Aire in Autoimmunity.

The role of the autoimmune regulator (Aire) in central immune tolerance and thymic self-representation was first described more than 20 years ago, but fascinating new insights into its biology continue to emerge, particularly in the era of advanced single-cell genomics. We briefly describe the role of human genetics in the discovery of Aire, as well as insights into its function gained from genotype-phenotype correlations and the spectrum of Aire-associated autoimmunity-including insights from patients with Aire mutations with broad and diverse implications for human health. We then highlight emerging trends in Aire biology, focusing on three topic areas. First, we discuss medullary thymic epithelial diversity and the role of Aire in thymic epithelial development. Second, we highlight recent developments regarding the molecular mechanisms of Aire and its binding partners. Finally, we describe the rapidly evolving biology of the identity and function of extrathymic Aire-expressing cells (eTACs), and a novel eTAC subset called Janus cells, as well as their potential roles in immune homeostasis.

Thymic Epithelial Cells.

Intrathymic T cell development is a complex process that depends upon continuous guidance from thymus stromal cell microenvironments. The thymic epithelium within the thymic stroma comprises highly specialized cells with a high degree of anatomic, phenotypic, and functional heterogeneity. These properties are collectively required to bias thymocyte development toward production of self-tolerant and functionally competent T cells. The importance of thymic epithelial cells (TECs) is evidenced by clear links between their dysfunction and multiple diseases where autoimmunity and immunodeficiency are major components. Consequently, TECs are an attractive target for cell therapies to restore effective immune system function. The pathways and molecular regulators that control TEC development are becoming clearer, as are their influences on particular stages of T cell development. Here, we review both historical and the most recent advances in our understanding of the cellular and molecular mechanisms controlling TEC development, function, dysfunction, and regeneration.

Thymic epithelial cells co-opt lineage-defining transcription factors to eliminate autoreactive T cells.

Medullary thymic epithelial cells (mTECs) ectopically express thousands of peripheral-tissue antigens (PTAs), which drive deletion or phenotypic diversion of self-reactive immature T cells during thymic differentiation. Failure of PTA expression causes multiorgan autoimmunity. By assaying chromatin accessibility in individual mTECs, we uncovered signatures of lineage-defining transcription factors (TFs) for skin, lung, liver, and intestinal cells-including Grhl, FoxA, FoxJ1, Hnf4, Sox8, and SpiB-in distinct mTEC subtypes. Transcriptomic and histologic analyses showed that these subtypes, which we collectively term mimetic cells, expressed PTAs in a biologically logical fashion, mirroring extra-thymic cell types while maintaining mTEC identity. Lineage-defining TFs bound to mimetic-cell open chromatin regions and were required for mimetic cell accumulation, whereas the tolerogenic factor Aire was partially and variably required. Expression of a model antigen in mimetic cells sufficed to induce cognate T cell tolerance. Thus, mTECs co-opt lineage-defining TFs to drive mimetic cell accumulation, PTA expression, and self-tolerance.

Primary and secondary defects of the thymus.

The thymus is the primary site of T-cell development, enabling generation, and selection of a diverse repertoire of T cells that recognize non-self, whilst remaining tolerant to self- antigens. Severe congenital disorders of thymic development (athymia) can be fatal if left untreated due to infections, and thymic tissue implantation is the only cure. While newborn screening for severe combined immune deficiency has allowed improved detection at birth of congenital athymia, thymic disorders acquired later in life are still underrecognized and assessing the quality of thymic function in such conditions remains a challenge. The thymus is sensitive to injury elicited from a variety of endogenous and exogenous factors, and its self-renewal capacity decreases with age. Secondary and age-related forms of thymic dysfunction may lead to an increased risk of infections, malignancy, and autoimmunity. Promising results have been obtained in preclinical models and clinical trials upon administration of soluble factors promoting thymic regeneration, but to date no therapy is approved for clinical use. In this review we provide a background on thymus development, function, and age-related involution. We discuss disease mechanisms, diagnostic, and therapeutic approaches for primary and secondary thymic defects.

Tracking the role of Aire in immune tolerance to the eye with a TCR transgenic mouse model.

