Extrathymic Aire-expressing Cells Research Articles

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.

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.

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.

Clinicopathological and immunohistochemical analysis of autoimmune regulator expression in patients with osteosarcoma.

Autoimmune regulator (AIRE) is a transcription factor that is expressed in medullary thymic epithelial cells. It plays an essential role in central tolerance by eliminating self-reactive T cells. Recently, extrathymic AIRE-expressing cells have been revealed, which are associated with peripheral tolerance. Moreover, AIRE expression has been demonstrated in skin tumors and breast cancer. However, the expression of AIRE in osteosarcoma is unknown. We used immunohistochemistry to investigate AIRE expression in biopsy samples from 43 patients with conventional osteosarcoma and statistically analyzed the association between AIRE expression and clinicopathological characteristics. High AIRE expression was detected in 25 patients (58.1%), and significantly associated with the presence of lung metastasis (P = 0.014) and an increased number of forkhead box P3-positive tumor-infiltrating lymphocytes (regulatory T cells) (P = 0.014). The overall survival rate for all osteosarcoma patients with high AIRE expression was significantly shorter than that for those with low AIRE expression (P = 0.046). In a subgroup analysis of American Joint Committee on Cancer stage II patients who underwent complete surgical resection and conventional chemotherapy, the overall survival and metastasis-free survival rates were significantly shorter for patients with high AIRE expression than for those with low AIRE expression (P = 0.019 and P < 0.01, respectively). High AIRE expression was confirmed to be an independent poor prognostic factor for both overall survival (hazard ratio: 3.841, P = 0.038) and metastasis-free survival (hazard ratio: 4.348, P = 0.022) in the multivariate analysis. The evaluation of AIRE expression may be useful for stratifying osteosarcoma patients for more effective clinical follow-up.

Identification of a novel cis-regulatory element essential for immune tolerance.

Thymic central tolerance is essential to preventing autoimmunity. In medullary thymic epithelial cells (mTECs), the Autoimmune regulator (Aire) gene plays an essential role in this process by driving the expression of a diverse set of tissue-specific antigens (TSAs), which are presented and help tolerize self-reactive thymocytes. Interestingly, Aire has a highly tissue-restricted pattern of expression, with only mTECs and peripheral extrathymic Aire-expressing cells (eTACs) known to express detectable levels in adults. Despite this high level of tissue specificity, the cis-regulatory elements that control Aire expression have remained obscure. Here, we identify a highly conserved noncoding DNA element that is essential for Aire expression. This element shows enrichment of enhancer-associated histone marks in mTECs and also has characteristics of being an NF-κB-responsive element. Finally, we find that this element is essential for Aire expression in vivo and necessary to prevent spontaneous autoimmunity, reflecting the importance of this regulatory DNA element in promoting immune tolerance.

A1.50 Extrathymic autoimmune regulator (AIRE) expression in rheumatoid arthritis synovial tissue

BackgroundAutoimmune Regulator (AIRE) is a transcription factor that is involved in the negative selection of self-reactive thymocytes in the thymus and therefore is pivotal in the establishment of central tolerance....

Function of Aire in central and peripheral immune tolerance

Negative selection induces central tolerance in which self-reactive T cells are deleted by medullary thymic epithelial cells (mTECs) to prevent autoimmunity. The transcriptional factor, autoimmune regulator (Aire), controls the expression of tissue-specific antigens (TSAs) by mTECs for negative selection. The mechanisms by which Aire targets loci which encode TSAs are unknown in detail; recently, however, the ATF7ip-MBD1 complex was identified as an Aire-interacting transcriptional protein complex required for its targeting the loci. Lineage tracing of Aire(+) mTECs identified that mTECs have a post-Aire stage during the development, where they lost maturation markers but maintained intermediate TSA expression, and Aire is required for the terminal differentiation of mTEC's. Extrathymic Aire-expressing cells (eTACs) are identified in murine and human secondary lymphoid organs. eTACs express major histocompatibility complex class II(hi), CD80(lo), CD86(lo), epithelial cell adhesion molecule(hi), CD45(lo) bone marrow-derived peripheral antigen-presenting cell population, which is distinct from mTECs and dendritic cells. They can induce activation-induced cell death of self-reactive CD8(+) T cells and unresponsiveness of self-reactive CD4(+) T cells through a mechanism that does not require regulatory T cells, suggesting that peripheral Aire plays a complementary role for central tolerance.

A Breath of Fresh Aire

The origin and function of extrathymic Aire-expressing cells (eTACs) is incompletely defined. In this issue of Immunity, Gardner et al. (2013) show that eTACs are a distinct tolerogenic cell population that functionally inactivates CD4⁺ T cells to induce peripheral tolerance.

