T cell homeostasis in Autoimmune polyendocrinopathy - candidiasis - ectodermal dystrophy (APECED)

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http://urn.fi/URN:ISBN:978-952-10-7729-6
Title: T cell homeostasis in Autoimmune polyendocrinopathy - candidiasis - ectodermal dystrophy (APECED)
Alternative title: T-soluryhmien ylläpidon häiriöt APECED-sairaudessa
Author: Laakso, Sini
Contributor organization: University of Helsinki, Faculty of Medicine, Haartman Institute, Bacteriology and Immunology
Helsingin yliopisto, lääketieteellinen tiedekunta, kliinisteoreettinen laitos
Helsingfors universitet, medicinska fakulteten, Haartman institutet
Publisher: Helsingin yliopisto
Date: 2012-03-30
Language: eng
URI: http://urn.fi/URN:ISBN:978-952-10-7729-6
http://hdl.handle.net/10138/31389
Thesis level: Doctoral dissertation (article-based)
Abstract: Autoimmune polyendocrinopathy - candidiasis - ectodermal dystrophy (APECED) is a rare but severe, recessively inherited monogenic autoimmune disease that is part of the Finnish disease heritage. Loss-of-function mutations in the Autoimmune regulator (AIRE) gene cause APECED, which thereby provides a unique model for studying human autoimmunity. The patients suffer from chronic mucocutaneous candidiasis (CMC) and organ-specific autoimmunity targeted against especially the endocrine organs; hypoparathyroidism and Addison s disease are the most common endocrinopathies, but many other disease components frequently occur. AIRE is a transcriptional regulator that is expressed the most in medullary thymic epithelial cells where it controls the expression of peripheral tissue restricted antigens. AIRE is therefore regarded an important tool of negative selection, which is the key step of central tolerance the process of generating T cells that are tolerant to self. AIRE has been described to have various other functions in the thymus and it is also expressed in the secondary lymphoid organs. Central tolerance is complemented in the immune system by the mechanisms of peripheral tolerance, such as the functions of regulatory T cells (Tregs) that suppress the responses of other T cells. In APECED, Treg functions have been found impaired. With the differences between the clinically healthy remaining Aire−⁄− mice and the severe human disease, the pathogenesis of clinical APECED is still unclear. The aim of this study was to discover mechanisms of autoimmunity in human APECED. This was approached first by studying further the population of Tregs in the patients, because understanding the reasons behind their functional defect could bring new knowledge on the impact AIRE has on peripheral tolerance. CD8+ T cell function in APECED was then addressed, because this population is thought to contain the highly autoreactive T cells in autoimmune diseases responsible for the direct tissue damage, and the pre-existing data on CD8+ T cells in the patients was scarce. The third study focused on the skin: the recently discovered autoantibodies against T helper 17 (Th17) class cytokines have implicated autoimmunity as a mechanism even behind CMC, which motivated a study on the in vivo immune response of Th cells in APECED patients. Flow cytometry was used to study the phenotype of T cells, which were for the most part isolated from peripheral blood samples of APECED patients and healthy controls. To generate an in vivo memory response, intradermal injections of purified protein derivative (PPD, tuberculin) were given to the skin of APECED patients and healthy controls, and the responding cells made retrievable by forming blisters on top of the reactions with a clinical suction pump. The isolated cells from peripheral blood and the skin blisters were analyzed also by quantitative polymerase chain reaction, to determine relative gene expression. This was preceded in some experiments with further separation of T cell subpopulations, performed by sorting or using immunomagnetic cell separation. The T cell receptor repertoire of CD8+ T cells was analyzed by spectratyping. Cell cultures were used to study the effects of polyclonal stimulation on peripheral blood Th cell function. The defect of Tregs in APECED was found to be more severe in the peripherally activated subpopulation than in the recently from the thymus emigrated (RTE) Tregs, indicating a defect of Treg peripheral homeostasis. This was associated with an increased attrition of RTE Tregs in the patients, most probably leading to insufficient long-term recruitment of functional Tregs in the periphery. The population of CD8+ T cells in the APECED patients was found to be under a severe interleukin 7 (IL-7) dysregulation, which is an important homeostatic cytokine in the immune system. This was associated with a notable skewing of the CD8+CD45RO- T cell subset of the patients, highly suggestive of