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 September 29, 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 Downloaded from http://www.jimmunol.org/content/174/5/2552

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

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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 http://www.jimmunol.org/ thus into both of the main conventional DC subtypes. The Journal of Immunology, 2005, 174: 2552–2562.

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, by guest on September 29, 2021 be delineated: bone marrow progenitor cells develop into cir- 8), but does not delineate DC origin (9–11). Plasmacytoid DC 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,

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. ELC chemokine (100 pression declines during DC differentiation. ng/ml) was added to the lower compartment to analyze migration toward gradient. After 90 min, 1 ϫ 104 Dynabeads (15-␮m diameter; Dynal Poly- mers) were added to the lower chamber to allow normalization for varia- Materials and Methods tions in the experimental procedure. Cells and beads were recovered, and Downloaded from Cells and cell culture samples were reacted with fluorescein-di-acetate to stain for living cells and analyzed by flow cytometry. By gating on beads, the ratio of beads to ϩ ϩ For generation of Flt3 CD11b progenitor cells and DC, bone marrow fluorescein-di-acetate-positive cells was determined and allowed a precise suspensions were prepared from C57BL/6 and BALB/c mice (Charles calculation of the number of transmigrated cells. River). Cells were seeded at 2 ϫ 106 cells/ml in RPMI 1640 medium supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin/ In vitro stimulation of TCR transgenic T cells streptomycin (all from Invitrogen Life Technologies), and 50 ␮M 2-ME ϩ CD8 T cells from OT-I mice express a transgenic TCR that recognizes http://www.jimmunol.org/ (Sigma-Aldrich) containing recombinant murine SCF (100 ng/ml), 25 b OVA257–264 peptide on H-2K (39, 40). Splenocytes of OT-I mice were ng/ml FL (PeproTech), 40 ng/ml recombinant long-range IGF-1 (Sigma- ϩ prepared and CD8 T cells were obtained by immunomagnetic bead pu- Aldrich), 5 ng/ml rIL-6/soluble IL-6R fusion protein (hyper-IL-6; Ref. 33), Ϫ6 rification using MACS anti-CD8 microbeads (Miltenyi Biotec) according 20 U/ml recombinant mouse GM-CSF, and 10 M dexamethasone. After ϩ to manufacturer’s instructions. CD4 T cells from DO11.10 mice express- 3 days in culture, cells were subjected to Ficoll-Hypaque density gradient ing a transgenic TCR that recognizes OVA peptide on I-Ad (41) centrifugation (density, 1.077 g/ml; Eurobio) to remove residual erythro- 323–339 were prepared accordingly using anti-CD4 microbeads (Miltenyi Biotec) cytes, dead cells, and debris. Medium with growth factors was replenished ϫ 6 for purification. every 2 days, and cells were maintained at 2 10 cells/ml cell density. ␮ Cells were pulsed with 0.1 M OVA257–264 peptide (SINNFEKL) for After 7 days, differentiation into DC was induced in culture medium sup- ␮ MHC class I or 0.1 M OVA323–339 peptide (ISQAVHAAHAEINEAGR) plemented with 200 U/ml recombinant mouse GM-CSF. Every 2 days, ϩ ϩ 6 for MHC class II-restricted T cell stimulation. CD8 OT-I T cells or CD4 medium was replenished, and cells were maintained at 2 ϫ 10 cells/ml by guest on September 29, 2021 DO11.10 T cells were then cocultured with irradiated APC (30 Gy) in cell density. Alternatively, DC were generated from mouse bone marrow 96-well microtiter plates. Untreated DC were used as control. At day 4 of using the protocol described by Inaba et al. (21, 34, 35). coculture, 0.75 ␮Ci/well [3H]thymidine (Amersham Biosciences) was In some experiments, 100 ng/ml TNF-␣ or 1 ␮g/ml LPS was added to added per well, cells were harvested 6 h later, and [3H]thymidine incor- induce DC maturation. Cell numbers were determined with an electronic poration was measured. cell counter device (CASY1; Scha¨rfe Systems). Hemopoietic stem cells from bone marrow of 8- to 12-wk-old NRMI CFSE labeling of OT-I splenocytes mice were isolated by depletion of lineage-positive cells using immunomagnetic beads (Miltenyi Biotec) and mAbs against B220 (RA3-6B2), Fluorescence labeling of OT-I splenocytes with CFSE (Molecular Probes) Mac-1 (M1/70), Gr-1 (RB6-8C5), CD4 (GK1.5), CD8 (53-6.72), and TER- was performed essentially as described (42). In brief, single-cell suspen- 119 (all Abs from BD Biosciences). Lineage-negative cells were then sions of splenocytes were isolated from OT-I mice and erythrocytes were sorted with a FACSVantage device (BD Biosciences) for c-kit (ACK45) lysed in hypotonic buffer. Cells were resuspended in PBS at 1 ϫ 107 and Sca-1 (E13-161.7) into linϪc-kitϩSca-1Ϫ and linϪc-kitϩSca-1ϩ popu- cells/ml, and labeling was performed with 10 ␮M CFSE for 10 min at lations, and linϪc-kitϩSca-1ϩ cells were used for further analysis. 37°C. Labeling was stopped by adding 1 vol of cell culture medium, and cells were washed an additional two times in medium. Cell proliferation assay Adoptive transfer and in vivo activation of OT-I T cells A total of 5 ϫ 104 cells were incubated in 200 ␮l of medium in a 96-well flat-bottom microtiter plate for 72 h at 37°C with the indicated growth A total of 5 ϫ 107 CFSE-labeled OT-I spleen cells in 500 ␮l of PBS was factors and combinations thereof. Samples were then pulsed for 2 h with injected into the tail vein of sex-matched C57BL/6 recipients. One day 3 ϫ 6 ␮ 0.75 ␮Ci/well [ H]thymidine (29 Ci/mmol; Amersham Biosciences) and later, 5 10 untreated DC, DC pulsed with 0.1 M OVA257–264 peptide harvested onto glass fiber filters. Radioactivity was measured by liquid (SINNFEKL) for 30 min, or PBS only were applied to recipient mice by scintillation counting in a Microbeta counter (Wallac). i.v. injection. As a positive control for effective activation, OVA protein in IFA was injected s.c. Three days later, single-cell suspensions were pre- Flow cytometry pared from spleen of recipients and stained with PE-labeled anti-V␤5.1/2 TCR (MR9-4) and biotinylated anti-V␣2 TCR (B20.1; both from BD Bio- Flow cytometry analysis for surface Ag expression was performed as pre- sciences) followed by streptavidin-TRI-COLOR staining. viously described (35) using the following Abs for staining and sorting: FITC-conjugated anti-MHC class II (I-A/I-E; clone 2G9), CD93-FITC Adoptive transfer and in vivo differentiation of Flt3ϩCD11bϩ (AA4.1), CD11c-PE (HL3), CD25-PE (3C7), CD11b-bio (M1/70), CD49f progenitors (GoH3), CD54 (3E2), CD80 (1G10), CD135/Flt3-PE (A2F10.1), and respective isotype controls (all purchased from BD Biosciences). CD8␣-TRI- Flt3ϩCD11bϩ progenitors were generated from C57BL/6J-Ly5.1 as de- COLOR (CT-CD8a), CD40-bio (2/23), and CD86 (RMMP2), isotype- scribed above. Amplified progenitors were i.v. injected at a ratio of 1:1 or matched FITC- or PE-labeled secondary Abs, streptavidin-FITC and 4:1 into lethally irradiated (5 Gy twice with a 3-h interval) C57BL Ly5.2 streptavidin-TRI-COLOR were from Caltag Laboratories. Anti-mouse recipients, along with 1 ϫ 106 unfractionated Ly5.2 bone marrow cells to CD83 (clone Michel 17) was obtained from Biocarta. Anti-mouse CXCR4/ ensure mouse survival (43). Alternatively, Flt3ϩCD11bϩ progenitors were CD184 Ab was a kind gift from R. Foerster (Hannover Medical School, cultured without FL for 16 h and sorted for Flt3 and CD11b into Hannover, Germany). Anti-prominin/AC133 Ab (36, 37) was kindly pro- Flt3ϩCD11bϩ and Flt3ϪCD11bϩ cells using a FACSVantage device. vided by W. Huttner and D. Corbeil (Max Planck Institute of Molecular Sorted cell populations were then i.v. injected at a ratio of 1:1 along with 2554 DC FROM Flt3ϩCD11bϩ PROGENITORS IN VITRO

