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

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

Thymic B Cells Are Licensed to Present Self Antigens for Central T Cell Tolerance Induction

Thymic antigen-presenting cells (APCs) such as dendritic cells and medullary thymic epithelial cells (mTECs) use distinct strategies of self-antigen expression and presentation to mediate central tolerance. The thymus also harbors B cells; whether they also display unique tolerogenic features and how they genealogically relate to peripheral B cells is unclear. Here, we found that Aire is expressed in thymic but not peripheral B cells. Aire expression in thymic B cells coincided with major histocompatibility class II (MHCII) and CD80 upregulation and immunoglobulin class-switching. These features were recapitulated upon immigration of naive peripheral B cells into the thymus, whereby this intrathymic licensing required CD40 signaling in the context of cognate interactions with autoreactive CD4(+) thymocytes. Moreover, a licensing-dependent neo-antigen selectively upregulated in immigrating B cells mediated negative selection through direct presentation. Thus, autoreactivity within the nascent T cell repertoire fuels a feed forward loop that endows thymic B cells with tolerogenic feature

Continuous T cell receptor signals maintain a functional regulatory t cell pool

Regulatory T (Treg) cells maintain immune homeostasis and prevent inflammatory and autoimmune responses. During development, thymocytes bearing a moderately self-reactive T cell receptor (TCR) can be selected to become Treg cells. Several observations suggest that also in the periphery mature Treg cells continuously receive self-reactive TCR signals. However, the importance of this inherent autoreactivity for Treg cell biology remains poorly defined. To address this open question, we genetically ablated the TCR of mature Treg cells in vivo. These experiments revealed that TCR-induced Treg lineage-defining Foxp3 expression and gene hypomethylation were uncoupled from TCR input in mature Treg cells. However, Treg cell homeostasis, cell-type-specific gene expression and suppressive function critically depend on continuous triggering of their TCR

Thymic CD4 T cell selection requires attenuation of March8-mediated MHCII turnover in cortical epithelial cells through CD83

Deficiency of CD83 in thymic epithelial cells (TECs) dramatically impairs thymic CD4 T cell selection. CD83 can exert cell-intrinsic and –extrinsic functions through discrete protein domains, but it remains unclear how CD83’s capacity to operate through these alternative functional modules relates to its crucial role in TECs. In this study, using viral reconstitution of gene function in TECs, we found that CD83’s transmembrane domain is necessary and sufficient for thymic CD4 T cell selection. Moreover, a ubiquitination-resistant MHCII variant restored CD4 T cell selection in Cd83[superscript −/−] mice. Although during dendritic cell maturation CD83 is known to stabilize MHCII through opposing the ubiquitin ligase March1, regulation of March1 did not account for CD83’s TEC-intrinsic role. Instead, we provide evidence that MHCII in cortical TECs (cTECs) is targeted by March8, an E3 ligase of as yet unknown physiological substrate specificity. Ablating March8 in Cd83[superscript −/−] mice restored CD4 T cell development. Our results identify CD83-mediated MHCII stabilization through antagonism of March8 as a novel functional adaptation of cTECs for T cell selection. Furthermore, these findings suggest an intriguing division of labor between March1 and March8 in controlling inducible versus constitutive MHCII expression in hematopoietic antigen-presenting cells versus TECs

Gut-resident CX3CR1hi macrophages induce tertiary lymphoid structures and IgA response in situ

Intestinal mononuclear phagocytes (MPs) are composed of heterogeneous dendritic cell (DC) and macrophage subsets necessary for the initiation of immune response and control of inflammation. Although MPs in the normal intestine have been extensively studied, the heterogeneity and function of inflammatory MPs remain poorly defined. We performed phenotypical, transcriptional, and functional analyses of inflammatory MPs in infectious Salmonella colitis and identified CX3CR1(+) MPs as the most prevalent inflammatory cell type. CX3CR1(+) MPs were further divided into three distinct populations, namely, Nos(2+)CX3CR1(lo), Ccr(7+)CX3CR1(int) (lymph migratory), and Cxcl(13+)CX3CR1(hi) (mucosa resident), all of which were transcriptionally aligned with macrophages and derived from monocytes. In follow-up experiments in vivo, intestinal CX3CR1(+) macrophages were superior to conventional DC1 (cDC1) and cDC2 in inducing Salmonella-specific mucosal IgA. We next examined spatial organization of the immune response induced by CX3CR1(+) macrophage subsets and identified mucosa-resident Cxcl73(+) CX3CR1(hi) macrophages as the antigen-presenting cells responsible for recruitment and activation of CD4(+) T and B cells to the sites of Salmonella invasion, followed by tertiary lymphoid structure formation and the local pathogen-specific IgA response. Using mice we developed with a floxed Ccr7 allele, we showed that this local IgA response developed independently of migration of the Ccr7(+)CX3CR1(int) population to the mesenteric lymph nodes and contributed to the total mucosal IgA response to infection. The differential activity of intestinal macrophage subsets in promoting mucosal IgA responses should be considered in the development of vaccines to prevent Salmonella infection and in the design of anti-inflammatory therapies aimed at modulating macrophage function in inflammatory bowel disease