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Deficiency for AIRE/Aire in both humans and mice results in the development of organ-specific autoimmune disease. We tested whether augmented and/or dysregulated AIRE/Aire expression might be also prone to the breakdown of self-tolerance. To define the effect of augmented Aire expression on the development of autoimmunity, antigen-specific clonal deletion and production of clonotypic regulatory T cells (Tregs) in the thymus were examined using mice expressing two additional copies of Aire in a heterozygous state (3xAire-knockin mice: 3xAire-KI). We found that both clonal deletion of autoreactive T cells and production of clonotypic Tregs in the thymus from 3xAire-KI were impaired in a T-cell receptor-transgenic system. Furthermore, 3xAire-KI females showed higher scores of experimental autoimmune encephalomyelitis induced by myelin oligodendrocyte glycoprotein than wild-type littermates, suggesting that augmented Aire expression exacerbates organ-specific autoimmunity under disease-prone conditions. In humans, we found that one patient with amyopathic dermatomyositis showed CD3–CD19– cells expressing AIRE in the peripheral blood before the treatment but not during the remission phase treated with immunosuppressive drugs. Thus, not only loss of function of AIRE/Aire but also augmented and/or dysregulated expression of AIRE/Aire should be considered for the pathogenesis of organ-specific autoimmunity. We suggest that further analyses should be pursued to establish a novel link between organ-specific autoimmune disease and dysregulated AIRE expression in clinical settings.

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.

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., Although the expression of Aire was initially considered to mark a terminal stage of mTEC maturation, recent studies have demonstrated that mTECs extend their differentiation beyond Aire. Integrating flow cytometry, functional, and transcriptomic analyses, Ferreirinha et al. provide an effective phenotypic‐based method to resolve Early‐, Late‐ and Post‐Aire mTECs.

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.

Diabetes can be ameliorated by additive AIRE expression in peripheral DCs, Tissue-specific autoimmune diseases are assumed to arise through malfunction of two checkpoints for immune tolerance: defective elimination of autoreactive T cells in the thymus and activation of these T cells by corresponding autoantigens in the periphery. However, evidence for this model and the outcome of such alterations in each or both of the tolerance mechanisms have not been sufficiently investigated. We studied these issues by expressing human AIRE (huAIRE) as a modifier of tolerance function in NOD mice wherein the defects of thymic and peripheral tolerance together cause type I diabetes (T1D). Additive huAIRE expression in the thymic stroma had no major impact on the production of diabetogenic T cells in the thymus. In contrast, huAIRE expression in peripheral antigen-presenting cells (APCs) rendered the mice resistant to T1D, while maintaining other tissue-specific autoimmune responses and antibody production against an exogenous protein antigen, because of the loss of Xcr1+ dendritic cells, an essential component for activating diabetogenic T cells in the periphery. These results contrast with our recent demonstration that huAIRE expression in both the thymic stroma and peripheral APCs resulted in the paradoxical development of muscle-specific autoimmunity. Our results reveal that tissue-specific autoimmunity is differentially controlled by a combination of thymic function and peripheral tolerance, which can be manipulated by expression of huAIRE/Aire in each or both of the tolerance mechanisms.

The thymus is the organ dedicated to the generation of T cells, which are key effector cells in immune clearance of pathogens and tumours. However, T cells can also react to our own tissues or turn into cancer cells, such as in the case of autoimmunity and leukaemia, respectively. Therefore, the development and selection of T cells is a tightly regulated process that proceeds within inductive thymic microenvironments formed by cortical (c) and medullary (m) thymic epithelial cells (TECs). Herein, we critically summarize our current knowledge on the molecular principles underlying the development and diversification of TEC compartments and highlight their specialized roles in specific stages of T cell development. Knowledge in this area is of fundamental and clinical relevance to understand how the immune system reaches the equilibrium between immunity and tolerance induction.

mTEC progenitors differentiate to mature Aire+ mTECs through a pathway that initiates with RANK and LtβR signaling via the nonclassical NF-κB, followed by TRAF-6–driven maturation., 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-β 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.

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.

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.

