Thymic Tolerance Research Articles

Identification of a novel cis-regulatory element essential for immune tolerance.

Thymic central tolerance is essential to preventing autoimmunity. In medullary thymic epithelial cells (mTECs), the Autoimmune regulator (Aire) gene plays an essential role in this process by driving the expression of a diverse set of tissue-specific antigens (TSAs), which are presented and help tolerize self-reactive thymocytes. Interestingly, Aire has a highly tissue-restricted pattern of expression, with only mTECs and peripheral extrathymic Aire-expressing cells (eTACs) known to express detectable levels in adults. Despite this high level of tissue specificity, the cis-regulatory elements that control Aire expression have remained obscure. Here, we identify a highly conserved noncoding DNA element that is essential for Aire expression. This element shows enrichment of enhancer-associated histone marks in mTECs and also has characteristics of being an NF-κB-responsive element. Finally, we find that this element is essential for Aire expression in vivo and necessary to prevent spontaneous autoimmunity, reflecting the importance of this regulatory DNA element in promoting immune tolerance.

Induction of central tolerance by the factor Aire: molecular and epigenetic regulation

The establishment of thymic central tolerance is a critical process to prevent the development of autoimmune diseases. Medullary thymic epithelial cells (mTEC) are essential to this process through the expression of the transcription factor Aire, which controls the transcription of many genes encoding tissue-restricted antigens. Mutations in the Aire gene are responsible for a rare autoimmune disorder called APECED (autoimmune polyendocrinopathy candidiasis ectodermal dystrophy). This review summarizes our current knowledge on the mode of action of Aire at the molecular and epigenetic levels in controlling the expression of tissue-restricted antigens. We also discuss recently described additional roles of this transcription factor in the induction of central T-cell tolerance.

Abstract SY07-04: The evolutionary landscape of CLL: Therapeutic implications

Abstract Clonal evolution is a key feature of cancer progression and relapse. Recent studies across cancers have demonstrated the extensive degree of intratumoral heterogeneity present within individual cancers. We hypothesized that evolutionary dynamics contribute to the variations in disease tempo and response to therapy that are highly characteristic of chronic lymphocytic leukemia (CLL). We have recently investigated this phenomenon by developing a pipeline that estimates the fraction of cancer cells harboring each somatic mutation within a tumor through integration of whole-exome sequence (WES) and local copy number data (Landau et al., Cell 2013). By applying this analysis approach to 149 CLL cases, we discovered earlier and later cancer drivers, uncovered patterns of clonal evolution in CLL and linked the presence of subclones harboring driver mutations with adverse clinical outcome. Thus, our study, generated from a heterogeneous sample cohort, strongly supports the concept that CLL clonal evolution arises from mass extinction and therapeutic bottlenecks which lead to the emergence of highly fit (and treatment resistant) subclones. We further hypothesized that epigenetic heterogeneity also shapes CLL clonal evolution through interrelation with genetic heterogeneity. Indeed, in recent work, we have uncovered stochastic methylation disorder as the primary cause of methylation changes in CLL and cancer in general, and that this phenomena impacts gene transcription, genetic evolution and clinical outcome. Thus, integrated studies of genetic and epigenetic heterogeneity in CLL have revealed the complex and diverse evolutionary trajectories of these cancer cells. Immunotherapy is exquisitely suited for specifically and simultaneously targeting multiple lesions. We have developed an approach that leverages whole-exome sequencing to systematically identify personal tumor mutations with immunogenic potential, which can be incorporated as antigen targets in multi-epitope personalized therapeutic vaccines. We are pioneering this approach in an ongoing trial in melanoma and will now expand this concept to address diverse malignancies. Our expectation is that the choice of tumor neoantigens for a vaccine bypasses thymic tolerance and thus generates highly specific and potent high-affinity T cell responses to eliminate tumors in any cancer, including both ‘trunk’ and ‘branch’ lesions. Citation Format: Catherine J. Wu. The evolutionary landscape of CLL: Therapeutic implications. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr SY07-04. doi:10.1158/1538-7445.AM2015-SY07-04

Thymic Crosstalk Coordinates Medulla Organization and T-Cell Tolerance Induction.

