After Injection into the Striatum, in Vitro-Differentiated Microglia- and Bone Marrow-Derived Dendritic Cells Can Leave the Central Nervous System via the Blood Stream
2008; Elsevier BV; Volume: 173; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2008.080234
ISSN1525-2191
AutoresSonja Hochmeister, Manuel Zeitelhofer, Jan Bauer, Eva-Maria Nicolussi, M Fischer, Bernhard Heinke, Edgar Selzer, Hans Lassmann, Monika Bradl,
Tópico(s)Single-cell and spatial transcriptomics
ResumoThe prototypic migratory trail of tissue-resident dendritic cells (DCs) is via lymphatic drainage. Since the central nervous system (CNS) lacks classical lymphatic vessels, and antigens and cells injected into both the CNS and cerebrospinal fluid have been found in deep cervical lymph nodes, it was thought that CNS-derived DCs exclusively used the cerebrospinal fluid pathway to exit from tissues. It has become evident, however, that DCs found in peripheral organs can also leave tissues via the blood stream. To study whether DCs derived from microglia and bone marrow can also use this route of emigration from the CNS, we performed a series of experiments in which we injected genetically labeled DCs into the striata of rats. We show here that these cells migrated from the injection site to the perivascular space, integrated into the endothelial lining of the CNS vasculature, and were then present in the lumen of CNS blood vessels days after the injection. Moreover, we also found these cells in both mesenteric lymph nodes and spleens. Hence, microglia- and bone marrow-derived DCs can leave the CNS via the blood stream. The prototypic migratory trail of tissue-resident dendritic cells (DCs) is via lymphatic drainage. Since the central nervous system (CNS) lacks classical lymphatic vessels, and antigens and cells injected into both the CNS and cerebrospinal fluid have been found in deep cervical lymph nodes, it was thought that CNS-derived DCs exclusively used the cerebrospinal fluid pathway to exit from tissues. It has become evident, however, that DCs found in peripheral organs can also leave tissues via the blood stream. To study whether DCs derived from microglia and bone marrow can also use this route of emigration from the CNS, we performed a series of experiments in which we injected genetically labeled DCs into the striata of rats. We show here that these cells migrated from the injection site to the perivascular space, integrated into the endothelial lining of the CNS vasculature, and were then present in the lumen of CNS blood vessels days after the injection. Moreover, we also found these cells in both mesenteric lymph nodes and spleens. Hence, microglia- and bone marrow-derived DCs can leave the CNS via the blood stream. In the intact central nervous system (CNS), dendritic cells (DCs) are contained within the meningeal and perivascular DC network, but not in the parenchyma proper.1McMenamin PG Distribution and phenotype of dendritic cells and resident tissue macrophages in the dura mater, leptomeninges, and choroid plexus of the rat brain as demonstrated in wholemount preparations.J Comp Neurol. 1999; 405: 553-562Crossref PubMed Scopus (273) Google Scholar, 2McMenamin PG Wealthall RJ Deverall M Cooper SJ Griffin B Macrophages and dendritic cells in the rat meninges and choroid plexus: three-dimensional localization by environmental scanning electron microscopy and confocal microscopy.Cell Tissue Res. 2003; 313: 259-269Crossref PubMed Scopus (120) Google Scholar Under pathological conditions like inflammation,3Kivisäkk P Mahad DJ Callahan MK Sikora K Trebst C Tucky B Wujek J Ravid R Staugaitis SM Lassmann H Ransohoff RM Expression of CCR7 in multiple sclerosis: implications for CNS immunity.Ann Neurol. 2004; 55: 627-638Crossref PubMed Scopus (233) Google Scholar degeneration,4Henkel JS Engelhardt JI Siklos L Simpson EP Kim SH Pan T Goodman JC Siddique T Beers DR Appel SH Presence of dendritic cells. 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Since brain and spinal cord lack lymphatic vessels, DCs cannot leave these organs using lymphatic drainage pathways.10Bradl M Flugel A The role of T cells in brain pathology.Curr Top Microbiol Immunol. 