Publications by authors named "Hervé Luche"

40 Publications

LymphoAtlas: a dynamic and integrated phosphoproteomic resource of TCR signaling in primary T cells reveals ITSN2 as a regulator of effector functions.

Mol Syst Biol 2020 07;16(7):e9524

Centre d'Immunologie de Marseille-Luminy, INSERM, CNRS, Aix Marseille Université, Marseille, France.

T-cell receptor (TCR) ligation-mediated protein phosphorylation regulates the activation, cellular responses, and fates of T cells. Here, we used time-resolved high-resolution phosphoproteomics to identify, quantify, and characterize the phosphorylation dynamics of thousands of phosphorylation sites in primary T cells during the first 10 min after TCR stimulation. Bioinformatic analysis of the data revealed a coherent orchestration of biological processes underlying T-cell activation. In particular, functional modules associated with cytoskeletal remodeling, transcription, translation, and metabolic processes were mobilized within seconds after TCR engagement. Among proteins whose phosphorylation was regulated by TCR stimulation, we demonstrated, using a fast-track gene inactivation approach in primary lymphocytes, that the ITSN2 adaptor protein regulated T-cell effector functions. This resource, called LymphoAtlas, represents an integrated pipeline to further decipher the organization of the signaling network encoding T-cell activation. LymphoAtlas is accessible to the community at: https://bmm-lab.github.io/LymphoAtlas.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/msb.20209524DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7333348PMC
July 2020

Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition).