The thymus ensures the generation of a functional and highly diverse T-cell repertoire. The thymic medulla, which is mainly composed of medullary thymic epithelial cells (mTECs) and dendritic cells (DCs), provides a specialized microenvironment dedicated to the establishment of T-cell tolerance. mTECs play a privileged role in this pivotal process by their unique capacity to express a broad range of peripheral self-antigens that are presented to developing T cells. Reciprocally, developing T cells control mTEC differentiation and organization. These bidirectional interactions are commonly referred to as thymic crosstalk. This review focuses on the relative contributions of mTEC and DC subsets to the deletion of autoreactive T cells and the generation of natural regulatory T cells. We also summarize current knowledge regarding how hematopoietic cells conversely control the composition and complex three-dimensional organization of the thymic medulla.

Bioengineering Thymus Organoids to Restore Thymic Function and Induce Donor-Specific Immune Tolerance to Allografts

One of the major obstacles in organ transplantation is to establish immune tolerance of allografts. Although immunosuppressive drugs can prevent graft rejection to a certain degree, their efficacies are limited, transient, and associated with severe side effects. Induction of thymic central tolerance to allografts remains challenging, largely because of the difficulty of maintaining donor thymic epithelial cells in vitro to allow successful bioengineering. Here, the authors show that three-dimensional scaffolds generated from decellularized mouse thymus can support thymic epithelial cell survival in culture and maintain their unique molecular properties. When transplanted into athymic nude mice, the bioengineered thymus organoids effectively promoted homing of lymphocyte progenitors and supported thymopoiesis. Nude mice transplanted with thymus organoids promptly rejected skin allografts and were able to mount antigen-specific humoral responses against ovalbumin on immunization. Notably, tolerance to skin allografts was achieved by transplanting thymus organoids constructed with either thymic epithelial cells coexpressing both syngeneic and allogenic major histocompatibility complexes, or mixtures of donor and recipient thymic epithelial cells. Our results demonstrate the technical feasibility of restoring thymic function with bioengineered thymus organoids and highlight the clinical implications of this thymus reconstruction technique in organ transplantation and regenerative medicine.

CCR4 is required for efficient thymocyte medullary entry and central tolerance induction (HEM7P.230)

Abstract Clonal deletion of auto-reactive thymocytes is essential for the establishment of central tolerance. The thymic medulla is a specialized microenvironment in which post-positive selection thymocytes are screened for reactivity against a wide range of self-antigens. Autoreactive cells are tolerized via clonal deletion or differentiation into the regulatory T cell lineage. Both thymic dendritic cells (DCs) and medullary thymic epithelial cells (mTECs) present auto-antigens to thymocytes to induce central tolerance. Thus, it is critical that post-positive selection thymocytes enter the medulla and interact efficiently with antigen-presenting cells therein to avoid export of autoreactive naïve T cells. We previously demonstrated that although CCR7 is required for medullary accumulation of thymocytes, additional GPCRs must be involved in medullary entry. Here we report that CCR4 is expressed by post-positive selection thymocytes, while CCR4 ligands are expressed by thymic DCs. Using two-photon time-lapse microscopy, we find that CCR4 is required for accumulation of post-positive selection thymocytes in the medulla, as well as efficient interactions between thymocytes and medullary DCs. In the absence of CCR4, thymocytes fail to undergo clonal deletion to low avidity antigens, autoreactive naïve T cells accumulate in secondary lymphoid organs, and autoimmunity ensues. Taken together, these data demonstrate an essential role for CCR4 in the establishment of thymic central tolerance.

Crossreactivity to vinculin and microbes provides a molecular basis for HLA-based protection against rheumatoid arthritis.

The HLA locus is the strongest risk factor for anti-citrullinated protein antibody (ACPA)(+) rheumatoid arthritis (RA). Despite considerable efforts in the last 35 years, this association is poorly understood. Here we identify (citrullinated) vinculin, present in the joints of ACPA(+) RA patients, as an autoantigen targeted by ACPA and CD4(+) T cells. These T cells recognize an epitope with the core sequence DERAA, which is also found in many microbes and in protective HLA-DRB1*13 molecules, presented by predisposing HLA-DQ molecules. Moreover, these T cells crossreact with vinculin-derived and microbial-derived DERAA epitopes. Intriguingly, DERAA-directed T cells are not detected in HLA-DRB1*13(+) donors, indicating that the DERAA epitope from HLA-DRB1*13 mediates (thymic) tolerance in these donors and explaining the protective effects associated with HLA-DRB1*13. Together our data indicate the involvement of pathogen-induced DERAA-directed T cells in the HLA-RA association and provide a molecular basis for the contribution of protective/predisposing HLA alleles.

