Intrathymic Expression Research Articles

Effect of Coxsackievirus B4 Infection on the Thymus: Elucidating Its Role in the Pathogenesis of Type 1 Diabetes.

The thymus gland is a primary lymphoid organ for T-cell development. Various viral infections can result in disturbance of thymic functions. Medullary thymic epithelial cells (mTECs) are important for the negative selection of self-reactive T-cells to ensure central tolerance. Insulin-like growth factor 2 (IGF2) is the dominant self-peptide of the insulin family expressed in mTECs and plays a crucial role in the intra-thymic programing of central tolerance to insulin-secreting islet β-cells. Coxsackievirus B4 (CVB4) can infect and persist in the thymus of humans and mice, thus hampering the T-cell maturation and differentiation process. The modulation of IGF2 expression and protein synthesis during a CVB4 infection has been observed in vitro and in vivo in mouse models. The effect of CVB4 infections on human and mouse fetal thymus has been studied in vitro. Moreover, following the inoculation of CVB4 in pregnant mice, the thymic function in the fetus and offspring was disturbed. A defect in the intra-thymic expression of self-peptides by mTECs may be triggered by CVB4. The effects of viral infections, especially CVB4 infection, on thymic cells and functions and their possible role in the pathogenesis of type 1 diabetes (T1D) are presented.

Semaphorin-3A-Related Reduction of Thymocyte Migration in Chemically Induced Diabetic Mice

Background: Previous work revealed the existence of a severe thymic atrophy with massive loss of immature CD4⁺CD8⁺ thymocytes in animals developing insulin-dependent diabetes, chemically induced by alloxan. Furthermore, the intrathymic expression of chemokines, such as CXCL12, is changed in these animals, suggesting that cell migration-related patterns may be altered. One molecular interaction involved in normal thymocyte migration is that mediated by soluble semaphorin-3A and its cognate receptor neuropilin-1. Objectives: We investigated herein the expression and role of semaphorin-3A in the migratory responses of thymocytes from alloxan-induced diabetic mice. We characterized semaphorin-3A and its receptor, neuropilin-1, in thymuses from control and diabetic mice as well as semaphorin-3A-dependent migration of developing thymocytes in both control and diabetic animals. Methods: Diabetes was chemically induced after a single injection of alloxan in young adult BALB/c mice. Thymocytes were excised from control and diabetic individuals and subjected to cytofluorometry for simultaneous detection of semaphorin-3A or neuropilin-1 in CD4/CD8-defined subsets. Cell migration in response to semaphorin-3A was performed using cell migration transwell chambers. Results: Confirming previous data, we observed a severe decrease in the total numbers of thymocytes in diabetic mice, which comprised alterations in both immature (double-negative subpopulations) and mature CD4/CD8-defined thymocyte subsets. These were accompanied by a decrease in the absolute numbers of semaphorin-3A-bearing thymocytes, comprising CD4^–CD8^–, CD4⁺CD8⁺, and CD4^–CD8⁺ cells. Additionally, immature CD4^–CD8^– and CD4⁺CD8⁺ developing T cells exhibited a decrease in the membrane density of semaphorin-3A. The relative and absolute numbers of neuropilin-1-positive thymocytes were also decreased in diabetic mouse thymocytes compared to controls, as seen in CD4^–CD8^–, CD4⁺CD8⁺, and CD4^–CD8⁺ cell subpopulations. Functionally, we observed a decrease in the chemorepulsive role of semaphorin-3A, as revealed by transwell migration chambers. Such an effect was seen in all immature and mature thymocyte subsets. Conclusions: Taken together, our data clearly unravel a disruption in the normal cell migration pattern of developing thymocytes following chemically induced insulin-dependent diabetes, as ascertained by the altered migratory response to sempahorin-3A. In conceptual terms, it is plausible to think that such disturbances in the migration pattern of thymocytes from these diabetic animals may exert an impact in the cell-mediated immune response of these mice.

Abnormal T-Cell Development in the Thymus of Non-obese Diabetic Mice: Possible Relationship With the Pathogenesis of Type 1 Autoimmune Diabetes.

