Age-related deregulation of Aire and peripheral tissue antigen genes in the thymic stroma of non-obese diabetic (NOD) mice is associated with autoimmune type 1 diabetes mellitus (DM-1)

Gene expression of peripheral tissue antigens (PTAs) in stromal medullary thymic epithelial cells (mTECs) is a key process to the negative selection of autoreactive thymocytes. This phenomenon was termed “promiscuous gene expression” (PGE), which is partially controlled by the Aire gene. Nevertheless, reasons for the correlation of Aire and PTAs with the emergence of autoimmune diseases are largely unknown, though it may be a result of a chronological effect. Although the effect of Aire mutations in pathogenic autoimmunity is well know, it could not be a unique cause for autoimmunity. Independently of mutations, temporal deregulation of Aire expression may imbalance Aire-dependent PTAs and/or wide PGE. This deregulation may be an early warning sign for autoimmune diseases as it guarantees autoantigen representation in the thymus. To assess this hypothesis, we studied the expression levels of Aire, Aire-dependent (Ins2) and Aire-independent (Gad67 and Col2a1) PTAs using real-time-PCR of the thymic stromal cells of NOD mice during the development of autoimmune type 1 diabetes mellitus (DM-1). Wide PGE was studied by microarrays in which the PTA genes were identified through parallel CD80⁺ mTEC 3.10 cell line expression profiling. The results show that Aire gene was down-regulated in young pre-autoimmune (pre-diabetic) NOD mice. PGE and specific PTA genes were down-regulated in adult autoimmune diabetic animals. These findings represent evidence indicating that chronological deregulation of genes important to negative selection may be associated with the development of an autoimmune disease (DM-1) in mice.     

The immune response to melanoma is limited by thymic selection of self-antigens

The expression of melanoma-associated antigens (MAA) being limited to normal melanocytes and melanomas, MAAs are ideal targets for immunotherapy and melanoma vaccines. As MAAs are derived from self, immune responses to these may be limited by thymic tolerance. The extent to which self-tolerance prevents efficient immune responses to MAAs remains unknown. The autoimmune regulator (AIRE) controls the expression of tissue-specific self-antigens in thymic epithelial cells (TECs). The level of antigens expressed in the TECs determines the fate of auto-reactive thymocytes. Deficiency in AIRE leads in both humans (APECED patients) and mice to enlarged autoreactive immune repertoires. Here we show increased IgG levels to melanoma cells in APECED patients correlating with autoimmune skin features. Similarly, the enlarged T cell repertoire in AIRE(-/-) mice enables them to mount anti-MAA and anti-melanoma responses as shown by increased anti-melanoma antibodies, and enhanced CD4(+) and MAA-specific CD8(+) T cell responses after melanoma challenge. We show that thymic expression of gp100 is under the control of AIRE, leading to increased gp100-specific CD8(+) T cell frequencies in AIRE(-/-) mice. TRP-2 (tyrosinase-related protein), on the other hand, is absent from TECs and consequently TRP-2 specific CD8(+) T cells were found in both AIRE(-/-) and AIRE(+/+) mice. This study emphasizes the importance of investigating thymic expression of self-antigens prior to their inclusion in vaccination and immunotherapy strategies.     

Medullary thymic epithelial cells, the indispensable player in central tolerance

Crosstalk between thymocytes and thymic epithelial cells is critical for T cell development and the establishment of central tolerance. Medullary thymic epithelial cells (mTECs) play important roles in the late stage of T cell development, especially negative selection and Treg generation. The function of mTECs is highly dependent on their characteristic features such as ectopic expression of peripheral tissue restricted antigens (TRAs) and their master regulator—autoimmune regulator (Aire), expression of various chemokines and cytokines. In this review, we summarize the current understanding of cellular and molecular mechanisms of mTEC development and its functions in T cell development and the establishment of central tolerance. The open questions in this field are also discussed. Understanding the function and underlying mechanisms of mTECs will contribute to the better control of autoimmune diseases and the improvement of immune reconstitution during aging or after infection, chemotherapy or radiotherapy.     