1 ϫ 106 unfractionated Ly5.2 bone marrow cells. At various times after cell ous differentiation (T. Jorgas, T. Hieronymus, and M. Zenke, un- transfer, single-cell suspensions were prepared from spleen of recipients, published data) as observed in other cell systems (29, 48, 49). and three-color staining with FITC-labeled anti-Ly5.1 (A20; BD Bio- Total number of cells had increased by ϳ100-fold within 3 wk sciences) and CD11c-PE in combination with CD8␣ or CD11b was performed to examine the generation of donor-derived DC. Donor-derived of culture (Fig. 1B). Cells retained their capacity to respond to hemopoietic lineage cells were distinguished by FITC-labeled anti-Ly5.1 growth factors (either individually or in combination) with time in in combination with CD11b-bio (M1/70), Gr-1-bio (RB6-8C5), CD19 culture (data not shown). Amplified progenitor cells were spherical (1D3), and CD3⑀-bio (145-2C11; all from BD Biosciences). Three recip- (Fig. 1C) and expressed Flt3/CD135 and CD11b (Fig. 1D and see ient mice were used for each setting in individual experiments. Fig. 4A). Surface Flt3 expression on cultured cells was not de- RNA isolation and DNA microarray analysis tected in FL-supplemented medium, presumably due to receptor RNA isolation, cDNA and cRNA synthesis, DNA microarray hybridization internalization in response to ligand, but was readily observed in and analysis for DC and Flt3ϩCD11bϩ progenitor cells were conducted the absence of FL. Cells were negative for lineage markers such as essentially as described before (18, 32). In brief, total RNA was isolated CD3, CD4, CD8, CD11c, CD14, CD34, B220, and Ter119 (see Fig. with RNeasy kits including DNase digestion (Qiagen). Five micrograms of 6, A and D, and data not shown). Therefore, we will in the fol- total RNA was used to generate cDNA according to the Expression Anal- lowing refer to these precursor cells as Flt3ϩCD11bϩ progenitors. ysis Technical Manual (Affymetrix). cRNA was generated with the Bio- ϩ ϩ Array High-Yield Transcript Labeling kit (ENZO). Total cellular RNA To address the question of the frequency of the Flt3 CD11b from a total of 75,000 linϪc-kitϩSca-1ϩ stem cells per experiment was progenitors in vivo, we analyzed isolated bone marrow cells for isolated with RNeasy Mini kit (Qiagen). In vitro transcription-based RNA expression of Flt3/CD135 and CD11b (Fig. 1E). Flt3 expression amplification was performed essentially as described (44), using the Am- was found on 3.8 Ϯ 0.9% (mean value Ϯ SEM; n ϭ 5) of total bion Megascript T7 kit for two rounds of in vitro transcription reaction. ␮ bone marrow in accordance with previous results (11). Impor- Downloaded from Finally, 10 g of cRNA per sample was hybridized to Affymetrix mouse ϩ MG-U74A arrays at 45°C for 16 h. DNA chips were stained, washed, and tantly, 5.4% of the CD11b cells (ranging from 2.8 to 8.6%; n ϭ ϩ ϩ scanned according to the manufacturer’s protocol. 5) coexpressed Flt3, demonstrating that the Flt3 CD11b progen- ϳ Bioinformatics itors are present in bone marrow with a frequency of 2–2.5%. Thus, the amplification rates achieved during in vitro culture are Scanned GeneChip .DAT files were analyzed by the Affymetrix Microar- ϳ4000- to 5000-fold corresponding to ϳ12 cell division cycles. ray Suite analysis software. Normalization and conversion of relative expression intensities into log ratios was performed with GeneSpring soft- http://www.jimmunol.org/ ware (Silicon Genetics) as described (18). The normalization procedure in Phenotypic and functional characterization of DC derived from GeneSpring converts negative values to 0. For further analysis, genes were ϩ ϩ filtered for the criteria to appear as abundant transcripts (expression call amplified Flt3 CD11b progenitors “present” by the Affymetrix Microarray Suite analysis software) in at least Flt3ϩCD11bϩ progenitors were then induced to differentiate into one sample per experiment. Classification of genes encoding for mouse CD molecules was done using information from various databases including DC by replacing growth-promoting factors (day 7) with a high Entrez PubMed (͗www.ncbi.nlm.nih.gov/entrez/query.fcgi͘), the mouse dose of GM-CSF only (Fig. 1B), thereby