2002; 265: 141-162PubMed Google Scholar As an alternative, emigration via cerebrospinal/interstitial fluid to deep cervical lymph nodes has been suggested.11Cserr HF Harling-Berg CJ Knopf PM Drainage of brain extracellular fluid into blood and deep cervical lymph and its immunological significance.Brain Pathol. 1992; 2: 269-276Crossref PubMed Scopus (346) Google Scholar, 12Hatterer E Davoust N Didier-Bazes M Vuaillat C Malcus C Belin M-F Nataf S How to drain without lymphatics? 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In the present study we conceived a series of experiments to specifically address these questions, and we show that microglia- and bone marrow-derived DCs can leave the CNS via the blood stream and home to mesenteric lymph nodes and spleen. Lewis rats, green fluorescent protein (GFP)-transgenic Lewis rats (≥ sixth back-cross generation of the GFP transgene onto the Lewis rat background) and Sprague-Dawley rats were used throughout this study. They were bred at the Decentral Facilities of the Institute for Biomedical Research (Medical University Vienna). Microglial cultures were established essentially as described.22Giulian D Baker TJ Characterization of ameboid microglia isolated from developing mammalian brain.J Neurosci. 1986; 6: 2163-2178Crossref PubMed Google Scholar Briefly, 0 to 1 day old GFP-transgenic and wild-type Lewis rats were sacrificed and their brains dissected. For each culture, the brains of 8 to 12 rats were dissociated in 2 to 3 ml of 1× trypsin. The resulting single cell suspensions were cultured for 5 to 7 days in poly-l-lysine-coated culture dishes, using RPMI 1640/10% fetal calf serum, and changing the medium every other day. After this time period, the mixed glial cell cultures consisted of a monolayer of astrocytes and some fibroblasts. On top of this monolayer, ramified microglial cells and glial progenitor cells were found. These cells were only loosely adherent. They were detached by shaking confluent mixed glial cultures for 12 to 15 hours (180 rpm, 37°C) and then plated for 5 to 10 minutes onto fresh, uncoated culture dishes. This led to the selective adherence of microglial cells, which were now no longer ramified, but had an amoeboid, macrophage-like phenotype. All other glial cells could not adhere and were removed by subsequent, vigorous washing in PBS. The resulting microglial cultures routinely had a purity of >99%. On average, 2 to 3 shake-offs, separated by 3 to 5 days, could be prepared from each mixed glial culture. For our studies, cells of the first through third shake-off were used. Microglial cultures were supplemented with 10 ng/ml each of recombinant rat granulocyte/monocyte colony stimulating factor (GM-CSF) and recombinant rat interleukin 4 (IL-4; all RnD Systems, Wiesbaden, Germany). Treatment of microglial cells with these factors changed the morphology of the microglial cells to a spindle-shaped phenotype. After 7 to 13 days of culture, micDCs detached from the adherent cell layer and were harvested from the supernatant of these cultures. Four to eight-week-old Lewis rats were irradiated (10 Gray) and immediately afterward injected i.p. with bone marrow cells derived from GFP-transgenic Lewis rats. For microglial cultures, these chimeric animals were sacrificed 4 to 15 months after bone marrow transfer. At the same time point, blood was taken to determine the degree of chimerism (= percentage of GFP-labeled white blood cells in the total white blood cell pool). The resulting microglial cells were then differentiated with GM-CSF/IL-4, and the percentage of GFP+ micDCs in the cell population with a morphological DC phenotype was determined by two different observers. One of these observers was blinded to the experiment. Bone marrow derived-DCs (bmDCs) were essentially generated as described,23Grauer O Wohlleben G Seubert S Weishaupt A Kämpgen E Gold R Analysis of maturation states of rat bone marrow-derived dendritic