Type 1 diabetes (T1D) is an autoimmune disease caused by the destruction of insulin-producing cells in the pancreas, by direct interactions with autoreactive pancreas infiltrating T lymphocytes (PILs). One of the most important animal models for this disease is the non-obese diabetic (NOD) mouse. Alterations in the NOD mouse thymus during the pathogenesis of the disease have been reported. From the initial migratory disturbances to the accumulation of mature thymocytes, including regulatory Foxp3+ T cells, important mechanisms seem to regulate the repertoire of T cells that leave the thymus to settle in peripheral lymphoid organs. A significant modulation of the expression of extracellular matrix and soluble chemoattractant molecules, in addition to integrins and chemokine receptors, may contribute to the progressive accumulation of mature thymocytes and consequent formation of giant perivascular spaces (PVS) that are observed in the NOD mouse thymus. Comparative large-scale transcriptional expression and network analyses involving mRNAs and miRNAs of thymocytes, peripheral T CD3+ cells and PILs provided evidence that in PILs chemokine receptors and mRNAs are post-transcriptionally regulated by miR-202-3p resulting in decreased activity of these molecules during the onset of T1D in NOD mice. In this review, we discuss the abnormal T-cell development in NOD mice in the context of intrathymic expression of different migration-related molecules, peptides belonging to the family of insulin and insulin-like growth factors as well as the participation of miRNAs as post-transcriptional regulators and their possible influence on the onset of aggressive autoimmunity during the pathogenesis of T1D.

Indirect presentation in the thymus limits naive and regulatory T-cell differentiation by promoting deletion of self-reactive thymocytes.

Acquisition of T-cell central tolerance involves distinct pathways of self-antigen presentation to thymocytes. One pathway termed indirect presentation requires a self-antigen transfer step from thymic epithelial cells (TECs) to bone marrow-derived cells before the self-antigen is presented to thymocytes. The role of indirect presentation in central tolerance is context-dependent, potentially due to variation in self-antigen expression, processing and presentation in the thymus. Here, we report experiments in mice in which TECs expressed a membrane-bound transgenic self-antigen, hen egg lysozyme (HEL), from either the insulin (insHEL) or thyroglobulin (thyroHEL) promoter. Intrathymic HEL expression was less abundant and more confined to the medulla in insHEL mice compared with thyroHEL mice. When indirect presentation was impaired by generating mice lacking MHC class II expression in bone marrow-derived antigen-presenting cells, insHEL-mediated thymocyte deletion was abolished, whereas thyroHEL-mediated deletion occurred at a later stage of thymocyte development and Foxp3+ regulatory T-cell differentiation increased. Indirect presentation increased the strength of T-cell receptor signalling that both self-antigens induced in thymocytes, as assessed by Helios expression. Hence, indirect presentation limits the differentiation of naive and regulatory T cells by promoting deletion of self-reactive thymocytes.

High-level intrathymic thyrotrophin receptor expression in thyroiditis-prone mice protects against the spontaneous generation of pathogenic thyrotrophin receptor autoantibodies.

The thyrotrophin receptor (TSHR) A-subunit is the autoantigen targeted by pathogenic autoantibodies that cause Graves' hyperthyroidism, a common autoimmune disease in humans. Previously, we reported that pathogenic TSHR antibodies develop spontaneously in thyroiditis-susceptible non-obese diabetic (NOD).H2h4 mice bearing a human TSHR A-subunit transgene, which is expressed at low levels in both the thyroid and thymus (Lo-expressor transgene). The present study tested recent evidence that high intrathymic TSHR expression protects against the development of pathogenic TSHR antibodies in humans. By successive back-crossing, we transferred to the NOD.H2h4 background a human TSHR A-subunit transgene expressed at high levels in the thyroid and thymus (Hi-expressor transgene). In the sixth back-cross generation (>98% NOD.H2h4 genome), only transgenic offspring produced spontaneously immunoglobulin (Ig)G class non-pathogenic human TSHR A-subunit antibodies. In contrast, both transgenic and non-transgenic offspring developed antibodies to thyroglobulin and thyroid peroxidase. However, non-pathogenic human TSHR antibody levels in Hi-expressor offspring were lower than in Lo-expressor transgenic mice. Moreover, pathogenic TSHR antibodies, detected by inhibition of TSH binding to the TSHR, only developed in back-cross offspring bearing the Lo-expressor, but not the Hi-expressor, transgene. High versus low expression human TSHR A-subunit in the NOD.H2h4 thymus was not explained by the transgene locations, namely chromosome 2 (127-147 Mb; Hi-expressor) and chromosome 1 (22.9-39.3 Mb; low expressor). Nevertheless, using thyroiditis-prone NOD.H2h4 mice and two transgenic lines, our data support the association from human studies that low intrathymic TSHR expression is associated with susceptibility to developing pathogenic TSHR antibodies, while high intrathymic TSHR expression is protective.