using conditions that are genome informatics database (͗www.informatics.jax.org/͘), and the PROW frequently used for bone marrow-derived DC (21, 34, 35). Fol- database for CD molecules (͗www.ncbi.nlm.nih.gov/prow/͘). Hierarchical lowing induction of differentiation, cells underwent two to three clustering (45) for those filtered CD molecules was done in GeneSpring additional cell division cycles (days 7–11), which led to a ϳ34- by guest on September 29, 2021 using the Pearson correlation with a separation ratio of 0.5 and a minimum ϭ distance of 0.001. Microarray data were submitted to GEO database fold increase in total cell numbers (n 8; ranging from 13.5- to (͗www.ncbi.nlm.nih.gov/geo/͘; series accession nos. GSE575, GSE692, 37-fold). At days 11–12, cells ceased proliferation and concomi- and GSE693). tantly acquired prominent dendritic processes and cytoplasmic projections that represent hallmarks of DC phenotype (Fig. 1C). Results ϩ ϩ Using this two-step culture system, we were able to obtain 0.7– In vitro growth of Flt3 CD11b progenitors from bone marrow 2.1 ϫ 109 DC from one bone marrow preparation. Mouse bone marrow cells were cultured for up to 3 wk in the DC development was monitored by assessing the expression presence of FL, SCF, IGF-1, hyper-IL-6, dexamethasone, and low profile of specific cell surface proteins by flow cytometry. Expres- concentrations of GM-CSF (Fig. 1, A and B). These factors were sion of MHC class II and CD11c, two commonly used cell surface chosen based on previous studies demonstrating expression of the Ags of mouse DC, was measured at various time points throughout respective receptors on hemopoietic linϪc-kitϩSca-1ϩ stem cells differentiation. As shown in Fig. 2, Flt3ϩCD11bϩ progenitor cells and DC precursor cells (Refs. 46 and 47; R. D. Kirsch, T. Hiero- expressed neither MHC class II nor CD11c. Importantly, expres- nymus, and M. Zenke, unpublished data; see also Fig. 5A). Next, sion of these cell surface proteins increased during DC develop- we quantitatively assessed the activity of FL, SCF, IGF-1, hyper- ment and, within 8–9 days of differentiation, resulted in a homog- IL-6, and GM-CSF on progenitor growth by using unfractionated enous population of CD11cϩ and MHC class IIϩ cells. To further bone marrow and established progenitor cells (day 7 of culture) characterize the progenitor population and the DC obtained, cells and by measuring DNA synthesis in response to factors. When were examined for expression of a variety of cell surface mole- individual factors were applied, SCF, FL, and GM-CSF were cules known to be involved in DC function. We found the found to be most effective (Fig. 1A). The activities of hyper-IL-6 Flt3ϩCD11bϩ progenitor cells to be negative for CD40, CD54, and IGF-1 were less pronounced individually, yet these factors CD80, CD86, and the DC activation marker CD25. Interestingly, exhibited synergistic effects when applied in various combinations. these molecules were also absent or expressed at low levels on FL together with either SCF or hyper-IL-6 plus IGF-1 clearly en- differentiated DC, indicating that the DC obtained are immature hanced proliferation rates. A maximal response on bone marrow (Figs. 1–3 and Table I). To induce DC maturation, cells were cells was observed when all factors were applied (Fig. 1A). This treated for 16 h with either 100 ng/ml TNF-␣ or 1 ␮g/ml LPS, and was different for established cultures where addition of GM-CSF expression of surface Ags was assessed. As expected, both stimuli decreased proliferation rates, presumably because GM-CSF down- effectively up-regulated the expression of MHC class II, CD25, regulates Flt3 and IGF-1 receptors (see Fig. 5, cluster IV, and data CD40, CD54, CD80, CD83, and CD86 on DC (Figs. 2 and 7, not shown). In addition, we noticed that an efficient outgrowth of Table I, and data not shown). Thus, with the differentiation con- progenitor cells from bone marrow was critically dependent on ditions applied, we consecutively generated homogenous popula- dexamethasone, which significantly reduced the rate of spontane- tions of immature and mature DC. The Journal of Immunology 2555 Downloaded from http://www.jimmunol.org/

