Publications by authors named "Hans Jörg Fehling"

33 Publications

The transcription factor Duxbl mediates elimination of pre-T cells that fail β-selection.

J Exp Med 2019 03 14;216(3):638-655. Epub 2019 Feb 14.

Developmental and Molecular Immunology, Department of Biomedicine, University of Basel, Basel, Switzerland

T cell development is critically dependent on successful rearrangement of antigen-receptor chains. At the β-selection checkpoint, only cells with a functional rearrangement continue in development. However, how nonselected T cells proceed in their dead-end fate is not clear. We identified low CD27 expression to mark pre-T cells that have failed to rearrange their β-chain. Expression profiling and single-cell transcriptome clustering identified a developmental trajectory through β-selection and revealed specific expression of the transcription factor Duxbl at a stage of high recombination activity before β-selection. Conditional transgenic expression of Duxbl resulted in a developmental block at the DN3-to-DN4 transition due to reduced proliferation and enhanced apoptosis, whereas RNA silencing of Duxbl led to a decrease in apoptosis. Transcriptome analysis linked Duxbl to elevated expression of the apoptosis-inducing Oas/RNaseL pathway. RNaseL deficiency or sustained Bcl2 expression led to a partial rescue of cells in Duxbl transgenic mice. These findings identify Duxbl as a regulator of β-selection by inducing apoptosis in cells with a nonfunctional rearrangement.
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http://dx.doi.org/10.1084/jem.20181444DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6400535PMC
March 2019

The H3K4 methyltransferase Setd1b is essential for hematopoietic stem and progenitor cell homeostasis in mice.

Elife 2018 06 19;7. Epub 2018 Jun 19.

Stem Cell Engineering, Biotechnology Center, Technische Universität Dresden, Dresden, Germany.

Hematopoietic stem cells require MLL1, which is one of six Set1/Trithorax-type histone 3 lysine 4 (H3K4) methyltransferases in mammals and clinically the most important leukemia gene. Here, we add to emerging evidence that all six H3K4 methyltransferases play essential roles in the hematopoietic system by showing that conditional mutagenesis of Setd1b in adult mice provoked aberrant homeostasis of hematopoietic stem and progenitor cells (HSPCs). Using both ubiquitous and hematopoietic-specific deletion strategies, the loss of Setd1b resulted in peripheral thrombo- and lymphocytopenia, multilineage dysplasia, myeloid-biased extramedullary hematopoiesis in the spleen, and lethality. By transplantation experiments and expression profiling, we determined that Setd1b is autonomously required in the hematopoietic lineages where it regulates key lineage specification components, including , and . Altogether, these data imply that the Set1/Trithorax-type epigenetic machinery sustains different aspects of hematopoiesis and constitutes a second framework additional to the transcription factor hierarchy of hematopoietic homeostasis.
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http://dx.doi.org/10.7554/eLife.27157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6025962PMC
June 2018

Epigenome analysis links gene regulatory elements in group 2 innate lymphocytes to asthma susceptibility.

J Allergy Clin Immunol 2018 12 2;142(6):1793-1807. Epub 2018 Mar 2.

Department of Pulmonary Medicine, Erasmus MC Rotterdam, The Netherlands. Electronic address:

Background: Group 2 innate lymphoid cells (ILC2s) are major producers of the cytokines driving allergic asthma, and increased ILC2 numbers have been detected in blood and sputum of asthmatic patients. Asthma susceptibility has a strong genetic component, but the underlying mechanisms and whether asthma genetics affect ILC2 biology remain unclear.

Objective: We sought to study the ILC2 transcriptome and epigenome during airway inflammation (AI) to couple these to genes and genetic variants associated with asthma pathogenesis.

Methods: Mice harboring a reporter for the key ILC2 transcription factor GATA-3 were subjected to IL-33-driven AI, and ILC2s were isolated from bronchoalveolar lavage fluid and mediastinal lymph nodes. Human ILC2s were purified from peripheral blood and activated in vitro. We used RNA sequencing, genome-wide identification of histone-3 lysine-4 dimethylation-marked chromatin, and computational approaches to study the ILC2 transcriptome and epigenome.

Results: Activated ILC2s in mice displayed a tissue-specific gene expression signature that emerged from remarkably similar epigenomes. We identified superenhancers implicated in controlling ILC2 identity and asthma-associated genes. More than 300 asthma-associated genetic polymorphisms identified in genome-wide association studies localized to H3K4Me2 gene regulatory elements in ILC2s. A refined set of candidate causal asthma-associated variants was uniquely enriched in ILC2, but not T2 cell, regulatory regions.

Conclusions: ILC2s in AI use a flexible epigenome that couples adaptation to new microenvironments with functional plasticity. Importantly, we reveal strong correlations between gene regulatory mechanisms in ILC2s and the genetic basis of asthma, supporting a pathogenic role for ILC2s in patients with allergic asthma.
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http://dx.doi.org/10.1016/j.jaci.2017.12.1006DOI Listing
December 2018

Group 2 Innate Lymphoid Cells Exhibit a Dynamic Phenotype in Allergic Airway Inflammation.

Front Immunol 2017 1;8:1684. Epub 2017 Dec 1.

Department of Pulmonary Medicine, Rotterdam, Netherlands.

