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Key Points Autoimmune regulator (AIRE) has a well-known role in preventing autoimmunity through upregulation of tissue-specific antigen (TSA) expression in medullary thymic epithelial cells (mTECs). Recognition of these thymic TSAs by self-reactive T cells leads to clonal deletion and/or diversion to the regulatory T cell lineage. Mutations in AIRE result in multi-organ autoimmune disease in both humans and mice. In humans, autosomal recessive mutations result in autoimmune polyendocrinopathy syndrome 1, whereas dominant mutations result in autoimmunity with a more narrow disease spectrum. AIRE expression is under strict spatiotemporal control. Regulation of AIRE expression is achieved through several mechanisms, including enhancer elements that regulate the transcription and alternative splicing of AIRE , which in turn control AIRE protein levels. The array of TSAs expressed by each individual mTEC is diverse. Nevertheless, clusters of TSAs are co-expressed, with distinct rules governing their co-expression. AIRE interacts with dozens of proteins with various functions, including the recruitment of AIRE to TSA genes, elongation of AIRE-dependent TSA transcripts and modification of AIRE itself. AIRE has important roles in conditions beyond autoimmunity, such as graft-versus-host disease and cancer. Thus, modulation of AIRE function may have potential therapeutic benefit in a wide range of diseases. Autoimmune regulator (AIRE) is best known for its role in immune tolerance. In this Review, the authors summarize the recent advances in our understanding of the diverse functions of AIRE, including its role in selection of regulatory T cells and modulation of non-autoimmune diseases. More than 15 years ago, mutations in the autoimmune regulator ( AIRE ) gene were identified as the cause of autoimmune polyglandular syndrome type 1 (APS1). It is now clear that this transcription factor has a crucial role in promoting self-tolerance in the thymus by regulating the expression of a wide array of self-antigens that have the commonality of being tissue-restricted in their expression pattern in the periphery. In this Review, we highlight many of the recent advances in our understanding of the complex biology that is related to AIRE, with a particular focus on advances in genetics, molecular interactions and the effect of AIRE on thymic selection of regulatory T cells. Furthermore, we highlight new areas of biology that are potentially affected by this key regulator of immune tolerance.

Regulatory T cells (T reg cells), a distinct subset of CD4 + T cells, are necessary for the maintenance of immune self-tolerance and homeostasis 1 , 2 . Recent studies have demonstrated that T reg cells exhibit a unique metabolic profile, characterized by an increase in mitochondrial metabolism relative to other CD4 + effector subsets 3 , 4 . Furthermore, the T reg cell lineage-defining transcription factor, Foxp3, has been shown to promote respiration 5 , 6 ; however, it remains unknown whether the mitochondrial respiratory chain is required for the T cell-suppression capacity, stability and survival of T reg cells. Here we report that T reg cell-specific ablation of mitochondrial respiratory chain complex III in mice results in the development of fatal inflammatory disease early in life, without affecting T reg cell number. Mice that lack mitochondrial complex III specifically in T reg cells displayed a loss of T cell-suppression capacity without altering T reg cell proliferation and survival. T reg cells deficient in complex III showed decreased expression of genes associated with T reg function, whereas Foxp3 expression remained stable. Loss of complex III in T reg cells increased DNA methylation as well as the metabolites 2-hydroxyglutarate (2-HG) and succinate that inhibit the ten-eleven translocation (TET) family of DNA demethylases 7 . Thus, T reg cells require mitochondrial complex III to maintain immune regulatory gene expression and suppressive function. Specific ablation of mitochondrial complex III subunits in T reg cells in mice results in inflammatory disease, altered T reg gene expression and defective T reg function, indicating a key functional role for mitochondrial complex III in T reg cells.

How expression of tissue-restricted self antigens is coordinated in medullary thymic epithelial cells has remained unclear. Benoist and colleagues use single-cell RNA sequencing to identify clusters of co-expressed tissue-restricted antigens. The transcription factor Aire controls immunological tolerance by inducing the ectopic thymic expression of many tissue-specific genes, acting broadly by removing stops on the transcriptional machinery. To better understand Aire's specificity, we performed single-cell RNA-seq and DNA-methylation analysis of Aire -sufficient and Aire -deficient medullary epithelial cells (mTECs). Each of Aire's target genes was induced in only a minority of mTECs, independently of DNA-methylation patterns, as small inter-chromosomal gene clusters activated in concert in a proportion of mTECs. These microclusters differed between individual mice. Thus, our results suggest an organization of the DNA or of the epigenome that results from stochastic determinism but is 'bookmarked' and stable through mTEC divisions, which ensures more effective presentation of self antigens and favors diversity of self-tolerance between individuals.

Immunological self-tolerance is established in the thymus by the expression of virtually all self-antigens, including tissue-restricted antigens (TRAs) and cell-type-restricted antigens (CRAs). Despite a wealth of knowledge about the transcriptional regulation of TRA genes, posttranscriptional regulation remains poorly understood. Here, we show that protein arginine methylation plays an essential role in central immune tolerance by maximizing the self-antigen repertoire in medullary thymic epithelial cells (mTECs). Protein arginine methyltransferase-5 (Prmt5) was required for pre-mRNA splicing of certain key genes in tolerance induction, including Aire as well as various genes encoding TRAs. Mice lacking Prmt5 specifically in thymic epithelial cells exhibited an altered thymic T cell selection, leading to the breakdown of immune tolerance accompanied by both autoimmune responses and enhanced antitumor immunity. Thus, arginine methylation and transcript splicing are essential for establishing immune tolerance and may serve as a therapeutic target in autoimmune diseases as well as cancer immunotherapy.