Authors:
Andrea Cossarizza Hyun-Dong Chang Andreas Radbruch Andreas Acs Dieter Adam Sabine Adam-Klages William W Agace Nima Aghaeepour Mübeccel Akdis Matthieu Allez Larissa Nogueira Almeida Giorgia Alvisi Graham Anderson Immanuel Andrä Francesco Annunziato Achille Anselmo Petra Bacher Cosima T Baldari Sudipto Bari Vincenzo Barnaba Joana Barros-Martins Luca Battistini Wolfgang Bauer Sabine Baumgart Nicole Baumgarth Dirk Baumjohann Bianka Baying Mary Bebawy Burkhard Becher Wolfgang Beisker Vladimir Benes Rudi Beyaert Alfonso Blanco Dominic A Boardman Christian Bogdan Jessica G Borger Giovanna Borsellino Philip E Boulais Jolene A Bradford Dirk Brenner Ryan R Brinkman Anna E S Brooks Dirk H Busch Martin Büscher Timothy P Bushnell Federica Calzetti Garth Cameron Ilenia Cammarata Xuetao Cao Susanna L Cardell Stefano Casola Marco A Cassatella Andrea Cavani Antonio Celada Lucienne Chatenoud Pratip K Chattopadhyay Sue Chow Eleni Christakou Luka Čičin-Šain Mario Clerici Federico S Colombo Laura Cook Anne Cooke Andrea M Cooper Alexandra J Corbett Antonio Cosma Lorenzo Cosmi Pierre G Coulie Ana Cumano Ljiljana Cvetkovic Van Duc Dang Chantip Dang-Heine Martin S Davey Derek Davies Sara De Biasi Genny Del Zotto Gelo Victoriano Dela Cruz Michael Delacher Silvia Della Bella Paolo Dellabona Günnur Deniz Mark Dessing James P Di Santo Andreas Diefenbach Francesco Dieli Andreas Dolf Thomas Dörner Regine J Dress Diana Dudziak Michael Dustin Charles-Antoine Dutertre Friederike Ebner Sidonia B G Eckle Matthias Edinger Pascale Eede Götz R A Ehrhardt Marcus Eich Pablo Engel Britta Engelhardt Anna Erdei Charlotte Esser Bart Everts Maximilien Evrard Christine S Falk Todd A Fehniger Mar Felipo-Benavent Helen Ferry Markus Feuerer Andrew Filby Kata Filkor Simon Fillatreau Marie Follo Irmgard Förster John Foster Gemma A Foulds Britta Frehse Paul S Frenette Stefan Frischbutter Wolfgang Fritzsche David W Galbraith Anastasia Gangaev Natalio Garbi Brice Gaudilliere Ricardo T Gazzinelli Jens Geginat Wilhelm Gerner Nicholas A Gherardin Kamran Ghoreschi Lara Gibellini Florent Ginhoux Keisuke Goda Dale I Godfrey Christoph Goettlinger Jose M González-Navajas Carl S Goodyear Andrea Gori Jane L Grogan Daryl Grummitt Andreas Grützkau Claudia Haftmann Jonas Hahn Hamida Hammad Günter Hämmerling Leo Hansmann Goran Hansson Christopher M Harpur Susanne Hartmann Andrea Hauser Anja E Hauser David L Haviland David Hedley Daniela C Hernández Guadalupe Herrera Martin Herrmann Christoph Hess Thomas Höfer Petra Hoffmann Kristin Hogquist Tristan Holland Thomas Höllt Rikard Holmdahl Pleun Hombrink Jessica P Houston Bimba F Hoyer Bo Huang Fang-Ping Huang Johanna E Huber Jochen Huehn Michael Hundemer Christopher A Hunter William Y K Hwang Anna Iannone Florian Ingelfinger Sabine M Ivison Hans-Martin Jäck Peter K Jani Beatriz Jávega Stipan Jonjic Toralf Kaiser Tomas Kalina Thomas Kamradt Stefan H E Kaufmann Baerbel Keller Steven L C Ketelaars Ahad Khalilnezhad Srijit Khan Jan Kisielow Paul Klenerman Jasmin Knopf Hui-Fern Koay Katja Kobow Jay K Kolls Wan Ting Kong Manfred Kopf Thomas Korn Katharina Kriegsmann Hendy Kristyanto Thomas Kroneis Andreas Krueger Jenny Kühne Christian Kukat Désirée Kunkel Heike Kunze-Schumacher Tomohiro Kurosaki Christian Kurts Pia Kvistborg Immanuel Kwok Jonathan Landry Olivier Lantz Paola Lanuti Francesca LaRosa Agnès Lehuen Salomé LeibundGut-Landmann Michael D Leipold Leslie Y T Leung Megan K Levings Andreia C Lino Francesco Liotta Virginia Litwin Yanling Liu Hans-Gustaf Ljunggren Michael Lohoff Giovanna Lombardi Lilly Lopez Miguel López-Botet Amy E Lovett-Racke Erik Lubberts Herve Luche Burkhard Ludewig Enrico Lugli Sebastian Lunemann Holden T Maecker Laura Maggi Orla Maguire Florian Mair Kerstin H Mair Alberto Mantovani Rudolf A Manz Aaron J Marshall Alicia Martínez-Romero Glòria Martrus Ivana Marventano Wlodzimierz Maslinski Giuseppe Matarese Anna Vittoria Mattioli Christian Maueröder Alessio Mazzoni James McCluskey Mairi McGrath Helen M McGuire Iain B McInnes Henrik E Mei Fritz Melchers Susanne Melzer Dirk Mielenz Stephen D Miller Kingston H G Mills Hans Minderman Jenny Mjösberg Jonni Moore Barry Moran Lorenzo Moretta Tim R Mosmann Susann Müller Gabriele Multhoff Luis Enrique Muñoz Christian Münz Toshinori Nakayama Milena Nasi Katrin Neumann Lai Guan Ng Antonia Niedobitek Sussan Nourshargh Gabriel Núñez José-Enrique O'Connor Aaron Ochel Anna Oja Diana Ordonez Alberto Orfao Eva Orlowski-Oliver Wenjun Ouyang Annette Oxenius Raghavendra Palankar Isabel Panse Kovit Pattanapanyasat Malte Paulsen Dinko Pavlinic Livius Penter Pärt Peterson Christian Peth Jordi Petriz Federica Piancone Winfried F Pickl Silvia Piconese Marcello Pinti A Graham Pockley Malgorzata Justyna Podolska Zhiyong Poon Katharina Pracht Immo Prinz Carlo E M Pucillo Sally A Quataert Linda Quatrini Kylie M Quinn Helena Radbruch Tim R D J Radstake Susann Rahmig Hans-Peter Rahn Bartek Rajwa Gevitha Ravichandran Yotam Raz Jonathan A Rebhahn Diether Recktenwald Dorothea Reimer Caetano Reis e Sousa Ester B M Remmerswaal Lisa Richter Laura G Rico Andy Riddell Aja M Rieger J Paul Robinson Chiara Romagnani Anna Rubartelli Jürgen Ruland Armin Saalmüller Yvan Saeys Takashi Saito Shimon Sakaguchi Francisco Sala-de-Oyanguren Yvonne Samstag Sharon Sanderson Inga Sandrock Angela Santoni Ramon Bellmàs Sanz Marina Saresella Catherine Sautes-Fridman Birgit Sawitzki Linda Schadt Alexander Scheffold Hans U Scherer Matthias Schiemann Frank A Schildberg Esther Schimisky Andreas Schlitzer Josephine Schlosser Stephan Schmid Steffen Schmitt Kilian Schober Daniel Schraivogel Wolfgang Schuh Thomas Schüler Reiner Schulte Axel Ronald Schulz Sebastian R Schulz Cristiano Scottá Daniel Scott-Algara David P Sester T Vincent Shankey Bruno Silva-Santos Anna Katharina Simon Katarzyna M Sitnik Silvano Sozzani Daniel E Speiser Josef Spidlen Anders Stahlberg Alan M Stall Natalie Stanley Regina Stark Christina Stehle Tobit Steinmetz Hannes Stockinger Yousuke Takahama Kiyoshi Takeda Leonard Tan Attila Tárnok Gisa Tiegs Gergely Toldi Julia Tornack Elisabetta Traggiai Mohamed Trebak Timothy I M Tree Joe Trotter John Trowsdale Maria Tsoumakidou Henning Ulrich Sophia Urbanczyk Willem van de Veen Maries van den Broek Edwin van der Pol Sofie Van Gassen Gert Van Isterdael René A W van Lier Marc Veldhoen Salvador Vento-Asturias Paulo Vieira David Voehringer Hans-Dieter Volk Anouk von Borstel Konrad von Volkmann Ari Waisman Rachael V Walker Paul K Wallace Sa A Wang Xin M Wang Michael D Ward Kirsten A Ward-Hartstonge Klaus Warnatz Gary Warnes Sarah Warth Claudia Waskow James V Watson Carsten Watzl Leonie Wegener Thomas Weisenburger Annika Wiedemann Jürgen Wienands Anneke Wilharm Robert John Wilkinson Gerald Willimsky James B Wing Rieke Winkelmann Thomas H Winkler Oliver F Wirz Alicia Wong Peter Wurst Jennie H M Yang Juhao Yang Maria Yazdanbakhsh Liping Yu Alice Yue Hanlin Zhang Yi Zhao Susanne Maria Ziegler Christina Zielinski Jakob Zimmermann Arturo Zychlinsky

Eur J Immunol 2019 Oct;49(10):1457-1973

Max Planck Institute for Infection Biology, Berlin, Germany.

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/eji.201970107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7350392PMC
October 2019

Quantitative interactomics in primary T cells unveils TCR signal diversification extent and dynamics.

Nat Immunol 2019 11 7;20(11):1530-1541. Epub 2019 Oct 7.

Centre d'Immunologie de Marseille-Luminy, Aix Marseille Université, INSERM, CNRS, Marseille, France.