Group 2 innate lymphoid cells (ILC2) are implicated in allergic asthma as an early innate source of the type 2 cytokines IL-5 and IL-13. However, their induction in house dust mite (HDM)-mediated airway inflammation additionally requires T cell activation. It is currently unknown whether phenotypic differences exist between ILC2s that are activated in a T cell-dependent or T cell-independent fashion. Here, we compared ILC2s in IL-33- and HDM-driven airway inflammation. Using flow cytometry, we found that surface expression levels of various markers frequently used to identify ILC2s were dependent on their mode of activation, highly variable over time, and differed between tissue compartments, including bronchoalveolar lavage (BAL) fluid, lung, draining lymph nodes, and spleen. Whereas IL-33-activated BAL fluid ILC2s exhibited an almost uniform CD25CD127T1/ST2ICOSKLRG1 phenotype, at a comparable time point after HDM exposure BAL fluid ILC2s had a very heterogeneous surface marker phenotype. A major fraction of HDM-activated ILC2s were CD25CD127T1/ST2 ICOSKLRG1, but nevertheless had the capacity to produce large amounts of type 2 cytokines. HDM-activated CD25 ILC2s in BAL fluid and lung rapidly reverted to CD25 ILC2s upon stimulation with IL-33. Genome-wide transcriptional profiling of BAL ILC2s revealed ~1,600 differentially expressed genes: HDM-stimulated ILC2s specifically expressed genes involved in the regulation of adaptive immunity through B and T cell interactions, whereas IL-33-stimulated ILC2s expressed high levels of proliferation-related and cytokine genes. In both airway inflammation models ILC2s were present in the lung submucosa close to epithelial cells, as identified by confocal microscopy. In chronic HDM-driven airway inflammation ILC2s were also found inside organized cellular infiltrates near T cells. Collectively, our findings show that ILC2s are phenotypically more heterogeneous than previously thought, whereby their surface marker and gene expression profile are highly dynamic.
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http://dx.doi.org/10.3389/fimmu.2017.01684DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5716969PMC
December 2017

RORγt+ cells selectively express redundant cation channels linked to the Golgi apparatus.

Sci Rep 2016 Mar 24;6:23682. Epub 2016 Mar 24.

INSERM UMR 1064, Center for Research in Transplantation and Immunology; Université de Nantes; CHU Nantes, Institut de Transplantation Urologie Néphrologie (ITUN); 44093 Nantes, France.

Retinoid-related orphan receptor gamma t (RORγt) is a master transcription factor central to type 17 immunity involving cells such as T helper 17, group 3 innate lymphoid cells or IL-17-producing γδ T cells. Here we show that the intracellular ion channel TMEM176B and its homologue TMEM176A are strongly expressed in these RORγt(+) cells. We demonstrate that TMEM176A and B exhibit a similar cation channel activity and mainly colocalise in close proximity to the trans-Golgi network. Strikingly, in the mouse, the loss of Tmem176b is systematically associated with a strong upregulation of Tmem176a. While Tmem176b single-deficiency has no effect on the course of experimental autoimmune encephalomyelitis, T cell or DSS-induced colitis, it significantly reduces imiquimod-induced psoriasis-like skin inflammation. These findings shed light on a potentially novel specific process linked to post-Golgi trafficking for modulating the function of RORγt(+) cells and indicate that both homologues should be simultaneously targeted to clearly elucidate the role of this intracellular ion flow.
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http://dx.doi.org/10.1038/srep23682DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4806298PMC
March 2016

Autoantibody-boosted T-cell reactivation in the target organ triggers manifestation of autoimmune CNS disease.

Proc Natl Acad Sci U S A 2016 Mar 8;113(12):3323-8. Epub 2016 Mar 8.

Institute of Neuroimmunology and Institute for Multiple Sclerosis Research, University Medical Centre Göttingen, D-37073 Göttingen, Germany; Max-Planck-Institute for Experimental Medicine Göttingen, D-37075 Göttingen, Germany

Multiple sclerosis (MS) is caused by T cells that are reactive for brain antigens. In experimental autoimmune encephalomyelitis, the animal model for MS, myelin-reactive T cells initiate the autoimmune process when entering the nervous tissue and become reactivated upon local encounter of their cognate CNS antigen. Thereby, the strength of the T-cellular reactivation process within the CNS tissue is crucial for the manifestation and the severity of the clinical disease. Recently, B cells were found to participate in the pathogenesis of CNS autoimmunity, with several diverse underlying mechanisms being under discussion. We here report that B cells play an important role in promoting the initiation process of CNS autoimmunity. Myelin-specific antibodies produced by autoreactive B cells after activation in the periphery diffused into the CNS together with the first invading pathogenic T cells. The antibodies accumulated in resident antigen-presenting phagocytes and significantly enhanced the activation of the incoming effector T cells. The ensuing strong blood-brain barrier disruption and immune cell recruitment resulted in rapid manifestation of clinical disease. Therefore, myelin oligodendrocyte glycoprotein (MOG)-specific autoantibodies can initiate disease bouts by cooperating with the autoreactive T cells in helping them to recognize their autoantigen and become efficiently reactivated within the immune-deprived nervous tissue.
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http://dx.doi.org/10.1073/pnas.1519608113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4812745PMC
March 2016

Identification of a novel lymphoid population in the murine epidermis.

Sci Rep 2015 Jul 30;5:12554. Epub 2015 Jul 30.

Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A*STAR), 138648, Singapore.