The cellularity of thymic regulatory T cells is regulated by CCR7 through a non-cell intrinsic mechanism (HEM7P.228)

Abstract To ensure central tolerance, developing T cells must enter the thymic medulla to encounter self-antigens that induce autoreactive cells to undergo apoptosis or differentiation into the regulatory T cell (Treg) lineage. Both dendritic cells (DCs) and medullary thymic epithelial cells (mTECs) present antigens to tolerize thymocytes. mTECs express a diverse array of self-antigens, while DCs express auto-antigens acquired both in the periphery and from mTECs. Thymocytes must interact efficiently with these medullary antigen presenting cells (APCs) to encounter the full spectrum of self- antigens. CCR7 guides thymocytes into the medulla, promoting encounters with medullary DCs and mTECs. In the absence of CCR7, negative selection of autoreactive thymocytes is impaired, and autoimmunity ensues. Surprisingly, thymic Treg cellularity is increased in Ccr7-/- mice. Diminished negative selection has previously been associated with a concomitant increase in Treg differentiation. Thus, we anticipated that a cell-intrinsic defect in CCR7-/- thymocytes would account for increased Treg generation. Instead, a series of bone marrow chimeras demonstrate that CCR7-deficient non-T, non-B lineage hematopoietic cells account for increased Treg cellularity. Expression profiling revealed expression of Ccr7 by thymic DCs. We present evidence that CCR7 deficiency in thymic DCs is responsible for increased Treg cellularity, suggesting that CCR7 has pleiotropic effects on thymic central tolerance.

An experiment study of thymus tolerance to treat surgical brain injury

Objective To explore the curative effect after surgical brain injury(SBI)by develop an immune system that is tolerant to auto- cerebral cells through thymus tolerance. Methods 24 male SD rats were randomly divided into three groups, 8 in each group: A group: SBI models and treatment group, B normal saline control group, C Sham group, functional outcome was evaluated using modified neurological severity score(MNSS), cerebral edema was detected by magnetic resonance imaging(MRI), transforming growth factor-β1(TGF-β1), interferon gamma(IFN-γ) were detected by enzyme linked- immuno- sorbent assay(ELISA), CD4+/CD8+ T ratio in peripheral blood detected by flow cytometry(FCM)at 1,3, 7, 14,21 d after SBI. Results A group MRI showed less cerebral edema, neurological evaluation confirmed functional outcomes were significantly improved compared with B group, cerebral edema volume respectively(mm3)were[(39.41±3.68, 80.91±7.50, 92.25±7.06, 74.53±7.05, 47.80±10.00),(42.21±4.11, 87.07±4.70, 108.56 ±10.29, 97.58±12.75,65.96±11.85)]; in A group, levels of TGF-β1 were obviously increased at 1, 3, 7 d and the mean were higher than B group(P< 0.05), concentration respectively(ng/L) were[(751.96±50.24, 780.99±50.23, 853.20±14.81),(704.55±36.89, 724.54±18.72, 804.13±50.07)]; Concentration of IFN-γsignificantly declined at 7 14 21 d, and the mean were lower than B group(P<0.05), concentration(ng/L) respectively were[(132.60±19.89,129.89± 21.12, 106.34±23.91),(164.43±32.52, 179.98±40.21, 157.46±50.68)]; CD4+/CD8+ T ratio in group A were significantly decreased at 7, 14 d, and the mean was lowest at 14 d time point (P< 0.05), the ratio were(1.43±0.20); B group showed a worsen functional outcomes compared with C Group, levels of TGF-β1 in group B were obviously increased at 3, 7, 14 d and the means were higher than C group(P< 0.05), concentration(ng/L) respectively were[(724.54±18.72, 804.13±50.07, 808.33±62.12),(646.66±94.53, 652.62±98.80, 619.39±89.20)], concentration of IFN-γsignificantly increasing at 1, 3, 7, 14 d and the means were higher than C group (P< 0.05) the concentration(ng/L) respectively were[(113.38±15.42, 133.56±26.10, 164.43 ±32.52, 179.98±40.21),(83.08±12.70, 87.76±14.10, 103.27±22.19, 97.29±13.98)], in B group CD4+/CD8+ T ratio continue to rise at 3, 7, 14 d, the ratio respectively were 1.68±0.31, 2.00±0.30, 2.20±0.27. Conclusion Developing immunological tolerance through thymus injected with auto- cerebral cells can reduce inflammation reaction, brain edema and mitigates the severity of brain damage after SBI. Key words: Immune tolerance; Surgical brain injury; Thymus; CD4+/CD8+ T ratio

Medullary thymic epithelial cells and central tolerance in autoimmune hepatitis development: novel perspective from a new mouse model.

