Defective Negative Selection Research Articles

Development of targeted biologics for systemic lupus erythematosus

The prototypic autoimmune disease, systemic lupus erythematosus (SLE), is known to be associated with a number of genetic polymorphisms that can affect B cell function as well as polyclonal B cell hyperactivity. Developing an understanding of the complex nature of human B cell activation and differentiation has permitted an assessment of whether specific stages of B cell maturation are affected by the tendency for polyclonal B cell activation. Moreover, the analysis of perturbations of the specific stages of B cell maturation has generated new information on whether abnormalities in B cell differentiation are primarily involved in SLE immunopathology or, rather, are secondary to the inflammatory environment characteristic of subjects with this autoimmune disease. Multivariant flow cytometric analysis has documented abnormalities in B cell maturation that are primarily associated with lupus, or, alternatively related to disease duration, disease activity and concomitant medication. Together, these analyses have provided new insights on the role of B cell over-reactivity in SLE. Characterization of peripheral blood B cell subsets in patients with SLE has provided unique opportunities to identify abnormalities among pre-naive, transitional, pre-naive, memory B cells and in particular plasmablasts/plasma cells including indications for defects in negative selection of autoreactive B cells at certain stages. The pathogenic impact of all of these individual disturbances remains less clear, although the findings have served to identify SLE as associated with numerous abnormalities in B cell development. Moreover, active SLE is characterized by overactive T cell dependent germinal center-like reactions that produce expanded and largely unregulated numbers of memory B cells and plasma cells, thereby contributing to SLE pathogenesis. Finally, an understanding of the nature of the B cell abnormalities in SLE has generated new targets for therapy of this disease. The first of these, belimumab, a monoclonal antibody to the B cell survival factor B Lymphocyte stimulator (BLyS), has proven successful in treating patients with SLE. Additional approaches that target specific stages of B cell activation or differentiation are in development for treatment of SLE.

Reduced TCR signaling potential impairs negative selection but does not result in autoimmune disease

Negative selection and regulatory T (T reg) cell development are two thymus-dependent processes necessary for the enforcement of self-tolerance, and both require high-affinity interactions between the T cell receptor (TCR) and self-ligands. However, it remains unclear if they are similarly impacted by alterations in TCR signaling potential. We generated a knock-in allele (6F) of the TCR ζ chain gene encoding a mutant protein lacking signaling capability whose expression is controlled by endogenous ζ regulatory sequences. Although negative selection was defective in 6F/6F mice, leading to the survival of autoreactive T cells, 6F/6F mice did not develop autoimmune disease. We found that 6F/6F mice generated increased numbers of thymus-derived T reg cells. We show that attenuation of TCR signaling potential selectively impacts downstream signaling responses and that this differential effect favors Foxp3 expression and T reg cell lineage commitment. These results identify a potential compensatory pathway for the enforcement of immune tolerance in response to defective negative selection caused by reduced TCR signaling capability.