The activation of T cells by the T cell antigen receptor (TCR) results in the formation of signaling protein complexes (signalosomes), the composition of which has not been analyzed at a systems level. Here, we isolated primary CD4 T cells from 15 gene-targeted mice, each expressing one tagged form of a canonical protein of the TCR-signaling pathway. Using affinity purification coupled with mass spectrometry, we analyzed the composition and dynamics of the signalosomes assembling around each of the tagged proteins over 600 s of TCR engagement. We showed that the TCR signal-transduction network comprises at least 277 unique proteins involved in 366 high-confidence interactions, and that TCR signals diversify extensively at the level of the plasma membrane. Integrating the cellular abundance of the interacting proteins and their interaction stoichiometry provided a quantitative and contextual view of each documented interaction, permitting anticipation of whether ablation of a single interacting protein can impinge on the whole TCR signal-transduction network.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41590-019-0489-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6859066PMC
November 2019

Diversity of innate immune cell subsets across spatial and temporal scales in an EAE mouse model.

Sci Rep 2018 03 23;8(1):5146. Epub 2018 Mar 23.

Institut des Neurosciences de la Timone, Aix-Marseille Université and CNRS UMR7289, Marseille, France.

In both multiple sclerosis and its model experimental autoimmune encephalomyelitis (EAE), the extent of resident microglia activation and infiltration of monocyte-derived cells to the CNS is positively correlated to tissue damage. To address the phenotype characterization of different cell subsets, their spatio-temporal distributions and contributions to disease development we induced EAE in Thy1-CFP//LysM-EGFP//CD11c-EYFP reporter mice. We combined high content flow cytometry, immunofluorescence and two-photon imaging in live mice and identified a stepwise program of inflammatory cells accumulation. First on day 10 after induction, EGFP neutrophils and monocytes invade the spinal cord parenchyma through the meninges rather than by extravasion. This event occurs just before axonal losses in the white matter. Once in the parenchyma, monocytes mature into EGFP/EYFP monocyte-derived dendritic cells (moDCs) whose density is maximal on day 17 when the axonal degradation and clinical signs stabilize. Meanwhile, microglia is progressively activated in the grey matter and subsequently recruited to plaques to phagocyte axon debris. LysM-EGFP//CD11c-EYFP mice appear as a powerful tool to differentiate moDCs from macrophages and to study the dynamics of immune cell maturation and phenotypic evolution in EAE.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-018-22872-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5865173PMC
March 2018

Administration of Drugs Targeting Microglia Improves the Neurodevelopmental Outcome Following Cytomegalovirus Infection of the Rat Fetal Brain.

Front Cell Neurosci 2018 6;12:55. Epub 2018 Mar 6.

INMED, French National Institute of Health and Medical Research INSERM U1249, Aix-Marseille University, Marseille, France.

Congenital cytomegalovirus (CMV) infections represent one leading cause of neurodevelopmental disorders. Recently, we reported on a rat model of CMV infection of the developing brain , characterized by early and prominent infection and alteration of microglia-the brain-resident mononuclear phagocytes. Besides their canonical function against pathogens, microglia are also pivotal to brain development. Here we show that CMV infection of the rat fetal brain recapitulated key postnatal phenotypes of human congenital CMV including increased mortality, sensorimotor impairment reminiscent of cerebral palsy, hearing defects, and epileptic seizures. The possible influence of early microglia alteration on those phenotypes was then questioned by pharmacological targeting of microglia during pregnancy. One single administration of clodronate liposomes in the embryonic brains at the time of CMV injection to deplete microglia, and maternal feeding with doxycyxline throughout pregnancy to modify microglia in the litters' brains, were both associated with dramatic improvements of survival, body weight gain, sensorimotor development and with decreased risk of epileptic seizures. Improvement of microglia activation status did not persist postnatally after doxycycline discontinuation; also, active brain infection remained unchanged by doxycycline. Altogether our data indicate that early microglia alteration, rather than brain CMV load , is instrumental in influencing survival and the neurological outcomes of CMV-infected rats, and suggest that microglia might participate in the neurological outcome of congenital CMV in humans. Furthermore this study represents a first proof-of-principle for the design of microglia-targeted preventive strategies in the context of congenital CMV infection of the brain.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fncel.2018.00055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5845535PMC
March 2018

Atp6ap2 ablation in adult mice impairs viability through multiple organ deficiencies.

Sci Rep 2017 08 29;7(1):9618. Epub 2017 Aug 29.

CELPHEDIA-PHENOMIN, Institut Clinique de la Souris (ICS), CNRS, INSERM, University of Strasbourg, 1 rue Laurent Fries, F-67404, Illkirch-Graffenstaden, France.

ATP6AP2 codes for the (pro)renin receptor and is an essential component of vacuolar H+ ATPase. Activating (pro)renin for conversion of Angiotensinogen to Angiotensin makes ATP6AP2 attractive for drug intervention. Tissue-specific ATP6AP2 inactivation in mouse suggested a strong impact on various organs. Consistent with this, we found that embryonic ablation of Atp6ap2 resulted in both male hemizygous lethality and female haploinsufficiency. Next, we examined the phenotype of an induced inactivation in the adult animal, most akin to detect potential effect of functional interference of ATP6AP2 through drug therapy. Induced ablation of Atp6ap2, even without equal efficiency in all tissues (aorta, brain and kidney), resulted in rapid lethality marked by weight loss, changes in nutritional as well as blood parameters, leukocyte depletion, and bone marrow hypoplasia. Upon Atp6ap2 ablation, the colon demonstrated a rapid disruption of crypt morphology, aberrant proliferation, cell-death activation, as well as generation of microadenomas. Consequently, disruption of ATP6AP2 is extremely poorly tolerated in the adult, and severely affects various organ systems demonstrating that ATP6AP2 is an essential gene implicated in basic cellular mechanisms and necessary for multiple organ function. Accordingly, any potential drug targeting of this gene product must be strictly assessed for safety.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-017-08845-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5575319PMC
August 2017

Natural Killer Defective Maturation Is Associated with Adverse Clinical Outcome in Patients with Acute Myeloid Leukemia.

Front Immunol 2017 29;8:573. Epub 2017 May 29.

Team Immunity and Cancer, Centre de Recherche en Cancérologie de Marseille (CRCM), Inserm, U1068; CNRS, UMR7258, Institut Paoli-Calmettes; Aix-Marseille University, UM 105, Marseille, France.

