Revisiting Aire and tissue-restricted antigens at single-cell resolution.

The thymus is a highly specialized organ that plays an indispensable role in the establishment of self-tolerance, a process characterized by the "education" of developing T-cells. To provide competent T-cells tolerant to self-antigens, medullary thymic epithelial cells (mTECs) orchestrate negative selection by ectopically expressing a wide range of genes, including various tissue-restricted antigens (TRAs). Notably, recent advancements in the high-throughput single-cell analysis have revealed remarkable heterogeneity in mTECs, giving us important clues for dissecting the mechanisms underlying TRA expression. We overview how recent single-cell studies have furthered our understanding of mTECs, with a focus on the role of Aire in inducing mTEC heterogeneity to encompass TRAs.

Cooperative but Distinct Role of Medullary Thymic Epithelial Cells and Dendritic Cells in the Production of Regulatory T Cells in the Thymus.

Regulatory T cells (Tregs) are produced in the thymus to establish self-tolerance, and agonistic stimuli by self-Ags play a pivotal role in this process. Although two types of APCs, medullary thymic epithelial cells (mTECs) and dendritic cells (DCs), are responsible for presenting self-Ags together with costimulatory/cytokine signals, the distinct role of each APC in producing Tregs remains enigmatic. We have approached this issue by depleting the mTECs and DCs using mice expressing diphtheria toxin receptors driven by Aire and CD11c promoters, respectively. Depletion of mTECs showed an effect on Treg production quantitatively and qualitatively more profound than that of DCs followed by the development of distinct organ-specific autoimmune lesions in the hosts. Because self-Ags produced by mTECs are transferable to DCs through a process known as Ag transfer, we monitored the process of Ag transfer using mice expressing GFP from TECs. Although GFP expressed from total TECs was effectively transferred to DCs, GFP expressed from cortical TECs was not, suggesting that mTECs are the predominant source of self-Ags. We also found that GFP expressed not only from mature mTECs but also from immature mTECs was transferred to DCs, suggesting that a broad spectrum of molecules were subjected to Ag transfer during mTEC development. Interestingly, the numbers of recirculating non-Tregs producing IL-2, an important source for Treg expansion in the thymus, were reduced only in the mTEC-depleted mice. These results suggested the cooperative but distinct role of mTECs and DCs in the production of Tregs to avoid autoimmunity.

AIRE illuminates the feature of medullary thymic epithelial cells in thymic carcinoma.

Despite the clear distinction between cortical (cTECs) and medullary thymic epithelial cells (mTECs) in physiology, the cell of origin of thymic carcinomas (TCs) and other thymic epithelial tumors remained enigmatic. We addressed this issue by focusing on AIRE, an mTEC-specific transcriptional regulator that is required for immunological self-tolerance. We found that a large proportion of TCs expressed AIRE with typical nuclear dot morphology by immunohistochemistry. AIRE expression in TCs was supported by the RNA-seq data in the TCGA-THYM database. Furthermore, our bioinformatics approach to the recent single-cell RNA-seq data on human thymi has revealed that TCs hold molecular characteristics of multiple mTEC subpopulations. In contrast, TCs lacked the gene signatures for cTECs. We propose that TCs are tumors derived from mTECs.

No Major Impact of Two Homologous Proteins Ly6C1 and Ly6C2 on Immune Homeostasis.

Ly6C comprises two homologous components of Ly6C1 and Ly6C2, and the expression of either of the Ly6C molecules defines unique functional subsets of monocytes. Ly6C is also expressed by other immune cell types, including Aire-expressing medullary thymic epithelial cells. Because the role of Ly6C expression in determining the functional subsets remains unclear, we generated mice deficient for both Ly6C1 and Ly6C2 with CRISPR-Cas9-mediated deletion. Mice deficient for Ly6C1/Ly6C2 showed no major alterations in the subsets and function of monocyte and other immune cells, including the cells involved in the dextran sulfate sodium salt-induced colitis model. By generating the mice deficient for Ly6C1 alone, we have also investigated the expression pattern of Ly6C1 and Ly6C2 in immune cells. Except for medullary thymic epithelial cells and CD4 single-positive T cells, immune cells predominantly expressed Ly6C2. Thus, despite the importance as a marker with a unique differential expression pattern, the Ly6C molecules have no major impact on determining the functional subsets and maintaining immune homeostasis.