Autoimmune hepatitis (AIH) is an immune-mediated disorder that affects the liver parenchyma. Diagnosis usually occurs at the later stages of the disease, complicating efforts towards understanding the causes of disease development. While animal models are useful for studying the etiology of autoimmune disorders, most of the existing animal models of AIH do not recapitulate the chronic course of the human condition. In addition, approaches to mimic AIH-associated liver inflammation have instead led to liver tolerance, consistent with the high tolerogenic capacity of the liver. Recently, we described a new mouse model that exhibited spontaneous and chronic liver inflammation that recapitulated the known histopathological and immunological parameters of AIH. The approach involved liver-extrinsic genetic engineering that interfered with the induction of T-cell tolerance in the thymus, the very process thought to inhibit AIH induction by liver-specific expression of exogenous antigens. The mutation led to depletion of specialized thymic epithelial cells that present self-antigens and eliminate autoreactive T-cells before they exit the thymus. Based on our findings, which are summarized below, we believe that this mouse model represents a relevant experimental tool towards elucidating the cellular and molecular aspects of AIH development and developing novel therapeutic strategies for treating this disease.

CD28-CD80/86 and CD40-CD40L Interactions Promote Thymic Tolerance by Regulating Medullary Epithelial Cell and Thymocyte Development.

Development and central tolerance of T lymphocytes in the thymus requires both TCR signals and collaboration with signals generated through costimulatory molecule interactions. In this review, we discuss the importance of CD28-CD80/86 and CD40-CD40L costimulatory interactions in promoting normal thymic development. This discussion includes roles in the generation of a normal thymic medulla, in the development of specific T-cells subsets, including iNKT and T regulatory cells, and in the generation of a tolerant mature T-cell repertoire. We discuss recent contributions to the understanding of CD28-CD80/86 and CD40-CD40L costimulatory interactions in thymic development, and we highlight the ways in which the many important roles mediated by these interactions collaborate to promote normal thymic development.

Breaking through the central tolerance ceiling to unleash anticancer immune responses

Central thymic tolerance mechanisms create a formidable barrier against the generation of self-reactive T cells. While preventing autoimmunity, this barrier also limits an effective antitumor immunological response. We recently reported that anti-RANKL blocking antibody breaches this central tolerance barrier, thus increasing the repertoire of melanoma reactive T cells. Thus, central tolerance blockade may be an effective therapeutic strategy to enhance anticancer immunity.

Epithelial lymphotoxin beta receptor regulates embryonic medullary thymic epithelial cell development for central tolerance (BA14P.212)

Abstract Medullary thymic epithelial cells (mTEC) play critical roles in central tolerance. Given multiple cell types involved and complex cellular and molecular crosstalk, the mechanisms underlying mTEC development remain intriguing. Previous studies showed reduced mTEC number in lymphotoxin beta receptor (LTβR) knockout (KO) mice, but exactly how LTβR functions is still unclear. Here, by using conditional knockout (cKO) mice, in which LTβR is specifically depleted from thymic epithelial cells, we found dramatically reduced number of mTECs in LTβR cKO adult mice. Unexpectedly, further experiments did not find any homeostatic proliferation or apoptosis defect of mTEC in postnatal cKO mice at any stages. Further analysis of embryonic thymi interestingly found mTEC number is reduced while the number of progenitor of mTEC (mTECp) is comparable between WT and cKO mice, suggesting differentiation and/or expansion defects of mTEC in cKO mice. To further investigate the biological impact of epithelial LTβR deficiency, thymic expression of peripheral tissue restricted antigens and self-reactivity at periphery were determined in adult mice. Thymic negative selection was also directly addressed using TRAMP and SV40-TCR tg mice. Current data suggest significant impairment of thymic tolerance and autoimmune phenotype at periphery. Collectively, our study has revealed that thymic epithelial LTβR is essential for embryonic mTEC development and establishment of central tolerance in adult mice.

