The thymus is responsible for generating a diverse yet self-tolerant pool of T cells. Although the thymic medulla consists mostly of developing and mature AIRE epithelial cells, recent evidence has suggested that there is far greater heterogeneity among medullary thymic epithelial cells than was previously thought. Here we describe in detail an epithelial subset that is remarkably similar to peripheral tuft cells that are found at mucosal barriers. Similar to the periphery, thymic tuft cells express the canonical taste transduction pathway and IL-25. However, they are unique in their spatial association with cornified aggregates, ability to present antigens and expression of a broad diversity of taste receptors. Some thymic tuft cells pass through an Aire-expressing stage and depend on a known AIRE-binding partner, HIPK2, for their development. Notably, the taste chemosensory protein TRPM5 is required for their thymic function through which they support the development and polarization of thymic invariant natural killer T cells and act to establish a medullary microenvironment that is enriched in the type 2 cytokine, IL-4. These findings indicate that there is a compartmentalized medullary environment in which differentiation of a minor and highly specialized epithelial subset has a non-redundant role in shaping thymic function.

SUMMARY Inducing immune tolerance to prevent rejection is a key step toward successful engraftment of stem-cell-derived tissue in a clinical setting. Using human pluripotent stem cells to generate thymic epithelial cells (TECs) capable of supporting T cell development represents a promising approach to reach this goal; however, progress toward generating functional TECs has been limited. Here, we describe a robust in vitro method to direct differentiation of human embryonic stem cells (hESCs) into thymic epithelial progenitors (TEPs) by precise regulation of TGFβ, BMP4, RA, Wnt, Shh, and FGF signaling. The hESC-derived TEPs further mature into functional TECs that support T cell development upon transplantation into thymus-deficient mice. Importantly, the engrafted TEPs produce T cells capable of in vitro proliferation as well as in vivo immune responses. Thus, hESC-derived TEP grafts may have broad applications for enhancing engraftment in cell-based therapies as well as restoring age-and stress-related thymic decline.

Summary The autoimmune regulator (AIRE) is essential for prevention of autoimmunity; its role is best understood in the thymus where it promotes self-tolerance through tissue-specific antigen (TSA) expression. Recently, extrathymic Aire-expressing cells (eTACs) have been described in murine secondary lymphoid organs, but the identity of such cells and their role in immune tolerance remains unclear. Here we have shown that eTACs are a discrete major histocompatibility complex class II (MHC II)hi, CD80lo, CD86lo, epithelial cell adhesion molecule (EpCAM)hi, CD45lo bone marrow-derived peripheral antigen presenting cell (APC) population. We also have demonstrated that eTACs can functionally inactivate CD4+ T cells through a mechanism that does not require regulatory T cells (Treg), and is resistant to innate inflammatory stimuli. Together these findings further define eTACs as a distinct tolerogenic cell population in secondary lymphoid organs.

Thymic epithelial cells in the medulla (mTECs) play a critical role in enforcing central tolerance through expression and presentation of tissue-specific antigens (TSAs) and deletion of autoreactive thymocytes. TSA expression requires autoimmune regulator (Aire), a transcriptional activator present in a subset of mTECs characterized by high CD80 and MHC II expression and a lack of potential for differentiation or proliferation. Here, using an Aire-DTR transgenic line, we show that short-term ablation specifically targets Aire+ mTECs, which quickly undergo RANK-dependent recovery. Repeated ablation also affects Aire− mTECs, and using an inducible Aire-Cre fate-mapping system, we find that this results from the loss of a subset of mTECs that showed prior expression of Aire, maintains intermediate TSA expression, and preferentially migrates towards the center of the medulla. These results clearly identify a distinct stage of mTEC development and underscore the diversity of mTECs that play a key role in maintaining tolerance.