Autophagy in Thymic epithelium shapes the T cell repertoire and is essential for tolerance

Aufgabe des Immunsystem ist es, den Organismus gegen eine Vielzahl von Krankheitserregern zu verteidigen und dabei die Toleranz gegenüber körpereigenen Strukturen aufrechtzuerhalten. T-Zellen sind eine der bedeutensten Komponenten der adaptiven Immunität, da sie sowohl für die humorale, als auch für die zelluläre Immunantwort essentiell sind. Während Ihrer Reifung im Thymus durchlaufen T-Zellen eine positive Selektion zur der Erkennung eigener MHC-Moleküle und eine negative Selektion, die sicherstellt, dass sich nur selbst-tolerante T-Zellen weiter entwickeln. Die Erkennung und Interaktion mit kortikalen und medullären Thymusepithelzellen (cTEC; mTEC) ermöglicht diese wichtigen Selektionsprozesse. Auswahl und Art der Prozessierung der für die Präsentation auf Thymusepitelzellen vorgesehenen Peptide sind noch weitgehend unbekannt. Obwohl MHC II Moleküle vorwiegend in Lysosomen mit Peptiden endozytotisch aufgenommener Polypeptide beladen werden, sind etwa 20% aller MHC II gebundenen Peptide auf hämatopoetischen APCs intrazellulären Ursprungs. Im Gegensatz dazu endozytieren und präsentieren TEC extrazelluläre Antigene äußerst ineffizient. Dies deutet auf eine Spezialisierung von TEC auf die Präsentation intrazellulärer Antigene auf MHC II Molekülen hin. Es wurden bereits mehrer Möglichkeiten für die nicht-klassische, endogene Beladung von MHC II Molekülen beschrieben, unter anderem die Chaperon-vermittelte Autophagie, Macroautophagie und der TAP-abhängige (Transporter assoziiert mit Antigen Prozessierung) Weg. Die Signifikanz dieser Prozesse in vivo ist jedoch noch unklar. In dieser Arbeit untersuchen wir Macroautophagie (im folgenden als Autophagie bezeichnet) im Thymusepithel und ihre Bedeutung für die Entwicklung von T-Zellen näher. Autophagie ist ein evolutionär konservierter Prozess, der das Recycling langlebiger, zytoplasmatischer Proteine und Organellen ermöglicht. Autophagosomen schließen Zellbestandteile ein, verschmelzen mit späten Endosomen oder Lysosomen und liefern so intrazelluläre Antigene in das MHC II Kompartment. Wir konnten zeigen, dass TEC, im Vergleich zu anderen Körperzellen, eine hohe, konstitutive Autophagierate aufweisen. Genetische Manipulation der Autophagie in TEC führte zu einer veränderten Positivselektion bestimmter MHC II restringierter T-Zell Spezifitäten, ohne die Selektion von MHC I restringierten T-Zellen zu beeinflussen. Athymische nude/nude Mäuse, denen ein Autophagie-defizienter Thymus transplantiert wurde, entwickelten schwere Colitis und wiesen Infiltrationen verschiedener Organe mit Lymphozyten auf. Aufgrund dieser Erkenntnisse glauben wir, dass Autophagie intrazelluläre Antigene für die Beladung von MHC II Molekülen in TEC bereit stellt und dadurch maßgeblich zur Effizienz der Positivselektion beziehungsweise Toleranzinduktion reifender CD4 T-Zellen beiträgt.The immune system has to fulfill the remarkable task of defending the organism against a plethora of pathogens, while at the same time remaining tolerant to self. T cells are the main mediators of adaptive immune responses, as they are essential for both cellular and humoral immunity. During their development in the thymus T cells pass through tightly controlled check points, namely positive and negative selection, that ensure that only T cells expressing antigen receptors that are self Major Histocompatibility Complex (MHC) restricted and self tolerant are generated. Recognition of MHC/peptide ligands (MHCp) on cortical thymic epithelial cells (cTEC) and medullary thymic epithelial cells (mTEC) serves non-redundant functions in positive and negative selection of T cells, respectively. However, the antigen sampling and processing pathways that shuttle peptides to the MHC class II loading compartment (MIIC) in thymic epithelial cells (TEC) remain elusive. Although it is generally accepted that MHCII/peptide epitopes (MHCII/p) are generated through lysosomal processing of endocytosed polypeptides about 20% of peptides bound by MHC class II molecules of hematopoietic antigen presenting cells (APC) originate from intracellular proteins. TEC, unlike hematopoietic APC, are inefficient in capturing and/or presenting extracellular antigens, implying that TEC might primarily concentrate on their intracellular milieu for the generation of MHCII/p repertoire. Several pathways have been implicated in non-classical, endogenous MHCII loading: chaperone mediated autophagy, macroautophagy and the transporter associated with antigen processing (TAP)-dependent pathway, but respective in vivo significance of these processes remained unknown. Here we addressed the role of macroautophagy (referred to as autophagy hereafter) in thymic epithelium in T cell development. Autophagy is an evolutionary conserved process responsible for the turnover of long lived cytoplasmic proteins and organelles. Autophagosomes fuse with late endosomes and lysosomes and thereby might deliver intracellular antigens to the MIIC compartment. We were able to show that TEC, unlike most other cells in the body, display a high constitutive autophagic activity. Genetic interference with autophagy specifically in thymic epithelium led to altered positive selection of certain MHC II- but not MHC I- restricted T cell specificities. Athymic nude mice grafted with an autophagy deficient thymus displayed severe colitis and immune mediated multi-organ tissue inflammation. On the basis of these findings we propose that autophagy shuttles intracellular antigens to MIIC in TEC and in that way contributes to the efficient positive selection and tolerance induction in developing CD4 T cells

