Explore the vast range of titles available.

The thymus is responsible for generating a diverse yet self-tolerant pool of T cells 1 . 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 2 . Here we describe in detail an epithelial subset that is remarkably similar to peripheral tuft cells that are found at mucosal barriers 3 . 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. A comprehensive analysis of the thymic medulla identifies a tuft-cell-like thymic epithelial cell population that is necessary for shaping thymic function.

Establishing effective central tolerance requires the promiscuous expression of tissue-restricted antigens by medullary thymic epithelial cells. However, whether central tolerance also extends to post-translationally modified proteins is not clear. Here we show a mouse model of autoimmunity in which disease development is dependent on post-translational modification (PTM) of the tissue-restricted self-antigen collagen type II. T cells specific for the non-modified antigen undergo efficient central tolerance. By contrast, PTM-reactive T cells escape thymic selection, though the PTM variant constitutes the dominant form in the periphery. This finding implies that the PTM protein is absent in the thymus, or present at concentrations insufficient to induce negative selection of developing thymocytes and explains the lower level of tolerance induction against the PTM antigen. As the majority of self-antigens are post-translationally modified, these data raise the possibility that T cells specific for other self-antigens naturally subjected to PTM may escape central tolerance induction by a similar mechanism. Post-translational modifications are associated with autoimmune diseases but definitive evidence of their contribution to escape from central tolerance mechanisms is needed. Here, the authors show that T cells specific for post-translational modifications of type II collagen escape intrathymic tolerance induction in a mouse model of rheumatoid arthritis.

Key Points Self-tolerance of T cells is commonly divided into central (thymic) and peripheral tolerance according to its site of induction. Self-tolerance to most peripheral parenchymal organs has been ascribed to peripheral tolerance. The finding that many tissue-specific genes are expressed by medullary thymic epithelial cells (mTECs) — known as promiscuous gene expression — has changed this view. Tissue-specific self-antigens expressed by mTECs are functionally and structurally highly diverse and encompass essentially all organs. This allows self-antigens, which are expressed in a spatially or temporally restricted manner (such as pregnancy- or puberty-associated self-antigens) to become continuously accessible to developing T cells. mTECs express self-antigens of both large and small organs at similar frequencies, therefore equating possible differences in the tolerogenic potential of organs of varying size. Both genetic and epigenetic mechanisms seem to account for this unorthodox mode of gene expression. Deficiencies in promiscuous gene expression can lead to organ-specific or multi-organ autoimmune syndromes. Promiscuous gene expression might have co-evolved with adaptive immunity in the wake of antigen-receptor diversity early during vertebrate development. The thymus has been viewed as the main site of tolerance induction to self-antigens that are specifically expressed by thymic cells and abundant blood-borne self-antigens, whereas tolerance to tissue-restricted self-antigens has been ascribed to extrathymic (peripheral) tolerance mechanisms. However, the phenomenon of promiscuous expression of tissue-restricted self-antigens by medullary thymic epithelial cells has led to a reassessment of the role of central T-cell tolerance in preventing organ-specific autoimmunity. Recent evidence indicates that both genetic and epigenetic mechanisms account for this unorthodox mode of gene expression. As we discuss here, these new insights have implications for our understanding of self-tolerance in humans, its breakdown in autoimmune diseases and the significance of this tolerance mode in vertebrate evolution.

How expression of tissue-restricted self-antigens (TRA) is coordinated in medullary thymic epithelial cells (mTECs) remains unclear. Steinmetz and colleagues use single-cell RNA-sequencing to describe clusters of co-expressed TRAs. Expression of tissue-restricted self antigens (TRAs) in medullary thymic epithelial cells (mTECs) is essential for the induction of self-tolerance and prevents autoimmunity, with each TRA being expressed in only a few mTECs. How this process is regulated in single mTECs and is coordinated at the population level, such that the varied single-cell patterns add up to faithfully represent TRAs, is poorly understood. Here we used single-cell RNA sequencing and obtained evidence of numerous recurring TRA–co-expression patterns, each present in only a subset of mTECs. Co-expressed genes clustered in the genome and showed enhanced chromatin accessibility. Our findings characterize TRA expression in mTECs as a coordinated process that might involve local remodeling of chromatin and thus ensures a comprehensive representation of the immunological self.