On death receptor 3 and its ligands…

The recent article in Immunology by Park et al.1 entitled ‘Interleukin-32 enhances cytotoxic effect of natural killer cells to cancer cells via activation of death receptor 3’ is very interesting; however, I believe that non-specialist readers would benefit from a more expansive and detailed discussion of its context. The authors have omitted much of the recent literature detailing the broader biological functions of Death Receptor 3 (DR3), most of which do not relate to regulating cell death. In addition, clarification is also required with regards to the ligands of DR3 because the older nomenclature can cause confusion and is particularly pertinent to the interpretation of this study. Towards the end of 1996 and beginning of 1997, DR3 (TNFRSF25) was reported simultaneously by a number of groups as a tumour necrosis factor receptor superfamily (TNFRSF) member with an intracellular, apoptosis-inducing death domain and was ascribed a variety of names – Apo3, LARD, TR3, TRAMP and WSL-1.2–7 In recent years, DR3 has emerged as a major regulator of inflammatory and autoimmune disease; research using knockout and transgenic mice has revealed that DR3 plays a critical role in the development of experimental autoimmune encephalomyelitis, allergic lung inflammation, inflammatory arthritis and experimental autoimmune uveoretinitis.8–12 Studies in vivo have also demonstrated a role in colitis and ileitis.13–17 DR3 regulates immunity to certain bacteria,18 viruses,19 tumours20 and intrinsically maintains neurological function.21 Research in humans has mirrored these findings, primarily showing that DR3 regulates inflammation and immunity through controlling the development of effector T cells and differentiation of myeloid subsets,22–30 but it may also have effects on other cell types such as neurons.31 Local and systemic increases of its ligand are associated with multiple human inflammatory disorders.32–35 In this respect, the designation ‘Death Receptor 3’ is a misnomer because many of the recognized functions of the gene are associated with cell expansion and differentiation, rather than death. Park et al.1 clearly describe an increase in cell viability of tumour cell lines following exposure to natural killer (NK) cells when DR3 expression was knocked down; results consistent with DR3 acting to trigger cell death. To my knowledge, this is the first functional demonstration of a pro-apoptotic role for DR3 in human tumour cell lines, but it is not unique as a general phenomenon. The original DR3 knockout mouse exhibited a defect in negative selection of thymocytes,36 while DR3-dependent apoptosis has been described in renal inflammation in vivo37 and osteoblast cell lines in vitro.38 Furthermore, a role in human cancer has been implied from the discovery that the DR3 gene is disrupted in ∼ 40% of neuroblastomas.39 It is in this context that clarification is useful on the nature of the DR3 ligand, as its identity is also complicated by a history of diverse nomenclature. Park et al.1 mention two ligands in their references, Apo3L and TL1A, both of which are distinct tumour necrosis factor superfamily (TNFSF) members. Apo3L was originally named as the ligand for DR3 (i.e. Apo3)40 and was also called TWEAK (TNFSF12). However, follow-up studies could not confirm this41 and indicated that TWEAK signalled in the absence of DR3.42 A second receptor for TWEAK, Fn14 (TNFRSF12A), was then identified,43 and TL1A (TNFSF15 and the full-length gene product of the vascular endothelial growth inhibitor, VEGI) was found to bind DR3.44 All-encompassing work from Bossen et al.45 involving flow cytometric binding assays between the majority of human and murine TNFSF:Fc proteins and cell lines transfected with TNFRSF members confirmed this, i.e. that TWEAK binds Fn14, whereas TL1A binds DR3 and there is minimal cross-reactivity, findings that have been borne out in later in vivo experiments using gene knockouts.10,11 TL1A is therefore currently the only recognized TNFSF ligand for DR3. Park et al.1 show quite elegantly with co-cultures and a series of small interfering RNA knockdown experiments that: (i) the NK cell line NK-92 could kill prostate and colon cancer cell lines dependent on interleukin-32 (IL-32) expression, (ii) DR3 was up-regulated on the cancer cells following co-culture, (iii) IL-32 induced Apo3L (TWEAK) expression on NK cells, and (iv) DR3 knockdown decreased susceptibility of the cancer cells to NK-92. However, their efforts to antagonize Apo3L and DR3 with antibodies demonstrate the action within their system of not one, but two distinct pathways, TWEAK/Fn14 and TL1A/DR3. The relative contribution of the two pathways, and the extent to which IL-32 triggers DR3 ligand (i.e. TL1A) release, remain areas of further research in this field.

Aire regulates immune tolerance towards melanoma (162.43)

Abstract Effective recognition of tumor antigens by circulating T cells constitutes an important part of successful immunosurveillance against cancer growth. Immune tolerance towards tumor antigens, therefore, is a barrier toward an effective anti-tumor immune response. Autoimmune regulator (Aire) is a transcriptional regulator that is critical for the development of central T cell tolerance by inducing self-antigen expression in the thymus. Aire has a well-known role in preventing autoimmunity, and Aire deficiency leads to multi-organ autoimmunity in both human patients and mice. Recently, Aire polymorphisms have been shown to be associated with melanoma development. Whether Aire may mediate immune responses against tumors and its mechanism, however, is not clear. We utilized a mouse harboring a dominant negative point mutation in Aire (Aire G228W) that results in hypomorphic Aire function to investigate the role of Aire deficiency in melanoma. Aire G228W mice develop more robust immune response against melanoma which leads to suppressed tumor growth. This enhanced immune response results from defective negative selection of T cells recognizing melanoma. Aire expression in the thymus is sufficient to mediate immune tolerance against melanoma. Our findings provide a biological mechanism for the epidemiological association between Aire polymorphisms and risk for melanoma and suggest that modulation of central tolerance mechanisms could be a potential strategy for cancer therapy.

