The Journal of Immunology TGF-b1 Accelerates Dendritic Cell Differentiation from Common Dendritic Cell Progenitors and Directs Subset Specification toward Conventional Dendritic Cells Piritta Felker,*,† Kristin Sere´,*,† Qiong Lin,*,† Christiane Becker,*,† Mihail Hristov,‡ Thomas Hieronymus,*,† and Martin Zenke*,† Dendritic cells (DCs) in lymphoid tissue comprise conventional DCs (cDCs) and plasmacytoid DCs (pDCs) that develop from com- mon DC progenitors (CDPs). CDPs are Flt3+c-kitintM-CSFR+ and reside in bone marrow. In this study, we describe a two-step culture system that recapitulates DC development from c-kithiFlt32/lo multipotent progenitors (MPPs) into CDPs and further into cDC and pDC subsets. MPPs and CDPs are amplified in vitro with Flt3 ligand, stem cell factor, hyper–IL-6, and insulin-like growth factor-1. The four-factor mixture readily induces self-renewal of MPPs and their progression into CDPs and has no self- renewal activity on CDPs. The amplified CDPs respond to all known DC poietins and generate all lymphoid tissue DCs in vivo and in vitro. Additionally, in vitro CDPs recapitulate the cell surface marker and gene expression profile of in vivo CDPs and possess a DC-primed transcription profile. TGF-b1 impacts on CDPs and directs their differentiation toward cDCs. Genome-wide gene expression profiling of TGF-b1–induced genes identified instructive transcription factors for cDC subset specification, such as IFN regulatory factor-4 and RelB. TGF-b1 also induced the transcription factor inhibitor of differentiation/DNA binding 2 that suppresses pDC development. Thus, TGF-b1 directs CDP differentiation into cDCs by inducing both cDC instructive factors and pDC inhibitory factors. The Journal of Immunology, 2010, 185: 5326–5335. + + + endritic cells (DCs) are professional APCs that are im- DC progenitors were described: a Flt3 c-kit CX3CR1 macrophage/ portant in inducing adaptive immune responses and main- DC progenitor (MDP) (7, 8) and an Flt3+c-kitintM-CSFR+ com- D taining T cell tolerance (1–3). DCs are divided into mon DC progenitor (CDP) (9, 10). MDPs give rise to macrophages several subsets according to their localization, phenotype, and and DCs, whereas CDPs are DC-restricted and do not generate other function. The main DC subsets are conventional DCs (cDCs) and cell types. Indeed, MDPs were shown to be more heterogeneous and plasmacytoid DCs (pDCs) found in lymphoid organs and migra- to contain CDPs (8, 11, 12). tory tissue DCs that are spread throughout peripheral organs (2, Frequently, DCs are generated from bone marrow or cord blood- by guest on September 30, 2021. Copyright 2010 Pageant Media Ltd. 3). DC development shows remarkable plasticity, and DCs have derived hematopoietic stem/progenitor cells or blood monocytes been shown to develop from both lymphoid and myeloid compart- in vitro. The major cytokines used in culturing DCs are Flt3 ligand ments via Flt3-expressing progenitors (4–6). Recently, clonogenic (Flt3L) and GM-CSF, and, more recently, M-CSF (13–17). Flt3L and M-CSF cultures give rise to cDC and pDC subsets, whereas hi *Department of Cell Biology, Institute for Biomedical Engineering, †Helmholtz In- GM-CSF induces the differentiation of CD11b DCs. According to stitute for Biomedical Engineering, and ‡Institute for Molecular Cardiovascular Re- the current view, Flt3L-generated DCs are considered as in vitro search, Medical Faculty, Rheinisch-Westfa¨lische Technische Hochschule, Aachen, Germany equivalents of steady state DCs, as found, for example, in spleen. Flt3L is the major cytokine for DC development in steady state Received for publication December 8, 2009. Accepted for publication August 20, 2010. in vivo; Flt3L is a potent inducer of both cDCs and pDCs in human https://www.jimmunol.org This work was supported by Grants SFB542 and SPP1356 from the German Research and mouse (15, 18, 19). Accordingly, Flt3L-deficient mice have Foundation (to M.Z.). a severe reduction in DC numbers in several lymphoid organs (15, 20, The microarray data presented in this article have been submitted to the Gene Ex- 21). Interestingly, administration of M-CSF induces cDC and pDC pression Omnibus database (http://www.ncbi.nlm.nih.gov/geo) under accession num- generation in vivo (17), and M-csf–deficient (CSF-1op/op)micehave ber GSE22432. reduced numbers of splenic DCs (22), indicating the in vivo rele- Address correspondence and reprint requests to Dr. Martin Zenke, Department of Cell Biology, Institute for Biomedical Engineering, Medical Faculty, Rheinisch- vance of M-CSF in DC development. In contrast, GM-CSF induces Downloaded from Westfa¨lische Technische Hochschule Aachen, Pauwelsstrasse 30, 52074 Aachen, differentiation of inflammatory DCs that have no steady-state Germany. E-mail address: [email protected] counterpart in vivo. GM-CSF appears to be involved in MDP and The online version of this article contains supplemental material. CDP homeostasis in bone marrow as well as in nonlymphoid tissue Abbreviations used in this paper: C, negative control; cDC, conventional dendritic cell; dermal DC development (21). However, Gm-csf– and Gm-csfr– CDP, common dendritic cell progenitor; CFU-G, CFU granulocyte; CFU-GEMM, deficient mice have only a very mild reduction in DC numbers in CFU granulocyte, erythrocyte, macrophage, megakaryocyte; CFU-GM, CFU granulo- cyte/macrophage; CFU-M, CFU macrophage; DC, dendritic cell; Flt3L, Flt3 ligand; lymphoid tissues where Flt3L is the more crucial factor (21, 23). GEMM, granulocyte, erythrocyte, macrophage, megakaryocyte; GO, Gene Ontology; TGF-b1 is a pleiotropic cytokine involved in a variety of bio- HLH, helix-loop-helix; HSC, hematopoietic stem cell; Id2, inhibitor of differentiation/ DNA binding 2; IGF-1, insulin-like growth factor-1; IRF, IFN regulatory factor; MDP, logical processes, such as development, differentiation, apoptosis, macrophage/DC progenitor; MHC-II, MHC class II; MPP, multipotent progenitor; and cell survival (24). TGF-b1 is crucial for development of PCA, principal component analysis; pDC, plasmacytoid dendritic cell; PI, propidium Langerhans cells, the cutaneous contingent of migratory DC, both iodide; SCF, stem cell factor. in vivo and in vitro (25–27). Yet, the influence of TGF-b1on Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 development of other DC subsets is so far unknown. www.jimmunol.org/cgi/doi/10.4049/jimmunol.0903950 The Journal of Immunology 5327 The current understanding of DC development is mainly based Flow cytometry and cell sorting on studies of genetically modified mice and knockout models that Abs directly conjugated to biotin, FITC, PE, PE-Cy5, PerCP-Cy5.5, PE- lack transcription factors (reviewed in Refs. 3, 28, and 29). STAT3 Cy7, allophycocyanin, or Pacific Blue specific for the following Ags were is a central transcription factor in Flt3 signaling, and deletion used: AA4.1/CD93/Ly68 (AA4.1), B220 (RA3-6B2), CD3ε (145-2C11), of STAT3 severely impairs DC development (30). PU.1, an Ets CD4 (GK1.5), CD8a (53-6.7), CD11b (M1/70), CD11c (N418 or HL3), family transcription factor, is expressed highly in hematopoetic CD19 (1D3), CD24 (M1/69), CD45.1 (A20), CD45.2 (104), CD115 (AFS98), CD117 (ACK2), CD127 (A7R34), CD135 (A2F10.1), Gr-1 progenitors and DCs (31). Mice reconstituted with Pu.1-deficient (RB6-C5), MHC class II (MHC-II; M5/114.15.2), PDCA-1 (JF05- hematopoietic cells have severe hematopoietic defects and lack 1C2.4.1), Sca-1 (D7), SiglecH (440c), and Ter119/Ly-76 (TER-119) (all cDCs (32, 33). Ikaros, a zinc finger transcriptional regulator, is from BD Biosciences, San Jose, CA; eBioscience, San Diego, CA; or required for the normal development of early hematopoietic pro- Miltenyi Biotec, Bergisch Gladbach, Germany). Pacific Blue-labeled F4/80 (Cl:A3-1) Ab was from AbD Serotec (Du¨sseldorf, Germany). Purified genitors (34). Ikaros deficiency leads to either the ablation of all CD49d (R1-2) and CD49f (GoH3) Abs were from BD Biosciences. DC subsets or the absence of specific DC subsets, which depends Lineage-positive cells were stained with biotin-conjugated Lineage Cell on the type of mutation introduced (35, 36). Other transcription Detection Cocktail (Miltenyi Biotec). Streptavidin-FITC, streptavidin-PE, factors have a more selected impact on DC subset specification. and streptavidin-allophycocyanin were from Invitrogen or eBioscience. The development of cDCs is influenced by the IFN regulatory Streptavidin-PerCP-Cy5.5 was from BD Biosciences. Secondary goat anti- rat Ab conjugated to PE was from Invitrogen. Cells were treated with anti- factors (IRFs) IRF-2, IRF-4, and IRF-8 (also known as ICSBP) FcR–blocking reagent (Miltenyi Biotec) and 3% mouse serum prestaining. (37), the helix-loop-helix (HLH) transcription factor inhibitor of differentiation/DNA binding 2 (Id2) (38), and the NF-kB/Rel family member RelB (39, 40), among others. pDC development depends on the basic HLH factor E2-2 (41, 42), the Ets-domain transcription factor Spi-B (43), and IRF-4 and IRF-8 (37). Al- though critically adding to our understanding of DC development, it is worthwhile to note that these studies so far concentrated on the phenotype in mature DCs. It is yet unclear at which stage and how these factors influence DC development. In this study, we investigated the early steps of DC develop- ment from multipotent progenitors (MPPs) and CDPs, and the impact of TGF-b1 on DC subset specification from CDPs. CDPs are very scarce cells in vivo; thus, we used an in vitro culture sys- tem to obtain high numbers of CDPs. We show consecutive dif- ferentiation of MPPs to CDPs and further to DC subtypes and provide functional and molecular analyses of this differentiation pathway. We also demonstrate that TGF-b1 accelerates DC dif- ferentiation and directs differentiation toward cDCs by inducing a cDC-affiliated transcription profile in CDPs. by guest on September 30, 2021. Copyright 2010 Pageant Media Ltd. Materials and Methods Mice C57BL/6 mice, CD45.1 (B6.SJL-Ptprca Pepcb/BoyJ), GFP transgenic mice (44), and CX3CR1-GFP knockin mice (45) were maintained under specific pathogen-free conditions in the central animal facility of RWTH Aachen University Hospital, Aachen, Germany.