CD8 + T cells and natural killer (NK) cells are central cellular components of immune responses against pathogens and cancer, which rely on interleukin (IL)-15 for homeostasis. Here we show that IL-15 also mediates homeostatic priming of CD8 + T cells for antigen-stimulated activation, which is controlled by a deubiquitinase, Otub1. IL-15 mediates membrane recruitment of Otub1, which inhibits ubiquitin-dependent activation of AKT, a kinase that is pivotal for T cell activation and metabolism. Otub1 deficiency in mice causes aberrant responses of CD8 + T cells to IL-15, rendering naive CD8 + T cells hypersensitive to antigen stimulation characterized by enhanced metabolic reprograming and effector functions. Otub1 also controls the maturation and activation of NK cells. Deletion of Otub1 profoundly enhances anticancer immunity by unleashing the activity of CD8 + T cells and NK cells. These findings suggest that Otub1 controls the activation of CD8 + T cells and NK cells by functioning as a checkpoint of IL-15-mediated priming. IL-15 has important functions in the activation and homeostasis of cytotoxic T lymphocytes (CTLs) and NK cells. Sun and colleagues demonstrate that the deubiquitinase Otub1 controls CTLs and NK cells in a cell-intrinsic manner by negatively regulating IL-15 signaling.

Protein tyrosine phosphatase nonreceptor type 22 (PTPN22) gene polymorphisms are associated with many autoimmune diseases. The major risk allele encodes an R620W amino acid change that alters B cell receptor (BCR) signaling involved in the regulation of central B cell tolerance. To assess whether this PTPN22 risk allele affects the removal of developing autoreactive B cells, we tested by ELISA the reactivity of recombinant antibodies isolated from single B cells from asymptomatic healthy individuals carrying one or two PTPN22 risk allele(s) encoding the PTPN22 R620W variant. We found that new emigrant/transitional and mature naive B cells from carriers of this PTPN22 risk allele contained high frequencies of autoreactive clones compared with those from non-carriers, revealing defective central and peripheral B cell tolerance checkpoints. Hence, a single PTPN22 risk allele has a dominant effect on altering autoreactive B cell counterselection before any onset of autoimmunity. In addition, gene array experiments analyzing mature naive B cells displaying PTPN22 risk allele(s) revealed that the association strength of PTPN22 for autoimmunity may be due not only to the impaired removal of autoreactive B cells but also to the upregulation of genes such as CD40, TRAF1, and IRF5, which encode proteins that promote B cell activation and have been identified as susceptibility genes associated with autoimmune diseases. These data demonstrate that early B cell tolerance defects in autoimmunity can result from specific polymorphisms and precede the onset of disease.

Self-tolerance by clonal anergy of B cells is marked by an increase in IgD and decrease in IgM antigen receptor surface expression, yet the function of IgD on anergic cells is obscure. Here we define the RNA landscape of the in vivo anergy response, comprising 220 induced sequences including a core set of 97. Failure to co-express IgD with IgM decreases overall expression of receptors for self-antigen, but paradoxically increases the core anergy response, exemplified by increased Sdc1 encoding the cell surface marker syndecan-1. IgD expressed on its own is nevertheless competent to induce calcium signalling and the core anergy mRNA response. Syndecan-1 induction correlates with reduction of surface IgM and is exaggerated without surface IgD in many transitional and mature B cells. These results show that IgD attenuates the response to self-antigen in anergic cells and promotes their accumulation. In this way, IgD minimizes tolerance-induced holes in the pre-immune antibody repertoire. Self-reactive B cells that are anergic express mainly IgD, yet the function of IgD is not clear. Here the authors analyse primary B cells from mice to show that IgD signalling attenuates self-antigen induced gene expression and promotes survival of anergic B cells that might go on to reactivate to foreign antigens and mutate away from self-reactivity.

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.

The MHC class I antigen presentation pathway allows the immune system to distinguish between self and nonself. Despite extensive research on the processing of antigenic peptides, little is known about their origin. Here, we show that mRNAs carrying premature stop codons that prevent the production of full-length proteins via the nonsense-mediated decay pathway still produce a majority of peptide substrates for the MHC class I pathway by a noncanonical mRNA translation process. Blocking the interaction of the translation initiation factor eIF4E with the cap structure suppresses the synthesis of full-length proteins but has only a limited effect on the production of antigenic peptides. These results reveal an essential cell biological function for a class of translation products derived during the pioneer round of mRNA translation and will have important implications for understanding how the immune system detects cells harboring pathogens and generates tolerance.

The DNA-binding factor SATB1 is known as a chromatin organizer. Schultze and colleagues show regulation of SATB1 expression by the transcription factor Foxp3 is necessary to confer suppression of effector cell activity. Regulatory T cells (T reg cells) are essential for self-tolerance and immune homeostasis. Lack of effector T cell (T eff cell) function and gain of suppressive activity by T reg cells are dependent on the transcriptional program induced by Foxp3. Here we report that repression of SATB1, a genome organizer that regulates chromatin structure and gene expression, was crucial for the phenotype and function of T reg cells. Foxp3, acting as a transcriptional repressor, directly suppressed the SATB1 locus and indirectly suppressed it through the induction of microRNAs that bound the SATB1 3′ untranslated region. Release of SATB1 from the control of Foxp3 in T reg cells caused loss of suppressive function, establishment of transcriptional T eff cell programs and induction of T eff cell cytokines. Our data support the proposal that inhibition of SATB1-mediated modulation of global chromatin remodeling is pivotal for maintaining T reg cell functionality.