Primary T Cell Immunodeficiencies and Autoimmunity

Abstract SCI-21There are a variety of primary T cell immunodeficiencies that can impair T cell differentiation or T cell activation. The latter include CD38δ deficiency, ZAP70 deficiency, Ca++ influx deficiency (ORAI1 and Stim1 deficiencies), and ITK deficiency as well. A new T cell activation deficiency as observed in a patient with defective T cell receptor triggered T cell activation and low CD4 T cell counts will be reported. A remarkable and quasi constant feature shared by all those T cell activation defects is the occurrence of autoimmune diseases, mostly related to autoantibodies, and inflammation such as colitis or panniculitis. Several mechanisms can account for these findings that include defective regulatory T cell development or function, defective negative selection impaired intrinsic feedback mechanism as well as non TCR-mediated T cell activation ultimately leading to proinflammatory cytokines release and autoantibody production by B cells. Another new form of primary T cell immunodeficiency with autosomal recessive inheritance observed in four patients from two families will be described. It is characterized by defective survival of naïve T cells. There again, autoimmunity appeared to be a significant component of the phenotype. Collectively, these results indicate that further insight into the role of key molecules in T cell activation/survival is provided by the analysis of new primary immunodeficiency phenotypes. In addition, the occurrence of autoimmunity in these settings stresses on one hand the role of T cells in the control of reactivity to self and, on the other hand, should be considered in the therapeutic strategy of these conditions. Disclosures:No relevant conflicts of interest to declare.

Autoimmune regulator (AIRE) contributes to Dectin-1–induced TNF-α production and complexes with caspase recruitment domain–containing protein 9 (CARD9), spleen tyrosine kinase (Syk), and Dectin-1

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) syndrome is a complex immunologic disease caused by mutation of the autoimmune regulator (AIRE) gene. Autoimmunity in patients with APECED syndrome has been shown to result from deficiency of AIRE function in transcriptional regulation of thymic peripheral tissue antigens, which leads to defective T-cell negative selection. Candidal susceptibility in patients with APECED syndrome is thought to result from aberrant adaptive immunity. To determine whether AIRE could function in anticandidal innate immune signaling, we investigated an extrathymic role for AIRE in the immune recognition of β-glucan through the Dectin-1 pathway, which is required for defense against Candida species. Innate immune signaling through the Dectin-1 pathway was assessed in both PBMCs from patients with APECED syndrome and a monocytic cell line. Subcellular localization of AIRE was assessed by using confocal microscopy. PBMCs from patients with APECED syndrome had reduced TNF-α responses after Dectin-1 ligation but in part used a Raf-1-mediated pathway to preserve function. In the THP-1 human monocytic cell line, reducing AIRE expression resulted in significantly decreased TNF-α release after Dectin-1 ligation. AIRE formed a transient complex with the known Dectin-1 pathway components phosphorylated spleen tyrosine kinase and caspase recruitment domain-containing protein 9after receptor ligation and localized with Dectin-1 at the cell membrane. AIRE can participate in the Dectin-1 signaling pathway, indicating a novel extrathymic role for AIRE and a defect that likely contributes to fungal susceptibility in patients with APECED syndrome.

Overexpression of short CYLD leads to autoimmunity and production of autoantibodies (47.20)

Abstract CYLD is a tumour suppressor gene which plays an important role in NF-kB signaling. To analyze the function of CYLD in vivo we used the CYLDex7/8 mouse strain, which is characterized by loss of the full-length transcript and overexpression of a naturally occurring short splice variant of the cyld gene (sCYLD). This overexpression resulted in splenomegaly and lymphadenopathy. Further, the B cell numbers in spleen and lymph nodes were increased at the expense of T cells. CYLDex7/8 mice showed a strong reduction of CD4 single positive (SP) and CD8 SP T cells in the thymus and periphery. In in vitro and in vivo studies we could show that CD4+ T cells display a hyperactive phenotype accompanied with an increased production of inflammatory cytokines. We could also demonstrate a defect in negative selection of CYLDex7/8 thymocytes by using the HY-TCR transgenic system. mTECs in the thymus, which are important for the negative selection of autoreactive T cells, were drastically reduced in number. Since abnormal T cell responses are often associated with chronic inflammations, we examined if CYLDex7/8 mice develop immunological abnormalities. Importantly, we could observe that CYLDex7/8 mice crossed onto TCR transgenic background developed severe colonic inflammation accompanied with high production of nuclear autoantibodies.