Accumulating evidence highlights natural killer (NK) cell parameters as potential prognostic factors in cancer patients, which provides a strong rationale for developing therapeutic strategies aiming at restoring NK cell. However, reaching this point warrants better characterization of tumor-induced NK cell alterations. Our group recently reported heterogeneous NK maturation in acute myeloid leukemia (AML) patients. However, the clinical significance of such observations remained to be assessed on a larger cohort of patients. NK maturation based on expression of CD56, CD57, and KIR was assessed by flow cytometry in newly diagnosed AML patients ( = 87 patients from GOELAMS-LAM-IR-2006 multicenter trial). Clinical outcome was evaluated with regard to NK maturation profiles. Unsupervised integrated analysis of NK maturation markers confirmed the existence of three distinct groups of patients [hypomaturation (24.1%), intermediate maturation (66.7%), and hypermaturation (9.2%)]. In univariate analysis, significant differences in overall survival (OS) ( = 0.0006) and relapse-free survival (RFS) ( < 0.0001) were observed among these different groups. Patients with hypomaturation profile had reduced OS, with 3-year OS rates of 12.5 vs 57.1 and 57.4% for patients with intermediate and hypermaturation, respectively. Consistently, patients with hypomaturation profile had reduced RFS, with 3-year RFS rates of 0 vs 52.6 and 73.3% for patients with intermediate and hypermaturation, respectively. In multivariate Cox regression models, NK hypomaturation remained significantly associated with reduced OS and RFS, independent of other factors [hazard ratio (HR) = 4.15,  = 0.004 and HR = 8.23,  = 0.003, respectively]. NK maturation defects were further explored by mass cytometry and revealed that NK hypomaturation profile is associated with a reduced frequency of memory-like NK cells. In conclusion, besides classical alterations of NK triggering and inhibitory receptors expression in AML, we confirm that the homeostasis of NK maturation can be modified in the context of AML, notably with a deep maturation blockade in almost 10% patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2017.00573DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5447002PMC
May 2017

Unsupervised High-Dimensional Analysis Aligns Dendritic Cells across Tissues and Species.

Immunity 2016 09 13;45(3):669-684. Epub 2016 Sep 13.

Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A(∗)STAR), 8A Biomedical Grove, IMMUNOS Building #3-4, BIOPOLIS, Singapore 138648, Singapore. Electronic address:

Dendritic cells (DCs) are professional antigen-presenting cells that hold great therapeutic potential. Multiple DC subsets have been described, and it remains challenging to align them across tissues and species to analyze their function in the absence of macrophage contamination. Here, we provide and validate a universal toolbox for the automated identification of DCs through unsupervised analysis of conventional flow cytometry and mass cytometry data obtained from multiple mouse, macaque, and human tissues. The use of a minimal set of lineage-imprinted markers was sufficient to subdivide DCs into conventional type 1 (cDC1s), conventional type 2 (cDC2s), and plasmacytoid DCs (pDCs) across tissues and species. This way, a large number of additional markers can still be used to further characterize the heterogeneity of DCs across tissues and during inflammation. This framework represents the way forward to a universal, high-throughput, and standardized analysis of DC populations from mutant mice and human patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5040826PMC
http://dx.doi.org/10.1016/j.immuni.2016.08.015DOI Listing
September 2016

Broad and Largely Concordant Molecular Changes Characterize Tolerogenic and Immunogenic Dendritic Cell Maturation in Thymus and Periphery.

Immunity 2016 08;45(2):305-18

Centre d'Immunologie de Marseille-Luminy, Aix Marseille Université, INSERM, CNRS, 13288 Marseille, France; Centre d'Immunophénomique, Aix Marseille Université, INSERM, CNRS, 13288 Marseille, France. Electronic address:

Dendritic cells (DCs) are instrumental in the initiation of T cell responses, but how thymic and peripheral tolerogenic DCs differ globally from Toll-like receptor (TLR)-induced immunogenic DCs remains unclear. Here, we show that thymic XCR1(+) DCs undergo a high rate of maturation, accompanied by profound gene-expression changes that are essential for central tolerance and also happen in germ-free mice. Those changes largely overlap those occurring during tolerogenic and, more unexpectedly, TLR-induced maturation of peripheral XCR1(+) DCs, arguing against the commonly held view that tolerogenic DCs undergo incomplete maturation. Interferon-stimulated gene (ISG) expression was among the few discriminators of immunogenic and tolerogenic XCR1(+) DCs. Tolerogenic XCR1(+) thymic DCs were, however, unique in expressing ISGs known to restrain virus replication. Therefore, a broad functional convergence characterizes tolerogenic and immunogenic XCR1(+) DC maturation in the thymus and periphery, maximizing antigen presentation and signal delivery to developing and to conventional and regulatory mature T cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.immuni.2016.07.019DOI Listing
August 2016

Cytomegalovirus Infection of the Rat Developing Brain In Utero Prominently Targets Immune Cells and Promotes Early Microglial Activation.

PLoS One 2016 29;11(7):e0160176. Epub 2016 Jul 29.

INSERM U901, Marseille, France.

Background: Congenital cytomegalovirus infections are a leading cause of neurodevelopmental disorders in human and represent a major health care and socio-economical burden. In contrast with this medical importance, the pathophysiological events remain poorly known. Murine models of brain cytomegalovirus infection, mostly neonatal, have brought recent insights into the possible pathogenesis, with convergent evidence for the alteration and possible involvement of brain immune cells.

Objectives And Methods: In order to confirm and expand those findings, particularly concerning the early developmental stages following infection of the fetal brain, we have created a model of in utero cytomegalovirus infection in the developing rat brain. Rat cytomegalovirus was injected intraventricularly at embryonic day 15 (E15) and the brains analyzed at various stages until the first postnatal day, using a combination of gene expression analysis, immunohistochemistry and multicolor flow cytometry experiments.

