Aire-expressing Cells Research Articles

A deep intronic splice-altering AIRE variant causes APECED syndrome through antisense oligonucleotide-targetable pseudoexon inclusion.

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a life-threatening monogenic autoimmune disorder primarily caused by biallelic deleterious variants in the autoimmune regulator (AIRE) gene. We prospectively evaluated 104 patients with clinically diagnosed APECED syndrome and identified 17 patients (16%) from 14 kindreds lacking biallelic AIRE variants in exons or flanking intronic regions; 15 had Puerto Rican ancestry. Through whole-genome sequencing, we identified a deep intronic AIRE variant (c.1504-818 G>A) cosegregating with the disease in all 17 patients. We developed a culture system of AIRE-expressing primary patient monocyte-derived dendritic cells and demonstrated that c.1504-818 G>A creates a cryptic splice site and activates inclusion of a 109-base pair frame-shifting pseudoexon. We also found low-level AIRE expression in patient-derived lymphoblastoid cell lines (LCLs) and confirmed pseudoexon inclusion in independent extrathymic AIRE-expressing cell lines. Through protein modeling and transcriptomic analyses of AIRE-transfected human embryonic kidney 293 and thymic epithelial cell 4D6 cells, we showed that this variant alters the carboxyl terminus of the AIRE protein, abrogating its function. Last, we developed an antisense oligonucleotide (ASO) that reversed pseudoexon inclusion and restored the normal AIRE transcript sequence in LCLs. Thus, our findings revealed c.1504-818 G>A as a founder APECED-causing AIRE variant in the Puerto Rican population and uncovered pseudoexon inclusion as an ASO-reversible genetic mechanism underlying APECED.

442.7: Aire-expressing cells are novel mediators of allograft tolerance in a murine heterotopic heart transplant model.

442.7: Aire-expressing cells are novel mediators of allograft tolerance in a murine heterotopic heart transplant model.

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. Expected final online publication date for the Annual Review of Immunology, Volume 42 is April 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Autoimmune regulator (Aire) deficiency results in reduced memory CD8+ T cells after Listeria monocytogenes infection in a murine model.

Homozygous mutations in the autoimmune regulator (AIRE) gene that cripple thymic negative selection of autoreactive T cells result in autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). However, how AIRE regulates the T cell response against foreign pathogens is not well understood. Here, we observed comparable primary CD8+ T cells but a markedly reduced memory T cell population and protective function in Aire-/- mice compared with wild-type after infection with a strain of recombinant Listeria monocytogenes. In adoptive transfer models, exogenous congenic CD8+ T cells transferred into Aire-/- mice also showed a reduction in the memory T cell population, indicating an important role for extrathymic Aire-expressing cells in shaping or sustaining memory T cells. Moreover, using a bone marrow chimeric model, we found that Aire expressed in radioresistant cells plays an important role in maintaining the memory phenotype. These results provide important insights into the role of extrathymic Aire in the T cell response to infection.

ILC3s select microbiota-specific T regcells to establish tolerance in the gut

Abstract Microbial colonization of the mammalian intestine elicits inflammatory or tolerogenic T cell responses, but the mechanisms controlling these distinct outcomes remain poorly understood, and accumulating evidence indicates that aberrant immunity to intestinal microbiota is causally associated with infectious, inflammatory and malignant diseases. Here we define a critical pathway controlling the fate of inflammatory versus tolerogenic T cells that respond to the microbiota and express the transcription factor RORγt. We profiled all RORγt +immune cells at single-cell resolution from the intestine-draining lymph nodes of mice and reveal a dominant presence of T regulatory (T reg) cells and lymphoid tissue inducer-like group 3 innate lymphoid cells (ILC3s), which co-localize at interfollicular regions. These ILC3s are distinct from but having interconverting potential with extrathymic AIRE-expressing cells, abundantly express major histocompatibility complex class II (MHCII), and are necessary and sufficient to promote microbiota-specific RORγt +T regcells and prevent their expansion as inflammatory T helper 17 (T H17) cells. This occurs through ILC3-mediated antigen presentation, αV integrin and competition for interleukin-2. Finally, single-cell analyses suggest that interactions between ILC3s and RORγt +T regcells are impaired in inflammatory bowel disease. Our results define a paradigm whereby ILC3s select for antigen-specific RORγt +Treg cells, and against T H17 cells, to establish immune tolerance to the microbiota and intestinal health. Crohn’s and Colitis Foundation Research Fellowship Award, 935259

Dispensable Role of Aire in CD11c+ Conventional Dendritic Cells for Antigen Presentation and Shaping the Transcriptome.