Autophagy in the Thymic Epithelium Is Dispensable for the Development of Self-Tolerance in a Novel Mouse Model

The thymic epithelium plays critical roles in the positive and negative selection of T cells. Recently, it was proposed that autophagy in thymic epithelial cells is essential for the induction of T cell tolerance to self antigens and thus for the prevention of autoimmune diseases. Here we have tested this hypothesis using mouse models in which autophagy was blocked specifically in epithelial cells expressing keratin 14 (K14), including the precursor of thymic epithelial cells. While the thymic epithelial cells of mice carrying the floxed Atg7 gene (ATG7 f/f) showed a high level of autophagy, as determined by LC3 Western blot analysis and fluorescence detection of the recombinant green fluorescent protein (GFP)-LC3 reporter protein on autophagosomes, autophagy in the thymic epithelium was efficiently suppressed by deletion of the Atg7 gene using the Cre-loxP system (ATG7 f/f K14-Cre). Suppression of autophagy led to the massive accumulation of p62/sequestosome 1 (SQSTM1) in thymic epithelial cells. However, the structure of the thymic epithelium as well as the organization and the size of the thymus were not altered in mutant mice. The ratio of CD4 to CD8-positive T cells, as well as the frequency of activated (CD69+) CD4 T cells in lymphoid organs, did not differ between mice with autophagy-competent and autophagy-deficient thymic epithelium. Inflammatory infiltrating cells, potentially indicative of autoimmune reactions, were present in the liver, lung, and colon of a similar fraction of ATG7 f/f and ATG7 f/f K14-Cre mice. In contrast to previously reported mice, that had received an autophagy-deficient thymus transplant, ATG7 f/f K14-Cre mice did not suffer from autoimmunity-induced weight loss. In summary, the results of this study suggest that autophagy in the thymic epithelium is dispensable for negative selection of autoreactive T cells

The thymic medulla: a unique microenvironment for intercellular self-antigen transfer