Extrathymic Aire-Expressing Cells Are a Distinct Bone Marrow-Derived Population that Induce Functional Inactivation of CD4+ T Cells

The autoimmune regulator (Aire) is essential for prevention of autoimmunity; its role is best understood in the thymus, where it promotes self-tolerance through tissue-specific antigen (TSA) expression. Recently, extrathymic Aire-expressing cells (eTACs) have been described in murine secondary lymphoid organs, but the identity of such cells and their role in immune tolerance remains unclear. Here we have shown that eTACs are a discrete major histocompatibility complex class II (MHC II)(hi), CD80(lo), CD86(lo), epithelial cell adhesion molecule (EpCAM)(hi), CD45(lo) bone marrow-derived peripheral antigen-presenting cell (APC) population. We also have demonstrated that eTACs can functionally inactivate CD4⁺ T cells through a mechanism that does not require regulatory T cells (Treg) and is resistant to innate inflammatory stimuli. Together, these findings further define eTACs as a distinct tolerogenic cell population in secondary lymphoid organs.

Control of central and peripheral tolerance by Aire.

The negative selection of self-reactive thymocytes depends on the expression of tissue-specific antigens by medullary thymic epithelial cells. The autoimmune regulator (Aire) protein plays an important role in turning on these antigens, and the absence of even one Aire-induced tissue-specific antigen in the thymus can lead to autoimmunity in the antigen-expressing target organ. Recently, Aire protein has been detected in peripheral lymphoid organs, suggesting that peripheral Aire plays a complementary role here. In these peripheral sites, Aire was found to regulate the expression of a group of tissue-specific antigens that is distinct from those expressed in the thymus. Furthermore, transgenic antigen expression in extrathymic Aire-expressing cells (eTACs) can mediate deletional tolerance, but the immunological relevance of Aire-dependent, endogenous tissue-specific antigens remains to be determined.

Mechanisms of tolerance

Mechanisms of tolerance

Aire expression in human lymph nodes: a comparison of type I diabetics and healthy controls. (148.14)

Abstract Autoimmune regulator (Aire) was identified in 1997 as the gene mutated in autoimmune polyendocrine syndrome type I (APS-1), a disease in which patients develop a wide range of autoimmune diseases including thyroiditis and type I diabetes (T1D). Aire is known to be highly expressed in medullary thymic epithelial cells where it upregulates tissue-specific antigens for negative selection. Recently, using transgenic reporter mice, our lab has characterized a rare population of cells in secondary lymphoid organs that also expresses Aire. These eTACs (extra-thymic Aire-expressing cells) displayed a “tolerizing” cell surface phenotype (MHC class IIhi, CD80-, CD86-) and promoted tolerance to prevent autoimmune diabetes. The goal of this study was to identify and characterize eTACs in human lymph node (LN) and to compare eTACs in T1D and healthy controls. Immuno-fluorescence revealed Aire-expressing cells in both pancreatic and non-pancreatic LN. Aire+ cells were relatively rare but showed the typical “nuclear speckling” pattern for staining. Co-staining was performed to examine both their cell linage and antigen presentation capabilities. Quantitative PCR was used to confirm the LN Aire expression. Finally, similar methods were used to compare the Aire expression in pancreatic LN of T1D and healthy controls. Overall, our data reinforces the fact that Aire is indeed expressed outside the thymus and that it may potentially have a role in peripheral tolerance in humans.

The role of Aire in clonal selection

In his clonal selection theory, Frank Macfarlane Burnet predicted that autoreactive lymphocytes are deleted to prevent autoimmunity. This and other principles of lymphocyte behavior outlined by Burnet guided many studies that lead to our current understanding of thymic selection. Thus, when the genetic mutation responsible for autoimmune polyglandular syndrome type 1 was mapped to the autoimmune regulator (AIRE) gene, and Aire was found to be highly expressed in thymic epithelium, studying the role of Aire in negative selection made sense in the context of modern models of thymic selection. We now know Aire is a transcription factor required for the expression of many tissue-specific antigens (TSAs) in the thymus. In the absence of functional Aire, human patients and mice develop multi-organ autoimmune disease because of a defect in thymic negative selection. In addition to its role in the thymus, recent work in our lab suggests that extrathymic Aire-expressing cells have an important role in the clonal deletion of autoreactive CD8+ T cells. In this review, we summarize the latest studies on thymic and peripheral Aire-expressing cells, as well as other TSA-expressing stromal cell populations in peripheral lymphoid organs. We also discuss theoretical differences in thymic and peripheral Aire function that warrant further studies.