T cell progenitors are known to arise from the foetal liver in embryos and the bone marrow in adults; however different studies have shown that a pool of T cell progenitors may also exist in the periphery. Here, we identified a lymphoid population resembling peripheral T cell progenitors which transiently seed the epidermis during late embryogenesis in both wild-type and T cell-deficient mice. We named these cells ELCs (Epidermal Lymphoid Cells). ELCs expressed Thy1 and CD2, but lacked CD3 and TCRαβ/γδ at their surface, reminiscent of the phenotype of extra- or intra- thymic T cell progenitors. Similarly to Dendritic Epidermal T Cells (DETCs), ELCs were radioresistant and capable of self-renewal. However, despite their progenitor-like phenotype and expression of T cell lineage markers within the population, ELCs did not differentiate into conventional T cells or DETCs in in vitro, ex vivo or in vivo differentiation assays. Finally, we show that ELC expressed NK markers and secreted IFN-γ upon stimulation. Therefore we report the discovery of a unique population of lymphoid cells within the murine epidermis that appears related to NK cells with as-yet-unidentified functions.
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http://dx.doi.org/10.1038/srep12554DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4519784PMC
July 2015

Conditional ablation of NKp46+ cells using a novel Ncr1(greenCre) mouse strain: NK cells are essential for protection against pulmonary B16 metastases.

Eur J Immunol 2014 Nov 19;44(11):3380-91. Epub 2014 Sep 19.

Département d'Immunologie, Unité d'Immunité Innée, Institut Pasteur, Paris, France; Institut Pasteur, INSERM U668, Paris, France; Cellule Pasteur, Sorbonne Paris Cité, Univ. Paris Diderot, Paris, France.

To study gene functions specifically in NKp46+ cells we developed novel Cre mice allowing for conditional gene targeting in cells expressing Ncr1 (encoding NKp46). We generated transgenic Ncr1(greenCre) mice carrying an EGFPcre fusion under the control of a proximal Ncr1 promoter that faithfully directed EGFPcre expression to NKp46+ cells from lymphoid and nonlymphoid tissues. This approach allowed for direct detection of Cre-expressing NKp46+ cells via their GFP signature by flow cytometry and histology. Cre was functional as evidenced by the NKp46+ cell-specific expression of RFP in Ncr1(greenCre) Rosa-dtRFP reporter mice. We generated Ncr1(greenCre) Il2rg(fl/fl) mice that lack NKp46+ cells in an otherwise intact hematopoietic environment. Il2rg encodes the common gamma chain (γc ), which is an essential receptor subunit for cytokines (IL-2, -4, -7, -9, -15, and -21) that stimulate lymphocyte development and function. In Ncr1(greenCre) Il2rg(fl/fl) mice, NK cells are severely reduced and the few remaining NKp46+ cells escaping γc deletion failed to express GFP. Using this new NK-cell-deficient model, we demonstrate that the homeostasis of NKp46+ cells from all tissues (including the recently described intraepithelial ILC1 subset) requires Il2rg. Finally, Ncr1(greenCre) Il2rg(fl/fl) mice are unable to reject B16 lung metastases demonstrating the essential role of NKp46+ cells in antimelanoma immune responses.
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http://dx.doi.org/10.1002/eji.201444643DOI Listing
November 2014

Improved method to retain cytosolic reporter protein fluorescence while staining for nuclear proteins.

Cytometry A 2014 Jul 19;85(7):621-7. Epub 2014 Feb 19.

Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg, University of Mainz, 55131, Mainz, Germany.

Staining of transcription factors (TFs) together with retention of fluorescent reporter proteins is hindered by loss of fluorescence using current available methods. In this study, it is shown that current TF staining protocols do not destroy fluorescent proteins (FPs) but rather that fixation is not sufficient to retain FPs in the cytosol of the permeabilized cells. In this article, a simple and reliable protocol is elaborated, which allows efficient TF and cytokine staining while retaining FPs inside fixed cells.
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http://dx.doi.org/10.1002/cyto.a.22451DOI Listing
July 2014

C-reactive protein (CRP) is essential for efficient systemic transduction of recombinant adeno-associated virus vector 1 (rAAV-1) and rAAV-6 in mice.

J Virol 2013 Oct 31;87(19):10784-91. Epub 2013 Jul 31.

Genethon, Evry, France.

The clinical relevance of gene therapy using the recombinant adeno-associated virus (rAAV) vectors often requires widespread distribution of the vector, and in this case, systemic delivery is the optimal route of administration. Humoral blood factors, such as antibodies or complement, are the first barriers met by the vectors administered systemically. We have found that other blood proteins, galectin 3 binding protein (G3BP) and C-reactive protein (CRP), can interact with different AAV serotypes in a species-specific manner. While interactions of rAAV vectors with G3BP, antibodies, or complement lead to a decrease in vector efficacy, systemic transduction of the CRP-deficient mouse and its respective control clearly established that binding to mouse CRP (mCRP) boosts rAAV vector 1 (rAAV-1) and rAAV-6 transduction efficiency in skeletal muscles over 10 times. Notably, the high efficacy of rAAV-6 in CRP-deficient mice can be restored by reconstitution of the CRP-deficient mouse with mCRP. Human CRP (hCRP) does not interact with either rAAV-1 or rAAV-6, and, consequently, the high efficiency of mCRP-mediated muscle transduction by these serotypes in mice cannot be translated to humans. No interaction of mCRP or hCRP was observed with rAAV-8 and rAAV-9. We show, for the first time, that serum components can significantly enhance rAAV-mediated tissue transduction in a serotype- and species-specific manner. Bioprocessing in body fluids should be considered when transfer of a preclinical proof of concept for AAV-based gene therapy to humans is planned.
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http://dx.doi.org/10.1128/JVI.01813-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3807418PMC
October 2013

In vivo fate mapping identifies pre-TCRα expression as an intra- and extrathymic, but not prethymic, marker of T lymphopoiesis.

J Exp Med 2013 Apr 18;210(4):699-714. Epub 2013 Mar 18.

Institute of Immunology, University Clinics Ulm, D-89081 Ulm, Germany.