Central tolerance is shaped by the array of self-antigens expressed and presented by various types of thymic antigen-presenting cells (APCs). Depending on the overall signal quality and/or quantity delivered in these interactions, self-reactive thymocytes either apoptose or commit to the T regulatory cell lineage. The cellular and molecular complexity underlying these events has only recently been appreciated. We analyzed the ex vivo presentation of ubiquitous or tissue-restricted self-antigens by medullary thymic epithelial cells (mTECs) and thymic dendritic cells (DCs), the two major APC types present in the medulla. We found that the ubiquitously expressed nuclear neo–self-antigen ovalbumin (OVA) was efficiently presented via major histocompatibility complex class II by mTECs and thymic DCs. However, presentation by DCs was highly dependent on antigen expression by TECs, and hemopoietic cells did not substitute for this antigen source. Accordingly, efficient deletion of OVA-specific T cells correlated with OVA expression by TECs. Notably, OVA was only presented by thymic but not peripheral DCs. We further demonstrate that thymic DCs are constitutively provided in situ with cytosolic as well as membrane-bound mTEC-derived proteins. The subset of DCs displaying transferred proteins was enriched in activated DCs, with these cells being most efficient in presenting TEC-derived antigens. These data provide evidence for a unique, constitutive, and unidirectional transfer of self-antigens within the thymic microenvironment, thus broadening the cellular base for tolerance induction toward promiscuously expressed tissue antigens

Monitoring Antigen Processing for MHC Presentation via Macroautophagy

Macroautophagy has recently emerged as an important catabolic process involved not only in innate immunity but also in adaptive immunity. Initially described to deliver intracellular antigens to MHC class II loading compartments, its molecular machinery has now also been described to impact the delivery of extracellular antigens to MHC class II loading compartments through the noncanonical use of the macroautophagy machinery during LC3-associated phagocytosis (LAP). Therefore, in pathological situations (viral or bacterial infections, tumorigenesis) the pathway might be involved in shaping CD4$^{+}$ T cell responses.In this chapter we describe three basic experiments for the monitoring and manipulation of macroautophagic antigen processing toward MHC class II presentation through the canonical pathway. Firstly, we will discuss how to monitor autophagic flux and autophagosome fusion with MHC class II loading compartments. Secondly, we will show how to target proteins to autophagosomes in order to monitor macroautophagy dependent antigen processing via their enhanced presentation on MHC class II molecules to CD4$^{+}$ T cells. And finally, we will describe how macroautophagy can be silenced in antigen presenting cells, like human monocyte-derived dendritic cells (DCs)

The multiple roles of autophagy in cancer

Autophagy is an evolutionarily conserved, catabolic process that involves the entrapment of cytoplasmic components within characteristic vesicles for their delivery to and degradation within lysosomes. Autophagy is regulated via a group of genes called AuTophaGy-related genes and is executed at basal levels in virtually all cells as a homeostatic mechanism for maintaining cellular integrity. The levels and cargos of autophagy can be modulated in response to a variety of intra- and extracellular cues to bring about specific and selective events. Autophagy is a multifaceted process and alterations in autophagic signalling pathways are frequently found in cancer and many other diseases. During tumour development and in cancer therapy, autophagy has paradoxically been reported to have roles in promoting both cell survival and cell death. In addition, autophagy has been reported to control other processes relevant to the aetiology of malignant disease, including oxidative stress, inflammation and both innate and acquired immunity. It is the aim of this review to describe the molecular basis and the signalling events that control autophagy in mammalian cells and to summarize the cellular functions that contribute to tumourigenesis when autophagy is perturbed

Thymic progenitor homing and lymphocyte homeostasis are linked via S1P-controlled expression of thymic P-selectin/CCL25