Enhancement of an anti-tumor immune response by transient blockade of central T cell tolerance.

Thymic central tolerance is a critical process that prevents autoimmunity but also presents a challenge to the generation of anti-tumor immune responses. Medullary thymic epithelial cells (mTECs) eliminate self-reactive T cells by displaying a diverse repertoire of tissue-specific antigens (TSAs) that are also shared by tumors. Therefore, while protecting against autoimmunity, mTECs simultaneously limit the generation of tumor-specific effector T cells by expressing tumor self-antigens. This ectopic expression of TSAs largely depends on autoimmune regulator (Aire), which is expressed in mature mTECs. Thus, therapies to deplete Aire-expressing mTECs represent an attractive strategy to increase the pool of tumor-specific effector T cells. Recent work has implicated the TNF family members RANK and RANK-Ligand (RANKL) in the development of Aire-expressing mTECs. We show that in vivo RANKL blockade selectively and transiently depletes Aire and TSA expression in the thymus to create a window of defective negative selection. Furthermore, we demonstrate that RANKL blockade can rescue melanoma-specific T cells from thymic deletion and that persistence of these tumor-specific effector T cells promoted increased host survival in response to tumor challenge. These results indicate that modulating central tolerance through RANKL can alter thymic output and potentially provide therapeutic benefit by enhancing anti-tumor immunity.

Clinical Spectrum of SCID: The Key is in the Thymus?

Clinical Spectrum of SCID: The Key is in the Thymus?

Recognition of Salmonella by Dectin-1 induces presentation of peptide antigen to type B T cells.

Type B T cells recognize peptide–MHC class II (pMHCII) isoforms that are structurally distinct from those recognized by conventional type A T cells. These alternative type B conformers result from peptide loading in the absence of HLA-DM. Type A conformers are more stable than type B pMHCII conformers but bind the same peptide in the same register. Here, we show that interaction of Salmonella Typhimurium with bone marrow derived dendritic cells (BMDCs) isolated from C3H/HeNCr1 mice results in enhanced presentation of peptide Ag to type B T cells. The effect could be mimicked by purified PAMPs, the most potent of which were curdlan and zymosan, β-(1,3)-glucan-containing polymers that are recognized by Dectin-1. Blocking of Dectin-1 with Ab and laminarin inhibited the induction of the type B T-cell response by BMDCs, confirming its role as a PRR for S. Typhimurium. Splenic DCs (sDCs) expressed Dectin-1 but were refractive to the induction of type B responses by S. Typhimurium and curdlan. Type B T cells have been shown to escape thymic tolerance and to transfer pathology in an autoimmune disease model. The induction of type B responses by gram-negative bacteria provides a mechanism by which autoreactive T cells may be produced during infection.

Late-onset myasthenia gravis – CTLA4low genotype association and low-for-age thymic output of naïve T cells

Late-onset myasthenia gravis (LOMG) has become the largest MG subgroup, but the underlying pathogenetic mechanisms remain mysterious. Among the few etiological clues are the almost unique serologic parallels between LOMG and thymoma-associated MG (TAMG), notably autoantibodies against acetylcholine receptors, titin, ryanodine receptor, type I interferons or IL-12. This is why we checked LOMG patients for two further peculiar features of TAMG – its associations with the CTLA4high/gain-of-function +49A/A genotype and with increased thymic export of naïve T cells into the blood, possibly after defective negative selection in AIRE-deficient thymomas. We analyzed genomic DNA from 116 Caucasian LOMG patients for CTLA4 alleles by PCR/restriction fragment length polymorphism, and blood mononuclear cells for recent thymic emigrants by quantitative PCR for T cell receptor excision circles. In sharp contrast with TAMG, we now find that: i) CTLA4low +49G(+) genotypes were more frequent (p = 0.0029) among the 69 LOMG patients with age at onset ≥60 years compared with 172 healthy controls; ii) thymic export of naïve T cells from the non-neoplastic thymuses of 36 LOMG patients was lower (p = 0.0058) at diagnosis than in 77 age-matched controls. These new findings are important because they suggest distinct initiating mechanisms in TAMG and LOMG and hint at aberrant immuno-regulation in the periphery in LOMG. We therefore propose alternate defects in central thymic or peripheral tolerance induction in TAMG and LOMG converging on similar final outcomes. In addition, our data support a 60-year-threshold for onset of ‘true LOMG’ and an LOMG/early-onset MG overlapping group of patients between 40 and 60.

