Biochemistry and Pharmacology - Research Publications
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ItemAutophagy and mechanisms of effective immunity(FRONTIERS MEDIA SA, 2012)Macroautophagy (autophagy) is a cellular pathway facilitating several critical functions. First, autophagy is a major pathway of degradation. It enables elimination of microbes that have invaded intracellular compartments. In addition, it promotes degradation of damaged cellular content, thereby acting to limit inflammatory signals. Second, autophagy is a major trafficking pathway, shuttling content between the cytosol and the lysosomal compartment. Given these two key roles, autophagy can have significant and sometimes unexpected consequences on mechanisms that initiate robust immunity. Here, we will discuss the impact of autophagy on pathways of innate and adaptive immune responses including microbe elimination, inflammatory cytokine production, antigen processing and T and B lymphocyte immunity.
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ItemFailure of CD4 T-Cells to Respond to Liver-Derived Antigen and to Provide Help to CD8 T-Cells(PUBLIC LIBRARY SCIENCE, 2011-07-14)CD4 T-cell help is required for the induction of efficient CD8 T-cells responses and the generation of memory cells. Lack of CD4 T-cell help may contribute to an exhausted CD8 phenotype and viral persistence. Little is known about priming of CD4 T-cells by liver-derived antigen. We used TF-OVA mice expressing ovalbumin in hepatocytes to investigate CD4 T-cell priming by liver-derived antigen and the impact of CD4 T-cell help on CD8 T-cell function. Naïve and effector CD4 T-cells specific for ovalbumin were transferred into TF-OVA mice alone or together with naïve ovalbumin-specific CD8 T-cells. T-cell activation and function were analyzed. CD4 T-cells ignored antigen presented by liver antigen-presenting cells (APCs) in vitro and in vivo but were primed in the liver-draining lymph node and the spleen. No priming occurred in the absence of bone-marrow derived APCs capable of presenting ovalbumin in vivo. CD4 T-cells primed in TF-OVA mice displayed defective Th1-effector function and caused no liver damage. CD4 T-cells were not required for the induction of hepatitis by CD8 T-cells. Th1-effector but not naïve CD4 T-cells augmented the severity of liver injury caused by CD8 T-cells. Our data demonstrate that CD4 T-cells fail to respond to liver-derived antigen presented by liver APCs and develop defective effector function after priming in lymph nodes and spleen. The lack of CD4 T-cell help may be responsible for insufficient CD8 T-cell function against hepatic antigens.
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ItemInitiation of autoimmune diabetes by developmentally regulated presentation of islet cell antigens in the pancreatic lymph nodes(ROCKEFELLER UNIV PRESS, 1999-01-18)Little is known about the events triggering lymphocyte invasion of the pancreatic islets in prelude to autoimmune diabetes. For example, where islet-reactive T cells first encounter antigen has not been identified. We addressed this issue using BDC2.5 T cell receptor transgenic mice, which express a receptor recognizing a natural islet beta cell antigen. In BDC2.5 animals, activated T cells were found only in the islets and the lymph nodes draining them, and there was a close temporal correlation between lymph node T cell activation and islet infiltration. When naive BDC2.5 T cells were transferred into nontransgenic recipients, proliferating cells were observed only in pancreatic lymph nodes, and this occurred significantly before insulitis was detectable. Surprisingly, proliferation was not seen in 10-day-old recipients. This age-dependent dichotomy was reproduced in a second transfer system based on an unrelated antigen artificially expressed on beta cells. We conclude that beta cell antigens are transported specifically to pancreatic lymph nodes, where they trigger reactive T cells to invade the islets. Systemic or extrapancreatic T cell priming, indicative of activation via molecular mimicry or superantigens, was not seen. Compromised presentation of beta cell antigens in the pancreatic lymph nodes of juvenile animals may be the root of a first "checkpoint" in diabetes progression.
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ItemDetection of Bcl-2 family member Bcl-G in mouse tissues using new monoclonal antibodies(NATURE PUBLISHING GROUP, 2012-08)Bcl-G is an evolutionarily conserved member of the Bcl-2 family of proteins that has been implicated in regulating apoptosis and cancer. We have generated monoclonal antibodies that specifically recognise mouse Bcl-G and have used these reagents to analyse its tissue distribution and subcellular localisation using western blotting, immunohistochemistry and immunofluorescence. We found that Bcl-G predominantly resides in the cytoplasm and is present in a wide range of mouse tissues, including the spleen, thymus, lung, intestine and testis. Immunohistochemical analyses revealed that Bcl-G is expressed highly in mature spermatids in the testis, CD8(+) conventional dendritic cells (DCs) in hematopoietic tissues and diverse epithelial cell types, including those lining the gastrointestinal and respiratory tracts. The Bcl-G monoclonal antibodies represent new tools for studying this protein, using a variety of techniques, including immunoprecipitation and flow cytometry.
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ItemBcl-2 family member Bcl-G is not a proapoptotic protein(NATURE PUBLISHING GROUP, 2012-10)The three major subgroups of the Bcl-2 family, including the prosurvival Bcl-2-like proteins, the proapoptotic Bcl-2 homology (BH)3-only proteins and Bax/Bak proteins, regulate the mitochondrial apoptotic pathway. In addition, some outliers within the Bcl-2 family do not fit into these subgroups. One of them, Bcl-G, has a BH2 and a BH3 region, and was proposed to trigger apoptosis. To investigate the physiological role of Bcl-G, we have inactivated the gene in the mouse and generated monoclonal antibodies to determine its expression. Although two isoforms of Bcl-G exist in human, only one is found in mice. mBcl-G is expressed in a range of epithelial as well as in dendritic cells. Loss of Bcl-G did not appear to affect any of these cell types. mBcl-G only binds weakly to prosurvival members of the Bcl-2 family, and in a manner that is independent of its BH3 domain. To understand what the physiological role of Bcl-G might be, we searched for Bcl-G-binding partners through immunoprecipitation/mass spectroscopy and yeast-two-hybrid screening. Although we did not uncover any Bcl-2 family member in these screens, we found that Bcl-G interacts specifically with proteins of the transport particle protein complex. We conclude that Bcl-G most probably does not function in the classical stress-induced apoptosis pathway, but rather has a role in protein trafficking inside the cell.