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 (β5t/Aire-transgenic) mice expressing Aire under control of the promoter of β5t, a thymoproteasome expressed exclusively in the cortex. Although Aire was expressed in cTECs as typical nuclear dot protein in β5t/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 β5t/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.

Hassall’s corpuscles (HCs) are composed of cornifying, terminally differentiated medullary thymic epithelial cells (mTECs) that are developed under the control of Aire. Here, we demonstrated that HC-mTECs show features of cellular senescence and produce inflammatory cytokines and chemokines including CXCL5, thereby recruiting and activating neutrophils to produce IL-23 in the thymic medulla. We further indicated that thymic plasmacytoid dendritic cells (pDCs) expressing IL-23 receptors constitutively produced Ifna, which plays a role in single positive (SP) cell maturation, in an Il23a-dependent manner. Neutrophil depletion with anti-Ly6G antibody injection resulted in a significant decrease of Ifna expression in the thymic pDCs, suggesting that thymic neutrophil activation underlies the Ifna expression in thymic pDCs in steady state conditions. A New Zealand White mouse strain showing HC hyperplasia exhibited greater numbers and activation of thymic neutrophils and pDCs than B6 mice, whereas Aire-deficient B6 mice with defective HC development and SP thymocyte maturation showed significantly compromised numbers and activation of these cells. These results collectively suggested that HC-mTECs with cell-senescence features initiate a unique cell activation cascade including neutrophils and pDCs leading to the constitutive IFNα expression required for SP T-cell maturation in the thymic medulla.

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 αβ 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.

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 CD80high mTECs seen in such mice. Of interest, increased CD80high 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.

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-β type II receptor (TGFβRII) 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 TGFβRII-knockout mice. Interestingly, the TGFβRII-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 TGFβRII-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 TGFβRII-knockout mice. The phosphorylated forms of ERK and JNK were also found in Hassall’s corpuscles of the TGFβRII-knockout mice. Taken together, we suggest that TGFβRII-mediated signaling in TECs inhibits their development into Hassall’s corpuscles in mice.

Summary Toll-like receptor-7 (TLR7) and 9, innate immune sensors for microbial RNA or DNA, have been implicated in autoimmunity. Upon activation, TLR7 and 9 are transported from the endoplasmic reticulum (ER) to endolysosomes for nucleic acid sensing by an ER-resident protein, Unc93B1. Little is known, however, about a role for sensor transportation in controlling autoimmunity. TLR9 competes with TLR7 for Unc93B1-dependent trafficking and predominates over TLR7. TLR9 skewing is actively maintained by Unc93B1 and reversed to TLR7 if Unc93B1 loses preferential binding via a D34A mutation. We here demonstrate that mice harboring a D34A mutation showed TLR7-dependent, systemic lethal inflammation. CD4 + T cells showed marked differentiation toward T helper 1 (Th1) or Th17 cell subsets. B cell depletion abolished T cell differentiation and systemic inflammation. Thus, Unc93B1 controls homeostatic TLR7 activation by balancing TLR9 to TLR7 trafficking.

In the mechanism of thymus-induced central tolerance, the transcription factor Aire has been demonstrated to promote the expression of a wide range of peripheral organ-specific antigens (Ags) in the medullary thymic epithelial cells (mTECs), which serve as self-Ags in negative selection. We examined the expression of desmoglein 3 (Dsg3), the autoantigen in pemphigus vulgaris (PV), in mouse thymus and the involvement of Aire in tolerance to Dsg3. Immunofluorescence and in situ hybridization revealed Dsg3 in single cells or in clusters in ∼3% of mTECs near the cortico-medullary junction of the thymus in C57BL/6 mice. Dsg3-expressing mTECs also expressed some Ags of skin-unrelated peripheral organs simultaneously. In contrast, Dsg3-positive mTECs were not detected in the Aire(-/-) thymus. Adoptive transfer of splenocytes from Aire(-/-) mice immunized with Dsg3 did not induce anti-Dsg3 IgG production or PV phenotype in Rag2(-/-) recipient mice. However, Aire(-/-) CD4(+) T cells, but not Aire(+/+) CD4(+) T cells, induced low levels of anti-Dsg3 IgG production when transferred with Dsg3(-/-) B cells. These findings indicate that Aire has an important role in Dsg3 expression as well as in selection of T cells that help B cells to produce anti-Dsg3 IgG in thymus.