Aire, the defect of which is responsible for the development of autoimmunity, is predominantly expressed in medullary thymic epithelial cells, and it controls a wide variety of genes, including those of tissue-restricted Ags, for establishing thymic tolerance. Aire is also expressed from APCs in the periphery, called extrathymic Aire-expressing cells (eTACs), and their complementing role to thymic tolerance has been suggested. eTACs are composed of two distinct classes of APCs, conventional dendritic cell (cDC)-type and group 3 innate lymphoid cell (ILC3)-like-type expressing retinoic acid receptor-related orphan receptor γt (RORγt). Although the essential role of Aire in the latter in the Th17-mediated immune response against Candida albicans has been reported, the role of Aire in the cDC-type eTACs for this action has not been examined. Furthermore, the significance of Aire in the production of the transcriptome of the cDC-type eTACs remains unknown. We have approached these issues using a high-fidelity Aire-reporter mouse strain. We found that although the cDC-type eTACs dominated ILC3-like-type eTACs in number and they served as efficient APCs for the immune response against an exogenous Ag as well as for the C. albicans-specific Th17 immune response, loss of Aire in cDC-type eTACs showed no clear effect on these functions. Furthermore, loss of Aire showed no major impact on the transcriptome from cDC-type eTACs. These results suggested that Aire in cDC-type eTACs may not have a cell-intrinsic role in the immune response in contrast to the role of Aire in ILC3-like-type eTACs.

ILC3s select microbiota-specific regulatory T cells to establish tolerance in the gut.

Microbial colonization of the mammalian intestine elicits inflammatory or tolerogenic T cell responses, but the mechanisms controlling these distinct outcomes remain poorly understood, and accumulating evidence indicates that aberrant immunity to intestinal microbiota is causally associated with infectious, inflammatory and malignant diseases1-8. Here we define a critical pathway controlling the fate of inflammatory versus tolerogenic T cells that respond to the microbiota and express the transcription factor RORγt. We profiled all RORγt+ immune cells at single-cell resolution from the intestine-draining lymph nodes of mice and reveal a dominant presence of Tregulatory (Treg) cells and lymphoid tissue inducer-like group 3 innate lymphoid cells (ILC3s), which co-localize at interfollicular regions. These ILC3s are distinct from extrathymic AIRE-expressing cells, abundantly express major histocompatibility complex class II, and are necessary and sufficient to promote microbiota-specific RORγt+ Treg cells and prevent their expansion as inflammatory T helper 17 cells. This occurs through ILC3-mediated antigen presentation, αV integrin and competition for interleukin-2. Finally, single-cell analyses suggest that interactions between ILC3s and RORγt+ Treg cells are impaired in inflammatory bowel disease. Our results define a paradigm whereby ILC3s select for antigen-specific RORγt+ Treg cells, and against T helper 17 cells, to establish immune tolerance to the microbiota and intestinal health.

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.

Single-cell multiomics defines tolerogenic extrathymic Aire-expressing cells with unique homology to thymic epithelium

Abstract The autoimmune regulator (Aire) protein, a key transcriptional regulator expressed in medullary thymic epithelial cells (mTECs), is crucial for central tolerance. Aire is also found in extrathymic Aire-expressing cells (eTACs) in secondary lymphoid organs. eTACs are hematopoietic antigen-presenting cells that are capable of inducing immune tolerance, but the precise identity of eTACs has remained unclear. To define eTACs, we utilized a combination of single-cell multiomics, transgenic murine models, and functional approaches. We found that eTACs consist of two similar cell types: CCR7+ Aire-expressing migratory dendritic cells and an Aire-high population co-expressing Aire and retinoic acid receptor–related orphan receptor γt (RORγt) that we termed Janus cells (JCs). eTACs, particularly JCs, have highly accessible chromatin and share remarkable homology with mTECs. Additionally, transgenic self-antigen expression by eTACs is sufficient to induce negative selection of cognate autoreactive T cells and prevent autoimmune diabetes. To better define the function of extrathymic Aire in eTACs and its contribution to peripheral immune tolerance, we have generated a Peripheral Aire KnockOut (PAKO) mouse (Vav1-Cre x Aire-fl/fl). Using intracellular Aire staining by flow cytometry, we validated that PAKO mice lack all eTACs in secondary lymphoid organs while maintaining normal thymic Aire expression in mTECs. Studying the biology of eTACs and peripheral Aire will help further elucidate the function of Aire and define basic peripheral tolerance mechanisms, which may have significance for a range of clinical applications from autoimmunity to tumor immunity to maternal-fetal tolerance. Supported by ARCS Foundation