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ItemDendritic cells and influenza A virus infection(LANDES BIOSCIENCE, 2012-11)Influenza A virus (IAV) is a dangerous virus equipped with the potential to evoke widespread pandemic disease. The 2009 H1N1 pandemic highlights the urgency for developing effective therapeutics against IAV infection. Vaccination is a major weapon to combat IAV and efforts to improve current regimes are critically important. Here, we will review the role of dendritic cells (DCs), a pivotal cell type in the initiation of robust IAV immunity. The complexity of DC subset heterogeneity in the respiratory tract and lymph node that drains the IAV infected lung will be discussed, together with the varied and in some cases, conflicting contributions of individual DC populations to presenting IAV associated antigen to T cells.
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ItemNeutrophils Require SHP1 To Regulate IL-1β Production and Prevent Inflammatory Skin Disease(AMER ASSOC IMMUNOLOGISTS, 2011-01-15)The regulation of neutrophil recruitment, activation, and disposal is pivotal for circumscribed inflammation. SHP1(Y208N/Y208N) mutant mice develop severe cutaneous inflammatory disease that is IL-1R dependent. Genetic reduction in neutrophil numbers and neutrophilic responses to infection is sufficient to prevent the spontaneous initiation of this disease. Neutrophils from SHP1(Y208N/Y208N) mice display increased pro-IL-1β production due to altered responses to MyD88-dependent and MyD88-independent signals. The IL-1R-dependent inflammatory disease in SHP1(Y208N/Y208N) mice develops independently of caspase 1 and proteinase 3 and neutrophil elastase. In response to Fas ligand, a caspase 1-independent inducer of IL-1β production, neutrophils from SHP1(Y208N/Y208N) mice produce elevated levels of IL-1β but display reduced caspase 3 and caspase 7 activation. In neutrophils deficient in SHP1, IL-1β induces high levels of pro-IL-1β suggesting the presence of a paracrine IL-1β loop. These data indicate that the neutrophil- and IL-1-dependent disease in SHP1(Y208N/Y208N) mice is a consequence of loss of negative regulation of TLR and IL-1R signaling.
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ItemInduction of antigen-specific effector-phase tolerance following vaccination against a previously ignored B-cell lymphoma(WILEY, 2011-07)The mechanisms of immune evasion during haematological malignancies are poorly understood. As lymphomas grow in lymphoid organs, it would be expected that if these lymphomas express neo-antigens they should be readily detected by the immune system. To test this assumption, we generated a new non-Hodgkin B-cell lymphoma model expressing the model tumour neo-antigen Ovalbumin (OVA), and analysed the endogenous antigen-specific CD8(+) T-cell response that it elicited in recipient mice. The OVA+ lymphoma cells were eliminated by cytotoxic T lymphocytes (CTL) in mice that had been previously vaccinated against OVA. In contrast, the immune system of naïve mice ignored the malignant cells even though these continuously expressed and presented OVA on their MHC class I molecules. This state of ignorance could be overcome by therapeutic vaccination, which led to the expansion of endogenous anti-OVA-specific CD8(+) T cells. However, the cytotoxic and interferon-γ secretion capacity of these T cells were impaired. The tumour model that we describe thus reproduces several key aspects of human lymphoma; tumor ignorance can be broken by vaccination but the ensuing immune response remains ineffective. This model can be exploited to further understand the mechanisms of lymphoma immunoevasion and devise effective immunotherapy.
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ItemThe Dendritic Cell Receptor Clec9A Binds Damaged Cells via Exposed Actin Filaments(CELL PRESS, 2012-04-20)The immune system must distinguish viable cells from cells damaged by physical and infective processes. The damaged cell-recognition molecule Clec9A is expressed on the surface of the mouse and human dendritic cell subsets specialized for the uptake and processing of material from dead cells. Clec9A recognizes a conserved component within nucleated and nonnucleated cells, exposed when cell membranes are damaged. We have identified this Clec9A ligand as a filamentous form of actin in association with particular actin-binding domains of cytoskeletal proteins. We have determined the crystal structure of the human CLEC9A C-type lectin domain and propose a functional dimeric structure with conserved tryptophans in the ligand recognition site. Mutation of these residues ablated CLEC9A binding to damaged cells and to the isolated ligand complexes. We propose that Clec9A provides targeted recruitment of the adaptive immune system during infection and can also be utilized to enhance immune responses generated by vaccines.
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ItemIntersection of autophagy with pathways of antigen presentation(SPRINGEROPEN, 2012-12)Traditionally, macroautophagy (autophagy) is viewed as a pathway of cell survival. Autophagy ensures the elimination of damaged or unwanted cytosolic components and provides a source of cellular nutrients during periods of stress. Interestingly, autophagy can also directly intersect with, and impact, other major pathways of cellular function. Here, we will review the contribution of autophagy to pathways of antigen presentation. The autophagy machinery acts to modulate both MHCI and MHCII antigen presentation. As such autophagy is an important participant in pathways that elicit host cell immunity and the elimination of infectious pathogens.