A specific anti-Aire antibody reveals aire expression is restricted to medullary thymic epithelial cells and not expressed in periphery

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy is an autoimmune disorder caused by mutations in the autoimmune regulator gene AIRE. We examined the expression of Aire in different organs (thymus, spleen, and lymph nodes) in C57BL/6 mice, using a novel rat mAb, specific for murine Aire. Using flow cytometry, directly fluorochrome-labeled mAb revealed Aire expression in a rare thymic cellular subset that was CD45(-), expressed low levels of Ly51, and was high for MHC-II and EpCam. This subset also expressed a specific pattern of costimulatory molecules, including CD40, CD80, and PD-L1. Immunohistochemical analysis revealed that Aire(+) cells were specifically localized to the thymus or, more precisely, to the cortico-medulla junction and medulla, correlating with the site of negative selection. Although in agreement with previous studies, low levels of Aire mRNA was detected in all dendritic cell subtypes however lacZ staining, immunohistochemistry and flow cytometry failed to detect Aire protein. At a cellular level, Aire was expressed in perinuclear speckles within the nucleus. This report provides the first detailed analysis of Aire protein expression, highlighting the precise location at both the tissue and cellular level.     

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.     

Defective autoimmune regulator-dependent central tolerance to myelin protein zero is linked to autoimmune peripheral neuropathy

Chronic inflammatory demyelinating polyneuropathy is a debilitating autoimmune disease characterized by peripheral nerve demyelination and dysfunction. How the autoimmune response is initiated, identity of provoking Ags, and pathogenic effector mechanisms are not well defined. The autoimmune regulator (Aire) plays a critical role in central tolerance by promoting thymic expression of self-Ags and deletion of self-reactive T cells. In this study, we used mice with hypomorphic Aire function and two patients with Aire mutations to define how Aire deficiency results in spontaneous autoimmune peripheral neuropathy. Autoimmunity against peripheral nerves in both mice and humans targets myelin protein zero, an Ag for which expression is Aire-regulated in the thymus. Consistent with a defect in thymic tolerance, CD4(+) T cells are sufficient to transfer disease in mice and produce IFN-γ in infiltrated peripheral nerves. Our findings suggest that defective Aire-mediated central tolerance to myelin protein zero initiates an autoimmune Th1 effector response toward peripheral nerves.     

Resveratrol ameliorates thymus senescence changes in D-galactose induced mice

The thymic microenvironment plays an important role in the development of T cells. A decrease of thymic epithelial cells is the main cause of age-related thymic atrophy or degeneration. Resveratrol (RSV), a phytoalexin produced from plants, has been shown to inhibit the adverse effects of dietary obesity on the structure and function of the thymus. D-Galactose (D-gal) can induce accelerated aging in mice. In the present study, young mice (2 months old) were injected with D-gal (120 mg/kg/day) for 8 consecutive weeks to construct an accelerated aging model. Compared with normal control mice, the thymus epithelium of the D-gal treated mice had structural changes, the number of senescent cells increased, the number of CD4+ T cells decreased, and CD8+ T cells increased. After RSV administration by gavage for 6 weeks, it was found that RSV improved the surface phenotypes of D-gal treated mice, and recovered thymus function by maintaining the ratio of CD4+ to CD8+ cells. It also indicated that RSV enhanced the cell proliferation and inhibited cell senescence. Increased autoimmune regulator (Aire) expression was present in the RSV treated mice. The lymphotoxin-beta receptor (LTβR) expression also increased. These findings suggested that RSV intake could restore the alterations caused by D-gal treatment in the thymus via stimulation of Aire expression.     

Subcellular location and expression pattern of autoimmune regulator (Aire), the mouse orthologue for human gene defective in autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED).

Autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED), also known as autoimmune polyglandular syndrome Type I (APS1), is an autosomal recessive autoimmune disease caused by mutations in a gene designated as AIRE (autoimmune regulator). Here we have studied the expression of Aire in transfected cell lines and in adult mouse tissues. Our results show that Aire has a dual subcellular location and that it is expressed in multiple immunologically relevant tissues such as the thymus, spleen, lymph nodes, and bone marrow. In addition, Aire expression was detected in various other tissues such as kidney, testis, adrenal glands, liver, and ovary. These findings suggest that APECED protein might also have a function(s) outside the immune system.(J Histochem Cytochem 49:197-208, 2001)     