FIGURE 2. Time course of MHC class II and CD11c surface expression on differentiating DC from Flt3ϩCD11bϩ progenitor cells. After 7 days of by guest on September 29, 2021 culture, Flt3ϩCD11bϩ progenitors were induced to differentiate into DC by applying GM-CSF (day 0). The kinetics of MHC class II and CD11c surface expression by ﬂow cytometry is shown (days 0–11). Cells treated with TNF-␣ at day 10 for an additional 24 h are also shown (day 11ϩTNF-␣). Open areas represent staining with isotype control Ab.

Cytokine and chemotactic responses of in vitro-differentiated DC In vitro-differentiated DC responded to various pathogen-associated molecules (LPS and CpG ODN) or inﬂuenza virus (Flu) and HSV (Fig. 3A). DC effectively produced IL-12p70 in response to LPS, whereas the response to CpG ODN was less pronounced. There was

in regular time intervals after Ficoll density gradient purification of progenitor cells at day 3 of culture. Results are mean values Ϯ SD of five and eight different experiments for growth of progenitor cells and differentiation of DC, respectively. The total number of cells increased ϳ2-fold within the first 3 days under growing conditions, indicating effective response to the factor combination used (mean value Ϯ SEM, 2.0 Ϯ 0.1; n ϭ FIGURE 1. Growth of bone marrow-derived Flt3ϩCD11bϩ progenitor 20). C, Morphology of Flt3ϩCD11bϩ progenitor cells at day 7 (inset) and cells and their differentiation into DC. A, Growth factor response of bone DC at day 17 of culture (day 10 differentiation condition) by phase contrast marrow cells (Ⅺ) and established progenitor cells at day 7 of culture (f). microscopy on fibronectin-coated coverslips. Bars represent 10 ␮m. D, Factors were applied to cells as indicated, and [3H]thymidine incorporation Flt3ϩCD11bϩ progenitor cells (day 7) were cultured with FL (ϩFL) or was determined 72 h later. Results (mean triplicate values Ϯ SD) from one without FL (ϪFL) for 6 and 16 h as indicated, and cells were analyzed for representative of three individual experiments are shown. B, Growth ki- Flt3/CD135 and CD11b surface expression by flow cytometry. Open areas netics of Flt3ϩCD11bϩ progenitor cells (E) and differentiated DC (f) represent staining with isotype control Ab. E, Flt3/CD135 and CD11b sur- from bone marrow. Cells were cultivated as described in Materials and face expression on isolated bone marrow cells was analyzed by flow cy- Methods. Differentiation of DC from Flt3ϩCD11bϩ progenitor cells was tometry. Proportion of cells in the quadrants is shown in percentages. Data induced at day 7 of cell culture. Cumulative cell numbers were determined are representative of five experiments. 2556 DC FROM Flt3ϩCD11bϩ PROGENITORS IN VITRO Downloaded from http://www.jimmunol.org/

FIGURE 3. Cytokine and chemotactic responses of DC and their Ag presentation capacity in vitro and in vivo. A, IL-12 and IFN-␣ production of DC in response to LPS (1 ␮g/ml), CpG ODN 1668 (100 ng/ml), or influenza virus (Flu) and HSV was determined by ELISA. For virus infection, an MOI of by guest on September 29, 2021 1, 3, and 10 was used as indicated. B, Migratory response to ELC chemokine was analyzed in Transwell assays (see Materials and Methods). Flt3ϩCD11bϩ progenitor cells were first cultivated for 7 days under growing conditions (ϭ day 0 of DC differentiation). DC were differentiated for the indicated time, and TNF-␣ stimulation (100 ng/ml) was performed after 9 days in culture for an additional 24 h. Values shown are means Ϯ SD for the indicated number of experiments. C and D, Ag-specific activation in vitro of OT-I CD8ϩ T cells (C) and DO11.10 CD4ϩ T cells (D) by OVA peptide-pulsed DC was analyzed by [3H]thymidine incorporation in DC/T cell cocultures. Peptide-pulsed DC in the presence (f) or absence (Ⅺ) of TNF-␣ (100 ng/ml) cocultured with T cells in various ratios were analyzed (upper panels). The lower panels show the Ag-specific T cell activation (10:1 T cell:APC ratio) of DC obtained by in vitro differentiation from Flt3ϩCD11bϩ progenitors (two-step DC) and of DC obtained with GM-CSF from bone marrow cell (one-step DC; Refs. 21, 34, and 35). E, Ag-specific activation of OT-I T cells in vivo. CFSE-labeled OT-I cells were injected into C57BL/6 recipient mice. One day later, DC pulsed with 0.1 ␮M OVA peptide, or PBS only were applied to recipient mice by i.v. injection. DC obtained from Flt3ϩCD11bϩ progenitors (two-step DC) or from bone marrow cells (one-step DC) were used as in C and D. Untreated DC were used as a negative control. As a positive control, 200 ␮g OVA protein in IFA was injected. Three days later, CFSE fluorescence was determined by flow cytometry on V␣2 and V␤5 double-positive T cells. One representative result of four individual experiments is shown. Open areas represent endogenous CFSE-negative V␣2 and V␤5 double-positive T cells of the negative control mice.

no IL-12p70 production in response to Flu and HSV viruses. How- In vitro-generated DC are fully active in Ag-specific T cell ever, these viruses induced production of IFN-␣ (Fig. 3A). activation in vitro and in vivo DC express chemokine receptors and respond to various che- One of the major hallmarks of DC is their potent capacity to ac- mokines (1, 2). CCR7 represents a chemokine receptor with a pivotal tivate naive T cells in an Ag-dependent manner. We therefore de- role in DC migration toward secondary lymphoid organs. Therefore, termined the Ag-specific MHC class I- and II-restricted T cell Transwell experiments were performed to evaluate the migratory ca- stimulation capacity of the Flt3ϩCD11bϩ progenitor cell-derived pacity of DC in response to CCR7 ligand ELC at different stages of DC in vitro and in vivo. Chicken OVA and Ag-specific CD8ϩ and differentiation. DC at day 10 of differentiation were highly active in ϩ migration toward ELC, whereas Flt3ϩCD11bϩ progenitor cells were CD4 T cells of TCR transgenic OT-I and DO11.10 mice, respec- as expected inactive (Fig. 3B). Stimulation of DC with TNF-␣ re- tively, were used. OT-I T cells recognize OVA257–264 peptide in b sulted in an increased number of transmigrated cells both in the pres- context of MHC class I H-2-K , whereas DO11.10 T cells detect d ence and absence of ELC (data not shown). DC at day 7 of differen- MHC class II I-A -presented OVA323–339 peptide. First, syngeneic tiation showed only a limited capacity to migrate in response to ELC, DC were activated with TNF-␣ for 16 h, or left untreated. Cells indicating the immature phenotype at this stage of DC differentiation were then pulsed with the respective peptides and analyzed in co- ϩ ϩ (Fig. 3B). cultures with CD8 or CD4 T cells of TCR transgenic mice for The Journal of Immunology 2557