Aire mediates peptide epitope-specific thymic deletion of self reactive T cells to prevent autoimmune uveitis (148.3)

Abstract Aire is a transcription factor required for the expression of many tissue specific antigens in the thymus. In the absence of functional Aire protein, human patients and mice develop multi-organ autoimmune disease, including autoimmune uveitis, due to a defect in negative selection. The development of spontaneous uveitis in the Aire KO mouse model is dependent on the eye antigen, interphotoreceptor retinoid-binding antigen (IRBP), which has Aire-regulated expression the thymus. To study the mechanism by which Aire prevents IRBP-dependent autoimmune uveitis, two tetramer reagents were generated to measure the frequency and phenotype of IRBP specific CD4+ T cells in the polyclonal repertoire of WT and Aire KO mice. Analysis of the polyclonal T cell repertoire of WT and Aire KO mice showed that T cells specific for both IRBP tetramers proliferated in Aire KO mice when they developed uveitis. However, tetramer staining of thymocytes showed that only one of the two tetramer binding populations were deleted via an Aire-dependent mechanism. These studies demonstrate that two types of IRBP-specific T cells participate in spontaneous autoimmune uveitis in Aire KO mice, those that are deleted in an Aire-dependent manner and those that are not deleted by Aire.

The pathophysiology of chronic graft-versus-host disease: the unveiling of an enigma

Chronic graft-versus-host disease (CGVHD) is one of the most significant complications of long-term survivors after allogeneic hematopoietic stem cell transplantation (allo-HSCT). CGVHD may have protean manifestations and can pose unique diagnostic and therapeutic challenges. New recommendations that emphasize the importance of qualitative differences, as opposed to time of onset after HSCT, are now being used to standardize the diagnosis and clinical assessment of CGVHD, but they require validation. During the past 3 decades, experimental studies and clinical observations have elucidated the mechanisms of acute GVHD, but its biology is much less well-understood. Experimental studies have generated at least 4 theories to explain the pathophysiology of CGVHD: (1) thymic damage and the defective negative selection of T cells, (2) regulatory T cell deficiencies, (3) auto-antibody production by aberrant B cells, and (4) the formation of profibrotic lesions. Mouse models have provided important insights into the pathophysiology of CGVHD, and these have helped improve clinical outcomes following allo-HSCT, but no animal model fully replicates all of the features of CGVHD in humans. In this article, recent clinical changes, the pathogenesis of CGHVD, the cellular and cytokine networks implicated in its pathogenesis, and the animal models used to devise strategies to prevent and treat CGVHD are reviewed.

Kinase suppressor of Ras 1 is required for full ERK activation in thymocytes but not for thymocyte selection

The scaffold protein kinase suppressor of Ras 1 (KSR1) is critical for efficient activation of ERK in a number of cell types. Consistent with this, we observed a defect in ERK activation in thymocytes that lack KSR1. Interestingly, we found that the defect was much greater after PMA stimulation than by CD3 activation. Since ERK activation is believed to be important for thymocyte development, we analyzed thymocyte selection in KSR1-deficient (KSR1(-/-) ) mice. We found that positive selection in two different TCR transgenic models, HY and AND, was normal. On the other hand, negative selection in the HY model was slightly impaired in KSR1(-/-) mice. However, a defect in negative selection was not apparent in the AND TCR model system or in an endogenous superantigen-mediated model of negative selection. These results suggest that, despite a requirement for KSR1 for full ERK activation in thymocytes, full and efficient ERK activation is not essential for the majority of thymocyte selection events.

Direct and indirect roles of the LTβR pathway in central tolerance induction

Medullary thymic epithelial cells (mTECs) play a critical role in thymic negative selection of autoreactive thymocytes, especially for thymocytes specific for peripheral tissue-restricted self-antigens (TRA). Deficiency in lymphotoxin b receptor (LTbetaR) is associated with peripheral tissue inflammation, but whether this is caused by defective negative selection has been unclear; the significance of the LTbetaR pathway for negative selection is evident in some models but not others. Here, we revisit the data and clarify the role of LTbetaR in mTEC development and function and thymic TRA expression. These processes are discussed as potential mechanisms for LTbetaR-mediated control of negative selection.

