Search

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, Nos2+CX3CR1lo, Ccr7+CX3CR1int (lymph migratory), and Cxcl13+CX3CR1hi (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 Cxcl13+CX3CR1hi 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+CX3CR1int 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.

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, Nos2(+)CX3CR1(lo), Ccr7(+)CX3CR1(int) (lymph migratory), and Cxcl13(+)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 Cxcl13(+)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.

Summary 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 features.

Work from the Klein lab shows that CD83 supports T cell selection in the thymus by restraining March8-mediated MHCII ubiquitination and internalization., 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−/− 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−/− 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.

Functional and biochemical assays indicate a substantial contribution of intracellularly derived peptides to the MHC class II 'ligandome'. Macroautophagy, a process traditionally known for its role in cellular housekeeping and adaptation to nutrient withdrawal, is an attractive candidate pathway for endogenous MHC class II loading. Work in cell culture systems, including antigen presentation assays, co-localization studies and sequencing of MHC class II bound peptides, demonstrates that substrates of autophagy can be loaded onto MHC class II. Advances in the development of mouse models to monitor or genetically disrupt macroautophagy now provide the basis for elucidating the immunological relevance of autophagy in vivo. Here, we will discuss recent findings suggesting a crucial role of macroautophagy in thymic epithelial cells for the generation of peptide/MHC class II ligands for positive selection and induction of T cell tolerance.

During intrathymic generation of the T cell repertoire, a series of selection processes ensures that only self-MHC (Major Histocompatibility Complex) restricted and self-tolerant T cells are allowed to survive. Interactions with MHC ligands on the surface of thymic epithelial cells (TECs) play a pivotal role in the decision-making of developing thymocytes. A number of distinct cell-biological features of TECs have emerged that may predispose them to serve non-redundant functions in thymocyte "education". Thus, cortical TECs express a rather unique set of proteolytic enzymes for antigen processing in the context of positive selection, whereas medullary TECs "ectopically" express a plethora of otherwise strictly tissue-restricted antigens (TRAs), a property that obviously has evolved to make these self-antigens "visible" to developing thymocytes for negative selection. One of the latest additions to this growing list of functional adaptations of TECs is their constitutively high rate of autophagy. Recently, we have provided evidence that autophagy in TECs shuttles cytoplasmic self-antigens into the MHC class II loading pathway for positive selection of T cells and tolerance induction.

T cell acute lymphoblastic leukemia (T-ALL) is an immature hematopoietic malignancy driven mainly by oncogenic activation of NOTCH1 signaling1. In this study we report the presence of loss-of-function mutations and deletions of the EZH2 and SUZ12 genes, which encode crucial components of the Polycomb repressive complex 2 (PRC2) 2,3, in 25% of T-ALLs. To further study the role of PRC2 in T-ALL, we used NOTCH1-dependent mouse models of the disease, as well as human T-ALL samples, and combined locus-specific and global analysis of NOTCH1-driven epigenetic changes. These studies demonstrated that activation of NOTCH1 specifically induces loss of the repressive mark Lys27 trimethylation of histone 3 (H3K27me3) 4 by antagonizing the activity of PRC2. These studies suggest a tumor suppressor role for PRC2 in human leukemia and suggest a hitherto unrecognized dynamic interplay between oncogenic NOTCH1 and PRC2 function for the regulation of gene expression and cell transformation. © 2012 Nature America, Inc. All rights reserved., J.N. was supported by the Damon Runyon Cancer Research Foundation. I.A. was supported by the US National Institutes of Health (RO1CA133379, RO1CA105129, R21CA141399, RO1CA149655 and RO1GM088847), The Leukemia & Lymphoma Society, The V Foundation, the American Cancer Society (RSG0806801) and the Dana Foundation. The Aifantis laboratory is also supported by a Feinberg Lymphoma Pilot grant. This study was also supported by the Fund for Scientific Research of Flanders (P.V.V. and K.D.K.); the US National Library of Medicine (1R01LM010140-01 to R.R.); the Eastern Cooperative Oncology Group tumor bank; a Northeast Biodefense Center American Recovery and Reinvestment Act award (U54-AI057158 to R.R.); the US National Institutes of Health (R01CA120196 and R01CA155743 to A.F.); the Stand Up To Cancer Innovative Research Award (A.F.), the Chemotherapy Foundation (I.A. and A.F.); the Rally Across America Foundation (A.F.) and the Swim Across America Foundation (A.F.). P.V.V. is an American Society of Hematology Scholar, and I.A. and A.F. are Leukemia & Lymphoma Society Scholars. M.D. is supported by grants from Spanish Ministerio de Ciencia e Innovación (BFU2009-09074 and MECCONSOLIDER CSD2007-00023), Generalitat Valenciana (PROMETEO2006/134) and an EU Research Grant (UE-HEALH-F2-2008-201666). F.P. is supported by the Institut du Cancer, the Association Laurette Fugain, the Ligue National Contre le Cancer and also by INSERM, CEA and StemPole. S.P. is supported by a fellowship by the Institut du Cancer. I.A. is a Howard Hughes Medical Institute Early Career Scientist.

