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)

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

Autonomous role of medullary thymic epithelial cells in central CD4+ T cell tolerance

International audienceMedullary thymic epithelial cells (mTECs) serve an essential function in central tolerance through expressing peripheral tissue-antigens. These antigens may be transferred to and presented by dendritic cells. Therefore, it is unclear whether mTECs, besides being an 'antigen-reservoir', also serve a mandatory function as antigen presenting cells. Here, we reduced MHC class II on mTECs through transgenic expression of a C2TA-specific 'designer miRNA'. This resulted in an enlarged polyclonal CD4 single-positive compartment and, among thymocytes specific for model-antigens expressed in mTECs, enhanced selection of regulatory T cells (Treg) at the expense of deletion. Our data document an autonomous contribution of mTECs to dominant and recessive mechanisms of CD4+ T cell tolerance and support an avidity model of Treg development versus deletion

MHC Class I Internalization via Autophagy Proteins

Macroautophagy is a ubiquitous degradative pathway involved in innate and adaptive immunity. Its molecular machinery has been described to deliver intracellular and extracellular antigens to MHC class II loading compartment by regulating autophagosome and phagosome maturation. We recently found that the respective Atg proteins can contribute to MHC class I-restricted antigen presentation to CD8$^{+}$ T cells by regulating MHC class I surface levels in mouse dendritic cell. Indeed, we determined that MHC class I molecules are stabilized on the cell surface of murine antigen presenting cells deficient for core components of the macroautophagy machinery such as Atg5 and Atg7. This stabilization seems to result from defective internalization of MHC class I molecules dependent on adaptor protein kinase 1 (AAK1), involved in clathrin-mediated endocytosis. Moreover, macroautophagy-dependent stabilization of MHC class I molecules leads to enhanced CD8$^{+}$ T cell priming during influenza A virus infection in vivo, resulting in decreased pathology. In this chapter, we describe four experiments to monitor, characterize, and quantify the effect of macroautophagy deficiency on MHC class I molecule trafficking and the subsequent CD8$^{+}$ T cell priming. First, we will show how to monitor MHC class I internalization in lung CD11c$^{+}$ cells from mice lacking key components of the macroautophagy machinery. Then, we will propose a method to characterize the interaction between either MHC class I or Atg8/LC3 with AAK1. Finally, we will describe how to evaluate the influenza A-specific CD8$^{+}$ T cell response in mice conditionally depleted for Atg5 in their DC compartment. This set of experiments allows to characterize MHC class I internalization with the help of the molecular machinery of macroautophagy

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.Panagiotis Ntziachristos, Aristotelis Tsirigos, G. Grant Welstead, Thomas Trimarchi, Sofia Bakogianni, Luyao Xu, Evangelia Loizou, Linda Holmfeldt, Alexandros Strikoudis, Bryan King, Jasper Mullenders, Jared Becksfort, Jelena Nedjic, Elisabeth Paietta, Martin S. Tallman, Jacob M. Rowe, Giovanni Tonon, Takashi Satoh, Laurens Kruidenier, Rab Prinjha, Shizuo Akira, Pieter Van Vlierberghe, Adolfo A. Ferrando, Rudolf Jaenisch, Charles G. Mullighan, Iannis Aifanti

Detection of an autoreactive T-cell population within the polyclonal repertoire that undergoes distinct autoimmune regulator (Aire)-mediated selection

The autoimmune regulator (Aire) plays a critical role in central tolerance by promoting the display of tissue-specific antigens in the thymus. To study the influence of Aire on thymic selection in a physiological setting, we used tetramer reagents to detect autoreactive T cells specific for the Aire-dependent tissue-specific antigen interphotoreceptor retinoid-binding protein (IRBP), in the polyclonal repertoire. Two class II tetramer reagents were designed to identify T cells specific for two different peptide epitopes of IRBP. Analyses of the polyclonal T-cell repertoire showed a high frequency of activated T cells specific for both IRBP tetramers in Aire−/− mice, but not in Aire+/+ mice. Surprisingly, although one tetramer-binding T-cell population was efficiently deleted in the thymus in an Aire-dependent manner, the second tetramer-binding population was not deleted and could be detected in both the Aire−/− and Aire+/+ T-cell repertoires. We found that Aire-dependent thymic deletion of IRBP-specific T cells relies on intercellular transfer of IRBP between thymic stroma and bone marrow-derived antigen-presenting cells. Furthermore, our data suggest that Aire-mediated deletion relies not only on thymic expression of IRBP, but also on proper antigen processing and presentation of IRBP by thymic antigen-presenting cells