cells using an improved culture technique.Histochem Cell Biol. 2002; 117: 351-362Crossref PubMed Scopus (51) Google Scholar using RPMI 1640/fetal calf serum containing 10 ng/ml each of recombinant rat GM-CSF and recombinant IL-4. BmDCs were harvested from these cultures after 7 to 13 days. For staining, the cells were incubated for 30 minutes at 4°C with antibodies against rat MHC class I (OX18), MHC class II (OX6), CD11b (OX42), CD11c, CD80 (B7.1), CD86 (B7.2), CD54 (intercellular adhesion molecule-1 [ICAM-1]), CD8α (OX8), CD4 (W3/25), and OX62 antigen (all from Serotec, Düsseldorf, Germany), diluted 1:100 in stain buffer (BD Pharmingen, San Diego, CA). Isotype matched control antibodies, mouse IgG1 and IgG2a (both from Dako, Glostrup, Denmark) were used. The cells were washed in stain buffer and incubated for 30 minutes at 4°C with polyclonal goat anti-mouse IgG-FITC (F[ab′]2, Dako, for wild-type cells) or polyclonal goat-anti-mouse IgG-RPE (F[ab′]2, Dako, for GFP-transgenic cells). MicDCs and bmDCs were cultured for 30 minutes in the presence of 100 μg/ml fluorescein isothiocyanate (FITC)-dextran or 0.2 μg/ml FITC-bovine serum albumin [BSA] (both from Sigma, Vienna, Austria), or 100 μg/ml fluorescein-conjugated E. coli K12 bioparticles (FITC-K12; Molecular Probes/Invitrogen, Glasgow, Scotland, UK). One aliquot of these cells was incubated at 4°C (control, to reveal unspecific binding of FITC-dextran or FITC-BSA), the other at 37°C (to reveal antigen capture and uptake). Antigen uptake was stopped by washing in ice-cold stain buffer, and evaluated by flow cytometry or fluorescence microscopy. MicDCs and bmDCs were used after 7 days of differentiation in GM-CSF/IL-4 containing medium. Then, the culture was continued for 48 hours in the presence (experimental cells) or absence (controls) of 100 ng/ml lipopolysaccharaide (LPS; from E. coli 0127:B8, Sigma). The surface properties of cells from both groups were characterized by flow cytometry. MicDCs and bmDCs were used 7 to 12 days after initiation of differentiation with GM-CSF/IL-4. T cells were isolated from the mesenteric lymph nodes of Sprague-Dawley rats. Graded doses of micDCs or bmDCs and T cells were cocultured as triplicates in a total volume of 200 μl. The DC:T cell ratios ranged from 0.5:1 to 0.0125:1. For the last 18 hours of a 48 hours incubation, [3H]-thymidine was added. The cells were harvested onto glass fiber filter membranes and the [3H]-thymidine incorporation was measured in a β-counter. cDNA from micDCs or bmDCs was used for PCR analysis, using primers specific for PU.1 (PU.1-for: 5′-TGGAAGGGTTTCCCCTCGTC-3′; PU.1-rev: 5′-TGCTGTCCTTCATGTCGCCG-3′; product 533 bp), Spi-B (Spi-B-for: 5′-GGCTTCG GTTTTTGAGATTGGG-3′; Spi-B-rev: 5′-TGACTGTAAAAGGGGGCTTTCC-3′; product 381 bp), and β-actin (β-act-for: 5′-ATGAAGTGTGACGTTGACATCC-3′; β-act-rev: 5′-GCCAGCTCAGTAACAGTCCGCC-3′; product 303 bp). For PCR reactions, 5 μl 10× PCR buffer (200 mmol/L Tris-HCl, pH 8.4, 500 mmol/L KCl), 1.5 μl 50 mmol/L MgCl2, 1 μl 10 mmol/L dNTP mix, 1 μl forward primer (100pmol/μl), 1 μl reverse primer (100pmol/μl), 1 μl Hot Gold Star polymerase (5U/μl, Eurogentec, Seraing, Belgium), 1 μl cDNA, and 38.5 μl H2O were mixed, heated for 11 minutes at 95°C (denaturation), and then subjected to 40 cycles of denaturation (30 seconds, 95°C), annealing (30s, 56°C for PU.1; 55°C for Spi-B; 53°C for β-act) and elongation (30s, 72°C). The MyCycler Thermal Cycler system with thermal gradient (Bio-Rad, München, Germany) was used. Final extension was made for 10 minutes at 72°C. The resulting PCR products were size-separated by agarose gel electrophoresis, excised, and sequenced. Oligo GEArray rat inflammatory cytokines & receptors microarrays and oligo GEArray rat chemokines & receptors microarrays (all from SuperArray, Frederick, MD) were used according to the instruction of the manufacturer. Six-week-old wild-type Lewis rats were anesthetized with Ketanest S/Rompun and placed in a stereotactic head frame. The incisor bar was adjusted until the plane defined by the lambda and bregma was parallel to the base plate. Then, the needle of a 0.5 μl Hamilton syringe was stereotactically guided into the left striatum (3 mm lateral to bregma, 4 mm below the dura). 