During T cell development in the thymus, scanning of peptide/major histocompatibility (MHC) molecule complexes on the surface of thymic epithelial cells ensures that only useful (self-MHC restricted) and harmless (self-tolerant) thymocytes survive. In recent years, a number of distinct cell-biological features of thymic epithelial cells have been unraveled that may have evolved to render these cells particularly suited for T cell selection, e.g., cortical epithelial cells use unique proteolytic enzymes for the generation of MHC/peptide complexes, whereas medullary epithelial cells "promiscuously" express otherwise tissue-restricted self-antigens. We recently showed that macroautophagy in thymic epithelial cells contributes to CD4 T cell selection and is essential for the generation of a self-tolerant T cell repertoire. We propose that the unusually high constitutive levels of autophagy in thymic epithelial cells deliver endogenous proteins to MHC class II molecules for both positive and negative selection of developing thymocytes.

Macro-autophagy in thymic epithelial cells is shown to contribute to endogenous antigen processing and selection of major histocompatibility complex class II (MHC II)-restricted CD4 T cells. This suggests that autophagy focuses the MHC class II/peptide repertoire of these T cells on their intracellular milieu, which is made up of a wide array of otherwise strictly 'tissue-specific' self-antigens. In doing so, it contributes to efficient positive selection and is essential for the generation of a self-tolerant T-cell repertoire. Recognition of self-antigen-derived epitopes presented by major histocompatibility complex class II (MHC II) molecules on thymic epithelial cells (TECs) is critical for the generation of a functional and self-tolerant CD4 T-cell repertoire. Whereas haematopoietic antigen-presenting cells generate MHC-II–peptide complexes predominantly through the processing of endocytosed polypeptides1, it remains unknown if and how TECs use unconventional pathways of antigen presentation. Here we address the role of macroautophagy, a process that has recently been shown to allow for endogenous MHC II loading2,3,4,5,6, in T-cell repertoire selection in the mouse thymus. In contrast to most other tissues, TECs had a high constitutive level of autophagy. Genetic interference with autophagy specifically in TECs led to altered selection of certain MHC-II-restricted T-cell specificities and resulted in severe colitis and multi-organ inflammation. Our findings indicate that autophagy focuses the MHC-II–peptide repertoire of TECs on their intracellular milieu, which notably comprises a wide array of otherwise strictly ‘tissue-specific’ self antigens7,8. In doing so, it contributes to T-cell selection and is essential for the generation of a self-tolerant T-cell repertoire.

During development in the thymus, each T lymphocyte is equipped with one, essentially unique, T cell receptor (TCR)-specificity. Due to its random nature, this process inevitably also leads to the emergence of potentially dangerous T lymphocytes that may recognize 'self.' Nevertheless, autoimmune tissue destruction, the cause of diseases such as multiple sclerosis and diabetes, is the exception rather than the rule. This state of immunological self-tolerance is to a large degree based upon a process called 'negative selection': prior to joining the circulating lymphocyte pool, immature T cells test their receptor on self-antigens within the thymic microenvironment, and TCR engagement at this immature stage elicits an apoptotic suicide program. We now find evidence that macroautophagy supports the tolerogenic presentation of self-antigens in the thymus.

Notch1 has an indispensable role in T cell development and NOTCH1 acts as a potent oncogene in T cell leukemia. New data now reveal the role of RNA-binding proteins in regulating the stability of Notch1 mRNA and the induction of T cell leukemia.

Macroautophagy supports central CD4+ T cell tolerance by facilitating the direct presentation of endogenous self-antigens by medullary thymic epithelial cells., Macroautophagy serves cellular housekeeping and metabolic functions through delivery of cytoplasmic constituents for lysosomal degradation. In addition, it may mediate the unconventional presentation of intracellular antigens to CD4+ T cells; however, the physiological relevance of this endogenous MHC class II loading pathway remains poorly defined. Here, we characterize the role of macroautophagy in thymic epithelial cells (TECs) for negative selection. Direct presentation for clonal deletion of MHC class II–restricted thymocytes required macroautophagy for a mitochondrial version of a neo-antigen, but was autophagy-independent for a membrane-bound form. A model antigen specifically expressed in Aire+ medullary TECs (mTECs) induced efficient deletion via direct presentation when targeted to autophagosomes, whereas interference with autophagosomal routing of this antigen through exchange of a single amino acid or ablation of an essential autophagy gene abolished direct presentation for negative selection. Furthermore, when this autophagy substrate was expressed by mTECs in high amounts, endogenous presentation and indirect presentation by DCs operated in a redundant manner, whereas macroautophagy-dependent endogenous loading was essential for clonal deletion at limiting antigen doses. Our findings suggest that macroautophagy supports central CD4+ T cell tolerance through facilitating the direct presentation of endogenous self-antigens by mTECs.