The current prevailing view regarding the role of Aire in self-tolerance is that it is involved in the transcriptional control of many tissue-restricted self-antigen genes in thymic epithelial cells in the medulla (mTECs); however, accumulating evidence also suggests that Aire has other roles, e.g. in mTEC differentiation, and furthermore that Aire can either promote or inhibit the mTEC differentiation program, i.e. Aire does not play a neutral role in mTEC differentiation. This review discusses when and how Aire plays an important role in controlling the organization of mTECs required for the expression of self-antigen genes.

Expression of the p16Ink4a tumor suppressor gene, a sensor of oncogenic stress, is up-regulated by a variety of potentially oncogenic stimuli in cultured primary cells. However, because p16Ink4a expression is also induced by tissue culture stress, physiological mechanisms regulating p16Ink4a expression remain unclear. To eliminate any potential problems arising from tissue culture–imposed stress, we used bioluminescence imaging for noninvasive and real-time analysis of p16Ink4a expression under various physiological conditions in living mice. In this study, we show that oncogenic insults such as ras activation provoke epigenetic derepression of p16Ink4a expression through reduction of DNMT1 (DNA methyl transferase 1) levels as a DNA damage response in vivo. This pathway is accelerated in the absence of p53, indicating that p53 normally holds the p16Ink4a response in check. These results unveil a backup tumor suppressor role for p16Ink4a in the event of p53 inactivation, expanding our understanding of how p16Ink4a expression is regulated in vivo.

SummaryThe thymic medulla provides a microenvironment where medullary thymic epithelial cells (mTECs) express autoimmune regulator and diverse tissue-restricted genes, contributing to launching self-tolerance. Positive selection is essential for thymic medulla formation via a previously unknown mechanism. Here we show that the cytokine RANK ligand (RANKL) was produced by positively selected thymocytes and regulated the cellularity of mTEC by interacting with RANK and osteoprotegerin. Forced expression of RANKL restored thymic medulla in mice lacking positive selection, whereas RANKL perturbation impaired medulla formation. These results indicate that RANKL produced by positively selected thymocytes is responsible for fostering thymic medulla formation, thereby establishing central tolerance.

The roles of autoimmune regulator (Aire) in the expression of the diverse arrays of tissue-restricted antigen (TRA) genes from thymic epithelial cells in the medulla (medullary thymic epithelial cells [mTECs]) and in organization of the thymic microenvironment are enigmatic. We approached this issue by creating a mouse strain in which the coding sequence of green fluorescent protein (GFP) was inserted into the Aire locus in a manner allowing concomitant disruption of functional Aire protein expression. We found that Aire+ (i.e., GFP+) mTECs were the major cell types responsible for the expression of Aire-dependent TRA genes such as insulin 2 and salivary protein 1, whereas Aire-independent TRA genes such as C-reactive protein and glutamate decarboxylase 67 were expressed from both Aire+ and Aire− mTECs. Remarkably, absence of Aire from mTECs caused morphological changes together with altered distribution of mTECs committed to Aire expression. Furthermore, we found that the numbers of mTECs that express involucrin, a marker for terminal epidermal differentiation, were reduced in Aire-deficient mouse thymus, which was associated with nearly an absence of Hassall's corpuscle-like structures in the medulla. Our results suggest that Aire controls the differentiation program of mTECs, thereby organizing the global mTEC integrity that enables TRA expression from terminally differentiated mTECs in the thymic microenvironment.