Table I. Mean fluorescence intensities (MFI) of selected surface Ags in ferentiation is highly synchronous and yields homogenous DC ϩ ϩ development of DC from Flt3 CD11b progenitor cellsa populations of the same maturation state. Second, the capability of DC to prime naive T cells in vivo was Surface Ags Progenitor DC DCϩTNF-␣ analyzed. CFSE-labeled OT-I T cells were transferred into synge- CD11c (n ϭ 9) 4.8 Ϯ 1.8 473 Ϯ 190 470 Ϯ 164 neic wild-type recipient mice and immunized 1 day later with un- MHC II (n ϭ 9) 4.7 Ϯ 1.4 284 Ϯ 51 1380 Ϯ 361 pulsed or OVA peptide-pulsed DC. Three days later, splenocytes CD25 (n ϭ 8) 5.0 Ϯ 2.3 32 Ϯ 4 138 Ϯ 25 were examined for proliferative response of transferred OT-I T CD40 (n ϭ 4) 4.8 Ϯ 1.9 27 Ϯ 6 226 Ϯ 147 cells by flow cytometry. Immunization with OVA peptide-pulsed CD54 (n ϭ 4) 18.0 Ϯ 799Ϯ 59 399 Ϯ 160 ϭ Ϯ Ϯ Ϯ DC effectively stimulated T cell proliferation as demonstrated by CD80 (n 6) 31.0 77637 837 264 Ϯ CD86 (n ϭ 6) 31.0 Ϯ 14 109 Ϯ 40 1301 Ϯ 588 a shift of CFSE-positive transgenic T cells (Fig. 3E) and a 1.5 Iso control I (n ϭ 9) 4.8 Ϯ 1.4 11 Ϯ 311Ϯ 3 0.2-fold increase in total cell number compared with control (mean Iso control II (n ϭ 6) 31.0 Ϯ 17 35 Ϯ 17 53 Ϯ 9 value Ϯ SEM of four independent experiments). This result is a Progenitors were grown for 7 days and differentiated into DC and stimulated similar to the effective stimulation of T cell proliferation using with TNF-␣ at day 10 of differentiation for 16 h (DC ϩ TNF-␣) or left untreated OVA-protein with Freund’s adjuvant (mean value Ϯ SEM, 1.9 Ϯ (DC). Cells were stained for the indicated surface Ags using either directly labeled 0.2; n ϭ 4). In addition, the potency for in vivo T cell stimulation mAbs or indirect staining as described in Materials and Methods. MFI of directly ϩ ϩ labeled (Iso control I) and indirectly stained (Iso control II) isotype control are shown. of DC generated from Flt3 CD11b progenitors was similar to Values represent mean MFI Ϯ SD from (n) independent experiments. that of DC obtained with GM-CSF from bone marrow (Fig. 3E) using the frequently used protocol by Inaba et al. (21, 34, 35). This ϩ ϩ result clearly demonstrate the potency of the Flt3 CD11b pro- Downloaded from Ag-specific T cell proliferation. DC effectively stimulated prolif- genitor cell-derived DC to prime naive T cells in vivo. eration of naive T cells in vitro, which was further enhanced by ϩ ϩ TNF-␣ treatment of DC, whereas progenitor cells and unpulsed Flt3 CD11b progenitor cells differentiate into DC in vivo DC were inactive (Fig. 3, C and D). DC obtained by the frequently Finally, we addressed the question of whether the in vitro-ampli- used one-step culture system with GM-CSF by Inaba et al. (21) fied Flt3ϩCD11bϩ progenitors have the capacity to differentiate ϩ ϩ

were analyzed in parallel and found to be 2- to 4-fold less efﬁcient into DC in vivo. Flt3 CD11b progenitor cells from Ly5.1 mice http://www.jimmunol.org/ in T cell activation than the DC generated by the two-step culture were generated and transferred into lethally irradiated Ly5.2 re- system described in this paper (Fig. 3, C and D, lower panels). cipient mice either as bulk cultures or as sorted populations of This might be because with the two-step culture system DC dif- Flt3ϩCD11bϩ or Flt3ϪCD11bϩ cells (Fig. 4, A and B). After 1 and

FIGURE 4. Generation of DC by guest on September 29, 2021 from Flt3ϩCD11bϩ progenitor cells in vivo. A, In vitro-ampliﬁed Flt3ϩCD11bϩ progenitors from Ly5.1 mice were sorted for Flt3 and CD11b expression into Flt3ϩCD11bϩ and Flt3ϪCD11bϩ cells and used for adoptive transfer experiments (B). B, Un- fractionated Flt3ϩCD11bϩ progenitor cells and sorted cell populations (A) were transferred i.v. into lethally irradiated Ly5.2 recipient mice. Single- cell suspensions from spleen of recipient mice were analyzed for the presence of donor-derived CD11cϩ DC 1 wk after transfer. Donor-derived DC were further characterized for expression of CD11b and CD8␣. The data shown are one representative of six individual experiments. C, In vitro-ampliﬁed Flt3ϩCD11bϩ progenitors from Ly5.1 mice were examined for the generation of hemopoietic lineage cells. One week after transfer, single-cell suspensions from spleen of recipient mice were analyzed for the presence of monocytes/macrophages (CD11b), granulocytes (Gr-1), B cells (CD19), and T cells (CD3⑀). The data shown are one representative of six individual experiments. 2558 DC FROM Flt3ϩCD11bϩ PROGENITORS IN VITRO