Expression of the pre-T cell receptor α (pTα) gene has been exploited in previous studies as a molecular marker to identify tiny cell populations in bone marrow (BM) and blood that were suggested to contain physiologically relevant thymus settling progenitors (TSPs). But to what extent these cells genuinely contribute to thymopoiesis has remained obscure. We have generated a novel pTα(iCre) knockin mouse line and performed lineage-tracing experiments to precisely quantitate the contribution of pTα-expressing progenitors to distinct differentiation pathways and to the genealogy of mature hematopoietic cells under physiological in vivo conditions. Using these mice in combination with fluorescent reporter strains, we observe highly consistent labeling patterns that identify pTα expression as a faithful molecular marker of T lineage commitment. Specifically, the fate of pTα-expressing progenitors was found to include all αβ and most γδ T cells but, in contrast to previous assumptions, to exclude B, NK, and thymic dendritic cells. Although we could detect small numbers of T cell progenitors with a history of pTα expression in BM and blood, our data clearly exclude these populations as physiologically important precursors of thymopoiesis and indicate that they instead belong to a pathway of T cell maturation previously defined as extrathymic.
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http://dx.doi.org/10.1084/jem.20122609DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3620354PMC
April 2013

Differential requirement for ROCK in dendritic cell migration within lymphatic capillaries in steady-state and inflammation.

Blood 2012 Sep 31;120(11):2249-58. Epub 2012 Jul 31.

Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology, ETH Zurich, Switzerland.

Dendritic cell (DC) migration via lymphatic vessels to draining lymph nodes (dLNs) is crucial for the initiation of adaptive immunity. We imaged this process by intravital microscopy (IVM) in the ear skin of transgenic mice bearing red-fluorescent vasculature and yellow-fluorescent DCs. DCs within lymphatic capillaries were rarely transported by flow, but actively migrated within lymphatics and were significantly faster than in the interstitium. Pharmacologic blockade of the Rho-associated protein kinase (ROCK), which mediates nuclear contraction and de-adhesion from integrin ligands, significantly reduced DC migration from skin to dLNs in steady-state. IVM revealed that ROCK blockade strongly reduced the velocity of interstitial DC migration, but only marginally affected intralymphatic DC migration. By contrast, during tissue inflammation, ROCK blockade profoundly decreased both interstitial and intralymphatic DC migration. Inhibition of intralymphatic migration was paralleled by a strong up-regulation of ICAM-1 in lymphatic endothelium, suggesting that during inflammation ROCK mediates de-adhesion of DC-expressed integrins from lymphatic-expressed ICAM-1. Flow chamber assays confirmed an involvement of lymphatic-expressed ICAM-1 and DC-expressed ROCK in DC crawling on lymphatic endothelium. Overall, our findings further define the role of ROCK in DC migration to dLNs and reveal a differential requirement for ROCK in intralymphatic DC crawling during steady-state and inflammation.
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http://dx.doi.org/10.1182/blood-2012-03-417923DOI Listing
September 2012

Transcriptional code and disease map for adult retinal cell types.

Nat Neurosci 2012 Jan 22;15(3):487-95, S1-2. Epub 2012 Jan 22.

Neural Circuit Laboratories, Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.

Brain circuits are assembled from a large variety of morphologically and functionally diverse cell types. It is not known how the intermingled cell types of an individual adult brain region differ in their expressed genomes. Here we describe an atlas of cell type transcriptomes in one brain region, the mouse retina. We found that each adult cell type expressed a specific set of genes, including a unique set of transcription factors, forming a 'barcode' for cell identity. Cell type transcriptomes carried enough information to categorize cells into morphological classes and types. Several genes that were specifically expressed in particular retinal circuit elements, such as inhibitory neuron types, are associated with eye diseases. The resource described here allows gene expression to be compared across adult retinal cell types, experimenting with specific transcription factors to differentiate stem or somatic cells to retinal cell types, and predicting cellular targets of newly discovered disease-associated genes.
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http://dx.doi.org/10.1038/nn.3032DOI Listing
January 2012

Salmonella gut invasion involves TTSS-2-dependent epithelial traversal, basolateral exit, and uptake by epithelium-sampling lamina propria phagocytes.

Cell Host Microbe 2012 Jan;11(1):19-32

Institute of Microbiology, D-BIOL, ETH Zürich, 8093 Zürich, Switzerland.

Salmonella Typhimurium causes diarrhea by infecting the epithelium and lamina propria of the intestinal mucosa and by secreting various effector proteins through type III secretion systems (TTSSs). However, the mechanisms by which Salmonella transverses the epithelium and is subsequently released into the lamina propria are poorly understood. Using a murine Salmonella-diarrhea model and in vivo microscopy, we show that epithelial traversal requires TTSS-1-mediated invasion and TTSS-2-dependent trafficking to the basolateral side. After being released into the lamina propria, the bacterium is transiently extracellular before being taken up by phagocytes, including CD11c(+)CX(3)CR1(high) monocytic phagocytes (MPs), which were found to constitutively sample cellular material shed from the basolateral side of the epithelium. Thus, Salmonella infects the cecal mucsa through a step-wise process wherein the bacterium transverses the epithelium through TTSS-2-dependent trafficking and then likely exploits lamina propria MPs, which are sampling the epithelium, to enter and replicate within the host.
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http://dx.doi.org/10.1016/j.chom.2011.11.013DOI Listing
January 2012

Cre-mediated cell ablation contests mast cell contribution in models of antibody- and T cell-mediated autoimmunity.

Immunity 2011 Nov;35(5):832-44

Division for Cellular Immunology, Deutsches Krebsforschungszentrum (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.