Although the role of p21 Waf1/Cip1 gene expression is well documented in various cell culture studies, its in vivo roles are poorly understood. To gain further insight into the role of p21 Waf1/Cip1 gene expression in vivo , we attempted to visualize the dynamics of p21 Waf1/Cip1 gene expression in living animals. In this study, we established a transgenic mice line (p21- p -luc) expressing the firefly luciferase under the control of the p21 Waf1/Cip1 gene promoter. In conjunction with a noninvasive bioluminescent imaging technique, p21- p -luc mice enabled us to monitor the endogenous p21 Waf1/Cip1 gene expression in vivo . By monitoring and quantifying the p21 Waf1/Cip1 gene expression repeatedly in the same mouse throughout its entire lifespan, we were able to unveil the dynamics of p21 Waf1/Cip1 gene expression in the aging process. We also applied this system to chemically induced skin carcinogenesis and found that the levels of p21 Waf1/Cip1 gene expression rise dramatically in benign skin papillomas, suggesting that p21 Waf1/Cip1 plays a preventative role(s) in skin tumor formation. Surprisingly, moreover, we found that the level of p21 Waf1/Cip1 expression strikingly increased in the hair bulb and oscillated with a 3-week period correlating with hair follicle cycle progression. Notably, this was accompanied by the expression of p63 but not p53. This approach, together with the analysis of p21 Waf1/Cip1 knockout mice, has uncovered a novel role for the p21 Waf1/Cip1 gene in hair development. These data illustrate the unique utility of bioluminescence imaging in advancing our understanding of the timing and, hence, likely roles of specific gene expression in higher eukaryotes.

Factors that determine the spectrum of target organs involved in autoimmune destruction are poorly understood. Although loss of function of autoimmune regulator (AIRE) in thymic epithelial cells is responsible for autoimmunity, the pathogenic roles of AIRE in regulating target-organ specificity remain elusive. In order to gain insight into this issue, we have established NOD mice, an animal model of type 1 diabetes caused by autoimmune attack against beta cell islets, in which Aire has been abrogated. Remarkably, acinar cells rather than beta cell islets were the major targets of autoimmune destruction in Aire-deficient NOD mice, and this alteration of intra-pancreatic target-organ specificity was associated with production of autoantibody against pancreas-specific protein disulfide isomerase (PDIp), an antigen expressed predominantly by acinar cells. Consistent with this pathological change, the animals were resistant to the development of diabetes. The results suggest that Aire not only is critical for the control of self-tolerance but is also a strong modifier of target-organ specificity through regulation of T cell repertoire diversification. We also demonstrated that transcriptional expression of PDIp was retained in the Aire-deficient NOD thymus, further supporting the concept that Aire may regulate the survival of autoreactive T cells beyond transcriptional control of self-protein expression in the thymus.

IκB kinase (IKK) α exhibits diverse biological activities through protein kinase-dependent and -independent functions, the former mediated predominantly through a noncanonical NF-κB activation pathway. The in vivo function of IKKα, however, still remains elusive. Because a natural strain of mice with mutant NF-κB-inducing kinase (NIK) manifests autoimmunity as a result of disorganized thymic structure with abnormal expression of Rel proteins in the thymic stroma, we speculated that the NIK-IKKα axis might constitute an essential step in the thymic organogenesis that is required for the establishment of self-tolerance. An autoimmune disease phenotype was induced in athymic nude mice by grafting embryonic thymus from IKKα-deficient mice. The thymic microenvironment that caused autoimmunity in an IKKα-dependent manner was associated with defective processing of NF-κB2, resulting in the impaired development of thymic epithelial cells. Thus, our results demonstrate a novel function for IKKα in thymic organogenesis for the establishment of central tolerance that depends on its protein kinase activity in cooperation with NIK.

The Toll-like receptor (TLR) family has important roles in microbial recognition and dendritic cell activation. TLRs 7 and 9 can recognize nucleic acids and trigger signalling cascades that activate plasmacytoid dendritic cells to produce interferon-alpha (IFN-alpha) (refs 7, 8). TLR7/9-mediated dendritic cell activation is critical for antiviral immunity but also contributes to the pathogenesis of systemic lupus erythematosus, a disease in which serum IFN-alpha levels are elevated owing to plasmacytoid dendritic cell activation. TLR7/9-induced IFN-alpha induction depends on a molecular complex that contains a TLR adaptor, MyD88, and IFN regulatory factor 7 (IRF-7) (refs 10-14), but the underlying molecular mechanisms are as yet unknown. Here we show that IkappaB kinase-alpha (IKK-alpha) is critically involved in TLR7/9-induced IFN-alpha production. TLR7/9-induced IFN-alpha production was severely impaired in IKK-alpha-deficient plasmacytoid dendritic cells, whereas inflammatory cytokine induction was decreased but still occurred. Kinase-deficient IKK-alpha inhibited the ability of MyD88 to activate the Ifna promoter in synergy with IRF-7. Furthermore, IKK-alpha associated with and phosphorylated IRF-7. Our results identify a role for IKK-alpha in TLR7/9 signalling, and highlight IKK-alpha as a potential target for manipulating TLR-induced IFN-alpha production.