2 wk, spleens of recipients were analyzed for donor-derived cells. CD11cϩ cells were already detectable 1 wk after precursor transfer. For unfractionated cell populations, 8.1–16.0% of total Ly5.1 donor cells were CD11cϩ (mean ϭ 12.3; n ϭ 6) and were found to be both CD11b positive and negative as well as CD8␣ positive and negative (Fig. 4B). Similarly, adoptive transfer of highly purified Flt3ϩCD11bϩ cells gave CD11cϩ cells that were CD11bϩ or CD11bϪ and CD8␣ϩ or CD8␣Ϫ (Fig. 4B, right panel). These results indicate that Flt3ϩCD11bϩ progenitor cells can give rise to the main conventional DC subtypes in vivo. CD11cϪ cells were also found comprising CD11bϩ macrophages and granulocytes, and no cells were found to express lymphoid-related lineage surface markers such as CD19 and CD3⑀ (Fig. 4, B, right panel, and C). Adoptive transfer of highly purified Flt3ϪCD11bϩ cells gave the same result, which might be due inefficient staining of these cells because of the absence of surface Flt3. In summary, our data clearly show that the two-step in vitro amplification/differentiation system described in this paper gener- ϩ ϩ ates large numbers of homogeneous Flt3 CD11b progenitor Downloaded from cells with sustained proliferative capacity. Additionally, these progenitor cells can be induced to undergo synchronous differentiation and develop into fully functional DC in vitro and in vivo. Thus, the FIGURE 5. Hierarchical cluster analysis of surface Ag expression. system provides an excellent means of generating DC at succes- Gene expression in Flt3ϩCD11bϩ progenitor cells, differentiated DC at sive stages of development that is most suitable for further cellular, days 7 and 10 of differentiation, and TNF-␣ (100 ng/ml)-treated DC was biochemical, and molecular studies. assessed by DNA microarray analysis. Hierarchical cluster analysis of sur- http://www.jimmunol.org/ face Ag genes encoding for mouse CD molecules was performed with Gene expression profiling of cell surface Ags during DC GeneSpring software, and one representative result is shown. Columns differentiation represent the different developmental steps in DC differentiation. Each gene is depicted by a single row of colored boxes. The color of the re- ϩ ϩ Flt3 CD11b progenitors and DC at different stages of develop- spective box in one row represents the expression value of the gene tran- ment were subjected to transcriptional profiling by DNA microar- script in one sample compared with the median expression level of the rays to gain new insights into differentially regulated genes in- gene’s transcript for all samples shown. Green, Transcript levels below volved in the DC differentiation program, thereby applying a median; black, transcript levels equal to median; red, transcript levels strategy that was successfully applied before for human DC (18, higher than median. The developmental stage-specific clusters and a se- by guest on September 29, 2021 32). RNA from Flt3ϩCD11bϩ progenitor cells and DC was pre- lection of CD molecule genes are shown. Cluster I contains genes which ␣ pared and subjected to DNA microarray analysis using Affymetrix are effectively induced by TNF- . Genes in cluster II have a high expression in DC and are not further up-regulated by TNF-␣. Cluster III com- GeneChip arrays that contain probe sets for ϳ6000 known genes ϳ prises genes that are up-regulated during early DC differentiation and are and 6500 expressed sequence tags. DC at days 7 and 10 of dif- down-regulated with further maturation. Cluster IV contains genes with ␣ ϩ ϩ ferentiation, and DC treated with TNF- at day 9 for an additional high expression in Flt3 CD11b progenitor cells only. A comprehensive 24 h (DC day 10 plus TNF-␣) were analyzed. Samples from three list of the clustered genes is provided as supplementary material. Microar- independent experiments were generated, and RNA probes were ray data are available in GEO database (series accession no. GSE 575). hybridized to the microarrays. Surface Ags are frequently used to characterize stem and early progenitor cells, and changes in surface Ag expression represent CD80 and CD86, and the chemokine receptor CCR7 (CD197; Fig. one of the hallmarks of DC differentiation. Approximately 200 5) (1, 2). Cluster II comprises genes expressed in DC that were not ␣ genes encoding CD molecules are represented on the DNA mi- further enhanced by TNF- , such as IL-4 and IL-7 receptors, FcR croarray, and 122 surface Ags were found to be expressed in pro- receptor, and chemokine receptor CCR5 (CD195). Cluster III com- genitor cells and/or differentiated DC. Hierarchical clustering (45) piles genes with highest expression in DC at day 7 of differenti- was used to unveil specific patterns inherent to the DC differentiation and that were down-regulated when cells further progressed ation program. This method revealed distinct clusters of differen- in differentiation. Interestingly, this cluster contains a larger num- tially regulated genes and also genes that were abundantly ex- ber of cell adhesion molecules, some of which have been impli- pressed in Flt3ϩCD11bϩ progenitors and DC and that did not cated in DC migration and/or homing like CD31 or CD44. More- significantly change during differentiation. Four major clusters of over, novel molecules, such as CD164/multi-glycosylated core regulated genes were identified: clusters I–III contain genes that protein 24 (MGC-24) and CD166/activated leukocyte cell adhe- were highly expressed in developing DC but not (or at low levels) sion molecule, which have so far not been related to DC phenotype in Flt3ϩCD11bϩ progenitor cells, and cluster IV compiles genes or function, were found in this cluster. CD164/MGC-24 and that were highly expressed in progenitors and down-regulated dur- CD166/activated leukocyte cell adhesion molecule were described ing DC differentiation (Fig. 5; a comprehensive list of the clustered originally to be expressed in hemopoietic progenitor cells and are genes is provided as supplementary material4). implicated as playing a role in homeostatic control of growth Cluster I contains genes with prominent expression in TNF-␣- and/or migration, including neural and organ development, hemo- treated DC, including molecules that specify activated and mature poiesis, immune response, and tumor progression (50, 51). As ex- DC such as CD40, CD54, and CD83, the costimulatory molecules pected, mannose receptor (CD206) is also found in cluster III. Finally, cluster IV comprises genes that were highly expressed in Flt3ϩCD11bϩ progenitor cells and down-regulated during DC 4 The online version of this article contains supplemental material. differentiation. The receptors for FL and IGF-1, CD135/Flt3, and The Journal of Immunology 2559