Single-cell multiomics defines tolerogenic extrathymic Aire-expressing populations with unique homology to thymic epithelium.

The autoimmune regulator (Aire), a well-defined transcriptional regulator in the thymus, is also found in extrathymic Aire-expressing cells (eTACs) in the secondary lymphoid organs. eTACs are hematopoietic antigen-presenting cells and inducers of immune tolerance, but their precise identity has remained unclear. Here, we use single-cell multiomics, transgenic murine models, and functional approaches to define eTACs at the transcriptional, genomic, and proteomic level. We find that eTACs consist of two similar cell types: CCR7+ Aire-expressing migratory dendritic cells (AmDCs) and an Airehi population coexpressing Aire and retinoic acid receptor–related orphan receptor γt (RORγt) that we term Janus cells (JCs). Both JCs and AmDCs have the highest transcriptional and genomic homology to CCR7+ migratory dendritic cells. eTACs, particularly JCs, have highly accessible chromatin and broad gene expression, including a range of tissue-specific antigens, as well as remarkable homology to medullary thymic epithelium and RANK-dependent Aire expression. Transgenic self-antigen expression by eTACs is sufficient to induce negative selection and prevent autoimmune diabetes. This transcriptional, genomic, and functional symmetry between eTACs (both JCs and AmDCs) and medullary thymic epithelium—the other principal Aire-expressing population and a key regulator of central tolerance—identifies a core program that may influence self-representation and tolerance across the spectrum of immune development.

Dual roles for LUBAC signaling in thymic epithelial cell development and survival.

Thymic epithelial cells (TECs) form a unique microenvironment that orchestrates T cell differentiation and immunological tolerance. Despite the importance of TECs for adaptive immunity, there is an incomplete understanding of the signalling networks that support their differentiation and survival. We report that the linear ubiquitin chain assembly complex (LUBAC) is essential for medullary TEC (mTEC) differentiation, cortical TEC survival and prevention of premature thymic atrophy. TEC-specific loss of LUBAC proteins, HOIL-1 or HOIP, severely impaired expansion of the thymic medulla and AIRE-expressing cells. Furthermore, HOIL-1-deficiency caused early thymic atrophy due to Caspase-8/MLKL-dependent apoptosis/necroptosis of cortical TECs. By contrast, deficiency in the LUBAC component, SHARPIN, caused relatively mild defects only in mTECs. These distinct roles for LUBAC components in TECs correlate with their function in linear ubiquitination, NFκB activation and cell survival. Thus, our findings reveal dual roles for LUBAC signaling in TEC differentiation and survival.

Extrathymic Aire-expressing cells support maternal-fetal tolerance.

Healthy pregnancy requires tolerance to fetal alloantigens as well as syngeneic embryonic and placental antigens. Given the importance of the autoimmune regulator (Aire) gene in self-tolerance, we investigated the role of Aire-expressing cells in maternal-fetal tolerance. We report that maternal ablation of Aire-expressing (Aire +) cells during early mouse pregnancy caused intrauterine growth restriction (IUGR) in both allogeneic and syngeneic pregnancies. This phenotype is immune mediated, as IUGR was rescued in Rag1-deficient mice, and involved a memory response, demonstrated by recurrence of severe IUGR in second pregnancies. Single-cell RNA sequencing demonstrated that Aire + cell depletion in pregnancy results in expansion of activated T cells, particularly T follicular helper cells. Unexpectedly, selective ablation of either Aire-expressing medullary thymic epithelial cells or extrathymic Aire-expressing cells (eTACs) mapped the IUGR phenotype exclusively to eTACs. Thus, we report a previously undescribed mechanism for the maintenance of maternal-fetal immune homeostasis and demonstrate that eTACs protect the conceptus from immune-mediated IUGR.