Roughly one-half of mice with partial defects in two immune tolerance pathways (AireGW/+Lyn-/- mice) spontaneously develop severe damage to their retinas due to T cell reactivity to Aire-regulated interphotoreceptor retinoid-binding protein (IRBP). Single-cell T cell receptor (TCR) sequencing of CD4+ T cells specific for a predominate epitope of IRBP showed a remarkable diversity of autoantigen-specific TCRs with greater clonal expansions in mice with disease. TCR transgenic mice made with an expanded IRBP-specific TCR (P2.U2) of intermediate affinity exhibited strong but incomplete negative selection of thymocytes. This negative selection was absent in IRBP-/- mice and greatly defective in AireGW/+ mice. Most P2.U2+/- mice and all P2.U.2+/-AireGW/+ mice rapidly developed inflammation of the retina and adjacent uvea (uveitis). Aire-dependent IRBP expression in the thymus also promoted Treg differentiation, but the niche for this fate determination was small, suggesting differences in antigen presentation leading to negative selection vs. thymic Treg differentiation and a stronger role for negative selection in preventing autoimmune disease in the retina.

Aire mediates tolerance to insulin through thymic trimming of high-affinity T cell clones.

Insulin is a central autoantigen in the pathogenesis of T1D, and thymic epithelial cell expression of insulin under the control of the Autoimmune Regulator (Aire) is thought to be a key component of maintaining tolerance to insulin. In spite of this general working model, direct detection of this thymic selection on insulin-specific T cells has been somewhat elusive. Here, we used a combination of highly sensitive T cell receptor transgenic models for detecting thymic selection and sorting and sequencing of Insulin-specific CD4+ T cells from Aire-deficient mice as a strategy to further define their selection. This analysis revealed a number of unique t cell receptor (TCR) clones in Aire-deficient hosts with high affinity for insulin/major histocompatibility complex (MHC) ligands. We then modeled the thymic selection of one of these clones in Aire-deficient versus wild-type hosts and found that this model clone could escape thymic negative selection in the absence of thymic Aire. Together, these results suggest that thymic expression of insulin plays a key role in trimming and removing high-affinity insulin-specific T cells from the repertoire to help promote tolerance.

Identification of Natural Regulatory T Cell Epitopes Reveals Convergence on a Dominant Autoantigen.

Regulatory T (Treg) cells expressing the transcription factor Foxp3 are critical for the prevention of autoimmunity and the suppression of anti-tumor immunity. The major self-antigens recognized by Treg cells remain undefined, representing a substantial barrier to the understanding of immune regulation. Here, we have identified natural Treg cell ligands in mice. We found that two recurrent Treg cell clones, one prevalent in prostate tumors and the other associated with prostatic autoimmune lesions, recognized distinct non-overlapping MHC-class-II-restricted peptides derived from the same prostate-specific protein. Notably, this protein is frequently targeted by autoantibodies in experimental models of prostatic autoimmunity. On the basis of these findings, we propose a model in which Treg cell responses at peripheral sites converge on those self-proteins that are most susceptible to autoimmune attack, and we suggest that this link could be exploited as a generalizable strategy for identifying the Treg cell antigens relevant to human autoimmunity.

Assembling the thymus medulla: Development and function of epithelial cell heterogeneity.

The thymus is a unique primary lymphoid organ that supports the production of self-tolerant T-cells essential for adaptive immunity. Intrathymic microenvironments are microanatomically compartmentalised, forming defined cortical, and medullary regions each differentially supporting critical aspects of thymus-dependent T-cell maturation. Importantly, the specific functional properties of thymic cortical and medullary compartments are defined by highly specialised thymic epithelial cells (TEC). For example, in the medulla heterogenous medullary TEC (mTEC) contribute to the enforcement of central tolerance by supporting deletion of autoreactive T-cell clones, thereby counterbalancing the potential for random T-cell receptor generation to contribute to autoimmune disease. Recent advances have further shed light on the pathways and mechanisms that control heterogeneous mTEC development and how differential mTEC functionality contributes to control self-tolerant T-cell development. Here we discuss recent findings in relation to mTEC development and highlight examples of how mTEC diversity contribute to thymus medulla function.

Thymic and extrathymic Aire-expressing cells in maternal-fetal tolerance.