Low-affinity IgG3 Abs to microbial membranes are important for primary immune defense against microbes, but little is known about the importance of TLRs in their production. IgG3 levels were extremely low in mice lacking radioprotective 105 (RP105), a B cell surface molecule structurally related to TLRs. RP105−/− B cells proliferated poorly in response to not only the TLR4 ligand LPS but also TLR2 ligand lipoproteins, both of which mediate the immunostimulatory activity of microbial membranes. RP105−/− mice were severely impaired in hapten-specific Ab production against LPS or lipoproteins. CD138 (syndecan-1)-positive plasma cells were detected after lipid A injection in wild-type spleen but much less in RP105−/− spleen. RP105 ligation in vivo induced plasma cell differentiation. RP105 expression was ∼3-fold higher on marginal zone B cells than on follicular and B1 cells and was down-regulated on germinal center cells. These results demonstrate that a signal via RP105 is uniquely important for regulating TLR-dependent Ab production to microbial membranes.

Autoimmune regulator (AIRE) is responsible for the development of organ-specific autoimmune disease in a monogenic fashion. Rare and low levels of tissue expression together with the lack of AIRE-expressing cell lines have hampered a detailed analysis of the molecular dynamics of AIRE. Here we have established cell lines stably transfected with AIRE and studied the regulatory mechanisms for its subcellular expression. We found that nuclear body (NB) formation by AIRE was dependent on the cell cycle. Biochemical fractionation revealed that a significant proportion of AIRE is associated with the nuclear matrix, which directs the functional domains of chromatin to provide sites for gene regulation. Upon proteasome inhibition, AIRE NBs were increased with concomitant reduced expression in the cytoplasm, suggesting that subcellular targeting of AIRE is regulated by a ubiquitin-proteasome pathway. We also found that AIRE NBs compete for cAMP-response element-binding protein-binding protein/p300, a common coactivator of transcription, with the promyelocytic leukemia gene product. These results suggest that the transcriptional regulating activities of AIRE within a cell are controlled and organized in a spatiotemporal manner.

Both nuclear factor (NF)-kappaB-inducing kinase (NIK) and inhibitor of kappaB (IkappaB) kinase (IKK) have been implicated as essential components for NF-kappaB activation in response to many external stimuli. However, the exact roles of NIK and IKKalpha in cytokine signaling still remain controversial. With the use of in vivo mouse models, rather than with enforced gene-expression systems, we have investigated the role of NIK and IKKalpha in signaling through the type I tumor necrosis factor (TNF) receptor (TNFR-I) and the lymphotoxin beta receptor (LTbetaR), a receptor essential for lymphoid organogenesis. TNF stimulation induced similar levels of phosphorylation and degradation of IkappaBalpha in embryonic fibroblasts from either wild-type or NIK-mutant mice. In contrast, LTbetaR stimulation induced NF-kappaB activation in wild-type mice, but the response was impaired in embryonic fibroblasts from NIK-mutant and IKKalpha-deficient mice. Consistent with the essential role of IKKalpha in LTbetaR signaling, we found that development of Peyer's patches was defective in IKKalpha-deficient mice. These results demonstrate that both NIK and IKKalpha are essential for the induction of NF-kappaB through LTbetaR, whereas the NIK-IKKalpha pathway is dispensable in TNFR-I signaling.