A novel method to identify Post-Aire stages of medullary thymic epithelial cell differentiation.

Autoimmune regulator+ (Aire) medullary thymic epithelial cells (mTECs) play a critical role in tolerance induction. Several studies demonstrated that Aire+mTECs differentiate further into Post‐Aire cells. Yet, the identification of terminal stages of mTEC maturation depends on unique fate‐mapping mouse models. Herein, we resolve this limitation by segmenting the mTEChi(MHCIIhiCD80hi) compartment into mTECA/hi (CD24−Sca1−), mTECB/hi (CD24+Sca1−), and mTECC/hi (CD24+Sca1+). While mTECA/hi included mostly Aire‐expressing cells, mTECB/hi contained Aire+ and Aire− cells and mTECC/hi were mainly composed of cells lacking Aire. The differential expression pattern of Aire led us to investigate the precursor‐product relationship between these subsets. Strikingly, transcriptomic analysis of mTECA/hi, mTECB/hi, and mTECC/hi sequentially mirrored the specific genetic program of Early‐, Late‐ and Post‐Aire mTECs. Corroborating their Post‐Aire nature, mTECC/hi downregulated the expression of tissue‐restricted antigens, acquired traits of differentiated keratinocytes, and were absent in Aire‐deficient mice. Collectively, our findings reveal a new and simple blueprint to survey late stages of mTEC differentiation.

Characterization of Aire-expressing DCs using a novel Aire-reporter strain

Abstract Aire is predominantly expressed by medullary thymic epithelial cells (mTECs), and it is essential for maintaining self-tolerance by controlling the expression of tissue-restricted self-antigens (TRAs). Besides mTECs, extra-thymic Aire-expressing cells (eTACs) have been reported. However, the exact nature of eTACs has been difficult to study because of the small numbers together with low expression levels of Aire within the cells. We have recently established a novel Aire reporter strain in which endogenous Aire was replaced by the human AIRE-GFP-Flag tag fusion gene (Aire/hAGF-knockin: J. Immunol. 2015). hAGF-reporter protein was efficiently produced and retained within the mTECs as authentic Aire nuclear dot protein. With this high-sensitivity and high-fidelity Aire-reporter strain, we found that DCs in the spleen and lymph nodes expressed Aire although their expression levels were rather low. Aire-expressing DCs showed high levels of MHC-II and CD80/CD86, and half of which expressed CD8. In the thymus, we found that there were two types of DCs expressing Aire: hAGF-low DCs and hAGF-high DCs. Interestingly, hAGF-low DCs had small hAGF dots within the nuclei, whereas hAGF-high DCs retained larger hAGF dots which were mainly present outside the nuclei, suggesting that hAGF-high DCs had acquired hAGF protein from mTECs. Acquisition of hAGF protein by hAGF-high DCs from mTECs was confirmed by the bone-marrow transfer experiment. Mechanisms underlying the transfer of nuclear protein (i.e., hAGF protein) from mTECs to thymic DCs, and immunological consequence of this event are now under investigation.

De novo generation of a functional human thymus from induced pluripotent stem cells

A proper functional thymus is required for generation of T cell–mediated immunity. This is dramatically illustrated by patients lacking a thymus, such as children with complete DiGeorge syndrome, which is fatal if left untreated.1-3 Therapeutic options for such patients are limited and confined to transplantation of small fragments from allogeneic neonatal thymi.

Pulling RANK on Cancer: Blocking Aire-Mediated Central Tolerance to Enhance Immunotherapy.

A major breakthrough in cancer treatment occurred with the development of strategies that overcome T-cell tolerance toward tumor cells. These approaches enhance antitumor immunity by overcoming mechanisms that are normally in place to prevent autoimmunity but simultaneously prevent rejection of tumor cells. Although tolerance mechanisms that restrict antitumor immunity take place both in the thymus and periphery, only immunotherapies that target peripheral tolerance mechanisms occurring outside of the thymus are currently available. We review here recent gains in our understanding of how thymic tolerance mediated by the autoimmune regulator (Aire) impedes antitumor immunity. It is now clear that transient depletion of Aire-expressing cells in the thymus can be achieved with RANKL blockade. Finally, we discuss key findings that support the repurposing of anti-RANKL as a cancer immunotherapy with a unique mechanism of action.