Lymphotoxin β Receptor Controls T Cell Progenitor Entry to the Thymus.

The recruitment of lymphoid progenitors to the thymus is essential to sustain T cell production throughout life. Importantly, it also limits T lineage regeneration following bone marrow transplantation, and so contributes to the secondary immunodeficiency that is caused by delayed immune reconstitution. Despite this significance, the mechanisms that control thymus colonization are poorly understood. In this study, we show that in both the steady-state and after bone marrow transplant, lymphotoxin β receptor (LTβR) controls entry of T cell progenitors to the thymus. We show that this requirement maps to thymic stroma, further underlining the key importance of this TNFR superfamily member in regulation of thymic microenvironments. Importantly, analysis of the requirement for LTβR in relationship to known regulators of thymus seeding suggests that it acts independently of its regulation of thymus-homing chemokines. Rather, we show that LTβR differentially regulates intrathymic expression of adhesion molecules known to play a role in T cell progenitor entry to the thymus. Finally, Ab-mediated in vivo LTβR stimulation following bone marrow transplant enhances initial thymus recovery and boosts donor-derived T cell numbers, which correlates with increased adhesion molecule expression by thymic stroma. Collectively, we reveal a novel link between LTβR and thymic stromal cells in thymus colonization, and highlight its potential as an immunotherapeutic target to boost T cell reconstitution after transplantation.

Co-ordination of intrathymic self-representation.

Intrathymic expression of self antigens is key for central tolerance. RNA-sequencing analysis of tissue-restricted antigens in individual medullary thymic epithelial cells reveals co-ordination in the gene-expression patterns that ensures effective self-representation for the production of self-tolerant T cells.

Autoimmune Predisposition in Down Syndrome May Result from a Partial Central Tolerance Failure due to Insufficient Intrathymic Expression of AIRE and Peripheral Antigens

Down syndrome (DS), or trisomy of chromosome 21, is the most common genetic disorder associated with autoimmune diseases. Autoimmune regulator protein (AIRE), a transcription factor located on chromosome 21, plays a crucial role in autoimmunity by regulating promiscuous gene expression (pGE). To investigate if autoimmunity in DS is promoted by the reduction of pGE owing to dysregulation of AIRE, we assessed the expression of AIRE and of several peripheral tissue-restricted Ag genes by quantitative PCR in thymus samples from 19 DS subjects and 21 euploid controls. Strikingly, despite the 21 trisomy, AIRE expression was significantly reduced by 2-fold in DS thymuses compared with controls, which was also confirmed by fluorescent microscopy. Allele-specific quantification of intrathymic AIRE showed that despite its lower expression, the three copies are expressed. More importantly, decreased expression of AIRE was accompanied by a reduction of pGE because expression of tissue-restricted Ags, CHRNA1, GAD1, PLP1, KLK3, SAG, TG, and TSHR, was reduced. Of interest, thyroid dysfunction (10 cases of hypothyroidism and 1 of Graves disease) developed in 11 of 19 (57.9%) of the DS individuals and in none of the 21 controls. The thymuses of these DS individuals contained significantly lower levels of AIRE and thyroglobulin, to which tolerance is typically lost in autoimmune thyroiditis leading to hypothyroidism. Our findings provide strong evidence for the fundamental role of AIRE and pGE, namely, central tolerance, in the predisposition to autoimmunity of DS individuals.