Results: Rat cytomegalovirus infection was increasingly seen in various brain areas including the choroid plexi and the ventricular and subventricular areas and was prominently detected in CD45low/int, CD11b+ microglial cells, in CD45high, CD11b+ cells of the myeloid lineage including macrophages, and in CD45+, CD11b- lymphocytes and non-B non-T cells. In parallel, rat cytomegalovirus infection of the developing rat brain rapidly triggered a cascade of pathophysiological events comprising: chemokines upregulation, including CCL2-4, 7 and 12; infiltration by peripheral cells including B-cells and monocytes at E17 and P1, and T-cells at P1; and microglia activation at E17 and P1.

Conclusion: In line with previous findings in neonatal murine models and in human specimen, our study further suggests that neuroimmune alterations might play critical roles in the early stages following cytomegalovirus infection of the brain in utero. Further studies are now needed to determine which role, whether favorable or detrimental, those putative double-edge swords events actually play.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0160176PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4966896PMC
August 2017

Phenotypic dynamics of microglial and monocyte-derived cells in glioblastoma-bearing mice.

Sci Rep 2016 05 19;6:26381. Epub 2016 May 19.

Institut des Neurosciences de la Timone, Marseille, Aix-Marseille Université and CNRS UMR7289, France.

Inflammatory cells, an integral component of tumor evolution, are present in Glioblastomas multiforme (GBM). To address the cellular basis and dynamics of the inflammatory microenvironment in GBM, we established an orthotopic syngenic model by grafting GL261-DsRed cells in immunocompetent transgenic LysM-EGFP//CD11c-EYFP reporter mice. We combined dynamic spectral two-photon imaging with multiparametric cytometry and multicolor immunostaining to characterize spatio-temporal distribution, morphology and activity of microglia and blood-derived infiltrating myeloid cells in live mice. Early stages of tumor development were dominated by microglial EYFP(+) cells invading the tumor, followed by massive recruitment of circulating LysM-EGFP(+) cells. Fluorescent invading cells were conventional XCR1(+) and monocyte-derived dendritic cells distributed in subpopulations of different maturation stages, located in different areas relative to the tumor core. The lethal stage of the disease was characterized by the progressive accumulation of EGFP(+)/EYFP(+) monocyte-derived dendritic cells. This local phenotypic regulation of monocyte subtypes marked a transition in the immune response.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep26381DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4872227PMC
May 2016

Evaluating the efficiency of isotope transmission for improved panel design and a comparison of the detection sensitivities of mass cytometer instruments.

Cytometry A 2015 Apr 20;87(4):357-68. Epub 2015 Feb 20.

CEA, Division of Immuno-Virology, IDMIT Center, Institute for Emerging Diseases and Innovative Therapies (iMETI), DSV, Fontenay-aux-Roses, France; Université Paris-Sud, UMR E1, Orsay, France; Vaccine Research Institute (VRI), Créteil, France.

The recent introduction of mass cytometry, a technique coupling a cell introduction system generating a stream of single cells with mass spectrometry, has greatly increased the number of parameters that can be measured per single cell. As with all new technology there is a need for dissemination of standardization and quality control procedures. Here, we characterize variations in sensitivity observed across the mass range of a mass cytometer, using different lanthanide tags. We observed a five-fold difference in lanthanide detection over the mass range and demonstrated that each instrument has its own sensitivity pattern. Therefore, the selection of lanthanide combinations is a key step in the establishment of a staining panel for mass cytometry-based experiments, particularly for multicenter studies. We propose the sensitivity pattern as the basis for panel design, instrument standardization and future implementation of normalization algorithms.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cyto.a.22648DOI Listing
April 2015

Promiscuous Foxp3-cre activity reveals a differential requirement for CD28 in Foxp3⁺ and Foxp3⁻ T cells.

Immunol Cell Biol 2015 Apr 23;93(4):417-23. Epub 2014 Dec 23.

1] Autoimmune Genetics Laboratory, VIB, Leuven, Belgium [2] Department of Microbiology and Immunology, University of Leuven, Leuven, Belgium.

Costimulatory signals by CD28 are critical for thymic regulatory T-cell (Treg) development. To determine the functional relevance of CD28 for peripheral Treg post thymic selection, we crossed the widely used Forkhead box protein 3 (Foxp3)-CreYFP mice to mice bearing a conditional Cd28 allele. Treg-specific CD28 deficiency provoked a severe autoimmune syndrome as a result of a strong disadvantage in competitive fitness and proliferation of CD28-deficient Tregs. By contrast, Treg survival and lineage integrity were not affected by the lack of CD28. This data demonstrate that, even after the initial induction requirement, Treg maintain a higher dependency on CD28 signalling than conventional T cells for homeostasis. In addition, we found the Foxp3-CreYFP allele to be a hypomorph, with reduced Foxp3 protein levels. Furthermore, we report here the stochastic activity of the Foxp3-CreYFP allele in non-Tregs, sufficient to recombine some conditional alleles (including Cd28) but not others (including R26-RFP). This hypomorphism and 'leaky' expression of the Foxp3-CreYFP allele should be considered when analysing the conditionally mutated Treg.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/icb.2014.108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4407013PMC
April 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cyto.a.22451DOI Listing
July 2014

Temporal lineage tracing of Aire-expressing cells reveals a requirement for Aire in their maturation program.

J Immunol 2014 Mar 10;192(6):2585-92. Epub 2014 Feb 10.