Healthy pregnancy requires maternal immune tolerance to both fetal and placental tissues which contain a range of self- and non-self-antigens. While many of the components and mechanisms of maternal-fetal tolerance have been investigated in detail and previously and thoroughly reviewed (Erlebacher A. Annu Rev Immunol. 2013;31:387-411), the role of autoimmune regulator (Aire), a critical regulator of central tolerance expressed by medullary thymic epithelial cells (mTECs), has been less explored. Aire is known to facilitate the expression of a range of otherwise tissue-specific antigens (TSAs) in mTECs, and here we highlight recent work showing a role for mTEC-mediated thymic selection in maintaining maternal-fetal tolerance. Recently, however, our group and others have identified additional populations of extrathymic Aire-expressing cells (eTACs) in the secondary lymphoid organs. These hematopoietic antigen-presenting cells possess the ability to induce functional inactivation and/or deletion of cognate T cells, and deletion of maternal eTACs during pregnancy increases T-cell activation in the lymph nodes and lymphocytic infiltration of the uterus, leading to pregnancy complications including intrauterine growth restriction (IUGR) and fetal resorption. In this review, we briefly summarize findings related to essential Aire biology, discuss the known roles of Aire-deficiency related to pregnancy complications and infertility, review the newly discovered role for eTACs in the maintenance of maternal-fetal tolerance-as well as recent work defining eTACs at the single-cell level-and postulate potential mechanisms by which eTACs may regulate this process.

Fetal-placental antigens and the maternal immune system: Reproductive immunology comes of age.

Reproductive physiology and immunology as scientific disciplines each have rich, largely independent histories. The physicians and philosophers of ancient Greece made remarkable observations and inferences to explain regeneration as well as illness and immunity. The scientific enlightenment of the renaissance and the technological advances of the past century have led to the explosion of knowledge that we are experiencing today. Breakthroughs in transplantation, immunology, and reproduction eventually culminated with Medawar's discovery of acquired immunological tolerance, which helped to explain the transplantation success and failure. Medawar's musings also keenly pointed out that the fetus apparently breaks these newly discovered rules, and with this, the field of reproductive immunology was launched. As a result of having stemmed from transplantation immunology, scientist still analogizes the fetus to a successful allograft. Although we now know of the fundamental differences between the two, this analogy remains a useful tool to understand how the fetus thrives despite its immunological disparity with the mother. Here, we review the history of reproductive immunology, and how major and minor histocompatibility antigens, blood group antigens, and tissue-specific "self" antigens from the fetus and transplanted organs parallel and differ.

Thymic mimetic cells: tolerogenic masqueraders.

Medullary thymic epithelial cells (mTECs) clonally delete or divert autoreactive T cells by ectopically expressing a diverse array of peripheral-tissue antigens (PTAs) within the thymus. Although thymic stromal cells with histological features of extra-thymic cell types, like myocytes or neurons, have been observed by light microscopy since the mid-1800s, most modern work on PTA expression has focused on the transcription factor Aire. Here, we highlight recent work that has refocused attention on such 'misplaced' thymic cells, referred to collectively as thymic mimetic cells. We review the molecular underpinnings of mimetic cells and their roles in establishing T cell tolerance, and we propose that mimetic cells play important roles in autoimmunity. Finally, we suggest future directions for this emerging area.

CTLA-4 on thymic epithelial cells complements Aire for T cell central tolerance.

Medullary thymic epithelial cells (mTECs) are essential for the establishment of T cell central tolerance. The transcription factor Aire plays a key role in this process, but other factors remain understudied. We found that a small population of mTECs expressed the coinhibitory receptor cytotoxic T lymphocyte-associated protein 4 (CTLA-4). These CTLA-4+ cells were detectable in perinates, peaked around young adulthood and expanded sixfold in the absence of Aire. Single-cell transcriptomics revealed CTLA-4+ mTECs to express a distinct gene signature encoding molecules associated with antigen presentation and interferon-gamma signaling. Mice conditionally lacking CTLA-4 in thymic epithelial cells had no major immunological deficiencies but displayed a mildly increased inflammatory tone and a partial defect in the generation of Foxp3+CD4+ regulatory T cells. Consequently, these mice developed modest levels of autoantibodies and lymphocytic infiltration of peripheral tissues. Thus, CTLA-4 expression in mTECs complements Aire to establish T cell central tolerance.