Epithelial cytoprotection sustains ectopic expression of tissue-restricted antigens in the thymus during murine acute GVHD

AbstractDevelopment of acute graft-versus-host disease (aGVHD) predisposes to chronic GVHD with autoimmune manifestations. A characteristic of experimental aGVHD is the de novo generation of autoreactive T cells. Central tolerance is dependent on the intrathymic expression of tissue-restricted peripheral self-antigens (TRA), which is in mature medullary thymic epithelial cells (mTEChigh) partly controlled by the autoimmune regulator (Aire). Because TECs are targets of donor T-cell alloimmunity, we tested whether murine aGVHD interfered with the capacity of recipient Aire+mTEChigh to sustain TRA diversity. We report that aGVHD weakens the platform for central tolerance induction because individual TRAs are purged from the total repertoire secondary to a decline in the Aire+mTEChigh cell pool. Peritransplant administration of an epithelial cytoprotective agent, fibroblast growth factor-7, maintained a stable pool of Aire+mTEChigh, with an improved TRA transcriptome despite aGVHD. Taken together, our data provide a mechanism for how autoimmunity may develop in the context of antecedent alloimmunity.

Prescience of a Surgeon in 1980

ThyroidVol. 23, No. 6 Letters to the EditorPrescience of a Surgeon in 1980Sandra M. McLachlanSandra M. McLachlanSearch for more papers by this authorPublished Online:6 Jun 2013https://doi.org/10.1089/thy.2012.0636AboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View article"Prescience of a Surgeon in 1980." , 23(6), pp. 773–774FiguresReferencesRelatedDetails Volume 23Issue 6Jun 2013 InformationCopyright 2013, Mary Ann Liebert, Inc.To cite this article:Sandra M. McLachlan.Prescience of a Surgeon in 1980.Thyroid.Jun 2013.773-774.http://doi.org/10.1089/thy.2012.0636Published in Volume: 23 Issue 6: June 6, 2013Online Ahead of Editing: January 25, 2013PDF download

Modeling the intrathymic pathogenesis of myasthenia gravis

Myasthenia gravis is (MG) a prototypic autoimmune disease; the immune effector mechanisms and autoantigenic target have been delineated. However, the events that lead to the abrogation of self-tolerance to neuromuscular acetylcholine receptors (nAChRs) remain a mystery. The thymus gland has long been considered to hold the key to solving this mystery, although the nature of its involvement remains to be elucidated. The nAChR was one of the first self-proteins associated with a defined autoimmune disease that was found to be expressed on thymic stromal populations. The studies described herein represent our efforts to determine how this “promiscuous” autoantigen expression may be involved in the immunopathogenesis of MG. We review our work, characterizing the expression of the nAChR alpha subunit in the thymus, and advance a hypothesis and experimental model, which explore how intrathymic expression of this autoantigen may contribute to the immunopathogenesis of this autoimmune disease.

Association Study of MAFA and MAFB Genes Related to Organ-Specific Autoimmunity, with Susceptibility to Type-1 Diabetes in Japanese and Caucasian Populations