Promiscuous expression of tissue-restricted autoantigens in medullary thymic epithelial cells (mTECs) imposes central T cell tolerance. The molecular regulation of this unusual gene expression is not understood, in particular its delineation from cell lineage-specific gene expression control remains unclear. Here, we compared the expression profile of the casein gene locus in mTECs and mammary gland epithelial cells by single cell PCR. Mammary gland cells showed highly correlated intra- and interchromosomal coexpression of milk proteins (the casein genes, lactalbumin-α and whey acidic protein) and one of its transcriptional regulators (Elf5). In contrast, coexpression of these genes in mature CD80hi mTECs was rarely observed and no pattern of gene expression in individual mTECs was discernible. The apparent stochastic expression pattern of genes within the casein locus, the lower mRNA levels compared with mammary gland cells in conjunction with frequent coexpression of insulin in single mTECs clearly delineates the molecular mechanism(s) of promiscuous gene expression from cell lineage-specific gene control.

Promiscuous expression of numerous tissue-restricted self-antigens (TRAs) in medullary thymic epithelial cells (mTECs) is essential to safeguard self-tolerance. A distinct feature of promiscuous gene expression is its mosaic pattern (i.e., at a given time, each self-antigen is expressed only in 1–3% of mTECs). How this mosaic pattern is generated at the single-cell level is currently not understood. Here, we show that subsets of human mTECs expressing a particular TRA coexpress distinct sets of genes. We identified three coexpression groups comprising overlapping and complementary gene sets, which preferentially mapped to certain chromosomes and intrachromosomal gene clusters. Coexpressed gene loci tended to colocalize to the same nuclear subdomain. The TRA subsets aligned along progressive differentiation stages within the mature mTEC subset and, in vitro, interconverted along this sequence. Our data suggest that single mTECs shift through distinct gene pools, thus scanning a sizeable fraction of the overall repertoire of promiscuously expressed self-antigens. These findings have implications for the temporal and spatial (re)presentation of self-antigens in the medulla in the context of tolerance induction.

Trigger for autoimmunity The human thymus is tasked to teach T-cells which antigens are foreign and which are 'self', a process that appears to go wrong in autoimmune disorders. A study of the variation in the promoter of one gene expressed in the thymus shows that a single nucleotide change can disrupt gene regulation, and increase susceptibility to autoimmune disease. The gene, CHRNA1 , encodes a subunit of the muscle acetylcholine receptor, a target for autoantibodies in the neuromuscular disease autoimmune myasthenia gravis. The human thymus is given the difficult task of teaching T-cells which antigens are 'foreign' and which are 'self', a process which seems to go wrong in autoimmune disorders. A study of variation in the promoter of one gene expressed in thymus shows that one nucleotide change can disrupt gene regulation, and might lead to increased risk for autoimmune disease. Promiscuous expression of tissue-restricted auto-antigens in the thymus imposes T-cell tolerance and provides protection from autoimmune diseases 1 , 2 , 3 . Promiscuous expression of a set of self-antigens occurs in medullary thymic epithelial cells 4 , 5 and is partly controlled by the autoimmune regulator (AIRE), a nuclear protein for which loss-of-function mutations cause the type 1 autoimmune polyendocrine syndrome 6 , 7 . However, additional factors must be involved in the regulation of this promiscuous expression. Here we describe a mechanism controlling thymic transcription of a prototypic tissue-restricted human auto-antigen gene, CHRNA1 . This gene encodes the α-subunit of the muscle acetylcholine receptor, which is the main target of pathogenic auto-antibodies in autoimmune myasthenia gravis 8 , 9 . On re-sequencing the CHRNA1 gene, we identified a functional bi-allelic variant in the promoter that is associated with early onset of disease in two independent human populations (France and United Kingdom). We show that this variant prevents binding of interferon regulatory factor 8 (IRF8) and abrogates CHRNA1 promoter activity in thymic epithelial cells in vitro . Notably, both the CHRNA1 promoter variant and AIRE modulate CHRNA1 messenger RNA levels in human medullary thymic epithelial cells ex vivo and also in a transactivation assay. These findings reveal a critical function of AIRE and the interferon signalling pathway in regulating quantitative expression of this auto-antigen in the thymus, suggesting that together they set the threshold for self-tolerance versus autoimmunity.

Thymic central tolerance comprehensively imprints the T-cell receptor repertoire before T cells seed the periphery. Medullary thymic epithelial cells (mTECs) play a pivotal role in this process by virtue of promiscuous expression of tissue-restricted autoantigens. The molecular regulation of this unusual gene expression, in particular the involvement of epigenetic mechanisms is only poorly understood. By studying promiscuous expression of the mouse casein locus, we report that transcription of this locus proceeds from a delimited region (“entry site”) to increasingly complex patterns along with mTEC maturation. Transcription of this region is preceded by promoter demethylation in immature mTECs followed upon mTEC maturation by acquisition of active histone marks and local locus decontraction. Moreover, analysis of two additional gene loci showed that promiscuous expression is transient in single mTECs. Transient gene expression could conceivably add to the local diversity of self-antigen display thus enhancing the efficacy of central tolerance.