Increased type 1 inflammation in gynecologic cervicovaginal samples in patients with APS-1.

BACKGROUND: Inborn errors of immunity offer important insights into mucosal immunity. In autoimmune polyendocrine syndrome type-1 (APS-1), chronic mucocutaneous candidiasis has been ascribed to neutralizing IL-17 autoantibodies. Recent evidence implicates excessive T-cell IFN-γ secretion and ensuing epithelial barrier disruption in predisposition to candidiasis, but these results remain to be replicated. Whether IL-17 paucity, increased type I inflammation, or their combination underlies susceptibility to chronic mucocutaneus candidiasis in APS-1 is debated. OBJECTIVE: Our aim was to characterize the immunologic features in the cervicovaginal mucosa of females with APS-1. METHODS: Vaginal fluid was collected with a flocked swab from 17 females with APS-1 and 18 controls, and cytokine composition was analyzed using Luminex (Luminex Corporation, Austin, Tex). Cervical cell samples were obtained with a cervix brush from 6 patients and 6 healthy controls and subjected to transcriptome analysis. RESULTS: The vaginal fluid samples from patients with APS-1 had IFN-γ concentrations comparable to those of the controls (2.6 vs 2.4 pg/mL) but high concentrations of the TH1 chemokines CXCL9 and CXCL10 (1094 vs 110 pg/mL [P < .001] and 4033 vs 273 pg/mL [P = .001], respectively), whereas the IL-17 levels in the samples from the 2 groups were comparable (28 vs 8.8 pg/mL). RNA sequencing of the cervical cells revealed upregulation of pathways related to mucosal inflammation and cell death in the patients with APS-1. CONCLUSION: Excessive TH1 cell response appears to underlie disruption of the mucosal immune responses in the genital tract of patients with APS-1 and may contribute to susceptibility to candidiasis in the genital tract as well.

Dysregulated germinal center reaction with expanded T follicular helper cells in autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy lymph nodes.

BACKGROUND: Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED, also called APS-1) is an inborn error of immunity with clear signs of B-cell autoimmunity such as neutralizing anti-IFN antibodies. In APECED, mutations in the AIRE gene impair thymic negative selection of T cells. The resulting T-cell alterations may then cause dysregulation of B-cell responses. However, no analysis of interactions of T and B cells in the germinal centers (GCs) in patients' secondary lymphatic tissues has been reported. OBJECTIVE: This study examined the relationship between B cells and follicular T helper cells (TfH) in peripheral blood and lymph node (LN) GCs in patients with APECED. METHODS: Immunophenotyping of peripheral blood B cells and TfH was performed for 24 patients with APECED. Highly multiplexed fluorescent immunohistochemical staining was performed on 7 LN biopsy samples from the patients to study spatial interactions of lymphocytes in the GCs at the single-cell level. RESULTS: The patients' peripheral B-cell phenotype revealed skewing toward a mature B-cell phenotype with marked loss of transitional and naive B cells. The frequency of circulating TfH cells was diminished in the patients, while in the LNs the TfH population was expanded. In LNs the overall frequency of Treg cells and interactions of Treg cells with nonfollicular T cells were reduced, suggesting that aberrant Treg cell function might fail to restrain TfH differentiation. CONCLUSIONS: GC reactions are disrupted in APECED as a result of defective T-cell control.

Neurological Diseases and Prevalence of Antineuronal Antibodies in Patients with Autoimmune Polyendocrine Syndrome Type 1 - A National Cohort Study.