The transcriptional factor MAFA is specifically expressed in beta cells of pancreatic islets, and activates tissuespecific transcription of insulin. We previously reported that MAFA is also expressed in the thymus and regulates intrathymic expression of insulin in the mouse. In humans, we identified a functional polymorphism of MAFA, Gly346Cys, which was suggested to be associated with type 1 diabetes. This study aimed to validate the association of MAFA with type 1 diabetes in a larger number of subjects. In addition, molecular scanning of MAFB another member of the large MAFA transcription family, and an association study with type 1 diabetes were also performed. A total of 1733 subjects, including newly recruited Japanese (346 controls and 532 cases) and Caucasians (223 controls and 228 cases), were studied. In newly recruited Japanese subjects, the minor allele frequency of MAFAGly346Cys was lower in cases than in controls (2.9 vs. 5.1%, odds ratio [95%CI]: 0.56 [0.34-0.91], p=0.02). Meta-analysis with our previous data showed a significant association of MAFA with type 1 diabetes (summary odds ratio [95%CI]: 0.49 [0.32-0.76], p=0.0013). When cases were limited to subjects with a risk genotype of INS, the association was further strengthened (odds ratio [95%CI]: 0.47 [0.30-0.74], p=0.00097). In the Caucasian population, the difference in minor allele frequency of MAFA between cases and controls was not significant (6.4% vs. 5.4%, odds ratio [95%CI]: 1.14 [0.66-1.99], NS). When data from Japanese and Caucasians were combined, summary odds ratio was 0.68 [95%CI: 0.48-0.95] (p G and 618C>A) were identified, but neither was significantly associated with type 1 diabetes. In conclusion, MAFA Gly346Cys is associated with type 1 diabetes, especially in the Japanese population, which possesses the high-risk INS genotype.

The Repertoires of Peptides Presented by MHC-II in the Thymus and in Peripheral Tissue: A Clue for Autoimmunity?

T-cell tolerance to self-antigens is established in the thymus through the recognition by developing thymocytes of self-peptide-MHC complexes and induced and maintained in the periphery. Efficient negative selection of auto-reactive T cells in the thymus is dependent on the in situ expression of both ubiquitous and tissue-restricted self-antigens and on the presentation of derived peptides. Weak or inadequate intrathymic expression of self-antigens increases the risk to generate an autoimmune-prone T-cell repertoire. Indeed, even small changes of self-antigen expression in the thymus affect negative selection and increase the predisposition to autoimmunity. Together with other mechanisms, tolerance is maintained in the peripheral lymphoid organs via the recognition by mature T cells of a similar set of self-peptides in homeostatic conditions. However, non-lymphoid peripheral tissue, where organ-specific autoimmunity takes place, often have differential functional processes that may lead to the generation of epitopes that are absent or non-presented in the thymus. These putative differences between peptides presented by MHC molecules in the thymus and in peripheral tissues might be a major key to the initiation and maintenance of autoimmune conditions.

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.

Characterization of Tolerance Induction through Prenatal Marrow Transplantation: The Requirement for a Threshold Level of Chimerism to Establish Rather than Maintain Postnatal Skin Tolerance

Hematopoietic chimerism resulting from prenatal marrow transplantation does not consistently result in allotolerance for unidentified causes. In a C57BL/6-into-FVB/N murine model, we transplanted T-cell-depleted adult marrow on gestational day 14 to elucidate the immunological significance of chimerism towards postnatal tolerance. Postnatally, chimerism was examined by flow cytometry, and tolerance by skin transplantation and mixed lymphocyte reaction. Regulatory T cells were quantified by FoxP3 expression. Peripheral chimerism linearly related to thymic chimerism, and predicted the degree of graft acceptance with levels >3% at skin placement, yielding consistent skin tolerance. Low- and high-level chimeras had lower intrathymic CD3(high) expression than microchimeras or untransplanted mice. Regardless of the skin tolerance status in mixed chimeras, donor-specific alloreactivity by lymphocytes was suppressed but could be partially restored by exogenous interleukin-2. Recipients that lost peripheral chimerism did not accept donor skin unless prior donor skin had engrafted at sufficient chimerism levels, suggesting that complete tolerance can develop as a consequence of chimerism-related immunosuppression of host lymphocytes and the tolerogenic effects of donor skin. Thus, hematopoietic chimerism exerted immunomodulatory effects on the induction phase of allograft tolerance. Once established, skin tolerance did not fade away along with spontaneous regression of peripheral and tissue chimerism, as well as removal of engrafted donor skin. Neither did it break following in vivo depletion of increased regulatory T cells, and subcutaneous interleukin-2 injection beneath the engrafted donor skin. Those observations indicate that the maintenance of skin tolerance is multifaceted, neither solely dependent upon hematopoietic chimerism and engrafted donor skin nor on the effects of regulatory T cells or clonal anergy. We conclude that hematopoietic chimerism generated by in utero hematopoietic stem cell transplantation is critical to establish rather than maintain postnatal skin tolerance. Therefore, the diminution of hematopoietic chimerism below a threshold level does not nullify an existing tolerance state, but lessens the chance of enabling complete tolerance.

