The thymic epithelial microRNA network elevates the threshold for infection-associated thymic involution via miR-29a mediated suppression of the IFN-α receptor.

Thymic output is a dynamic process, with high activity at birth punctuated by transient periods of involution during infection. Interferon-α (IFN-α) is a critical molecular mediator of pathogen-induced thymic involution, yet despite the importance of thymic involution, relatively little is known about the molecular integrators that establish sensitivity. Here we found that the microRNA network dependent on the endoribonuclease Dicer, and specifically microRNA miR-29a, was critical for diminishing the sensitivity of the thymic epithelium to simulated infection signals, protecting the thymus against inappropriate involution. In the absence of Dicer or the miR-29a cluster in the thymic epithelium, expression of the IFN-α receptor by the thymic epithelium was higher, which allowed suboptimal signals to trigger rapid loss of thymic cellularity.

Adult thymus contains FoxN1(-) epithelial stem cells that are bipotent for medullary and cortical thymic epithelial lineages.

Within the thymus, two major thymic epithelial cell (TEC) subsets-cortical and medullary TECs-provide unique structural and functional niches for T cell development and establishment of central tolerance. Both lineages are believed to originate from a common progenitor cell, yet the cellular and molecular identity of these bipotent TEC progenitors/stem cells remains ill defined. Here we identify rare stromal cells in the murine adult thymus, which under low-attachment conditions formed spheres (termed "thymospheres"). These thymosphere-forming cells (TSFCs) displayed the stemness features of being slow cycling, self-renewing, and bipotent. TSFCs could be significantly enriched based on their distinct surface antigen phenotype. The FoxN1 transcription factor was dispensable for TSFCs maintenance in situ and for commitment to the medullary and cortical TEC lineages. In summary, this study presents the characterization of the adult thymic epithelial stem cells and demonstrates the dispensability of FoxN1 function for their stemness.

An evolutionarily conserved mutual interdependence between Aire and microRNAs in promiscuous gene expression.

The establishment and maintenance of central tolerance depends to a large extent on the ability of medullary thymic epithelial cells to express a variety of tissue-restricted antigens, the so-called promiscuous gene expression (pGE). Autoimmune regulator (Aire) is to date the best characterised transcriptional regulator known to at least partially coordinate pGE. There is accruing evidence that the expression of Aire-dependent and -independent genes is modulated by higher order chromatin configuration, epigenetic modifications and post-transcriptional control. Given the involvement of microRNAs (miRNAs) as potent post-transcriptional modulators of gene expression, we investigated their role in the regulation of pGE in purified mouse and human thymic epithelial cells (TECs). Microarray profiling of TEC subpopulations revealed evolutionarily conserved cell type and differentiation-specific miRNA signatures with a subset of miRNAs being significantly upregulated during terminal medullary thymic epithelial cell differentiation. The differential regulation of this subset of miRNAs was correlated with Aire expression and some of these miRNAs were misexpressed in the Aire knockout thymus. In turn, the specific absence of miRNAs in TECs resulted in a progressive reduction of Aire expression and pGE, affecting both Aire-dependent and -independent genes. In contrast, the absence of miR-29a only affected the Aire-dependent gene pool. These findings reveal a mutual interdependence of miRNA and Aire.

Aire-dependent genes undergo Clp1-mediated 3'UTR shortening associated with higher transcript stability in the thymus.

The ability of the immune system to avoid autoimmune disease relies on tolerization of thymocytes to self-antigens whose expression and presentation by thymic medullary epithelial cells (mTECs) is controlled predominantly by Aire at the transcriptional level and possibly regulated at other unrecognized levels. Aire-sensitive gene expression is influenced by several molecular factors, some of which belong to the 3'end processing complex, suggesting they might impact transcript stability and levels through an effect on 3'UTR shortening. We discovered that Aire-sensitive genes display a pronounced preference for short-3'UTR transcript isoforms in mTECs, a feature preceding Aire's expression and correlated with the preferential selection of proximal polyA sites by the 3'end processing complex. Through an RNAi screen and generation of a lentigenic mouse, we found that one factor, Clp1, promotes 3'UTR shortening associated with higher transcript stability and expression of Aire-sensitive genes, revealing a post-transcriptional level of control of Aire-activated expression in mTECs.

A Thymic Epithelial Stem Cell Pool Persists throughout Ontogeny and Is Modulated by TGF-ß.

Adult tissue-specific stem cells (SCs) mediate tissue homeostasis and regeneration and can give rise to all lineages in the corresponding tissue, similar to the early progenitors that generate organs in the first place. However, the developmental origins of adult SCs are largely unknown. We recently identified thymosphere-forming stem cells (TSFCs) in the adult mouse thymus, which display genuine stemness features and can generate the two major thymic epithelial cell lineages. Here, we show that embryonic TSFCs possess stemness features but differ from adult TSFCs in surface marker profile. Our findings support the model of a continuous thymic SC lineage that is maintained throughout ontogeny. TGF-ß signaling differentially affects embryonic versus adult thymosphere formation, suggesting that thymic epithelial SC potency depends on both developmental stage and environmental signals. Collectively, our findings suggest that embryonic TSFCs contribute to an adult SC pool and that TSFC plasticity is controlled by TGF-ß signaling.

Promiscuous Gene Expression in the Thymus: A Matter of Epigenetics, miRNA, and More?

The induction of central tolerance in the course of T cell development crucially depends on promiscuous gene expression (pGE) in medullary thymic epithelial cells (mTECs). mTECs express a genome-wide variety of tissue-restricted antigens (TRAs), preventing the escape of autoreactive T cells to the periphery, and the development of severe autoimmunity. Most of our knowledge of how pGE is controlled comes from studies on the autoimmune regulator (Aire). Aire activates the expression of a large subset of TRAs by interacting with the general transcriptional machinery and promoting transcript elongation. However, further factors regulating Aire-independent TRAs must be at play. Recent studies demonstrated that pGE in general and the function of Aire in particular are controlled by epigenetic and post-transcriptional mechanisms. This mini-review summarizes current knowledge of the regulation of pGE by miRNA and epigenetic regulatory mechanisms such as DNA methylation, histone modifications, and chromosomal topology.