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Progenitors in Vitro + Cd11b + Mouse Dendritic Cells from Flt3

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Progenitors in Vitro + Cd11b + Mouse Dendritic Cells from Flt3 Progressive and Controlled Development of Mouse Dendritic Cells from Flt3 +CD11b+ Progenitors In Vitro This information is current as Thomas Hieronymus, Tatjana C. Gust, Ralf D. Kirsch, of October 2, 2021. Thorsten Jorgas, Gitta Blendinger, Mykola Goncharenko, Kamilla Supplitt, Stefan Rose-John, Albrecht M. Müller and Martin Zenke J Immunol 2005; 174:2552-2562; ; doi: 10.4049/jimmunol.174.5.2552 http://www.jimmunol.org/content/174/5/2552 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2005/02/23/174.5.2552.DC1 Material http://www.jimmunol.org/ References This article cites 58 articles, 30 of which you can access for free at: http://www.jimmunol.org/content/174/5/2552.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on October 2, 2021 • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Progressive and Controlled Development of Mouse Dendritic Cells from Flt3؉CD11b؉ Progenitors In Vitro1 Thomas Hieronymus,*† Tatjana C. Gust,† Ralf D. Kirsch,† Thorsten Jorgas,*† Gitta Blendinger,† Mykola Goncharenko,*† Kamilla Supplitt,* Stefan Rose-John,‡ Albrecht M. Mu¨ller,§ and Martin Zenke2*† Dendritic cells (DC) represent key regulators of the immune system, yet their development from hemopoietic precursors is poorly defined. In this study, we describe an in vitro system for amplification of a Flt3؉CD11b؉ progenitor from mouse bone marrow .with specific cytokines. Such progenitor cells develop into both CD11b؉ and CD11b؊ DC, and CD8␣؉ and CD8␣؊ DC in vivo Furthermore, with GM-CSF, these progenitors synchronously differentiated into fully functional DC in vitro. This two-step culture system yields homogeneous populations of Flt3؉CD11b؉ progenitor cells in high numbers and allows monitoring the consecutive steps of DC development in vitro under well-defined conditions. We used phenotypic and functional markers and transcriptional Downloaded from profiling by DNA microarrays to study the Flt3؉CD11b؉ progenitor and differentiated DC. We report here on an extensive analysis of the surface Ag expression of Flt3؉CD11b؉ progenitor cells and relate that to surface Ag expression of hemopoietic stem cells. Flt3؉CD11b؉ progenitors studied exhibit a broad overlap of surface Ags with stem cells and express several stem cell Ags ؉ ؉ ␣ such as Flt3, IL-6R, c-kit/SCF receptor, and CD93/AA4.1, CD133/AC133, and CD49f/integrin 6. Thus, Flt3 CD11b progenitors express several stem cell surface Ags and develop into both CD11b؉ and CD11b؊ DC, and CD8␣؉ and CD8␣؊ DC in vivo, and thus into both of the main conventional DC subtypes. The Journal of Immunology, 2005, 174: 2552–2562. http://www.jimmunol.org/ endritic cells (DC)3 are the most professional APC of Different DC subsets have been identified both in lymphoid and the immune system and represent key components for nonlymphoid organs, but their relationship and developmental or- D induction of primary immune responses and mainte- igins remain unclear or highly controversial (4–7). It has been nance of immunological tolerance (1–3). DC originate from he- proposed that mature DC originate from both myeloid and lym- mopoietic stem cells in bone marrow through consecutive steps phoid-committed precursors. In mouse, CD11cϩCD11bϩ DC rep- of differentiation, which can be distinguished by phenotype, resent the classical myeloid tissue DC that are found in almost all localization, and function. Four steps of DC development can tissues. CD8␣ expression was used to subdivide DC subsets (4–6, be delineated: bone marrow progenitor cells develop into cir- 8), but does not delineate DC origin (9–11). Plasmacytoid DC by guest on October 2, 2021 culating precursor DC in blood, which enter tissues where they (pDC) represent yet another DC subclass, which was initially char- reside as sentinel immature DC and encounter pathogens. Fol- acterized by production of large amounts of type 1 IFN in response lowing Ag uptake and activation by inflammatory signals, im- to virus and bacteria (12, 13). mature DC undergo a process of maturation, which is tightly Recent studies on gene knockout mice revealed novel insights linked with their migration to secondary lymphoid organs, into how DC develop. RelBϪ/Ϫ mice lack CD11cϩCD11bϩ DC, where they initiate primary immune responses (1, 2). whereas other DC subsets are apparently normal (14–16). Mice deficient for IFN consensus sequence binding protein lack pDC (17). Id2Ϫ/Ϫ mice lack Langerhans cells, the cutaneous contingent of DC, and also the CD8␣ϩ splenic DC subset (18, 19). B cells and *Institute for Biomedical Engineering—Cell