Division of Molecular Immunology, Institute for Enzyme Research, University of Tokushima, Tokushima 770-8503, Japan;

Understanding the cellular dynamics of Aire-expressing lineage(s) among medullary thymic epithelial cells (AEL-mTECs) is essential for gaining insight into the roles of Aire in establishment of self-tolerance. In this study, we monitored the maturation program of AEL-mTECs by temporal lineage tracing, in which bacterial artificial chromosome transgenic mice expressing tamoxifen-inducible Cre recombinase under control of the Aire regulatory element were crossed with reporter strains. We estimated that the half-life of AEL-mTECs subsequent to Aire expression was ∼7-8 d, which was much longer than that reported previously, owing to the existence of a post-Aire stage. We found that loss of Aire did not alter the overall lifespan of AEL-mTECs, inconsistent with the previous notion that Aire expression in medullary thymic epithelial cells (mTECs) might result in their apoptosis for efficient cross-presentation of self-antigens expressed by AEL-mTECs. In contrast, Aire was required for the full maturation program of AEL-mTECs, as exemplified by the lack of physiological downregulation of CD80 during the post-Aire stage in Aire-deficient mice, thus accounting for the abnormally increased CD80(high) mTECs seen in such mice. Of interest, increased CD80(high) mTECs in Aire-deficient mice were not mTEC autonomous and were dependent on cross-talk with thymocytes. These results further support the roles of Aire in the differentiation program of AEL-mTECs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4049/jimmunol.1302786DOI Listing
March 2014

Self-antigen-driven activation induces instability of regulatory T cells during an inflammatory autoimmune response.

Immunity 2013 Nov;39(5):949-62

Diabetes Center, University of California, San Francisco, San Francisco, CA 94143, USA.

Stable Foxp3 expression is crucial for regulatory T (Treg) cell function. We observed that antigen-driven activation and inflammation in the CNS promoted Foxp3 instability selectively in the autoreactive Treg cells that expressed high amounts of Foxp3 before experimental autoimmune encephalitis induction. Treg cells with a demethylated Treg-cell-specific demethylated region in the Foxp3 locus downregulated Foxp3 transcription in the inflamed CNS during the induction phase of the response. Stable Foxp3 expression returned at the population level with the resolution of inflammation or was rescued by IL-2-anti-IL-2 complex treatment during the antigen priming phase. Thus, a subset of fully committed self-antigen-specific Treg cells lost Foxp3 expression during an inflammatory autoimmune response and might be involved in inadequate control of autoimmunity. These results have important implications for Treg cell therapies and give insights into the dynamics of the Treg cell network during autoreactive CD4(+) T cell effector responses in vivo.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.immuni.2013.10.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3912996PMC
November 2013

Origins and functional specialization of macrophages and of conventional and monocyte-derived dendritic cells in mouse skin.

Immunity 2013 Nov 31;39(5):925-38. Epub 2013 Oct 31.

Centre d'Immunologie de Marseille-Luminy (CIML), UM2 Aix-Marseille Université, 13288 Marseille Cedex 9, France; INSERM U1104, 13288 Marseille Cedex 9, France; CNRS UMR7280, 13288 Marseille Cedex 9, France.

In the skin, the lack of markers permitting the unambiguous identification of macrophages and of conventional and monocyte-derived dendritic cells (DCs) complicates understanding of their contribution to skin integrity and to immune responses. By combining CD64 and CCR2 staining, we successfully identified each of these cell types and studied their origin, transcriptomic signatures, and migratory and T cell stimulatory properties. We also analyzed the impact of microbiota on their development and their contribution to skin inflammation during contact hypersensitivity. Dermal macrophages had a unique scavenging role and were unable to migrate and activate T cells. Conventional dermal DCs excelled both at migrating and activating T cells. In the steady-state dermis, monocyte-derived DCs are continuously generated by extravasated Ly-6C(hi) monocytes. Their T cell stimulatory capacity combined with their poor migratory ability made them particularly suited to activate skin-tropic T cells. Therefore, a high degree of functional specialization occurs among the mononuclear phagocytes of the skin.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.immuni.2013.10.004DOI Listing
November 2013

Identification of a new stromal cell type involved in the regulation of inflamed B cell follicles.

PLoS Biol 2013 Oct 1;11(10):e1001672. Epub 2013 Oct 1.

Centre d'Immunologie de Marseille-Luminy (CIML), Aix-Marseille University, UM2, Marseille, France ; Institut National de la Santé et de la Recherche Médicale (INSERM), U1104, Marseille, France ; Centre National de la Recherche Scientifique (CNRS), UMR7280, Marseille, France.

Lymph node (LN) stromal cells provide survival signals and adhesive substrata to lymphocytes. During an immune response, B cell follicles enlarge, questioning how LN stromal cells manage these cellular demands. Herein, we used a murine fate mapping system to describe a new stromal cell type that resides in the T cell zone of resting LNs. We demonstrated that upon inflammation, B cell follicles progressively trespassed into the adjacent T cell zone and surrounded and converted these stromal cells into CXCL13 secreting cells that in return delineated the new boundaries of the growing follicle. Acute B cell ablation in inflamed LNs abolished CXCL13 secretion in these cells, while LT-β deficiency in B cells drastically affected this conversion. Altogether, we reveal the existence of a dormant stromal cell subset that can be functionally awakened by B cells to delineate the transient boundaries of their expanding territories upon inflammation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pbio.1001672DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3794863PMC
October 2013

TRAF6 is essential for maintenance of regulatory T cells that suppress Th2 type autoimmunity.

PLoS One 2013 13;8(9):e74639. Epub 2013 Sep 13.

Department of Microbiology and Immunology, Keio University School of Medicine, Tokyo, Japan.