Autoimmune polyendocrine syndrome type 1 (APS-1) is a rare monogenic disease caused by mutations in the autoimmune regulator gene. Although the disease-associated autoantibodies mostly target endocrine organs, autoantibodies from patients with APS-1 bind also to rat brain structures. The patients often have GAD65-antibodies, that can cause autoimmune encephalitis. However, neurological manifestations of APS-1 have not been systematically explored. We conducted a retrospective chart review on 44 Finnish patients with APS-1 (median age 38 years, 61% females) and collected all their neurological diagnoses. To assess the prevalence of serum antineuronal antibodies in APS-1, serum samples of 24 patients (median age 36 years, 63% females) were analyzed using a fixed cell-based assay. Of the 44 APS-1 patients, 10 (23%) had also received a diagnosis of a neurological disease. Of these neurological comorbidities, migraine (n = 7; 16%), central nervous system infections (n = 3; 7%), and epilepsy (n = 2; 5%) were the most prevalent. Other diagnoses recorded for single patients were axonal sensorimotor polyneuropathy, essential tremor, idiopathic intracranial hypertension, ischemic stroke, and trigeminal neuralgia. Serum antineuronal antibodies were detected in 42% of patients tested (10/24, 50% females, median age 42 years), GAD65 antibodies being the most common finding. Antibodies against glycine and aquaporin 4 were found in low titers. In four patients, relatively high titers of GAD65 antibodies without coexisting type 1 diabetes were found, but none presented with GAD65-encephalitis. Our study suggests an association between APS-1 and neurological disorders, the mechanisms of which are to be further investigated.

A novel missense mutation in the AIRE gene underlying autoimmune polyglandular syndrome type 1.

The immune regulator gene AIRE plays an essential role in the establishment of immune tolerance and the prevention of autoimmunity. This transcription factor plays a critical role in promoting self-tolerance in the thymus by regulating the expression of a large number of self-antigens that share the common feature of being tissue-restricted in their expression pattern in the periphery. Dysfunction of AIRE in humans causes a rare disease, autoimmune polyglandular syndrome type 1 (APS1), characterized by an autoimmune response against peripheral tissues, particularly endocrine tissues. Although a few dominant mutations have been described, the inactivation of AIRE is usually caused by recessive mutations. Recent data suggests that alterations in AIRE function contribute not only to APS1 but also to more common forms of autoimmune disease. Here, we present a previously unreported missense mutation (NM_000383.2:c.260 T > C) in exon 2 of the AIRE gene, predicted to cause the substitution (p.(Leu87Pro)) in the CARD domain of the AIRE protein. When inherited in conjunction with another dysfunctional AIRE allele, this mutation was associated with immune dysregulation in a pediatric patient. The presence of hypergammaglobulinemia, malabsorption syndrome, ectodermal dysplasia, mucocutaneous candidiasis, vitiligo, and hypothyroidism as well as the presence of multiple autoantibodies allowed us to confirm an APS1 diagnosis.

T-cell receptor repertoire analysis of CD4-positive T cells from blood and an affected organ in an autoimmune mouse model.

One hallmark of some autoimmune diseases is the variability of symptoms among individuals. Organs affected by the disease differ between patients, posing a challenge in diagnosing the affected organs. Although numerous studies have investigated the correlation between T cell antigen receptor (TCR) repertoires and the development of infectious and immune diseases, the correlation between TCR repertoires and variations in disease symptoms among individuals remains unclear. This study aimed to investigate the correlation of TCRα and ß repertoires in blood T cells with the extent of autoimmune signs that varies among individuals. We sequenced TCRα and ß of CD4+ CD44high CD62Llow T cells in the blood and stomachs of mice deficient in autoimmune regulator (Aire) (AIRE KO), a mouse model of human autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy. Data analysis revealed that the degree of similarity in TCR sequences between the blood and stomach varied among individual AIRE KO mice and reflected the extent of T cell infiltration in the stomach. We identified a set of TCR sequences whose frequencies in blood might correlate with extent of the stomach manifestations. Our results propose a potential of using TCR repertoires not only for diagnosing disease development but also for diagnosing affected organs in autoimmune diseases.

Aire-dependent transcripts escape Raver2-induced splice-event inclusion in the thymic epithelium.