Aire deficiency during early pregnancy leads to maternal T cell imbalance and embryo loss

Abstract Healthy pregnancy relies on maternal tolerance to a unique set of pregnancy associated self-antigens (PAAs), which are encoded in the maternal genome, but have not been produced since the mother herself was a fetus with a placenta. We hypothesize that Aire-mediated expression of PAAs promotes deletion of PAA-reactive T cells and induction of PAA-specific Tregs, and that Aire-deficiency may play a role in pregnancy complications. We used an Aire-diphtheria toxin receptor (DTR) transgenic mouse model to ablate maternal Aire-expressing cells only during the first nine days of pregnancy. AireDTR+DT plugged females were 6.5 times as likely to show complete embryo resorption by E9.5, compared to WT+DT dams (RR 6.5; 95% CI 1.61–26.1; p<0.01), and twice as likely to show no signs of implantation at E9.5 compared to WT+DT dams (RR 2.0; 95% CI 1.05–3.94; p<0.05). AireDTR+DT dams had significantly fewer FoxP3+ Tregs (p<0.0001) and more CD4+ T cells (p<0.0001) in the maternal thymus, and increased frequency of CD3+ CD4− CD8− T cells in the uterus (p<0.05). AireKO mice are known to develop ovarian insufficiency and subsequent progesterone (P4) deficiency. However, we found no significant differences in serum P4 levels of AireDTR+DT compared to WT+DT dams. P4 supplementation failed to prevent embryo resorption in AireDTR+DT dams or to change the alteration in thymic Tconv:Treg. Thus, Aire deficiency during early pregnancy leads to maternal T cell imbalance and embryo loss without inducing ovarian insufficiency, suggesting a novel mechanism for autoimmune mediated infertility.

The Aire family expands.

T cell tolerance depends upon Aire-expressing cells to purge the T cell repertoire of autoreactive clones. Once thought to be the exclusive domain of thymic epithelial cells, a new study by Yamano et al. (https://doi.org/10.1084/jem.20181430) in this issue of JEM identifies ILC3-like cells in the lymph nodes with similar properties.

Aire-expressing ILC3-like cells in the lymph node display potent APC features

The autoimmune regulator (Aire) serves an essential function for T cell tolerance by promoting the "promiscuous" expression of tissue antigens in thymic epithelial cells. Aire is also detected in rare cells in peripheral lymphoid organs, but the identity of these cells is poorly understood. Here, we report that Aire protein-expressing cells in lymph nodes exhibit typical group 3 innate lymphoid cell (ILC3) characteristics such as lymphoid morphology, absence of "classical" hematopoietic lineage markers, and dependence on RORγt. Aire+ cells are more frequent among lineage-negative RORγt+ cells of peripheral lymph nodes as compared with mucosa-draining lymph nodes, display a unique Aire-dependent transcriptional signature, express high surface levels of MHCII and costimulatory molecules, and efficiently present an endogenously expressed model antigen to CD4+ T cells. These findings define a novel type of ILC3-like cells with potent APC features, suggesting that these cells serve a function in the control of T cell responses.

Aire Controls in Trans the Production of Medullary Thymic Epithelial Cells Expressing Ly-6C/Ly-6G.

Medullary thymic epithelial cells (mTECs), which express a wide range of tissue-restricted Ags (TRAs), contribute to the establishment of self-tolerance by eliminating autoreactive T cells and/or inducing regulatory T cells. Aire controls a diverse set of TRAs within Aire-expressing cells by employing various transcriptional pathways. As Aire has a profound effect on transcriptomes of mTECs, including TRAs not only at the single-cell but also the population level, we suspected that Aire (Aire+ mTECs) might control the cellular composition of the thymic microenvironment. In this study, we confirmed that this is indeed the case by identifying a novel mTEC subset expressing Ly-6 family protein whose production was defective in Aire-deficient thymi. Reaggregated thymic organ culture experiments demonstrated that Aire did not induce the expression of Ly-6C/Ly-6G molecules from mTECs as Aire-dependent TRAs in a cell-intrinsic manner. Instead, Aire+ mTECs functioned in trans to maintain Ly-6C/Ly-6G+ mTECs. Thus, Aire not only controls TRA expression transcriptionally within the cell but also controls the overall composition of mTECs in a cell-extrinsic manner, thereby regulating the transcriptome from mTECs on a global scale.