Apoptosis is involved in both the cellular and humoral immune system destroying tumors. An apoptosis-inducing factor from HL-60 myeloid leukemia cells was obtained, purified, and sequenced. The protein found has been identified as a human complement factor B-derived fragment Bb, although it is known that factor B is able to induce apoptosis in several leukemia cell lines. Monoclonal antibodies against fragment Ba and Bb inhibited the apoptotic activity of factor B. When the purified fragment Bb was used for apoptosis induction, only the anti-Bb antibody inhibited Bb-induced apoptosis, and not the anti-Ba antibody. The apoptosis-inducing activity was found to be enhanced under conditions facilitating the formation of Bb. Blocking TNF/TNFR or FasL/Fas interactions did not interfere with the factor B-induced apoptosis. CD11c (iC3bR) acts as the main subunit of a heterodimer binding to fragment Bb in the apoptosis pathway, and the factor B-derived fragment Bb was found to possess the previously unknown function of inducing apoptosis in leukemic cells through a suicide mechanism of myeloid lineage cells during the differentiation stage.

TRAF5 [tumor necrosis factor (TNF) receptor-associated factor 5] is implicated in NF-κB and c-Jun NH2-terminal kinase/stress-activated protein kinase activation by members of the TNF receptor superfamily, including CD27, CD30, CD40, and lymphotoxin-β receptor. To investigate the functional role of TRAF5in vivo, we generated TRAF5-deficient mice by gene targeting. Activation of either NF-κB or c-Jun NH2-terminal kinase/stress-activated protein kinase by tumor necrosis factor, CD27, and CD40 was not abrogated intraf5−/−mice. However,traf5−/−B cells showed defects in proliferation and up-regulation of various surface molecules, including CD23, CD54, CD80, CD86, and Fas in response to CD40 stimulation. Moreover,in vitroIg production oftraf5−/−B cells stimulated with anti-CD40 plus IL-4 was reduced substantially. CD27-mediated costimulatory signal also was impaired intraf5−/−T cells. Collectively, these results demonstrate that TRAF5 is involved in CD40- and CD27-mediated signaling.

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., The clearance of apoptotic cells by macrophages is important for tissue homoeostasis. Here Nakaya et al. reveal a role for GRK6 in the regulation of apoptotic engulfment and show that GRK6 deficiency in mice leads to autoimmune disease and iron accumulation in the spleen.

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.

Parkin is a multifunctional protein, including maintaining mitochondrial homeostasis. Recent evidence suggests that Parkin is recruited from the cytoplasm to damaged mitochondria with low membrane potential. We found that intracellular localization of Parkin changed with cellular growth phase. Parkin was preferentially localized in the mitochondria of cultured cells. The mitochondria with large amounts of Parkin showed preserved membrane potentials even during treatment with carbonyl cyanide m-chlorophenylhydrazone. Here we report a novel protein named Klokin 1 that transports Parkin to the mitochondria. Klokin 1 was localized to the mitochondria and enhanced mitochondrial expression of Parkin. Klokin 1 enhanced cell viability in Parkin-silenced cells. Klokin 1 expression was enhanced in the brains of Parkin-deficient mice but not in an autopsied PARK2 brain. Our findings indicate that mitochondrial Parkin prevents mitochondrial depolarization and that Klokin 1 may compensate for Parkin deficiency.

It has recently become clear that signals mediated by members of the TNFR superfamily, including lymphotoxin-β receptor (LTβR), receptor activator for NF-κB (RANK), and CD40, play essential roles in organizing the integrity of medullary thymic epithelial cells (mTECs) required for the establishment of self-tolerance. However, details of the mechanism responsible for the unique and cooperative action of individual and multiple TNFR superfamily members during mTEC differentiation still remain enigmatic. In this study, we show that the LTβR signal upregulates expression of RANK in the thymic stroma, thereby promoting accessibility to the RANK ligand necessary for mTEC differentiation. Cooperation between the LTβR and RANK signals for optimal mTEC differentiation was underscored by the exaggerated defect of thymic organogenesis observed in mice doubly deficient for these signals. In contrast, we observed little cooperation between the LTβR and CD40 signals. Thus, the LTβR signal exhibits a novel and unique function in promoting RANK activity for mTEC organization, indicating a link between thymic organogenesis mediated by multiple cytokine signals and the control of autoimmunity.