Immunological functions of mast cells remain poorly understood. Studies in Kit mutant mice suggest key roles for mast cells in certain antibody- and T cell-mediated autoimmune diseases. However, Kit mutations affect multiple cell types of both immune and nonimmune origin. Here, we show that targeted insertion of Cre-recombinase into the mast cell carboxypeptidase A3 locus deleted mast cells in connective and mucosal tissues by a genotoxic Trp53-dependent mechanism. Cre-mediated mast cell eradication (Cre-Master) mice had, with the exception of a lack of mast cells and reduced basophils, a normal immune system. Cre-Master mice were refractory to IgE-mediated anaphylaxis, and this defect was rescued by mast cell reconstitution. This mast cell-deficient strain was fully susceptible to antibody-induced autoimmune arthritis and to experimental autoimmune encephalomyelitis. Differences comparing Kit mutant mast cell deficiency models to selectively mast cell-deficient mice call for a systematic re-evaluation of immunological functions of mast cells beyond allergy.
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http://dx.doi.org/10.1016/j.immuni.2011.09.015DOI Listing
November 2011

No reduction of atherosclerosis in C-reactive protein (CRP)-deficient mice.

J Biol Chem 2011 Feb 11;286(8):6272-9. Epub 2010 Dec 11.

Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, Leipzig Research Center for Civilization Diseases, University Leipzig, Liebigstrasse 27, D-04103 Leipzig, Germany.

C-reactive protein (CRP), a phylogenetically highly conserved plasma protein, is the classical acute phase reactant in humans. Upon infection, inflammation, or tissue damage, its plasma level can rise within hours >1000-fold, providing an early, nonspecific disease indicator of prime clinical importance. In recent years, another aspect of CRP expression has attracted much scientific and public attention. Apart from transient, acute phase-associated spikes in plasma concentration, highly sensitive measurements have revealed stable interindividual differences of baseline CRP values in healthy persons. Strikingly, even modest elevations in stable baseline CRP plasma levels have been found to correlate with a significantly increased risk of future cardiovascular disease. These observations have triggered intense controversies about potential atherosclerosis-promoting properties of CRP. To directly assess potential effects of CRP on atherogenesis, we have generated CRP-deficient mice via gene targeting and introduced the inactivated allele into atherosclerosis-susceptible ApoE(-/-) and LDLR(-/-) mice, two well established mouse models of atherogenesis. Morphometric analyses of atherosclerotic plaques in CRP-deficient animals revealed equivalent or increased atherosclerotic lesions compared with controls, an experimental result, which does not support a proatherogenic role of CRP. In fact, our data suggest that mouse CRP may even mediate atheroprotective effects, adding a cautionary note to the idea of targeting CRP as therapeutic intervention against progressive cardiovascular disease.
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http://dx.doi.org/10.1074/jbc.M110.161414DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3057833PMC
February 2011

Lineage relationship analysis of RORgammat+ innate lymphoid cells.

Science 2010 Oct 23;330(6004):665-9. Epub 2010 Sep 23.

Lymphoid Tissue Development Unit, Institut Pasteur, 75724 Paris, France.

Lymphoid tissue-inducer (LTi) cells initiate the development of lymphoid tissues through the activation of local stromal cells in a process similar to inflammation. LTi cells express the nuclear hormone receptor RORγt, which also directs the expression of the proinflammatory cytokine interleukin-17 in T cells. We show here that LTi cells are part of a larger family of proinflammatory RORγt(+) innate lymphoid cells (ILCs) that differentiate from distinct fetal liver RORγt(+) precursors. The fate of RORγt(+) ILCs is determined by mouse age, and after birth, favors the generation of cells involved in intestinal homeostasis and defense. Contrary to RORγt(+) T cells, however, RORγt(+) ILCs develop in the absence of microbiota. Our study indicates that RORγt(+) ILCs evolve to preempt intestinal colonization by microbial symbionts.
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http://dx.doi.org/10.1126/science.1194597DOI Listing
October 2010

Preferential localization of IgG memory B cells adjacent to contracted germinal centers.

Proc Natl Acad Sci U S A 2010 Jul 14;107(27):12192-7. Epub 2010 Jun 14.

Laboratory for Lymphocyte Differentiation, RIKEN Research Center for Allergy and Immunology, Yokohama, Kanagawa 230-0045, Japan.

It has long been presumed that after leaving the germinal centers (GCs), memory B cells colonize the marginal zone or join the recirculating pool. Here we demonstrate the preferential localization of nitrophenol-chicken gamma-globulin-induced CD38(+)IgG1(+) memory B cells adjacent to contracted GCs in the spleen. The memory B cells in this region proliferated after secondary immunization, a response that was abolished by depletion of CD4(+) T cells. We also found that these IgG1(+) memory B cells could present antigen on their surface, and that this activity was required for their activation. These results implicate this peri-GC region as an important site for survival and reactivation of memory B cells.
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http://dx.doi.org/10.1073/pnas.1005443107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2901464PMC
July 2010

Dicer-dependent microRNAs control maturation, function, and maintenance of Langerhans cells in vivo.

J Immunol 2010 Jul 7;185(1):400-9. Epub 2010 Jun 7.

Institute for Immunology, Ludwig-Maximilian-University Munich, Munich, Germany.