0.3 μl solution (containing ∼10000 GFP+ cells) in sterile endotoxin-free PBS was slowly injected. The needle was left in place for an additional 10 minutes before it was removed. The animals were sacrificed 0.5, 1, 3, and 6 days later for histological analyses. Initially, we used GFP+ micDCs after 7 days of differentiation, or after 7 days of differentiation and an additional stimulation for 48 hours with TNF-α to obtain more mature cells. Since both types of cells showed an absolute identical migratory behavior, we refer to them as one single group. All animal experiments were approved by the local animal welfare committee and the Austrian Ministry for Education, Science, and Culture. For histological examination, the animals were euthanized with CO2 and then perfused with 4% paraformaldehyde in phosphate buffered saline pH 7.4. The brains, lymph nodes and spleens were dissected, postfixed in paraformaldehyde/PBS and paraffin-embedded. Serial sections were cut on a microtome. Antigen was retrieved by steaming the tissue sections in 1 mmol/L citric acid buffer pH 6.0 (for usage in double stainings with Ox6 and CM1) or 1 mmol/L EDTA buffer, pH 8.5 (all other antibodies and antibody combinations) for 60 minutes. Immunohistochemical staining and confocal microscopy were done as described,24Aboul-Enein F Bauer J Klein M Schubart A Flugel A Ritter T Kawakami N Siedler F Linington C Wekerle H Lassmann H Bradl M Selective and antigen-dependent effects of myelin degeneration on central nervous system inflammation.J Neuropathol Exp Neurol. 2004; 63: 1284-1296PubMed Google Scholar using rabbit anti-GFP antibodies (gift of W. Sieghart, Med. University Vienna, Center for Brain Research), mouse anti-rat MHCII (OX6), rabbit anti-activated caspase 3 (CM-1, antibody kindly provided by Thomas Deckwerth, Idun Pharmaceuticals, San Diego, CA), mouse anti-rat T cells (W3/13), and rabbit anti-human von Willebrand factor (vWF, cross-reactive with rat, DAKO, Glostrup, Denmark) as primary antibodies. For the detection of apoptotic cells, tissue sections were pretreated with 1 mmol/L citric acid buffer, and then incubated over night at 4°C with OX-6 (1:250) and CM-1 (1:50,000). Bound OX-6 was detected by alkaline phosphatase conjugated anti-mouse IgG (1:200) followed by Fast Blue B base (Sigma), and bound CM-1 was visualized using biotinylated donkey-anti-rabbit (Amersham Pharmacia Biotech, Uppsala, Sweden) in fetal calf serum/PBS (1 hour, room temperature) followed by avidin peroxidase (1:100; Sigma; 1 hour, room temperature) and stained with 3,3′diaminobenzidine-tetra-hydrochloride (Sigma). Sections were inspected using conventional light microscopy. For determining the location of injected cells, double-staining was performed with OX6 (1:100) and anti-vWF (1:500), which were applied over night at 4°C. Bound OX6 was detected with a donkey anti-mouse Cy2 (green), bound anti-vWF antibodies with biotinylated sheep anti-rabbit antibodies (Amersham Pharmacia Biotech, 1:200) and then with streptavidin-Cy3 (red) (Jackson Immuno Research Laboratories, West Grove, PA, 1:75). Alternatively, anti-GFP (1:2500) and anti-vWF (1:500) antibodies were applied over night at 4°C. Bound anti-vWF antibodies were detected with goat anti-rabbit Cy3 (Jackson Immuno Research Laboratories, 1:100). Since they also reacted with the biotinylated sheep anti-rabbit antibodies (1:200) and the streptavidin-Cy2 (Jackson Immuno Research Laboratories, 1:75) used to detect bound anti-GFP antibodies, blood vessel endothelium appears yellow, and GFP+ cells green. Sections were inspected with a laser confocal microscope (LSM-410, Carl Zeiss, Jena, Germany). We isolated RNA from paraformaldehyde-fixed paraffin embedded tissue sections from mesenteric lymph nodes and spleens, using the Paradise Whole Transcript RT Reagent System (Arcturus, Mountain View, CA) according to the instructions of the manufacturer. This