autoreactivity. Naive CD8+ T cells were also found severely altered, as well as the CD8+ T cells bearing a marker of recent thymic emigrancy, which suggested that the IL-7 dysregulation had begun already in the thymus. Studying the in vivo immune response, a selective defect of the Th17 class cytokine IL-22 production emerged, which was evident both at the site of antigen challenge as well as on the unexposed skin, and appeared also in stimulated cells of the peripheral blood. Production of the cytokine IL-17 was not significantly altered, which suggested that the Th population affected in APECED might be the emerging Th22 cells, especially involved in maintaining the barrier function and attracting proper immune responses in the skin. This study reveals multiple defects of T cell homeostasis in APECED that can explain the severity of the disease in humans and the appearance of additional disease components with aging: The defect of Tregs in APECED has a temporal component, which is likely to result from alterations imprinted in them in the thymus, and their peripheral activation fails, possibly relating to the lack of functional AIRE in the periphery. Therefore the autoreactive T cells, which gain further momentum from the IL-7 dysregulation, can cause damage to additional target organs. The IL-7 dysregulation might be the consequence of increased type 1 interferon exposure inside the thymus, which has been suggested to be causing the autoantibodies against these cytokines in APECED. The IL-22 defect, in turn, increases microbial exposure and can imbalance the immune regulation even further. The findings offer novel insights into the pathogenesis of organ-specific autoimmunity, and may have implications for more common autoimmune diseases where Tregs, IL-7 regulation and the effects of IL-22 are indicated. Furthermore, the study provides support for seeking therapies for APECED that balance the homeostatic milieu of T cells, a strategy perhaps easier to implement than repairing the process of negative selection.Autoimmuuni polyendokrinopatia - kandidiaasi - ektodermaalinen dystrofia (APECED) on harvinainen mutta vakava, yhden geenin mutaatioiden aiheuttama autoimmuunisairaus, joka kuuluu suomalaiseen tautiperimään. Mutaatiot esiintyvät Autoimmune regulator (AIRE) -geenissä. AIRE on geeniluennan säätelijä ennen kaikkea kateenkorvassa, jossa se mahdollistaa kateenkorvan ulkopuolisten, kehon omien rakenteiden ilmentämisen. Täten AIRE on tärkeä kehittyvien T-solujen kouluttamisessa reagoimattomiksi omia rakenteita vastaan. AIRElla on kuitenkin kuvattu olevan monia muita tehtäviä kateenkorvassa, ja AIRE ilmenee myös kateenkorvan ulkopuolisissa imusolujärjestelmän kudoksissa. Säätelijä-T-solut on erityinen T-soluryhmä, jotka estävät muiden T-solujen toimintaa, ja APECED-sairaudessa myös näiden solujen toiminnassa on havaittu puutteita. Tämän tutkimuksen tavoite oli löytää autoimmuniteetin mekanismeja APECED-potilaissa. Tätä tavoitetta lähestyttiin ensin tutkimalla tarkemmin säätelijä-T-solujen populaatiota. CD8+ T-solujen toiminta oli seuraava tutkimuksen kohde, koska tämä soluryhmä useimmiten aikaansaa autoimmuunisairauksissa suoran kudostuhon, ja olemassaoleva tieto näiden solujen toiminnasta APECEDissa oli niukkaa. Kolmas osatyö kohdistui ihoon: auttaja-T-solujen tuottamia välittäjäaineita vastaan kohdistuvat vasta-aineet ovat viimeaikainen löydös APECED-potilaissa, ja ne ovat muuttaneet käsitystä ihon ja limakalvojen hiivatulehduksen syntymekanismista autoimmuunipohjaisen mekanismin suuntaan. Tämä innosti tutkimaan in vivo -asetelmassa ihon auttaja-T-solujen immuunivastetta APECED-potilaissa. Tämä tutkimus paljastaa useita T-solujen populaatioiden ylläpidon häiriöitä APECEDissa: Säätelijä-T-solujen häiriöllä on ajallinen osatekijä, joka todennäköisesti johtuu häiriöistä solujen kehityksessä kateenkorvassa, ja säätelijä-T-solujen aktivaatio kateenkorvan ulkopuolella on puutteellista, johtuen mahdollisesti perifeerisen AIREn puuttumisesta. Näin ollen kehoa vastaan reagoivat CD8+ T-solut, jotka vahvistuvat IL-7 säätelyhäiriöstä, pääsevät aiheuttamaan vauriota uusille kohde-elimille. IL-7 säätelyhäiriö saattaa alkaa jo kateenkorvassa. IL-22 häiriö iholla johtaa puolestaan lisääntyneeseen mikrobialtistukseen, mikä voi edelleen johtaa immuunisäätelyn epätasapainoon. Löydökset tuovat uutta kohde-elinspesifien autoimmuunisairauksien kehittymisen ymmärrykseen, ja voivat olla hyödyllisiä yleisempien autoimmuunisairauksien tutkimukselle, missä säätelijä-T-solut, IL-7 säätely ja IL-22n vaikutukset on alustavasti tunnistettu. Tutkimus myös kannustaa etsimään hoitoja APECED-potilaille T-solujen ylläpitoa tasapainottavista tekijöistä, mikä saattaa olla helpompaa kuin puutteellisen valintaprosessin korjaaminen kateenkorvassa.
Subject: lääketiede
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