Dendritic cells (DCs) are central for the induction of T cell immunity and tolerance. Fundamental for DCs to control the immune system is their differentiation from precursors into various DC subsets with distinct functions and locations in lymphoid organs and tissues. In contrast to the differentiation of epidermal Langerhans cells (LCs) and their seeding into the epidermis, LC maturation, turnover, and MHC class II Ag presentation capacities are strictly dependent on the presence of Dicer, which generates mature microRNAs (miRNAs). Absence of miRNAs caused a strongly disturbed steady-state homeostasis of LCs by increasing their turnover and apoptosis rate, leading to progressive ablation of LCs with age. The failure to maintain LCs populating the epidermis was accompanied by a proapoptotic gene expression signature. Dicer-deficient LCs showed largely increased cell sizes and reduced expression levels of the C-type lectin receptor Langerin, resulting in the lack of Birbeck granules. In addition, LCs failed to properly upregulate MHC class II, CD40, and CD86 surface molecules upon stimulation, which are critical hallmarks of functional DC maturation. This resulted in inefficient induction of CD4 T cell proliferation, whereas Dicer-deficient LCs could properly stimulate CD8 T cells. Taken together, Dicer-dependent generation of miRNAs affects homeostasis and function of epidermal LCs.
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http://dx.doi.org/10.4049/jimmunol.0903912DOI Listing
July 2010

Characterizing light-regulated retinal microRNAs reveals rapid turnover as a common property of neuronal microRNAs.

Cell 2010 May;141(4):618-31

Friedrich Miescher Institute for Biomedical Research, PO Box 2543, 4002 Basel, Switzerland.

Adaptation to different levels of illumination is central to the function of the retina. Here, we demonstrate that levels of the miR-183/96/182 cluster, miR-204, and miR-211 are regulated by different light levels in the mouse retina. Concentrations of these microRNAs were downregulated during dark adaptation and upregulated in light-adapted retinas, with rapid decay and increased transcription being responsible for the respective changes. We identified the voltage-dependent glutamate transporter Slc1a1 as one of the miR-183/96/182 targets in photoreceptor cells. We found that microRNAs in retinal neurons decay much faster than microRNAs in nonneuronal cells. The high turnover is also characteristic of microRNAs in hippocampal and cortical neurons, and neurons differentiated from ES cells in vitro. Blocking activity reduced turnover of microRNAs in neuronal cells while stimulation with glutamate accelerated it. Our results demonstrate that microRNA metabolism in neurons is higher than in most other cells types and linked to neuronal activity.
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http://dx.doi.org/10.1016/j.cell.2010.03.039DOI Listing
May 2010

Fate mapping reveals separate origins of T cells and myeloid lineages in the thymus.

Immunity 2010 Mar 18;32(3):426-36. Epub 2010 Mar 18.

Institute for Immunology, University of Ulm, Ulm, Germany.

The cellular differentiation pathway originating from the bone marrow leading to early T lymphocytes remains poorly understood. The view that T cells branch off from a lymphoid-restricted pathway has recently been challenged by a model proposing a common progenitor for T cell and myeloid lineages. We generated interleukin-7 receptor alpha (Il7r) Cre recombinase knockin mice and traced lymphocyte development by visualizing the history of Il7r expression. Il7r fate mapping labeled all T cells but few myeloid cells. More than 85% of T cell progenitors were Il7r reporter(+) and, hence, had arisen from an Il7r-expressing pathway. In contrast, the overwhelming majority of myeloid cells in the thymus were derived from Il7r reporter(-) cells. Thus, lymphoid-restricted progenitors are the major route to T cells, and distinct origins of lymphoid and myeloid lineages represent a fundamental hallmark of hematopoiesis.
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http://dx.doi.org/10.1016/j.immuni.2010.03.005DOI Listing
March 2010

Asynchronous RAG-1 expression during B lymphopoiesis.

J Immunol 2009 Dec;183(12):7768-77

Immunobiology & Cancer Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA.

Changes in cell surface markers and patterns of gene expression are commonly used to construct sequences of events in hematopoiesis. However, the order may not be as rigid as once thought and it is unclear which changes represent the best milestones of differentiation. We developed a fate-mapping model where cells with a history of RAG-1 expression are permanently marked by red fluorescence. This approach is valuable for appreciating lymphoid-lineage relationships without need for irradiation and transplantation. Hematopoietic stem cells (HSC) as well as myeloid and dendritic cell progenitors were unlabeled. Also as expected, most previously identified RAG-1(+) early lymphoid progenitors in bone marrow and all lymphoid-affiliated cells were marked. Of particular interest, there was heterogeneity among canonical common lymphoid progenitors (CLP) in bone marrow. Labeled CLP expressed slightly higher levels of IL-7Ralpha, displayed somewhat less c-Kit, and generated CD19(+) lymphocytes faster than the unlabeled CLP. Furthermore, CLP with a history of RAG-1 expression were much less likely to generate dendritic and NK cells. The RAG-1-marked CLP were lineage stable even when exposed to LPS, while unlabeled CLP were redirected to become dendritic cells in response to this TLR4 ligand. These findings indicate that essential events in B lymphopoiesis are not tightly synchronized. Some progenitors with increased probability of becoming lymphocytes express RAG-1 while still part of the lineage marker-negative Sca-1(+)c-Kit(high) (LSK) fraction. Other progenitors first activate this locus after c-Kit levels have diminished and cell surface IL-7 receptors are detectable.
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http://dx.doi.org/10.4049/jimmunol.0902333DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2796202PMC
December 2009

Intestinal lamina propria dendritic cell subsets have different origin and functions.

Immunity 2009 Sep 3;31(3):502-12. Epub 2009 Sep 3.

Department of Immunology, The Weizmann Institute of Science, Rehovot, Israel 76100.