The Role of Photopheresis in the Treatment of Graft-Versus-Host Disease

Hematopoietic stem-cell transplantation [...]

A hypomorphic allele of ZAP-70 reveals a distinct thymic threshold for autoimmune disease versus autoimmune reactivity

ZAP-70 is critical for T cell receptor (TCR) signaling. Tyrosine to phenylalanine mutations of Y315 and Y319 in ZAP-70 suggest these residues function to recruit downstream effector molecules, but mutagenesis and crystallization studies reveal that these residues also play an important role in autoinhibition ZAP-70. To address the importance of the scaffolding function, we generated a zap70 mutant mouse (YYAA mouse) with Y315 and Y319 both mutated to alanines. These YYAA mice reveal that the scaffolding function is important for normal development and function. Moreover, the YYAA mice have many similarities to a previously identified ZAP-70 mutant mouse, SKG, which harbors a distinct hypomorphic mutation. Both YYAA and SKG mice have impaired T cell development and hyporesponsiveness to TCR stimulation, markedly reduced numbers of thymic T regulatory cells and defective positive and negative selection. YYAA mice, like SKG mice, develop rheumatoid factor antibodies, but fail to develop autoimmune arthritis. Signaling differences that result from ZAP-70 mutations appear to skew the TCR repertoire in ways that differentially influence propensity to autoimmunity versus autoimmune disease susceptibility. By uncoupling the relative contribution from T regulatory cells and TCR repertoire during thymic selection, our data help to identify events that may be important, but alone are insufficient, for the development of autoimmune disease.

A hypomorphic allele of ZAP-70 reveals a distinct thymic threshold for autoimmune disease versus autoimmune reactivity (49.6)

Abstract ZAP-70, a Syk family protein tyrosine kinase, is critical for efficient signaling downstream of the TCR. Our lab and others have previously shown that tyrosine 315 and 319 in the interdomain B positively regulate ZAP-70 function by recruiting Vav1 and Lck, respectively. Surprisingly, in addition to their scaffolding function, we recently discovered that Y315 and Y319 play an important role in the autoinhibition ZAP-70 kinase activity. To address the relative importance of the scaffolding function of these residues versus their autoinhibition function, we generated a knock-in ZAP-70 mutant mouse (YYAA mouse) with Y315 and Y319 both mutated to ananine residues. These YYAA mice showed many similarities to a previously identified ZAP-70 mutant mouse, SKG, which harbors a distinct hypomorphic mutation. Both YYAA and SKG mice have impaired T cell development, hyporesponsiveness to TCR stimulation, and defective positive and negative selection. YYAA mice, like SKG mice, develop rheumatoid factor antibodies, but fail to develop autoimmune arthritis. Analysis of TCR repertoire revealed that qualitative difference in TCR signaling may skew the TCR repertoire in ways that differentially influence propensity to autoimmunity versus autoimmune disease susceptibility.

Biology of Chronic Graft-versus-Host Disease: Implications for a Future Therapeutic Approach

Hematopoietic cell transplantation (HCT) is frequently complicated by graft-versus-host disease (GVHD). During the past three decades, experimental studies and clinical observations have elucidated the pathophysiology of acute GVHD, but the biology of chronic GVHD is much less well understood. Recommendations of the NIH Consensus Development Project on Criteria for Clinical Trials in Chronic GVHD have begun to standardize the diagnosis and clinical assessment of the disease. These criteria have emphasized the importance of qualitative differences, as opposed to time of onset after HCT, in making the distinction between acute and chronic GVHD. Experimental studies have generated at least four theories to explain the pathophysiology of chronic GVHD. These theories include 1) thymic damage and defective negative selection of T cells generated from marrow progenitors after HCT, 2) aberrant production of transforming growth factor-beta, 3) auto-antibody production, and 4) deficiency of T-regulatory cells. Recent studies in humans have corroborated a possible role for each of these mechanisms in humans. No animal model fully replicates all of the features of chronic GVHD in humans, and it appears likely that multiple biological mechanisms account for the diverse features the disease. Chronic GVHD may represent a "syndrome" with diverse causes among individual patients. In the future, it might become possible to tailor specific therapeutic interventions for patients as individually needed for each distinct pathophysiologic mechanism involved in development of the disease.