Aire suppresses CTLA-4 expression from the thymic stroma to control autoimmunity.

Impaired production of thymic regulatory T cells (Tregs) is implicated in the development of Aire-dependent autoimmunity. Because Tregs require agonistic T cell receptor stimuli by self-antigens to develop, reduced expression of self-antigens from medullary thymic epithelial cells (mTECs) has been considered to play a major role in the reduced Treg production in Aire deficiency. Here, we show that mTECs abnormally express co-inhibitory receptor CTLA-4 if Aire is non-functional. Upon binding with CD80/CD86 ligands expressed on thymic dendritic cells (DCs), the ectopically expressed CTLA-4 from Aire-deficient mTECs removes the CD80/CD86 ligands from the DCs. This attenuates the ability of DCs to provide co-stimulatory signals and to present self-antigens transferred from mTECs, both of which are required for Treg production. Accordingly, impaired production of Tregs and organ-specific autoimmunity in Aire-deficient mice are rescued by the depletion of CTLA-4 expression from mTECs. Our studies illuminate the significance of mTEC-DC interaction coordinated by Aire for the establishment of thymic tolerance.

Aire Controls Heterogeneity of Medullary Thymic Epithelial Cells for the Expression of Self-Antigens.

The deficiency of Aire, a transcriptional regulator whose defect results in the development of autoimmunity, is associated with reduced expression of tissue-restricted self-Ags (TRAs) in medullary thymic epithelial cells (mTECs). Although the mechanisms underlying Aire-dependent expression of TRAs need to be explored, the physical identification of the target(s) of Aire has been hampered by the low and promiscuous expression of TRAs. We have tackled this issue by engineering mice with augmented Aire expression. Integration of the transcriptomic data from Aire-augmented and Aire-deficient mTECs revealed that a large proportion of so-called Aire-dependent genes, including those of TRAs, may not be direct transcriptional targets downstream of Aire. Rather, Aire induces TRA expression indirectly through controlling the heterogeneity of mTECs, as revealed by single-cell analyses. In contrast, Ccl25 emerged as a canonical target of Aire, and we verified this both in vitro and in vivo. Our approach has illuminated the Aire's primary targets while distinguishing them from the secondary targets.

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 (MHCIIhi CD80hi ) 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.

Development of a new monoclonal antibody specific to mouse Vγ6 chain.

There are seven Vγ gene segments in the TCR γ chain loci of mice. We developed monoclonal antibodies (mAbs) specific to the Vγ6 chain (Heilig & Tonegawa nomenclature). By immunizing Vγ4/6 KO mice with complementarity-determining region peptides in Vγ6 chains, we generated three hybridomas. These hybridomas produced mAbs capable of cell surface staining of Vγ6/Vδ1 gene-transfected T-cell line lacking TCR as well as of Vγ1- Vγ4- Vγ5- Vγ7- γδ T cells and the CD3high TCRδint γδ T cells in various organs. The location of Vγ6+ γδ T cells, which peaked in the newborn thymus, was associated with mTEC. In vivo administration of clone 1C10-1F7 mAb impaired protection against Klebsiella pneumoniae infection but ameliorated psoriasis-like dermatitis induced by imiquimod treatment. These new mAbs are useful to elucidate the development, location, and functions of Vγ6 γδ T cells in mice.

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.

Mode of Tolerance Induction and Requirement for Aire Are Governed by the Cell Types That Express Self-Antigen and Those That Present Antigen.

Aire controls the fate of autoreactive thymocytes (i.e., clonal deletion or development into regulatory T cells [Tregs]) through transcriptional control of the expression of tissue-restricted self-antigens (TRAs) from medullary thymic epithelial cells (mTECs) and bone marrow (BM)-derived cells. Although TRAs expressed by mTECs and BM-derived cells are suggested to complement each other to generate a full spectrum of TRAs, little is known about the relative contribution of TRAs from each component for establishment of self-tolerance. Furthermore, the precise role of Aire in specific types of Aire-expressing APCs remains elusive. We have approached these issues by generating two different types of transgenic mouse (Tg) model, which express a prefixed model self-antigen driven by the insulin promoter or the Aire promoter. In the insulin-promoter Tg model, mTECs alone were insufficient for clonal deletion, and BM-derived APCs were required for this action by utilizing Ag transferred from mTECs. In contrast, mTECs alone were able to induce Tregs, although at a much lower efficiency in the absence of BM-derived APCs. Importantly, lack of Aire in mTECs, but not in BM-derived APCs, impaired both clonal deletion and production of Tregs. In the Aire-promoter Tg model, both mTECs and BM-derived APCs could independently induce clonal deletion without Aire, and production of Tregs was impaired by the lack of Aire in mTECs, but not in BM-derived APCs. These results suggest that the fate of autoreactive thymocytes together with the requirement for Aire depend on the cell types that express self-antigens and the types of APCs involved in tolerance induction.