The intestinal immune system discriminates between tolerance toward the commensal microflora and robust responses to pathogens. Maintenance of this critical balance is attributed to mucosal dendritic cells (DCs) residing in organized lymphoid tissue and dispersed in the subepithelial lamina propria. In situ parameters of lamina propria DCs (lpDCs) remain poorly understood. Here, we combined conditional cell ablation and precursor-mediated in vivo reconstitution to establish that lpDC subsets have distinct origins and functions. CD103(+) CX(3)CR1(-) lpDCs arose from macrophage-DC precursors (MDPs) via DC-committed intermediates (pre-cDCs) through a Flt3L growth-factor-mediated pathway. CD11b(+) CD14(+) CX(3)CR1(+) lpDCs were derived from grafted Ly6C(hi) but not Ly6C(lo) monocytes under the control of GM-CSF. Mice reconstituted exclusively with CX(3)CR1(+) lpDCs when challenged in an innate colitis model developed severe intestinal inflammation that was driven by graft-derived TNF-alpha-secreting CX(3)CR1(+) lpDCs. Our results highlight the critical importance of the lpDC subset balance for robust gut homeostasis.
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http://dx.doi.org/10.1016/j.immuni.2009.06.025DOI Listing
September 2009

The patch-like pattern of OR37 receptors is formed by turning off gene expression in non-appropriate areas.

Mol Cell Neurosci 2009 Aug 20;41(4):474-85. Epub 2009 May 20.

University of Hohenheim, Institute of Physiology, Garbenstrasse 30, 70593 Stuttgart, Germany.

Olfactory sensory neurons (OSNs) expressing the same odorant receptor (OR) gene are generally widely dispersed throughout the olfactory epithelium (OE). In contrast, OSNs expressing any member from the special OR37 subfamily are concentrated in a small patch in the centre of the OE. To evaluate whether transcription of OR37 genes is only possible in the patch region, or if they can generally be chosen also in non-appropriate areas, a transgenic approach was employed that permanently labelled all cells which ever transcribed a representative OR37 gene. It was found that - in addition to cells inside the patch - numerous cells outside were labelled, indicating that they had transcribed the OR37 gene, but then turned it off while choosing another OR gene. Permanent expression of the OR37 gene was exclusively maintained in the patch. The results suggest that mechanisms acting downstream of an initial OR gene choice restrict OR37 expression to the patch.
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http://dx.doi.org/10.1016/j.mcn.2009.05.005DOI Listing
August 2009

The pluripotency-associated gene Dppa4 is dispensable for embryonic stem cell identity and germ cell development but essential for embryogenesis.

Mol Cell Biol 2009 Jun 30;29(11):3186-203. Epub 2009 Mar 30.

Institute of Immunology, University Clinics Ulm, Ulm, Germany.

Dppa4 (developmental pluripotency-associated 4) has been identified in several high-profile screens as a gene that is expressed exclusively in pluripotent cells. It encodes a nuclear protein with an SAP-like domain and appears to be associated preferentially with transcriptionally active chromatin. Its exquisite expression pattern and results of RNA interference experiments have led to speculation that Dppa4, as well as its nearby homolog Dppa2, might play essential roles in embryonic stem (ES) cell function and/or germ cell development. To rigorously assess suggested roles, we have generated Dppa4-deficient and Dppa4/Dppa2 doubly deficient ES cells, as well as mice lacking Dppa4. Contrary to predictions, we find that Dppa4 is completely dispensable for ES cell identity and germ cell development. Instead, loss of Dppa4 in mice results in late embryonic/perinatal death and striking skeletal defects with partial penetrance. Thus, surprisingly, Dppa4-deficiency affects tissues that apparently never transcribed the gene, and at least some loss-of-function defects manifest phenotypically at an embryonic stage long after physiologic Dppa4 expression has ceased. Concomitant with targeted gene inactivation, we have introduced into the Dppa4 locus a red fluorescent marker (tandem-dimer red fluorescent protein) that is compatible with green fluorescent proteins and allows noninvasive visualization of pluripotent cells and reprogramming events.
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http://dx.doi.org/10.1128/MCB.01970-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2682008PMC
June 2009

Deletion of Notch1 converts pro-T cells to dendritic cells and promotes thymic B cells by cell-extrinsic and cell-intrinsic mechanisms.

Immunity 2009 Jan 24;30(1):67-79. Epub 2008 Dec 24.

Institute for Immunology, University of Ulm, D-89081 Ulm, Germany.

Notch1 signaling is required for T cell development and has been implicated in fate decisions in the thymus. We showed that Notch1 deletion in progenitor T cells (pro-T cells) revealed their latent developmental potential toward becoming conventional and plasmacytoid dendritic cells. In addition, Notch1 deletion in pro-T cells resulted in large numbers of thymic B cells, previously explained by T-to-B cell fate conversion. Single-cell genotyping showed, however, that the majority of these thymic B cells arose from Notch1-sufficient cells by a cell-extrinsic pathway. Fate switching nevertheless exists for a subset of thymic B cells originating from Notch1-deleted pro-T cells. Chimeric mice lacking the Notch ligand delta-like 4 (Dll4) in thymus epithelium revealed an essential role for Dll4 in T cell development. Thus, Notch1-Dll4 signaling fortifies T cell commitment by suppressing non-T cell lineage potential in pro-T cells, and normal Notch1-driven T cell development repels excessive B cells in the thymus.
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http://dx.doi.org/10.1016/j.immuni.2008.10.016DOI Listing
January 2009

Impaired function of primitive hematopoietic cells in mice lacking the Mixed-Lineage-Leukemia homolog MLL5.

Blood 2009 Feb 24;113(7):1444-54. Epub 2008 Oct 24.

Institute of Immunology, University Clinics Ulm, Ulm, Germany.