Key Points The cell fate decisions of developing thymocytes are coordinated by interactions with self-peptide–MHC complexes that are displayed by various types of thymic antigen presenting cells (APCs). Different thymic APCs use cell type-specific strategies of self antigen sampling and processing. Cortical thymic epithelial cells (cTECs) use unique proteolytic pathways to generate MHC class I-bound and MHC class II-bound peptides, and these 'private' peptides expressed by cTECs are critical for the positive selection of a fully functional T cell repertoire. Several types of haematopoieteic and non-haematopoietic APCs cooperatively present self antigens for central tolerance induction. Medullary thymic epithelial cells (mTECs) promiscuously express peripheral self antigens and autonomously present these to thymocytes. Different subsets of dendritic cells sample blood-borne and mTEC-derived self antigens within the thymus or transport peripheral self antigens into the thymus. Here, the authors describe the key characteristics of the different antigen-presenting cell (APC) populations that govern T cell development in the thymus. They discuss how the interactions that occur between thymocytes and thymic APCs shape the mature T cell repertoire, and how they subsequently affect the nature of peripheral immune responses. The fate of developing T cells is specified by the interaction of their antigen receptors with self-peptide–MHC complexes that are displayed by thymic antigen-presenting cells (APCs). Various subsets of thymic APCs are strategically positioned in particular thymic microenvironments and they coordinate the selection of a functional and self-tolerant T cell repertoire. In this Review, we discuss the different strategies that these APCs use to sample and process self antigens and to thereby generate partly unique, 'idiosyncratic' peptide–MHC ligandomes. We discuss how the particular composition of the peptide–MHC ligandomes that are presented by specific APC subsets not only shapes the T cell repertoire in the thymus but may also indelibly imprint the behaviour of mature T cells in the periphery.

Key Points Interactions with self-peptide–MHC complexes on thymic epithelial cells are crucial for thymocyte survival (positive selection) and CD4 versus CD8 lineage commitment, but can also result in apoptotic cell death (negative selection). It is increasingly recognized that individual subsets of haematopoietic and epithelial antigen-presenting cells (APCs), residing within distinct thymic microenvironments, use partly unique strategies of antigen processing and handling and thus support T cell selection in a cooperative rather than a redundant manner. Recent data suggest that cortical thymic epithelial cells (cTECs) use unique pathways of self-antigen processing to generate peptide–MHC complexes for positive selection. Thymic dendritic cells (DCs) are more heterogeneous than previously appreciated. Migratory DCs carry peripheral self antigens into the thymus and thereby may extend the scope of intrathymically presented self antigens. Other biological implications of the heterogeneity of thymic DCs and the way in which the various DC subsets may differentially contribute to the intrathymic representation of peripheral tissues are just beginning to emerge. Medullary thymic epithelial cells (mTECs) are not only unique in their ability to promiscuously express tissue-restricted antigens, but they also have adapted their cell biology to focus their MHC class II-bound peptides on this endogenous antigen pool, thus fulfilling an autonomous APC function not only in CD8 + but also in CD4 + T cell tolerance. Constitutive and unidirectional transfer of mTEC-derived self antigens to thymic DCs increases the probability that autoreactive T cells encounter self antigens expressed by rare mTECs. Recognition of self-peptide–MHC complexes in the thymus is necessary for thymocyte survival, but can also result in cell death. Here, the authors provide a unique insight into this apparent paradox, describing how the repertoire of self-peptide–MHC complexes that support T cell selection is shaped. Understanding how thymic selection imparts self-peptide–MHC complex restriction and a high degree of self tolerance on the T cell repertoire requires a detailed description of the parameters that shape the MHC ligand repertoire of distinct thymic antigen-presenting cells and of how these cells communicate with T cells. Several recent discoveries pertaining to cortex-specific pathways of antigen processing, the heterogeneity of thymic dendritic cells and the intercellular transfer of self antigens have uncovered surprising and unique aspects of antigen presentation in the thymic microenvironment. Here, we discuss these new findings in the context of how individual thymic stromal cell types support T cell selection in a cooperative rather than a redundant manner.