Insulin Transactivator MafA Regulates Intrathymic Expression of Insulin and Affects Susceptibility to Type 1 Diabetes

OBJECTIVETissue-specific self-antigens are ectopically expressed within the thymus and play an important role in the induction of central tolerance. Insulin is expressed in both pancreatic islets and the thymus and is considered to be the primary antigen for type 1 diabetes. Here, we report the role of the insulin transactivator MafA in the expression of insulin in the thymus and susceptibility to type 1 diabetes.RESEARCH DESIGN AND METHODSThe expression profiles of transcriptional factors (Pdx1, NeuroD, Mafa, and Aire) in pancreatic islets and the thymus were examined in nonobese diabetic (NOD) and control mice. Thymic Ins2 expression and serum autoantibodies were examined in Mafa knockout mice. Luciferase reporter assay was performed for newly identified polymorphisms of mouse Mafa and human MAFA. A case-control study was applied for human MAFA polymorphisms.RESULTSMafa, Ins2, and Aire expression was detected in the thymus. Mafa expression was lower in NOD thymus than in the control and was correlated with Ins2 expression. Targeted disruption of MafA reduced thymic Ins2 expression and induced autoantibodies against pancreatic islets. Functional polymorphisms of MafA were newly identified in NOD mice and humans, and polymorphisms of human MAFA were associated with susceptibility to type 1 diabetes but not to autoimmune thyroid disease.CONCLUSIONSThese data indicate that functional polymorphisms of MafA are associated with reduced expression of insulin in the thymus and susceptibility to type 1 diabetes in the NOD mouse as well as human type 1 diabetes.

Cutting Edge: A Thymocyte-Thymic Epithelial Cell Cross-Talk Dynamically Regulates Intrathymic IL-7 Expression In Vivo

Thymic epithelial cells (TECs) are the predominant intrathymic source of the essential thymopoietin IL-7. Whether thymocyte-TEC interactions have a role in the regulation of IL-7 expression is not known. By exploiting IL-7 reporter mice in which yellow fluorescent protein expression identifies TECs expressing high levels of IL-7 (Il7(+) TECs), we show that Il7(+) TECs segregate from emerging medullary TECs during thymic organogenesis. Although Il7(+) TECs normally diminish with age, we found that Il7(+) TECs are markedly retained in alymphoid Rag2(-/-)Il2rg(-/-) IL-7 reporter mice that manifest a profound thymopoietic arrest. Transfer of Tcra(-/-) or wild-type (but not Rag2(-/-)) hematopoietic progenitors to alymphoid IL-7 reporter recipients normalizes the frequency of Il7(+) TECs and re-establishes cortical TEC/medullary TEC segregation. Although thymocyte-derived signals are often considered stimulatory for TEC maturation, our findings identify a negative feedback mechanism in which signals derived from TCRbeta-selected thymocytes modulate TEC-dependent IL-7 expression.

The role of AIRE polymorphisms in melanoma

Polymorphisms of AIRE, a transcription factor that up-regulates intrathymic expression of tissue-specific antigens including melanoma-associated antigens (MAAs), may variably affect the selection of MAAs-specific thymocytes, generating T-cell repertoires protecting or predisposing individuals to melanoma. We found that AIRE single nucleotide polymorphisms (SNPs) rs1055311, rs1800520 and rs1800522 were significantly more frequent in healthy subjects than in melanoma patients, independently from sex, age and stages of melanoma. The presence of these SNPs was associated with increased frequency of two T-cell clonotypes specific for MAGE-1 linking their protective effect to selection/expansion of MAA-specific T cells. Interestingly, mRNA transcribed on the rs1800520 SNP showed increased free energy than the wild type suggesting that its reduced stability may be responsible for the different activity of the polymorphic AIRE molecule. This finding may contribute at identifying subjects with increased risk of developing melanoma or patients with melanoma that may take benefit from immunotherapy.