The human Mixed-Lineage-Leukemia-5 (MLL5) gene is located in a genomic region frequently deleted in patients with myeloid malignancies and encodes a widely expressed nuclear protein most closely related to MLL1, a Trithorax transcriptional regulator with established involvement in leukemogenesis. Although the physiologic function of MLL5 is completely unknown, domain structure and homology to transcriptional regulators with histone methyltransferase activity suggest a role in epigenetic gene regulation. To investigate physiologic functions of Mll5, we have generated a knockout mouse mutant using Cre/loxP technology. Adult homozygous Mll5-deficient mice are obtained at reduced frequency because of postnatal lethality. Surviving animals display a variety of abnormalities, including male infertility, retarded growth, and defects in multiple hematopoietic lineages. Interestingly, Mll5(-/-) mice die of sublethal whole-body irradiation but can be rescued with wild-type bone marrow grafts. Flow cytometric ana-lysis, bone marrow reconstitution, and in vivo BrdU-labeling experiments reveal numerical, functional, and cell-cycle defects in the lineage-negative Sca-1(+), Kit(+) (LSK) population, which contains short- and long-term hematopoietic stem cells. Together, these in vivo findings establish several nonredundant functions for Mll5, including an essential role in regulating proliferation and functional integrity of hematopoietic stem/progenitor cells.
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http://dx.doi.org/10.1182/blood-2008-02-142638DOI Listing
February 2009

Faithful activation of an extra-bright red fluorescent protein in "knock-in" Cre-reporter mice ideally suited for lineage tracing studies.

Eur J Immunol 2007 Jan;37(1):43-53

Institute of Immunology, University Clinics Ulm, Ulm, Germany.

The considerable potential of Cre recombinase as a tool for in vivo fate-mapping studies depends on the availability of reliable reporter mice. By targeting a tandem-dimer red fluorescent protein (tdRFP) with advanced spectral and biological properties into the ubiquitously expressed ROSA26 locus of C57BL/6-ES cells, we have generated a novel inbred Cre-reporter mouse with several unique characteristics. We directly demonstrate the usefulness of our reporter strain in inter-crosses with a "universal Cre-deleter" strain and with mice expressing Cre recombinase in a T lineage-specific manner. Cytofluorometric and histological analyses illustrate: (i) non-toxicity and extraordinary brightness of the fluorescent reporter, allowing quantitative detection and purification of labeled cells with highest accuracy, (ii) reliable Cre-mediated activation of tdRFP from an antisense orientation relative to ROSA26 transcription, effectively excluding "leaky" reporter expression, (iii) absence of gene expression variegation effects, (iv) quantitative detection of tdRFP-expressing cells even in paraformaldehyde-fixed tissue sections, and (v) full compatibility with GFP/YFP-based fluorescent markers in multicolor experiments. Taken together, the data show that our C57BL/6-inbred reporter mice are ideally suited for sophisticated lineage-tracing experiments requiring sensitive and quantitative detection/purification of live Cre-expressing cells and their progeny.
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http://dx.doi.org/10.1002/eji.200636745DOI Listing
January 2007

The ubiquitin-proteasome system plays essential roles in presenting an 8-mer CTL epitope expressed in APC to corresponding CD8+ T cells.

Int Immunol 2006 May 28;18(5):679-87. Epub 2006 Mar 28.

Department of Microbiology and Immunology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.

MUT1 is an H-2Kb-restricted 8-mer CTL epitope expressed in Lewis lung carcinoma (3LL) tumor cells derived from C57BL/6 (B6) mice. We constructed a chimeric gene encoding ubiquitin-fused MUT1 (pUB-MUT1). By using a gene gun, B6 mice were immunized with the gene prior to challenge with 3LL tumor cells. Tumor growth and lung metastasis were prominently suppressed in mice immunized with pUB-MUT1 but only slightly in those immunized with the MUT1 gene (pMUT) alone. CD8+ T cells were confirmed to be the final effector by in vitro experiments and in vivo removal of the cells with a corresponding antibody. Anti-tumor immunity was profoundly suppressed in mice deficient in an immuno-subunit of proteasome, LMP7. Furthermore, mice deficient in a proteasome regulator, PA28alpha/beta, failed to acquire protective immunity. Thus, application of the ubiquitin-fusion degradation pathway was useful even in immunization with genes encoding a single CTL epitope for induction of specific and active CD8+ T cells.
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http://dx.doi.org/10.1093/intimm/dxl005DOI Listing
May 2006

The involvement of immunoproteasomes in induction of MHC class I-restricted immunity targeting Toxoplasma SAG1.

Microbes Infect 2006 Apr 18;8(4):1045-53. Epub 2006 Jan 18.

Department of Parasitology, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.

The ubiquitin-proteasome system (UPS) plays an indispensable role in inducing MHC class I-restricted CD8+ T cells and was exploited in the development of a DNA vaccine against the intracellular protozoan Toxoplasma gondii by constructing a chimeric DNA encoding a fusion protein between murine ubiquitin and the toxoplasma antigen SAG1. The SAG1 peptide was promptly degraded in antigen-presenting cells (APCs) transfected with the chimeric DNA. Degradation, however, was hampered by incubating the APCs with the proteasome inhibitor epoxomicin. Mice vaccinated with the DNA acquired potent protective immunity mediated by MHC class I-restricted CD8+ T cells against infection by the highly virulent Toxoplasma. The accelerated degradation and induction of immunity were dependent on the UPS since mice lacking an immuno-subunit of 20S proteasome, LMP7, lost these functions, although they were independent of the proteasome regulator PA28alpha/beta complex.
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http://dx.doi.org/10.1016/j.micinf.2005.10.023DOI Listing
April 2006