Aire allows medullary thymic epithelial cells (mTECs) to express and present a large number of self-antigens for central tolerance. Although mTECs express a high diversity of self-antigen splice isoforms, the extent and regulation of alternative splicing events (ASEs) in their transcripts, notably in those induced by Aire, is unknown. In contrast to Aire-neutral genes, we find that transcripts of Aire-sensitive genes show only a low number of ASEs in mTECs, with about a quarter present in peripheral tissues excluded from the thymus. We identify Raver2, as a splicing-related factor overexpressed in mTECs and dependent on H3K36me3 marks, that promotes ASEs in transcripts of Aire-neutral genes, leaving Aire-sensitive ones unaffected. H3K36me3 profiling reveals its depletion at Aire-sensitive genes and supports a mechanism that is preceding Aire expression leading to transcripts of Aire-sensitive genes with low ASEs that escape Raver2-induced alternative splicing. The lack of ASEs in Aire-induced transcripts would result in an incomplete Aire-dependent negative selection of autoreactive T cells, thus highlighting the need of complementary tolerance mechanisms to prevent activation of these cells in the periphery.

Cohort profile: The Assessing Imperial Valley Respiratory Health and the Environment (AIRE) study.

BACKGROUND: The Children's Assessing Imperial Valley Respiratory Health and the Environment (AIRE) study is a prospective cohort study of environmental influences on respiratory health in a rural, southeastern region of California (CA), which aims to longitudinally examine the contribution of a drying saline lake to adverse health impacts in children. OBJECTIVES: This cohort was established through a community-academic partnership with the goal of assessing the health effects of childhood exposures to wind-blown particulate matter (PM) and inform public health action. We hypothesize that local PM sources are related to poorer children's respiratory health. POPULATION: Elementary school children in Imperial Valley, CA. DESIGN: Prospective cohort study. METHODS: Between 2017 and 2019, we collected baseline information on 731 children, then follow-up assessments yearly or twice-yearly since 2019. Data have been collected on children's respiratory health, demographics, household characteristics, physical activity and lifestyle, via questionnaires completed by parents or primary caregivers. In-person measurements, conducted since 2019, repeatedly assessed lung function, height, weight and blood pressure. Exposure to air pollutants has been assessed by multiple methods and individually assigned to participants using residential and school addresses. Health data will be linked to ambient and local sources of PM, during and preceding the study period to understand how spatiotemporal trends in these environmental exposures may relate to respiratory health. PRELIMINARY RESULTS: Analyses of respiratory symptoms indicate a high prevalence of allergies, bronchitic symptoms and wheezing. Asthma diagnosis was reported in 24% of children at enrolment, which exceeds both CA state and US national prevalence estimates for children. CONCLUSIONS: The Children's AIRE cohort, while focused on the health impacts of the drying Salton Sea and air quality in Imperial Valley, is poised to elucidate the growing threat of drying saline lakes and wind-blown dust sources to respiratory health worldwide, as sources of wind-blown dust emerge in our changing climate.

Spontaneous development of an autoimmune hepatitis - primary biliary cholangitis overlap syndrome in dnTGFßRII Aire-/- mice.

Autoimmune regulator (Aire) and TGF-ß signaling play important roles in central tolerance and peripheral tolerance, respectively, by eliminating or suppressing the activity of autoreactive T cells. We previously demonstrated that dnTGFßRII mice develop a defect in peripheral tolerance and a primary biliary cholangitis (PBC)-like disease. We hypothesized that by introducing the Aire gene to this model, we would observe a more severe PBC phenotype. Interestingly, however, we demonstrated that, while dnTGFßRII Aire-/- mice do manifest key histological and serological features of autoimmune cholangitis, they also develop mild to moderate interface hepatitis and show high levels of alanine transaminase (ALT) and antinuclear antibodies (ANA), characteristics of autoimmune hepatitis (AIH). To further understand this unique phenotype, we performed RNA sequencing (RNA-seq) and flow cytometry to explore the functional pathways and immune cell pathways in the liver of dnTGFßRII Aire-/- mice. Our data revealed enrichments of programmed cell death pathways and predominant CD8+ T cell infiltrates. Depleting CD8+ T cells using an anti-CD8α antibody significantly alleviated hepatic inflammation and prolonged the life span of these mice. Finally, RNA-seq data indicated the clonal expansion of hepatic CD8+ T cells. In conclusion, these mice developed an autoreactive CD8+ T-cell-mediated autoimmune cholangitis with concurrent hepatitis that exhibited key histological and serological features of the AIH-PBC overlap syndrome, representing a novel model for the study of tolerance and autoimmune liver disease. © 2023 The Pathological Society of Great Britain and Ireland.