Thymic T cell progenitor (TCP) importation is a periodic, gated event that is dependent on the expression of functional P-selectin ligands on TCPs. Occupancy of intrathymic TCP niches is believed to negatively regulate TCP importation, but the nature of this feedback mechanism is not yet resolved. We show that P-selectin and CCL25 are periodically expressed in the thymus and are essential parts of the thymic gate-keeping mechanism. Periodicity of thymic TCP receptivity and the size of the earliest intrathymic TCP pool were dependent on the presence of functional P-selectin ligand on TCPs. Furthermore, we show that the numbers of peripheral blood lymphocytes directly affected thymic P-selectin expression and TCP receptivity. We identified sphingosine-1-phosphate (S1P) as one feedback signal that could mediate influence of the peripheral lymphocyte pool on thymic TCP receptivity. Our findings suggest a model whereby thymic TCP importation is controlled by both early thymic niche occupancy and the peripheral lymphocyte pool via S1P

Contrasting roles of histone 3 lysine 27 demethylases in acute lymphoblastic leukaemia

T-cell acute lymphoblastic leukaemia (T-ALL) is a haematological malignancy with a dismal overall prognosis, including a relapse rate of up to 25%, mainly because of the lack of non-cytotoxic targeted therapy options. Drugs that target the function of key epigenetic factors have been approved in the context of haematopoietic disorders, and mutations that affect chromatin modulators in a variety of leukaemias have recently been identified; however, ‘epigenetic’ drugs are not currently used for T-ALL treatment. Recently, we described that the polycomb repressive complex 2 (PRC2) has a tumour-suppressor role in T-ALL. Here we delineated the role of the histone 3 lysine 27 (H3K27) demethylases JMJD3 and UTX in T-ALL. We show that JMJD3 is essential for the initiation and maintenance of T-ALL, as it controls important oncogenic gene targets by modulating H3K27 methylation. By contrast, we found that UTX functions as a tumour suppressor and is frequently genetically inactivated in T-ALL. Moreover, we demonstrated that the small molecule inhibitor GSKJ4 (ref. 5) affects T-ALL growth, by targeting JMJD3 activity. These findings show that two proteins with a similar enzymatic function can have opposing roles in the context of the same disease, paving the way for treating haematopoietic malignancies with a new category of epigenetic inhibitors.National Institutes of Health (U.S.) (Grant R37-HD04502

Central CD4+ T cell tolerance: deletion versus regulatory T cell differentiation

The diversion of MHC class II-restricted thymocytes into the regulatory T (Treg) cell lineage, similarly to clonal deletion, is driven by intrathymic encounter of agonist self-antigens. Somewhat paradoxically, it thus seems that the expression of an autoreactive T cell receptor is a shared characteristic of T cells that are subject to clonal deletion and those that are diverted into the Treg cell lineage. Here, we discuss how thymocyte-intrinsic and -extrinsic determinants may specify the choice between these two fundamentally different T cell fates

The mucosal immune system and its regulation by autophagy

The gastrointestinal tract presents a unique challenge to the mucosal immune system, which has to constantly monitor the vast surface for the presence of pathogens, while at the same time maintaining tolerance to beneficial or innocuous antigens. In the intestinal mucosa, specialized innate and adaptive immune components participate in directing appropriate immune responses toward these diverse challenges. Recent studies provide compelling evidence that the process of autophagy influences several aspects of mucosal immune responses. Initially described as a “self-eating” survival pathway that enables nutrient recycling during starvation, autophagy has now been connected to multiple cellular responses, including several aspects of immunity. Initial links between autophagy and host immunity came from the observations that autophagy can target intracellular bacteria for degradation. However, subsequent studies indicated that autophagy plays a much broader role in immune responses, as it can impact antigen processing, thymic selection, lymphocyte homeostasis, and the regulation of immunoglobulin and cytokine secretion. In this review, we provide a comprehensive overview of mucosal immune cells and discuss how autophagy influences many aspects of their physiology and function. We focus on cell type-specific roles of autophagy in the gut, with a particular emphasis on the effects of autophagy on the intestinal T cell compartment. We also provide a perspective on how manipulation of autophagy may potentially be used to treat mucosal inflammatory disorders