be explained? It is possible that antagomiR-10a had off-target effects, e.g. inhibition of miR-10b and miR-125a, which has sequence homology or interaction with other targets. In fact, antagomiR-125a did reduce FoxP3 expression to some extent (although to a lesser degree than antagomiR-10a) but the combination of antagomiR-10a and antagomiR-125a did not further enhance FoxP3 downregulation (data not shown). Alternatively, miR-10b might compensate for the absence of miR-10a in miR-10a-deficient Treg, since miRNAs are often redundant 
. Temporally controlled conditional miR-10a ablation might reveal its function 
. Finally, miR-10a might be expressed and functional only in a subset of Treg as it has become clear that CD4+
cells are heterogeneous and may be functionally diverse, depending on tissue location and activation status 
In summary, further studies are required to investigate miR-10a function in vivo
. Despite an intriguing expression pattern, we were not able to define the miR-10a function in Treg in vivo. Importantly, our results suggest that phenotypic effects obtained using antagomiRs and possibly other classes of pharmacological miRNA inhibitors should be interpreted with a certain degree of caution. Of note, similar discrepancies between pharmacological miRNA inhibition and genetic ablation have been described for another miRNA and other pharmacologic inhibitors 
In summary, it appears that Treg do not express a specific miRNA essential for Treg function. Although absence of all miRNAs leads to spontaneous scurfy-like disease 
, no individual miRNA required for Treg function under homeostatic conditions has been described yet. The Treg-enriched miRNAs miR-155 and miR-10a are on their own largely dispensable for global Treg function under homeostatic conditions in vivo
and data not shown. Even the role of miR-146a remains unclear as the studies of miR-146a-deficient Tregs involved the co-transfer of miR-146a-deficient Treg along with miR-146a-deficient hyper-reactive IFN-γ secreting Teff and scurfy bone marrow as well as a pre-transplant preconditioning regimen 
. Therefore, the function of miR-146a in Tregs under homeostatic conditions has not been determined. Thus, the absence of the protective layer of multiple miRNAs may predispose Treg to dysfunction, particularly in an inflammatory milieu and might constitute a risk factor for the development of autoimmune disease but absence of several individual miRNAs does not lead to complete Treg dysfunction.