The autoimmune regulator (AIRE) is essential for the establishment of central tolerance and prevention of autoimmunity. Interestingly, different AIRE mutations cause autoimmunity in either recessive or dominant-negative manners. Using engineered mouse models, we establish that some monoallelic mutants, including C311Y and C446G, cause breakdown of central tolerance. By using RNAseq, ATACseq, ChIPseq, and protein analyses, we dissect the underlying mechanisms for their dominancy. Specifically, we show that recessive mutations result in a lack of AIRE protein expression, while the dominant mutations in both PHD domains augment the expression of dysfunctional AIRE with altered capacity to bind chromatin and induce gene expression. Finally, we demonstrate that enhanced AIRE expression is partially due to increased chromatin accessibility of the AIRE proximal enhancer, which serves as a docking site for AIRE binding. Therefore, our data not only elucidate why some AIRE mutations are recessive while others dominant, but also identify an autoregulatory mechanism by which AIRE negatively modulates its own expression.

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Foxp3+ regulatory T cells (Tregs) are potent suppressor cells, essential for the maintenance of immune homeostasis. Most Tregs develop in the thymus and are then released into the immune periphery. However, some Tregs populate the thymus and constitute a major subset of yet poorly understood cells. Here we describe a subset of thymus recirculating IL18R+ Tregs with molecular characteristics highly reminiscent of tissue-resident effector Tregs. Moreover, we show that IL18R+ Tregs are endowed with higher capacity to populate the thymus than their IL18R– or IL18R–/– counterparts, highlighting the key role of IL18R in this process. Finally, we demonstrate that IL18 signaling is critical for the induction of the key thymus-homing chemokine receptor – CCR6 on Tregs. Collectively, this study provides a detailed characterization of the mature Treg subsets in the mouse thymus and identifies a key role of IL18 signaling in controlling the CCR6-CCL20-dependent migration of Tregs into the thymus.

FOXN1 is a transcription factor critical for the development of both thymic epithelial cell (TEC) and hair follicle cell (HFC) compartments. However, mechanisms controlling its expression remain poorly understood. To address this question, we performed thorough analyses of the evolutionary conservation and chromatin status of the Foxn1 locus in different tissues and states and identified several putative cis-regulatory regions unique to TECs versus HFCs. Furthermore, experiments using genetically modified mice with specific deletions in the Foxn1 locus and additional bioinformatic analyses helped us identify key regions and transcription factors involved in either positive or negative regulation of Foxn1 in both TECs and HFCs. Specifically, we identified SIX1 and FOXN1 itself as key factors inducing Foxn1 expression in embryonic and neonatal TECs. Together, our data provide important mechanistic insights into the transcriptional regulation of the Foxn1 gene in TEC versus HFC and highlight the role of FOXN1 in its autoregulation.

Thymic epithelial cells (TEC) play an indispensable role in the development and selection of immunocompetent, yet self-tolerant T cells. To provide further insights into TEC functional and developmental diversity, we utilized multiome analysis, which revealed a detailed atlas of the TEC compartment based on their transcriptional states and chromatin landscapes. The analysis also highlighted numerous unconventional TEC subsets, which shared striking similarities with functionally well-defined parenchymal populations, including endocrine cells, microfold cells or myocytes. Moreover, our fate mapping experiments revealed that most of these rare TEC “parenchymal analogues” differentiated from Csnb+ MHCIIhi mTEC precursors, with varying impacts of Aire on their development. By further focusing on the endocrine- and the microfold-TEC populations, we found that both subsets play different and non-redundant functional roles and require different transcription factors for their terminal differentiation. Specifically, while the endocrine-TEC required Insm1 for their development, and were critical for induction of self-tolerance to various endocrine tissues, the microfold-TEC required Spib for their development, and were essential for the generation of thymic IgA+ plasma cells. Collectively, our study reveals that MHCIIhi mTEC have the potential to differentiate into various types of molecularly functionally defined cells, which not only contribute to the induction of central tolerance, but also regulate homeostasis of other thymus-resident populations.

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