Identification of embryonic precursor cells that differentiate into thymic epithelial cells expressing autoimmune regulator.

Medullary thymic epithelial cells (mTECs) expressing autoimmune regulator (Aire) are critical for preventing the onset of autoimmunity. However, the differentiation program of Aire-expressing mTECs (Aire(+) mTECs) is unclear. Here, we describe novel embryonic precursors of Aire(+) mTECs. We found the candidate precursors of Aire(+) mTECs (pMECs) by monitoring the expression of receptor activator of nuclear factor-κB (RANK), which is required for Aire(+) mTEC differentiation. pMECs unexpectedly expressed cortical TEC molecules in addition to the mTEC markers UEA-1 ligand and RANK and differentiated into mTECs in reaggregation thymic organ culture. Introduction of pMECs in the embryonic thymus permitted long-term maintenance of Aire(+) mTECs and efficiently suppressed the onset of autoimmunity induced by Aire(+) mTEC deficiency. Mechanistically, pMECs differentiated into Aire(+) mTECs by tumor necrosis factor receptor-associated factor 6-dependent RANK signaling. Moreover, nonclassical nuclear factor-κB activation triggered by RANK and lymphotoxin-ß receptor signaling promoted pMEC induction from progenitors exhibiting lower RANK expression and higher CD24 expression. Thus, our findings identified two novel stages in the differentiation program of Aire(+) mTECs.

Switching on the Aire conditioner.

Aire has been cloned as the gene responsible for a hereditary type of organ-specific autoimmune disease. Aire controls the expression of a wide array of tissue-restricted Ags by medullary thymic epithelial cells (mTECs), thereby leading to clonal deletion and Treg-cell production, and ultimately to the establishment of self-tolerance. However, relatively little is known about the mechanism responsible for the control of Aire expression itself. In this issue of the European Journal of Immunology, Haljasorg et al. [Eur. J. Immunol. 2015. 45: 3246-3256] have reported the presence of an enhancer element for Aire that binds with NF-κB components downstream of the TNF receptor family member, RANK (receptor activator of NF-κB). The results suggest that RANK has a dual mode of action in Aire expression: one involving the promotion of mTEC differentiation and the other involving activation of the molecular switch for Aire within mature mTECs.

Aire Expression Is Inherent to Most Medullary Thymic Epithelial Cells during Their Differentiation Program.

Aire in medullary thymic epithelial cells (mTECs) plays an important role in the establishment of self-tolerance. Because Aire(+) mTECs appear to be a limited subset, they may constitute a unique lineage(s) among mTECs. An alternative possibility is that all mTECs are committed to express Aire in principle, but Aire expression by individual mTECs is conditional. To investigate this issue, we established a novel Aire reporter strain in which endogenous Aire is replaced by the human AIRE-GFP-Flag tag (Aire/hAGF-knockin) fusion gene. The hAGF reporter protein was produced and retained very efficiently within mTECs as authentic Aire nuclear dot protein. Remarkably, snapshot analysis revealed that mTECs expressing hAGF accounted for >95% of mature mTECs, suggesting that Aire expression does not represent a particular mTEC lineage(s). We confirmed this by generating Aire/diphtheria toxin receptor-knockin mice in which long-term ablation of Aire(+) mTECs by diphtheria toxin treatment resulted in the loss of most mature mTECs beyond the proportion of those apparently expressing Aire. These results suggest that Aire expression is inherent to all mTECs but may occur at particular stage(s) and/or cellular states during their differentiation, thus accounting for the broad impact of Aire on the promiscuous gene expression of mTECs.

Ectopic Aire Expression in the Thymic Cortex Reveals Inherent Properties of Aire as a Tolerogenic Factor within the Medulla.