Allogeneic Hematopoietic Stem Cell Transplantation For Severe Neuromyelitis Optica

BackgroundNeuromyelitis Optica (NMO), also known as Devic's disease, is a rare inflammatory and demyelinating disorder of the central nervous system characterized by recurrent attacks of optic neuritis (ON) and longitudinally extensive trasverse myelitis (LETM). Previously considered as a severe variant of multiple sclerosis, NMO has been recently recognized as a distinct disorder associated with peculiar pathogenic auto-antibodies to aquaporin-4 (AQP4). Despite transient control of disease activity, standard treatments are often followed by relapses with accumulating disability and ultimately a poor prognosis. Thus, new therapeutic strategies for NMO are warranted. Hematopoietic Stem Cell Transplantation (HSCT) can induce stable remissions in patients with severe treatment-refractory Autoimmune Diseases. Here we report our investigational experience on allogeneic HSCT in two patients with severe and refractory forms of the disease. MethodsBoth treated patients had aggressive forms of NMO and serum positivity for AQP4 antibodies. Upn#1 is a 30 yrs old male affected by severe relapsing LETM, with paraparesis and thoracic spinal cord lesion on magnetic resonance imaging (MRI). Upn#2 is a 28 yrs female with recurrent attacks of ON and LETM. Both had received several lines of treatment without benefit, including high-dose corticosteroids, cyclophosphamide, rituximab, natalizumab, alemtuzumab, plasma exchange, high-dose thiotepa/cyclophosphamide and/or BEAM with Anti-Thymocyte Globulin (ATG) and cyclosporine followed by autologous HSCT rescue. Before HSCT the two patients showed an Kurtzke Expanded Disability Status Score (EDSS) of 6 (assistance required to walk) and 8.5 (restricted to bed, paraplegia) respectively, while both of them presented active contrast enhancing lesions on MRI and positivity for the pathogenic AQP4 auto-antibodies. Upn#1 underwent allogeneic HSCT from his HLA-identical sibling, while Upn#2 from a matched unrelated donor. Conditioning regimen consisted of full-dose treosulfan and fludarabine. Graft versus host disease (GvHD) prophylaxis combined ATG-Fresenius with cyclosporine and a short course of methotrexate (Upn#1) or mycophenolate and rapamycin (Upn#2); B cell depletion of both patients and graft was obtained by rituximab. ResultsHematopoietic recovery occurred in both patients within day 30, and was accompanied by rapid achievement of full donor chimerism. Each patient experienced an episode of febrile neutropenia and one of them a CMV reactivation, both responsive to medical therapy; no serious adverse events were reported. None of the patients experienced neither acute nor chronic GvHD. MRI demonstrated the disappearance of the inflammatory lesions, without evidence of new ones. The immunological data obtained from the follow-up of these two patients suggest that allogeneic HSCT can alter the course of NMO through several concurring mechanisms: the eradication of autoreactive cell clones by high-dose chemotherapy and by the in vivo T and B cell depletion used for conditioning, the disappearance of the pathogenetic anti-AQP4 antibodies and achievement of a stable full-donor hematopoietic chimerism by donor T cell-mediated alloreactivity; the re-establishment of thymic central tolerance and renewal of the immune repertoire. Strikingly, these results correlated with a marked improvement of neurological functions in both treated patients. In UPN#1 EDSS dropped from 6 to 3.5 (fully ambulatory but with moderate disability in one functional neurological system), while in UPN#2 EDSS decreased from 8.5 to 7.5 (unable to take more than a few steps, restricted to wheelchair). At last follow-up (48 and 36 months after HSCT, respectively), both patients are alive, well and relapse-free. ConclusionsThese findings suggest that allogeneic HSCT may be beneficial in patients who have aggressive forms of NMO, by renewing the immune repertoire of T- and B-cells, thus reducing disease activity and arresting disability progression. The preliminary evidence of long-term disease control in these two refractory patients paves the way for further prospective phase I-II clinical trials to validate allogeneic HSCT as promising novel option for aggressive forms of NMO. Disclosures:Bonini:MolMed S.p.A: Consultancy.

A novel cell-based assay for measuring neutralizing autoantibodies against type I interferons in patients with autoimmune polyendocrine syndrome type 1.