IL-7 dysregulation and loss of CD8+ T cell homeostasis in the monogenic human disease autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a monogenic autoimmune disease that is caused by mutations in the AIRE gene. Murine studies have linked AIRE to thymocyte selection and peripheral deletional tolerance, but the pathogenesis of the human disease remains unclear. In this study, we show that APECED patients have elevated IL-7 levels and a drastically decreased expression of IL-7R on CD8(+) T cells. This is associated with increased proliferation and a decreased expression of the negative TCR regulator CD5 in the CD45RO(-) subset. The CD45RO(-) cells also display oligoclonal expansions, decreased expression of the lymph node homing factors CCR7 and CD62L, and increased expression of perforin, consistent with the accumulation of highly differentiated effector cells. The CD45RO(-)CCR7(+)CD8(+) population of cells with markers characteristic of naive phenotype is also skewed, as shown by decreased expression of CD5 and increased expression of perforin. The putative CD31(+) recent thymic emigrant population is likewise affected. These data are consistent with IL-7 dysregulation inducing a decreased threshold of TCR signaling and self-antigen-driven proliferation, probably in synergy with the failed thymic selection. The resultant loss of CD8(+) T cell homeostasis is likely to play a significant role in the pathogenesis of APECED. Our findings may also hold lessons for other diseases in which the IL-7-IL-7R pathway has emerged as a risk factor.     

Chagasic thymic atrophy does not affect negative selection but results in the export of activated CD4+CD8+ T cells in severe forms of human disease

Extrathymic CD4+CD8+ double-positive (DP) T cells are increased in some pathophysiological conditions, including infectious diseases. In the murine model of Chagas disease, it has been shown that the protozoan parasite Trypanosoma cruzi is able to target the thymus and induce alterations of the thymic microenvironment and the lymphoid compartment. In the acute phase, this results in a severe atrophy of the organ and early release of DP cells into the periphery. To date, the effect of the changes promoted by the parasite infection on thymic central tolerance has remained elusive. Herein we show that the intrathymic key elements that are necessary to promote the negative selection of thymocytes undergoing maturation during the thymopoiesis remains functional during the acute chagasic thymic atrophy. Intrathymic expression of the autoimmune regulator factor (Aire) and tissue-restricted antigen (TRA) genes is normal. In addition, the expression of the proapoptotic Bim protein in thymocytes was not changed, revealing that the parasite infection-induced thymus atrophy has no effect on these marker genes necessary to promote clonal deletion of T cells. In a chicken egg ovalbumin (OVA)-specific T-cell receptor (TCR) transgenic system, the administration of OVA peptide into infected mice with thymic atrophy promoted OVA-specific thymocyte apoptosis, further indicating normal negative selection process during the infection. Yet, although the intrathymic checkpoints necessary for thymic negative selection are present in the acute phase of Chagas disease, we found that the DP cells released into the periphery acquire an activated phenotype similar to what is described for activated effector or memory single-positive T cells. Most interestingly, we also demonstrate that increased percentages of peripheral blood subset of DP cells exhibiting an activated HLA-DR+ phenotype are associated with severe cardiac forms of human chronic Chagas disease. These cells may contribute to the immunopathological events seen in the Chagas disease.     

NF-kappaB2 is required for the control of autoimmunity by regulating the development of medullary thymic epithelial cells

Medullary thymic epithelial cells function as antigen-presenting cells in negative selection of self-reactive T cell clones, a process essential for the establishment of central self-tolerance. These cells mirror peripheral tissues through promiscuous expression of a diverse set of tissue-restricted self-antigens. The genes and signaling pathways that regulate the development of medullary thymic epithelial cells are not fully understood. Here we show that mice deficient in NF-kappaB2, a member of the NF-kappaB family, display a marked reduction in the number of mature medullary thymic epithelial cells that express CD80 and bind the lectin Ulex europaeus agglutinin-1, leading to a significant decrease in the extent of promiscuous gene expression in the thymus of NF-kappaB2(-/-) mice. Moreover, NF-kappaB2(-/-) mice manifest autoimmunity characterized by multiorgan infiltration of activated T cells and high levels of autoantibodies to multiple organs. A subpopulation of the mice also develops immune complex glomerulonephritis. These findings identify a physiological function of NF-kappaB2 in the development of medullary thymic epithelial cells and, thus, the control of self-tolerance induction.