Cortical thymic epithelial cells (cTECs) and medullary thymic epithelial cells (mTECs) play essential roles in the positive and negative selection of developing thymocytes, respectively. Aire in mTECs plays an essential role in the latter process through expression of broad arrays of tissue-restricted Ags. To determine whether the location of Aire within the medulla is absolutely essential or whether Aire could also function within the cortex for establishment of self-tolerance, we used bacterial artificial chromosome technology to establish a semiknockin strain of NOD-background (ß5t/Aire-transgenic) mice expressing Aire under control of the promoter of ß5t, a thymoproteasome expressed exclusively in the cortex. Although Aire was expressed in cTECs as typical nuclear dot protein in ß5t/Aire-Tg mice, cTECs expressing Aire ectopically did not confer transcriptional expression of either Aire-dependent or Aire-independent tissue-restricted Ag genes. We then crossed ß5t/Aire-Tg mice with Aire-deficient NOD mice, generating a strain in which Aire expression was confined to cTECs. Despite the presence of Aire(+) cTECs, these mice succumbed to autoimmunity, as did Aire-deficient NOD mice. The thymic microenvironment harboring Aire(+) cTECs, within which many Aire-activated genes were present, also showed no obvious alteration of positive selection, suggesting that Aire's unique property of generating a self-tolerant T cell repertoire is functional only in mTECs.

NF-κB-inducing kinase in thymic stroma establishes central tolerance by orchestrating cross-talk with not only thymocytes but also dendritic cells.

Essential roles of NF-κB-inducing kinase (NIK) for the development of medullary thymic epithelial cells (mTECs) and regulatory T cells have been highlighted by studies using a strain of mouse bearing a natural mutation of the NIK gene (aly mice). However, the exact mechanisms underlying the defect in thymic cross-talk leading to the breakdown of self-tolerance in aly mice remain elusive. In this study, we demonstrated that production of regulatory T cells and the final maturation process of positively selected conventional αß T cells are impaired in aly mice, partly because of a lack of mature mTECs. Of note, numbers of thymic dendritic cells and their expression of costimulatory molecules were also affected in aly mice in a thymic stroma-dependent manner. The results suggest a pivotal role of NIK in the thymic stroma in establishing self-tolerance by orchestrating cross-talk between mTECs and dendritic cells as well as thymocytes. In addition, we showed that negative selection was impaired in aly mice as a result of the stromal defect, which accounts for the development of organ-specific autoimmunity through a lack of normal NIK.

Temporal lineage tracing of Aire-expressing cells reveals a requirement for Aire in their maturation program.

Understanding the cellular dynamics of Aire-expressing lineage(s) among medullary thymic epithelial cells (AEL-mTECs) is essential for gaining insight into the roles of Aire in establishment of self-tolerance. In this study, we monitored the maturation program of AEL-mTECs by temporal lineage tracing, in which bacterial artificial chromosome transgenic mice expressing tamoxifen-inducible Cre recombinase under control of the Aire regulatory element were crossed with reporter strains. We estimated that the half-life of AEL-mTECs subsequent to Aire expression was ∼7-8 d, which was much longer than that reported previously, owing to the existence of a post-Aire stage. We found that loss of Aire did not alter the overall lifespan of AEL-mTECs, inconsistent with the previous notion that Aire expression in medullary thymic epithelial cells (mTECs) might result in their apoptosis for efficient cross-presentation of self-antigens expressed by AEL-mTECs. In contrast, Aire was required for the full maturation program of AEL-mTECs, as exemplified by the lack of physiological downregulation of CD80 during the post-Aire stage in Aire-deficient mice, thus accounting for the abnormally increased CD80(high) mTECs seen in such mice. Of interest, increased CD80(high) mTECs in Aire-deficient mice were not mTEC autonomous and were dependent on cross-talk with thymocytes. These results further support the roles of Aire in the differentiation program of AEL-mTECs.

TGF-ß type II receptor expression in thymic epithelial cells inhibits the development of Hassall's corpuscles in mice.