Regulatory T cells (Tregs) maintain immune homeostasis by limiting inflammatory responses. TRAF6 plays a key role in the regulation of innate and adaptive immunity by mediating signals from various receptors including the T-cell receptor (TCR). T cell-specific deletion of TRAF6 has been shown to induce multiorgan inflammatory disease, but the role of TRAF6 in Tregs remains to be investigated. Here, we generated Treg-specific TRAF6-deficient mice using Foxp3-Cre and TRAF6-flox mice. Treg-specific TRAF6-deficient (cKO) mice developed allergic skin diseases, arthritis, lymphadenopathy and hyper IgE phenotypes. Although TRAF6-deficient Tregs possess similar in vitro suppression activity compared to wild-type Tregs, TRAF6-deficient Tregs did not suppress colitis in lymphopenic mice very efficiently due to reduced number of Foxp3-positive cells. In addition, the fraction of TRAF6-deficient Tregs was reduced compared with wild-type Tregs in female cKO mice without inflammation. Moreover, adoptive transfer of Foxp3 (+) Tregs into Rag2(-/-) mice revealed that TRAF6-deficient Tregs converted into Foxp3(-) cells more rapidly than WT Tregs under lymphopenic conditions. Fate-mapping analysis also revealed that conversion of Tregs from Foxp3(+) to Foxp3(-) (exFoxp3 cells) was accelerated in TRAF6-deficient Tregs. These data indicate that TRAF6 in Tregs plays important roles in the maintenance of Foxp3 in Tregs and in the suppression of pathogenic Th2 type conversion of Tregs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0074639PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3772853PMC
June 2014

Multicolor fate mapping of Langerhans cell homeostasis.

J Exp Med 2013 Aug 12;210(9):1657-64. Epub 2013 Aug 12.

Centre d'Immunologie de Marseille-Luminy (CIML), Aix-Marseille Université, Marseille, France.

Langerhans cells (LCs) constitute a network of immune sentinels in the skin epidermis that is seeded during embryogenesis. Whereas the development of LCs has been extensively studied, much less is known about the homeostatic renewal of adult LCs in "nonmanipulated" animals. Here, we present a new multicolor fluorescent fate mapping system and quantification approach to investigate adult LC homeostasis. This novel approach enables us to propose and provide evidence for a model in which the adult epidermal LC network is not formed by mature coequal LCs endowed with proliferative capabilities, but rather constituted by adjacent proliferative units composed of "dividing" LCs and their terminally differentiated daughter cells. Altogether, our results demonstrate the general utility of our novel fate-mapping system to follow cell population dynamics in vivo and to establish an alternative model for LC homeostasis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1084/jem.20130403DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3754858PMC
August 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood-2012-03-417923DOI Listing
September 2012

Plasticity of Foxp3(+) T cells reflects promiscuous Foxp3 expression in conventional T cells but not reprogramming of regulatory T cells.

Immunity 2012 Feb 9;36(2):262-75. Epub 2012 Feb 9.

Research Unit for Immune Homeostasis, RIKEN Research Center for Allergy and Immunology, Yokohama, Kanagawa 230-0045, Japan.

The emerging notion of environment-induced reprogramming of Foxp3(+) regulatory T (Treg) cells into helper T (Th) cells remains controversial. By genetic fate mapping or adoptive transfers, we have identified a minor population of nonregulatory Foxp3(+) T cells exhibiting promiscuous and transient Foxp3 expression, which gave rise to Foxp3(-) ("exFoxp3") Th cells and selectively accumulated in inflammatory cytokine milieus or in lymphopenic environments including those in early ontogeny. In contrast, Treg cells did not undergo reprogramming under those conditions irrespective of their thymic or peripheral origins. Moreover, although a few Treg cells transiently lose Foxp3 expression, such "latent" Treg cells retained their memory and robustly re-expressed Foxp3 and suppressive function upon activation. This study establishes that Treg cells constitute a stable cell lineage, whose committed state in a changing environment is ensured by DNA demethylation of the Foxp3 locus irrespectively of ongoing Foxp3 expression.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.immuni.2011.12.012DOI Listing
February 2012

Differential processing of self-antigens by subsets of thymic stromal cells.

Curr Opin Immunol 2012 Feb 30;24(1):99-104. Epub 2012 Jan 30.

Centre de Physiopathologie de Toulouse Purpan, Université Toulouse III Paul-Sabatier, Toulouse F-31300, France.

The stromal network of the thymus provides a unique environment that supports the development of mature CD4(+) and CD8(+) T cells expressing a very diverse repertoire of T cell receptors (TCR) with limited reactivity to self-antigens. Thymic cortical epithelial cells (cTECs) are specialized antigen-presenting cells (APCs) that promote the positive selection of developing thymocytes while medullary thymic epithelial cells (mTECs) and thymic dendritic cells (tDCs) induce central tolerance to self-antigens. Recent studies showed that cTECs express a unique set of proteases involved in the generation of self-peptides presented by major-histocompatibility encoded molecules (pMHC) and consequently may express a unique set of pMHC complexes. Conversely, the stromal cells of the medulla developed several mechanisms to mirror as closely as possible the constellation of self-peptides derived from peripheral tissues. Here, we discuss how these different features allow for the development of a highly diverse but poorly self-reactive repertoire of functional T cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.coi.2012.01.008DOI Listing
February 2012

CD64 expression distinguishes monocyte-derived and conventional dendritic cells and reveals their distinct role during intramuscular immunization.

J Immunol 2012 Feb 18;188(4):1751-60. Epub 2012 Jan 18.

Centre d'Immunologie de Marseille-Luminy, Université de la Méditerranée, Case 906, 13288 Marseille Cedex 9, France.

Although most vaccines are administered i.m., little is known about the dendritic cells (DCs) that are present within skeletal muscles. In this article, we show that expression of CD64, the high-affinity IgG receptor FcγRI, distinguishes conventional DCs from monocyte-derived DCs (Mo-DCs). By using such a discriminatory marker, we defined the distinct DC subsets that reside in skeletal muscles and identified their migratory counterparts in draining lymph nodes (LNs). We further used this capability to analyze the functional specialization that exists among muscle DCs. After i.m. administration of Ag adsorbed to alum, we showed that alum-injected muscles contained large numbers of conventional DCs that belong to the CD8α(+)- and CD11b(+)-type DCs. Both conventional DC types were capable of capturing Ag and of migrating to draining LNs, where they efficiently activated naive T cells. In alum-injected muscles, Mo-DCs were as numerous as conventional DCs, but only a small fraction migrated to draining LNs. Therefore, alum by itself poorly induces Mo-DCs to migrate to draining LNs. We showed that addition of small amounts of LPS to alum enhanced Mo-DC migration. Considering that migratory Mo-DCs had, on a per cell basis, a higher capacity to induce IFN-γ-producing T cells than conventional DCs, the addition of LPS to alum enhanced the overall immunogenicity of Ags presented by muscle-derived DCs. Therefore, a full understanding of the role of adjuvants during i.m. vaccination needs to take into account the heterogeneous migratory and functional behavior of muscle DCs and Mo-DCs revealed in this study.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4049/jimmunol.1102744DOI Listing
February 2012

Fate mapping analysis of lymphoid cells expressing the NKp46 cell surface receptor.