Signal transduction pathways regulating NF-κB activation essential for microenvironment formation in secondary lymphoid organs remain to be determined. We investigated the effect of a deficiency of TNFR-associated factor 6 (TRAF6), which activates the classical NF-κB pathway, in splenic microenvironment formation. Two-week-old TRAF6-deficient mice showed severe defects in B cell follicle and marginal zone formation, similar to mutant mice defective in lymphotoxin (Lt) β receptor (LtβR) signal induction of nonclassical NF-κB activation. However, analysis revealed a TRAF6 role in architecture formation distinct from its role in the early neonatal Lt signaling pathway. LtβR signal was essential for primary B cell cluster formation with initial differentiation of follicular dendritic cells (FDCs) in neonatal mice. In contrast, TRAF6 was dispensable for progression to this stage but was required for converting B cell clusters to B cell follicles and maintaining FDCs through to later stages. Fetal liver transfer experiments suggested that TRAF6 in radiation-resistant cells is responsible for follicle formation. Despite FDC-specific surface marker expression, FDCs in neonatal TRAF6-deficient mice had lost the capability to express CXCL13. These data suggest that developmentally regulated activation of TRAF6 in FDCs is required for inducing CXCL13 expression to maintain B cell follicles.

Thymic epithelial cells (TECs) play pivotal roles in the establishment of self tolerance through critical dialogue with developing thymocytes. Unique actions of two transcriptional regulators within TECs, NF-kappaB-inducing kinase (NIK) and an autoimmune regulator (AIRE), for the establishment of self tolerance have recently been highlighted by studies using a strain of mouse bearing a natural mutation of the NIK gene (aly mice) and gene-targeted mice, respectively. Previous studies have demonstrated essential roles of NIK downstream of the lymphotoxin-beta receptor (LTbetaR), which is essential for the development of secondary lymphoid organs; aly mice lack all lymph nodes and Peyer's patches because of the defective LTbetaR signaling. Additional roles of NIK in thymic organogenesis downstream of LTPR, mainly through the developmental regulation of TECs, have now emerged, although the corresponding ligand(s) for LTbetaR participating in this action have not been fully characterized. In contrast, AIRE, a gene responsible for the development of an organ-specific autoimmune disease that demonstrates monogenic autosomal recessive inheritance, contributes to the establishment of self tolerance probably by controlling the expression of self antigens through yet undetermined molecular mechanisms. Thus, it is highly likely that a group of genes control self tolerance within TECs through unique and coordinated actions, and that an understanding of this process would help to unravel the pathogenesis of autoimmune disease.

The microenvironments of the thymus are generated by thymic epithelial cells (TECs) and are essential for inducing immune self-tolerance or developing T cells. However, the molecular mechanisms that underlie the differentiation of TECs and thymic compartmentalization are not fully understood. Here we show that deficiency in the tumor necrosis factor receptor–associated factor (TRAF) 6 results in disorganized distribution of medullary TECs (mTECs) and the absence of mature mTECs. Engraftment of thymic stroma of TRAF6 -/- embryos into athymic nude mice induced autoimmunity. Thus, TRAF6 directs the development of thymic stroma and represents a critical point of regulation for self-tolerance and autoimmunity.

Nucling is a novel protein isolated from murine embryonal carcinoma cells with an up-regulated expression during cardiac muscle differentiation. We show here that Nucling was up-regulated by proapoptotic stimuli and important for the induction of apoptosis after cytotoxic stress. We further demonstrated that overexpressed Nucling was able to induce apoptosis. In Nucling-deficient cells, the expression levels of Apaf-1 and cytochrome c, which are the major components of an apoptosis-promoting complex named apoptosome, were both down-regulated under cellular stress. A deficiency of Nucling also conferred resistance to apoptotic stress on the cell. After UV irradiation, Nucling was shown to reside in an Apaf-1/pro-caspase-9 complex, suggesting that Nucling might be a key molecule for the formation and maintenance of this complex. Nucling induced translocation of Apaf-1 to the nucleus, thereby distributing the Nucling/Apaf-1/pro-caspase-9 complex to the nuclear fraction. These findings suggest that Nucling recruits and transports the apoptosome complex during stress-induced apoptosis.