Biology, University Medical School Rheinisch-Westfa¨lische Technische Hochschule Aachen, Aachen, Germany; †Max pDC were increased in these mice (18, 20). These studies suggest Delbru¨ck Center for Molecular Medicine, Berlin, Germany; ‡Department of Bio- that specific DC subsets develop through independent pathways. chemistry, Christian Albrechts University, Kiel, Germany; and §Institute of Medical ϩ Radiation and Cell Research, University of Wurzburg, Wurzburg, Germany Similarly, a bone marrow Flt3 precursor population contains early DC precursors for all DC subsets and comprises Flt3ϩ com- Received for publication January 12, 2004. Accepted for publication December ϩ 8, 2004. mon lymphoid precursors (CLP) and Flt3 common myeloid pre- The costs of publication of this article were defrayed in part by the payment of page cursors (CMP; Ref. 7). charges. This article must therefore be hereby marked advertisement in accordance A large number of protocols have been developed for generating with 18 U.S.C. Section 1734 solely to indicate this fact. DC in vitro. Frequently, mouse DC are obtained in vitro from bone 1 This work was supported by grants of the Fond der Chemischen Industrie and the marrow based on the protocol initially described by Inaba et al. German Research Foundation (Deutsche Forschungsgemeinschaft Ze432/1 and Ze432/2) (to M.Z.). T.H. received a Max Delbru¨ck Center for Molecular Medicine (21, 22) using GM-CSF. Flt3 ligand (FL) was observed to increase postdoctoral fellowship. DC numbers in vivo and in vitro, and this relates well to Flt3 2 Address correspondence and reprint requests to Dr. Martin Zenke, University Med- expression on early DC precursor in mouse bone marrow (Ref. 7 ical School Rheinisch-Westfa¨lische Technische Hochschule Aachen, Institute for Bio- medical Engineering—Cell Biology, Pauwelsstrasse 30, 52074 Aachen, Germany. and references therein, 23–26). Furthermore, FL and GM-CSF E-mail address: [email protected] were demonstrated to differentially regulate development of ϩ ϩ 3 Abbreviations used in this paper: DC, dendritic cell; pDC, plasmacytoid DC; CLP, CD11c CD11b DC and pDC, respectively (25). common lymphoid precursor; CMP, common myeloid precursor; FL, Flt3 ligand; A variety of cytokines and growth factors have been reported to SCF, stem cell factor; IGF-1, insulin-like growth factor 1; hyper-IL-6, IL-6/soluble IL-6R fusion protein; ODN, oligodeoxynucleotide; MOI, multiplicity of infection; efficiently expand hemopoietic progenitors in vitro, including stem MGC-24, multi-glycosylated core protein 24. cell factor (SCF), FL, insulin-like growth factor 1 (IGF-1), IL-3, Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 The Journal of Immunology 2553 IL-6 and soluble IL-6R fusion protein (hyper-IL-6), and different Cell Biology and Genetics, Dresden, Germany). Cells were analyzed by combinations thereof (Refs. 27–32, and references therein). DC FACSCalibur flow cytometer using CellQuest software (BD Biosciences). precursors were first amplified by SCF, FL, Tpo, and hyper-IL-6, Cytokine production in response to LPS, CpG and then induced to differentiate into DC by GM-CSF and IL-4 oligodeoxynucleotide (ODN), and viruses (18, 32). Using this culture system, fully functional DC were ob- ␣ tained and used to monitor the changes in gene expression during For analyzing IL-12 and IFN- production in response to LPS, CpG ODN, or viruses, 1 ϫ 105 DC/ml (day 10 of differentiation) were stimulated with DC development by DNA microarrays (18, 32). either LPS (1 ␮g/ml), CpG ODN 1668 (100 ng/ml), influenza virus (mul- In this study, we have extended these studies to mouse DC and tiplicity of infection (MOI), 1 and 10), and HSV (MOI, 1 and 3), and developed a two-step amplification/differentiation system to study supernatants were collected after 24-h incubation. ELISA for detection of ϩ ϩ ␣ a Flt3 CD11b progenitor from mouse bone marrow. This pro- IFN- (PBL Biomedical Laboratories) and IL-12 p70 (eBiosciences) was performed according to the manufacturer’s instructions. genitor was first amplified in vitro under the aegis of a specific stem cell cytokine/growth factor mixture and then differentiated Chemotaxis assay into DC with GM-CSF. By transcriptional profiling, we identified Chemotaxis assay was performed as described by Kellermann et al. (38) several stem cell marker genes including Flt3, IL-6R, c-kit/SCF with minor modifications (35). Briefly, Transwell inserts (5-␮m pore size; receptor, and the stem cell Ags CD93/AA4.1 and CD133/AC133, Costar) were preincubated in medium to block unspecific binding, and 2 ϫ which are expressed on Flt3ϩCD11bϩ progenitors, and their ex- 105 cells were seeded in the upper compartment.
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