Proc Natl Acad Sci U S A 2011 Nov 21;108(45):18324-9. Epub 2011 Oct 21.

Centre d'Immunologie de Marseille-Luminy, Université de la Méditerranée UM 631, Campus de Luminy Case 906, 13288 Marseille, France.

NKp46 is a cell surface receptor expressed on natural killer (NK) cells, on a minute subset of T cells, and on a population of innate lymphoid cells that produce IL-22 and express the transcription factor retinoid-related orphan receptor (ROR)-γt, referred to as NK cell receptor (NKR)(+)ROR-γt(+) cells. Here we describe Nkp46(iCre) knock-in mice in which the gene encoding the improved Cre (iCre) recombinase was inserted into the Nkp46 locus. This mouse was used to noninvasively trace cells expressing NKp46 in vivo. Fate mapping experiments demonstrated the stable expression of NKp46 on NK cells and allowed a reappraisal of the sequential steps of NK cell maturation. NKp46 genetic tracing also showed that gut NKR(+)ROR-γt(+) and NK cells represent two distinct lineages. In addition, the genetic heterogeneity of liver NK cells was evidenced. Finally, Nkp46(iCre) mice also represent a unique mouse model of conditional mutagenesis specifically in NKp46(+) cells, paving the way for further developments in the biology of NKp46(+) NK, T, and NKR(+)ROR-γt(+) cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1112064108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3215049PMC
November 2011

Cutting edge: expression of XCR1 defines mouse lymphoid-tissue resident and migratory dendritic cells of the CD8α+ type.

J Immunol 2011 Nov 23;187(9):4411-5. Epub 2011 Sep 23.

Centre d'Immunologie Marseille-Luminy, Université de la Méditerranée, 13288 Marseille, France.

Subsets of dendritic cells (DCs) have been described according to their functions and anatomical locations. Conventional DC subsets are defined by reciprocal expression of CD11b and CD8α in lymphoid tissues (LT), and of CD11b and CD103 in non-LT (NLT). Spleen CD8α(+) and dermal CD103(+) DCs share a high efficiency for Ag cross-presentation and a developmental dependency on specific transcription factors. However, it is not known whether all NLT-derived CD103(+) DCs and LT-resident CD8α(+) DCs are similar despite their different anatomical locations. XCR1 was previously described as exclusively expressed on mouse spleen CD8α(+) DCs and human blood BDCA3(+) DCs. In this article, we showed that LT-resident CD8α(+) DCs and NLT-derived CD103(+) DCs specifically express XCR1 and are characterized by a unique transcriptional fingerprint, irrespective of their tissue of origin. Therefore, CD8α(+) DCs and CD103(+) DCs belong to a common DC subset which is unequivocally identified by XCR1 expression throughout the body.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4049/jimmunol.1101717DOI Listing
November 2011

The earliest intrathymic precursors of CD8α(+) thymic dendritic cells correspond to myeloid-type double-negative 1c cells.

Eur J Immunol 2011 Aug 4;41(8):2165-75. Epub 2011 Jul 4.

Centre d'Immunologie de Marseille-Luminy, Université de la Méditerranée, Marseille, France.

The dendritic cells (DCs) present in lymphoid and non-lymphoid organs are generated from progenitors with myeloid-restricted potential. However, in the thymus a major subset of DCs expressing CD8α and langerin (CD207) appears to stand apart from all other DCs in that it is thought to derive from progenitors with lymphoid potential. Using mice expressing a fluorescent reporter and a diphtheria toxin receptor under the control of the cd207 gene, we demonstrated that CD207(+) CD8α(+) thymic DCs do not share a common origin with T cells but originate from intrathymic precursors that express markers that are normally present on all (CD11c(+) and MHCII molecules) or on some (CD207, CD135, CD8α, CX3CR1) DC subsets. Those intrathymic myeloid-type precursors correspond to CD44(+) CD25(-) double-negative 1c (DN1c) cells and are continuously renewed from bone marrow-derived canonical DC precursors. In conclusion, our results demonstrate that the earliest intrathymic precursors of CD8α(+) thymic DCs correspond to myeloid-type DN1c cells and support the view that under physiological conditions myeloid-restricted progenitors generate the whole constellation of DCs present in the body including the thymus.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/eji.201141728DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4291128PMC
August 2011

Specific erythroid-lineage defect in mice conditionally deficient for Mediator subunit Med1.

Proc Natl Acad Sci U S A 2010 Dec 23;107(50):21541-6. Epub 2010 Nov 23.

Max-Planck-Institute of Immunobiology, 79108 Freiburg, Germany.

The Mediator complex forms the bridge between transcriptional activators and the RNA polymerase II. Med1 (also known as PBP or TRAP220) is a key component of Mediator that interacts with nuclear hormone receptors and GATA transcription factors. Here, we show dynamic recruitment of GATA-1, TFIIB, Mediator, and RNA polymerase II to the β-globin locus in induced mouse erythroid leukemia cells and in an erythropoietin-inducible hematopoietic progenitor cell line. Using Med1 conditional knockout mice, we demonstrate a specific block in erythroid development but not in myeloid or lymphoid development, highlighted by the complete absence of β-globin gene expression. Thus, Mediator subunit Med1 plays a pivotal role in erythroid development and in β-globin gene activation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1005794107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3003004PMC
December 2010