Physical contact between thymocytes and the thymic stroma is essential for T cell maturation and shapes the T cell repertoire in the periphery. Stromal elements that control these processes still remain elusive. We used a mouse strain with mutant NF-κB-inducing kinase (NIK) to examine the mechanisms underlying the breakdown of self-tolerance. This NIK-mutant strain manifests autoimmunity and disorganized thymic structure with abnormal expression of Rel proteins in the stroma. Production of immunoregulatory T cells that control autoreactive T cells was impaired in NIK-mutant mice. The autoimmune disease seen in NIK-mutant mice was reproduced in athymic nude mice by grafting embryonic thymus from NIK-mutant mice, and this was rescued by supply of exogenous immunoregulatory T cells. Impaired production of immunoregulatory T cells by thymic stroma without normal NIK was associated with altered expression of peripheral tissue-restricted Ags, suggesting an essential role of NIK in the thymic microenvironment in the establishment of central tolerance.

NF-κB-inducing kinase (NIK) is involved in lymphoid organogenesis in mice through lymphotoxin-β receptor signaling. To clarify the roles of NIK in T cell activation through TCR/CD3 and costimulation pathways, we have studied the function of T cells from aly mice, a strain with mutant NIK. NIK mutant T cells showed impaired proliferation and IL-2 production in response to anti-CD3 stimulation, and these effects were caused by impaired NF-κB activity in both mature and immature T cells; the impaired NF-κB activity in mature T cells was also associated with the failure of maintenance of activated NF-κB. In contrast, responses to costimulatory signals were largely retained in aly mice, suggesting that NIK is not uniquely coupled to the costimulatory pathways. When NIK mutant T cells were stimulated in the presence of a protein kinase C (PKC) inhibitor, proliferative responses were abrogated more severely than in control mice, suggesting that both NIK and PKC control T cell activation in a cooperative manner. We also demonstrated that NIK and PKC are involved in distinct NF-κB activation pathways downstream of TCR/CD3. These results suggest critical roles for NIK in setting the threshold for T cell activation, and partly account for the immunodeficiency in aly mice.

Alymphoplasia (aly) mice, a natural strain with a mutant NF-κB-inducing kinase (NIK) gene, manifest a unique phenotype; they lack lymph nodes and Peyer’s patches, have a disturbed spleen architecture, and exhibit defects in both Ab and cellular immune responses. Although a stromal defect caused by impaired lymphotoxin-β receptor signaling accounts for their abnormal lymphoid organogenesis, the exact mechanisms underlying the development of immunodeficiency in aly mice are poorly understood. We therefore investigated the contribution of hemopoietic cells with the aly NIK mutation to the development of immunodeficiency. Transfer of aly/aly bone marrow cells into aly/+ mice resulted in poorly developed B cell follicles and lack of support for the development of germinal centers and isotype switching, indicating that the hemopoietic cells of aly mice contain an autonomous defect. However, follicular dendritic cell clusters were maintained in the spleens of these bone marrow chimeras, suggesting that the lack of follicular dendritic cell clusters in aly mice is probably due to the stromal defect. The aly mice lacked marginal zone B cells in their spleens, and aly/aly B cells showed an impaired proliferative response after in vitro stimulation. IL-2 production by activated T cells was also impaired. By contrast, the dendritic cells of aly mice exhibited grossly normal development and function. Supporting the concept of an autonomous cell defect, Rel protein expression was altered in aly/aly spleens. Thus, the aly NIK mutation affects hemopoietic cell function in an intrinsic fashion and, together with the stromal defect, may contribute to the development of immunodeficiency in aly mice.

Medullary thymic epithelial cells (mTECs) establish T cell self-tolerance through the expression of autoimmune regulator (Aire) and peripheral tissue-specific self-antigens. However, signals underlying mTEC development remain largely unclear. Here, we demonstrate crucial regulation of mTEC development by receptor activator of NF-kappaB (RANK) and CD40 signals. Whereas only RANK signaling was essential for mTEC development during embryogenesis, in postnatal mice, cooperation between CD40 and RANK signals was required for mTEC development to successfully establish the medullary microenvironment. Ligation of RANK or CD40 on fetal thymic stroma in vitro induced mTEC development in a tumor necrosis factor-associated factor 6 (TRAF6)-, NF-kappaB inducing kinase (NIK)-, and IkappaB kinase beta (IKKbeta)-dependent manner. These results show that developmental-stage-dependent cooperation between RANK and CD40 promotes mTEC development, thereby establishing self-tolerance.