Event Abstract Back to Event A novel cell-based assay for measuring neutralizing autoantibodies against type I interferons in patients with autoimmune polyendocrine syndrome type 1. Lars Breivik1*, Bergithe E. Oftedal1, Anette S. Wolff1, Elizaveta Orlova2 and Eystein S. Husebye1, 3 1 University of Bergen, Department of Clinical Science, Norway 2 Endocrinology Research Center, Institute of Paediatric Endocrinology, Russia 3 Haukeland University hospital,, Department of Medicine, Norway Autoimmune polyendocrine syndrome type 1 (APS-1) is a unique monogenic disorder caused by loss-of-function mutations in the autoimmune regulator (AIRE) gene. The loss of a functional AIRE protein presumably causes defects in central thymic tolerance and the escape of pathogenic autoreactive T cells into peripheral tissues. The most prevalent clinical manifestations are chronic mucocutaneous candidiasis, hypoparathyroidism and primary adrenocortical failure although a wide range of other autoimmune manifestations may occur. An important characteristic feature of APS-1 is the presence of neutralizing serum autoantibodies against type I interferons (subtypes –α and -ω) at frequencies approaching 100 %. These autoantibodies may therefore serve as an important diagnostic tool, complementing genetic mutation analysis of AIRE when APS-1 is suspected. Several techniques for measuring these antibodies are currently available, including fluid-phase binding assays using recombinant antigens labeled with radioactivity (radioactive immunoassay; RIA) or luciferase, and assays measuring the actual interferon-neutralizing capabilities of the antibodies such as the antiviral interferon neutralizing assay (AVINA). We report here on a cell-based assay using a commercially available reporter cell-line that provides a simple, sensitive, rapid and reliable way of assaying neutralizing autoantibodies against the type I interferons subtypes –α and –ω as compared to a RIA assay. Acknowledgements We would like to thank all the doctors and patients that contributed to this work. The technical help from Elisabeth T. Halvorsen is greatly acknowledged. The study has been supported by grants from Helse-Vest and The Norwegian Research Council. Keywords: Autoantibodies, Interferon Type I, Assay development, APS-1, biomarker Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Translational immunology and immune intervention Citation: Breivik L, Oftedal BE, Wolff AS, Orlova E and Husebye ES (2013). A novel cell-based assay for measuring neutralizing autoantibodies against type I interferons in patients with autoimmune polyendocrine syndrome type 1.. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00920 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Jun 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Lars Breivik, University of Bergen, Department of Clinical Science, BERGEN, N-5021, Norway, larsbreivik@hotmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Lars Breivik Bergithe E Oftedal Anette S Wolff Elizaveta Orlova Eystein S Husebye Google Lars Breivik Bergithe E Oftedal Anette S Wolff Elizaveta Orlova Eystein S Husebye Google Scholar Lars Breivik Bergithe E Oftedal Anette S Wolff Elizaveta Orlova Eystein S Husebye PubMed Lars Breivik Bergithe E Oftedal Anette S Wolff Elizaveta Orlova Eystein S Husebye Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Aire mediates thymic expression and tolerance of pancreatic antigens via an unconventional transcriptional mechanism

The autoimmune regulator (Aire), mediates central tolerance of peripheral self. Its activity in thymic epithelial cells (TECs) directs the ectopic expression of thousands of tissue-restricted antigens (TRAs), causing the deletion of autoreactive thymocytes. The molecular mechanisms orchestrating the breadth of transcriptional regulation by Aire remain unknown. One prominent model capable of explaining both the uniquely high number of Aire-dependent targets and their specificity posits that tissue-specific transcription factors induced by Aire directly activate their canonical targets, exponentially adding to the total number of Aire-dependent TRAs. To test this "Hierarchical Transcription" model, we analysed mice deficient in the pancreatic master transcription factor pancreatic and duodenal homeobox 1 (Pdx1), specifically in TECs (Pdx1(ΔFoxn1) ), for the expression and tolerance of pancreatic TRAs. Surprisingly, we found that lack of Pdx1 in TECs did not reduce the transcription of insulin or somatostatin, or alter glucagon expression. Moreover, in a model of thymic deletion driven by a neo-TRA under the control of the insulin promoter, Pdx1 in TECs was not required to affect thymocyte deletion or the generation of regulatory T (Treg) cells. These findings suggest that the capacity of Aire to regulate expression of a huge array of TRAs relies solely on an unconventional transcriptional mechanism, without intermediary transcription factors.