Hassall's corpuscles are concentric clusters of keratinized epithelial cells located within the thymic medulla of humans and guinea pigs but are scant in mouse and rat. They are considered to be the terminally differentiated stages of medullary thymic epithelial cells (mTECs) but the mechanisms of their origin are unclear. We have previously deleted the TGF-ß type II receptor (TGFßRII) specifically in mouse TECs and reported that these mice have mitigated thymic involution and exhibit earlier reconstitution post-irradiation. In this study, we analyzed the differentiation of mTECs in the TGFßRII-knockout mice. Interestingly, the TGFßRII-knockout mice display enhanced development of Hassall's corpuscles. The expression of Aire, stromal-cell-derived factor 1 and thymic stromal lymphopoietin in the thymi of the TGFßRII-knockout mice was similar to that previously reported for the human thymus. In addition, the putative epithelial progenitor markers MTS20 and MTS24 labeled Hassall's corpuscles in normal mice, but the extent and intensity of this staining were greatly enhanced in Hassall's corpuscles of the TGFßRII-knockout mice. The phosphorylated forms of ERK and JNK were also found in Hassall's corpuscles of the TGFßRII-knockout mice. Taken together, we suggest that TGFßRII-mediated signaling in TECs inhibits their development into Hassall's corpuscles in mice.

Androgen receptor promotes sex-independent angiogenesis in response to ischemia and is required for activation of vascular endothelial growth factor receptor signaling.

BACKGROUND: Hypoandrogenemia is associated with an increased risk of ischemic diseases. Because actions of androgens are exerted through androgen receptor (AR) activation, we studied hind-limb ischemia in AR knockout mice to elucidate the role of AR in response to ischemia. METHODS AND RESULTS: Both male and female AR knockout mice exhibited impaired blood flow recovery, more cellular apoptosis, and a higher incidence of autoamputation after ischemia. In ex vivo and in vivo angiogenesis studies, AR-deficient vascular endothelial cells showed reduced angiogenic capability. In ischemic limbs of AR knockout mice, reductions in the phosphorylation of the Akt protein kinase and endothelial nitric oxide synthase were observed despite a robust increase in hypoxia-inducible factor 1α and vascular endothelial cell growth factor (VEGF) gene expression. In in vitro studies, siRNA-mediated ablation of AR in vascular endothelial cells blunted VEGF-stimulated phosphorylation of Akt and endothelial nitric oxide synthase. Immunoprecipitation experiments documented an association between AR and kinase insert domain protein receptor that promoted the recruitment of downstream signaling components. CONCLUSIONS: These results document a physiological role of AR in sex-independent angiogenic potency and provide evidence of novel cross-talk between the androgen/AR signaling and VEGF/kinase insert domain protein receptor signaling pathways.

GRK6 deficiency in mice causes autoimmune disease due to impaired apoptotic cell clearance.

Efficient engulfment of apoptotic cells is critical for maintaining tissue homoeostasis. When phagocytes recognize 'eat me' signals presented on the surface of apoptotic cells, this subsequently induces cytoskeletal rearrangement of phagocytes for the engulfment through Rac1 activation. However, the intracellular signalling cascades that result in Rac1 activation remain largely unknown. Here we show that G-protein-coupled receptor kinase 6 (GRK6) is involved in apoptotic cell clearance. GRK6 cooperates with GIT1 to activate Rac1, which promotes apoptotic engulfment independently from the two known DOCK180/ELMO/Rac1 and GULP1/Rac1 engulfment pathways. As a consequence, GRK6-deficient mice develop an autoimmune disease. GRK6-deficient mice also have increased iron stores in splenic red pulp in which F4/80(+) macrophages are responsible for senescent red blood cell clearance. Our results reveal previously unrecognized roles for GRK6 in regulating apoptotic engulfment and its fundamental importance in immune and iron homoeostasis.

Rab13 small G protein and junctional Rab13-binding protein (JRAB) orchestrate actin cytoskeletal organization during epithelial junctional development.

During epithelial junctional development, both vesicle transport and reorganization of the actin cytoskeleton must be spatiotemporally regulated. Coordination of these cellular functions is especially important, but the precise mechanism remains elusive. Previously, we identified junctional Rab13-binding protein (JRAB)/molecules interacting with CasL-like 2 (MICAL-L2) as an effector of the Rab13 small G protein, and we found that the Rab13-JRAB system may be involved in the formation of cell-cell adhesions via transport of adhesion molecules. Here, we showed that JRAB interacts with two actin-binding proteins, actinin-1 and -4, and filamentous actin via different domains and regulates actin cross-linking and stabilization through these interactions. During epithelial junctional development, JRAB is prominently enriched in the actin bundle at the free border; subsequently, JRAB undergoes a Rab13-dependent conformational change that is required for maturation of cell-cell adhesion sites. These results suggest that Rab13 and JRAB regulate reorganization of the actin cytoskeleton throughout epithelial junctional development from establishment to maturation of cell-cell adhesion.