CD221/IGF1R, respectively, are found in this cluster, which is in poietic stem cells and Flt3ϩCD11bϩ progenitor cells, whereas line with the prominent effect of these cytokines on Flt3ϩCD11bϩ only 10 and 24 were solely found in stem cells and progenitor progenitor cell growth and FACS analysis (Fig. 1, A, B, and D). cells, respectively (Fig. 6B). CD molecules that were expressed by Transcripts for the IL-6 signal transducer gp130 (CD130) and the Flt3ϩCD11bϩ progenitor cells only include, e.g., the integrins GM-CSF receptor ␣- and ␤-chains (CD116 and CD131) were up- CD11b and CD18 (see Fig. 1D and also Fig. 7). regulated during differentiation and are therefore contained in clus- Flt3ϩCD11bϩ progenitors express several cytokine receptors ter I. Interestingly, cluster IV comprises also known stem cell Ags that are related to the stem cell phenotype, such as receptors for FL ␣ like CD49f/integrin 6, CD93/Ly68/AA4.1, and CD133/AC133/ (Flt3/CD135), SCF (c-kit/CD117), and stromal-cell derived fac- prominin (Refs. 36, 37, 46, and 47; R. D. Kirsch, T. Hieronymus, tor-1 (CXCR4/CD184), and also the IL-6 signal transducer gp130/ and M. Zenke, unpublished data). CD130 (Fig. 6D). Flt3ϩCD11bϩ progenitors express also the stem

ϩ ϩ cell Ags CD93 (Ly68/AA4.1), CD133 (AC133/Prominin), and in- Flt3 CD11b progenitor and stem cells exhibit an overlapping ␣ tegrin 6 (CD49f). surface Ag repertoire ϩ ϩ To further extend this analysis, we compared the surface Ag rep- Stem cell Ag expression on Flt3 CD11b progenitor cells ertoire of Flt3ϩCD11bϩ progenitor cells to that of linϪc- To further extend these observations, surface Ag expression of kitϩSca-1ϩ hemopoietic stem cells (46, 47); R. D. Kirsch, T. Hi- Flt3ϩCD11bϩ progenitors was analyzed by ﬂow cytometry. Pro- eronymus, and M. Zenke, unpublished data). Flt3ϩCD11bϩ genitors express surface CD49f, CD93/AA4.1, and CD133/Promi- progenitor cells expressed 73 of the 206 CD molecules analyzed, nin, and their expression was lost when cells differentiate into DC whereas hemopoietic stem cells expressed 59 (Fig. 6). Surpris- (Fig. 7). CXCR4 chemokine receptor (CD184) was expressed on Downloaded from ingly, the majority of surface Ags (49) were found in both hemo- Flt3ϩCD11bϩ progenitor cells and further up-regulated during DC http://www.jimmunol.org/

FIGURE 6. Comparative gene expression analysis for CD molecules of Flt3ϩCD11bϩ progenitor cells and linϪc-kitϩSca-1ϩ hemopoietic stem cells. Gene expression in Flt3ϩCD11bϩ by guest on September 29, 2021 progenitor cells and linϪc-kitϩSca-1ϩ stem cells was assessed by DNA microarrays. Results shown are mean in- tensity values Ϯ SD of normalized data from two independent replicate results of stem cell hybridization data and ﬁve independent experiments for Flt3ϩ CD11bϩ progenitor cells, respectively. Only those genes are shown that were recognized as abundant transcripts by the Microarray Suite analysis software. A, Genes found to be expressed both in linϪc-kitϩSca-1ϩ stem cells (Ⅺ) and Flt3ϩCD11bϩ progenitor cells (f). B, Summary of expressed genes encoding mouse CD molecules in Flt3ϩCD11bϩ progenitor cells and stem cells. C and D, Genes exclusively found to be expressed in stem cells (Ⅺ) or progenitor cells (f), respectively. A comprehensive list of the analyzed genes is provided as supplementary material. All microarray data are available in GEO database (series accession nos. GSE 692 and GSE 693). 2560 DC FROM Flt3ϩCD11bϩ PROGENITORS IN VITRO

FIGURE 7. Cell surface Ag expression on Flt3ϩCD11bϩ progenitor cells, DC, and DC plus TNF-␣. Expression of DC lineage markers (CD11b, CD83), chemokine receptor CXCR4 (CD184), integrin ␣ 6 (CD49f), and stem/progenitor cell markers (CD93/ AA4.1 and CD133/AC133/prominin) in Flt3ϩCD11bϩ progenitor cells, differentiated DC (day 10), and DC (day 10) treated with TNF-␣ (100 ng/ml for 16 h). Open areas represent staining with isotype control Abs.

development, which is in line with previously published reports on sen based on microarray studies of linϪc-kitϩSca-1ϩ hemopoietic human DC (52, 53). Flt3ϩCD11bϩ progenitors abundantly ex- stem cells that demonstrated expression of their cognate receptors. pressed the myeloid Ags CD11b and CD16/32, but were negative These cytokines yield an effective outgrowth and ampliﬁcation of ϩ ϩ for B220 (Fig. 7 and data not shown). As expected, several well- Flt3 CD11b progenitors and overcome former limitations in Downloaded from characterized DC markers such as MHC class II, CD11c, CD25, number, purity, and homogenicity for such progenitors. However, CD40, CD80, CD83, and CD86 were highly induced during DC whether other cytokines contribute to the generation and mainte- differentiation (Figs. 2 and 7, Table I). Differentiated DC were nance of hemopoietic precursors with the same or even broader CD4Ϫ and CD8␣Ϫ (data not shown) and thus represent classical differentiation potential is currently being investigated. myeloid CD11cϩCD11bϩCD8␣Ϫ DC. Addition of glucocorticoid receptor antagonist dexamethasone ϩ ϩ ϩ ϩ