Impaired responsiveness to T-cell receptor stimulation and defective negative selection of thymocytes in CCR7-deficient mice

The chemokine receptor CCR7 has been implicated in maintenance of thymus morphology and establishment of tolerance to self-antigens. In this study, we provide direct evidence that negative selection of maturing thymocytes is defective in CCR7-deficent mice. Impaired negative selection was observed after TCR/CD3 complex stimulation in vivo as well as in vitro and was prominent in both double-positive and semimature single positive cells (CD4(+)CD8(-)CD24(high)). It is noteworthy that thymocytes of CCR7(-/-) mice display defective negative selection in response to endogenous superantigens, demonstrating that the defect also occurs under physiological conditions. Disturbed negative selection was correlated with delayed activation kinetics and decreased calcium flux response of CCR7(-/-) thymocytes after in vitro TCR/CD3 stimulation, suggesting that an impaired response of CCR7(-/-) thymocytes via TCR-mediated signaling is responsible for defective negative selection in these mice.

Lymphotoxin β Receptor Is Required for the Migration and Selection of Autoreactive T Cells in Thymic Medulla

How organ-specific central tolerance is established and regulated has been an intriguing question. Lymphotoxin beta receptor (LTbetaR) deficiency is associated with autoimmune phenotypes characterized by humoral and cellular autoreactivity to peripheral organs. Whether this results from defective negative selection of T cells directed at tissue-restricted Ags has not been well understood. By tracing the development of OT-I thymocytes in rat insulin 2 promoter-mOVA transgenic mice on either Ltbr+/+ or Ltbr-/- background, we demonstrate that LTbetaR is necessary for thymic negative selection. LTbetaR deficiency resulted in a dramatic escape of "neo-self" specific OT-I cells that persist in circulation and lead to development of peri-insulitis. When the underlying mechanism was further explored, we found interestingly that LTbetaR deficiency did not result in reduced thymic expression of mOVA. Instead, LTbetaR was revealed to control the expression of thymic medullary chemokines (secondary lymphoid tissue chemokine (SLC) and EBV-induced molecule 1 ligand chemokine (ELC)) which are required for thymocytes migration and selection in medulla. Furthermore, RIP-mOVA transgenic mice on SLC/ELC deficient background (plt) demonstrated significant impaired negative selection of OT-I cells, suggesting that the dysregulation of SLC/ELC- expression alone in Ltbr-/- thymi can be sufficient to impair thymic negative selection. Thus, LTbetaR has been revealed to play an important role in thymic negative selection of organ-specific thymocytes through thymic medullary chemokines regulation.

The NOD allele of the Idd5 locus on chromosome 1 mediates a non-cell-autonomous defect in negative selection of T cells

Recent data have suggested that non-obese diabetic (NOD) mice display a defect in negative thymic selection. Using mixed bone marrow chimeras, we demonstrate that the NOD allele of the diabetes susceptibility region 5 ( Idd5) locus on chromosome 1, confers defective negative selection in response to endogenous superantigens (SAg) Mtv8 and Mtv9. We generated mixed bone marrow (BM) chimeras in which the donor cells of NOD and C3H or NOD.Idd5 b10 and C3H coexist and are similarly exposed to the Mtv8 and Mtv9 SAg. Under these conditions, SAg-mediated deletion of Vβ11 + T cells is less efficient in chimeric mice reconstituted with NOD + C3H BM, compared with chimeras reconstituted with NOD.Idd5 b10 + C3H BM. Interestingly, the observed discrepancy was not T cell autonomous but was found to be mediated by a BM derived cellular subset, and under control of a gene(s) in the Idd5 region.

Impairment of organ-specific T cell negative selection by diabetes susceptibility genes: genomic analysis by mRNA profiling

A transcription profiling study, together with genetic linkage data, provides a molecular map of the T-cell negative selection response in vivo.

The autoimmune regulator (Aire) controls iNKT cell development and maturation

The mechanism underlying the autoimmune polyglandular syndrome type-1 (APS1) has been attributed to defective T-cell negative selection resulting from reduced expression and presentation of autoantigens in thymic medullary epithelial cells (MECs). It has also been postulated that Aire is involved in development of regulatory T cells, although supporting evidence is lacking. Here we show that expression of Aire in MECs is required for development of iNKT cells, suggesting a role for iNKT cells in APS1.