Thyroid Antigens, Not Central Tolerance, Control Responses to Immunization in BALB/c Versus C57BL/6 Mice

Vaccination with cDNA for the human thyrotropin receptor (TSHR) in a plasmid, without adjuvant, induces TSHR antibodies in C57BL/6 but rarely in BALB/c mice. This outcome could be due to a difference between "high" versus "low" antibody responder mouse strains. However, unlike their poor response to TSHR-DNA vaccination, BALB/c mice vaccinated with thyroid peroxidase (TPO)-cDNA readily develop antibodies to TPO. We hypothesized that insight into these conundrums would be provided by the following differences in central tolerance: (i) between two mouse strains (C57BL/6 versus BALB/c) for the TSHR; and (ii) between two thyroid autoantigens (TPO and the TSHR) in one mouse strain (BALB/c). We studied autoantigen expression using real-time polymerase chain reaction to quantify mRNA transcripts for the TSHR, TPO, and thyroglobulin (Tg) in thymic tissue (as well as in thyroid) of young mice. Our hypothesis was not confirmed. Intrathymic TSHR transcript expression was similar in BALB/c and C57BL/6 mice. Moreover, thymic mRNA transcripts for TSHR and TPO were comparable. Unlike the 10-fold differences for the autoantigens in thyroid tissue (Tg greater than TPO which, in turn was greater than the TSHR), intrathymic transcripts for TPO and the TSHR were similar, both being slightly lower than the level for Tg. Central tolerance, assessed by measuring intrathymic transcripts of thyroid autoantigens, does not explain the different outcome of TSHR-DNA vaccination in BALB/c and C57BL/6 mice, or even susceptibility versus resistance to hyperthyroidism induced by TSHR-adenovirus. Instead, differences in MHC and TSHR T-cell epitopes likely contribute to TSHR antibody development (or not) following DNA plasmid immunization. The greater immunogenicity of TPO versus TSHR probably relates to the greater number of nonhomologous amino acids in the human and mouse TPO ectodomains (78 amino acids) than in the human and mouse TSHR ectodomains (58 amino acids). Overall, the autoantigens themselves, not central tolerance, control DNA plasmid-induced immunity to TPO and the TSHR.

Studies in mice deficient for the autoimmune regulator (Aire) and transgenic for the thyrotropin receptor reveal a role for Aire in tolerance for thyroid autoantigens.

The autoimmune regulator (Aire) mediates central tolerance for many autoantigens, and autoimmunity occurs spontaneously in Aire-deficient humans and mice. Using a mouse model of Graves' disease, we investigated the role of Aire in tolerance to the TSH receptor (TSHR) in Aire-deficient and wild-type mice (hyperthyroid-susceptible BALB/c background). Mice were immunized three times with TSHR A-subunit expressing adenovirus. The lack of Aire did not influence T-cell responses to TSHR protein or TSHR peptides. However, antibody levels were higher in Aire-deficient than wild-type mice after the second (but not the third) immunization. After the third immunization, hyperthyroidism persisted in a higher proportion of Aire-deficient than wild-type mice. Aire-deficient mice were crossed with transgenic strains expressing high or low-intrathyroidal levels of human TSHR A subunits. In the low-expressor transgenics, Aire deficiency had the same effect on the pattern of the TSHR antibody response to immunization as in nontransgenics, although the amplitude of the response was lower in the transgenics. High-expressor A-subunit transgenics were unresponsive to immunization. We examined intrathymic expression of murine TSHR, thyroglobulin, and thyroid peroxidase (TPO), the latter two being the dominant autoantigens in Hashimoto's thyroiditis (particularly TPO). Expression of the TSHR and thyroglobulin were reduced in the absence of Aire. Dramatically, thymic expression of TPO was nearly abolished. In contrast, the human A-subunit transgene, lacking a potential Aire-binding motif, was unaffected. Our findings provide insight into how varying intrathymic autoantigen expression may modulate thyroid autoimmunity and suggest that Aire deficiency may contribute more to developing Hashimoto's thyroiditis than Graves' disease.