Taken together, the Flt3 CD11b progenitors obtained express to Flt3 CD11b progenitor cultures significantly increased cell http://www.jimmunol.org/ several stem cell Ags and effectively differentiate into myeloid DC numbers and ensured homogenicity of the progenitor population. in vitro. Dexamethasone appears to impose a differentiation block (T. Jor- gas, T. Hieronymus, and M. Zenke, unpublished data) similar to Discussion that observed in other in vitro culture systems (29, 48, 49). ϩ ϩ In this paper, we used a two-step culture system for amplification During the amplification phase, up to 108 Flt3 CD11b pro- of Flt3ϩCD11bϩ progenitor cells and their differentiation into genitor cells are readily obtained from one bone marrow prepara- fully functional DC. Transcriptional profiling with DNA microar- tion. Further cultivation with GM-CSF results in an ordered and rays was used to study the gene expression repertoire of the highly synchronized differentiation of precursor cells into DC, Flt3ϩCD11bϩ progenitor and the ongoing changes during DC dif- which thus enables the discrimination of consecutive steps of mat- by guest on September 29, 2021 ferentiation. One of the major findings is the broad overlap of uration. DC are fully functional as APCs both in vitro and in vivo, surface marker expression of Flt3ϩCD11bϩ progenitors and he- which qualifies this two-step culture system for various applica- mopoietic stem cells. Flt3ϩCD11bϩ progenitors express, e.g., Flt3, tions including vaccination studies (T. Hieronymus, T. C. Gust, IL-6R and c-kit/SCF receptor, the CD34-like molecule CD164/ and M. Zenke, unpublished data). MGC-24 and the ABC transporter CD243/MDR1, and also the An interesting question is how the Flt3ϩCD11bϩ progenitor stem cell Ags CD93/AA4.1, CD133/AC133, and CD49f/integrin cells described in this paper relate to hemopoietic stem cells and to ␣ 6, and their expression declines during DC differentiation. Addi- various hemopoietic precursors, such as CLP and CMP (9, 10, 27, tionally, following administration of GM-CSF, Flt3ϩCD11bϩ pro- 59) and Flt3ϩ DC precursors (7). genitors synchronously differentiate into fully functional DC, and Gene expression profiling by high density oligonucleotide mi- this allows studying the consecutive steps that determine DC croarrays provides a powerful means of exploring transcriptional differentiation. patterns on a genome-wide scale. By using microarray analysis, we DC constitute a complex system of heterogeneous cells varying recently identified the basic helix-loop-helix transcription factor in their tissue distribution in the organism and differing in surface Id2 as one major determinant for DC development (18). Hemo- phenotype and/or specific functions (1, 2). It has been reasoned poietic cells are commonly characterized by their surface Ag ex- that different DC subsets are derived from different hemopoietic pression. Therefore, microarray data of Flt3ϩCD11bϩ progenitors lineages (4, 6). However, their lineage origin and the underlying were evaluated for their surface Ag expression pattern, and this molecular mechanisms that determine DC development have re- was compared with that of linϪc-kitϩSca-1ϩ hemopoietic stem mained controversial. In recent years, several in vitro systems that cells (46, 47); R. D. Kirsch, T. Hieronymus, and M. Zenke, unpub- recapitulate DC differentiation in cell culture have been established lished data). It was found that Flt3ϩCD11bϩ progenitors exhibit a (54–56). Although most in vitro culture systems focus on human considerable overlap of the surface markers with hemopoietic stem DC, similar systems for mouse DC are just being developed (24, cells including Flt3, IL-6R, c-kit/SCF receptor, and the stem cell ␣ 57, 58). In this study, we describe a novel two-step culture system Ags CD93/AA4.1, CD133/AC133, and CD49f/integrin 6. ϩ ϩ ␣ ϩ ϩ for mouse DC where a Flt3 CD11b progenitor from mouse bone The integrin 6 (CD49f) was also detectable on Flt3 CD11b marrow is first amplified with a specific cytokine combination and progenitor cells as expected from the microarray data, and its ex- then differentiated into fully functional DC with GM-CSF. This pression declined with differentiation. Interestingly, in a recent pa- approach follows a strategy that we successfully used before for per, Melton and coworkers (47) propose the interaction with ex- ␣ ␤ human DC (18, 32, 56). tracellular matrix via integrin 6/ 1 as an essential stem cell Our two-step culture system for mouse Flt3ϩCD11bϩ progen- property. itor cells employs SCF, FL, IGF-1, GM-CSF, hyper-IL-6, and the Flt3ϩCD11bϩ progenitors do not express IL-7R, which repre- glucocorticoid dexamethasone. This factor combination was cho- sents a hallmark of CLP (59). Initial evidence suggests that The Journal of Immunology 2561

ϩ ϩ ϩ Flt3 CD11b progenitors are also different from the Flt3 CMP 10. Manz, M. G., D. Traver, T. Miyamoto, I. L. Weissman, and K. Akashi. 2001. identified by D’Amico and Wu (7). Whether Flt3ϩCD11bϩ pro- Dendritic cell potentials of early lymphoid and myeloid progenitors. Blood 97:3333. genitors give rise to hemopoietic lineages other than DC is cur- 11. Wu, L., A. D’Amico, H. Hochrein, M. O’Keeffe, K. Shortman, and K. Lucas. rently being investigated. An interesting question relates to the in 2001. Development of thymic and splenic dendritic cell populations from differ- vivo frequency of the Flt3ϩCD11bϩ progenitor, and we demon- ent hemopoietic precursors. Blood 98:3376. 12. Asselin-Paturel, C., A. Boonstra, M. Dalod, I. Durand, N. Yessaad, strate that its frequency in bone marrow is ϳ2–2.5%. C. Dezutter-Dambuyant, A. Vicari, A. O’Garra, C. Biron, F. Briere, and An Flt3ϩ macrophage precursor has been described that is G. Trinchieri. 2001. 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