RNA was transcribed to cDNA and then subjected to PCR analysis, using primers specific for GFP (GFP-for: 5′-GCTGACCCTGAAGTTCATCTGC-3′; GFP-rev: 5′-GTGGCTGTTGTAGTTGTACTCC-3′; product 302 bp), and β-actin. For PCR reactions, 5 μl 10× PCR buffer (200 mmol/L Tris-HCl, pH 8.4, 500 mmol/L KCl), 1.5 μl 50 mmol/L MgCl2, 1 μl 10 mmol/L dNTP mix, 1 μl forward primer (100pmol/μl), 1 μl reverse primer (100pmol/μl), 1 μl polymerase (5U/μl), 1 μl cDNA, and 38.5 μl H2O were mixed together. The reaction mix was heated for 11 minutes at 95°C (denaturation), and then subjected to 40 cycles of denaturation (30s, 95°C), annealing (30s, 55°C for GFP; 53°C for β-act) and elongation (30s, 72°C). The final extension was made for 10 minutes at 72°C. The size of the resulting PCR products was determined by agarose gel electrophoresis. Afterward, the PCR products were purified and sequenced. All T cells used were memory T cells specific for myelin basic protein (MBP, Sigma), derived from Lewis rats, and were "resting," ie, readily activatable in response to MBP and syngenic antigen presenting cells. We used the Vybrant CFDA SE Cell tracer kit (Invitrogen, Lofer, Austria) to obtain carboxyfluorescein succinimidyl ester (CFSE)-labeled MBP-specific T cells, and CellTracker Orange 5- and-6-[4-chloromethyl] benzoyl amino (CMTMR) tetramethylrhodamine (Invitrogen) to obtain CellTracker Orange labeled MBP-specific T cells. In both cases, the labeling procedure followed the instructions of the manufacturer. GFP+ microglia-derived dendritic cells in 1 ml culture medium were pre-incubated for 45′at 37°C with 10 μl MBP as the cognate or 10 μl Ovalbumin (Sigma) as an irrelevant antigen. The stock concentrations of the antigens used were 1 mg/ml. After the incubation, the cells were carefully washed to remove any traces of unbound antigens. Then, ∼10,000 GFP+ microglia-derived dendritic cells per rat were injected into the striatum, as outlined in the material and methods section. Two rats per group were injected. Immediately after the intrastriatal injection of the antigen-loaded dendritic cells, 2.5 × 106 CFSE-labeled and 2.5 × 106 unlabeled resting MBP-specific T cells were injected i.p./i.v. Six days later, splenocytes of these animals were analyzed by flow cytometry to search for proliferation of CFSE-labeled T cells. Remaining splenocytes were used for ELISPOTS and T cell proliferation assays. ELISPOT analyses were performed on spleens of Lewis rats that had been injected with GFP+ MBP- or ovalbumin-loaded microglia-derived dendritic cells into the striatum, and with CFSE-labeled and unlabeled resting MBP-specific T cells i.p./i.v. (see above). Six days after these manipulations, the animals were sacrificed, and 1.25, 2.5, 5, and 10 × 104 erythrocyte-depleted splenocytes were seeded in triplicate in the absence of any antigen (negative control), or in the presence of MBP, OVA (both final concentration 10 μg/100 μl), or Concanavalin A (ConA, Sigma, final concentration 2.5 μg/100 μl, positive control), using the rat interferon-γ ELISPOT (U-CyTech, Utrecht, Netherlands) according to the instructions of the manufacturer. For T cell proliferation assays, 1 × 106 erythrocyte-depleted splenocytes/well were seeded in triplicate in the absence of any antigen (negative control), or in the presence of MBP, OVA (both final concentration 10 μg/100 μl), or ConA (final concentration 2.5 μg/100 μl, positive control). The cells were cultured for 72 hours. [3H]-thymidine was added during the last 18 hours of culture to reveal de novo DNA synthesis during the S-phase of the cell cycle of activated T cells. Approximately 10,000 microglia-derived GFP+ dendritic cells/rat were injected into the striatum of five Lewis rats, as outlined in the material and methods section. 0.5 to 1 × 107 resting CellTracker Orange labeled MBP-specific T cells were immediately afterward injected i.p./i.v. The animals were sacrificed 1 (2 rats), 2 (2 rats), or 3 days (1 rat) after the injection, and perfused with 4% paraformaldehyde. Cryosections of the spleens were made and analyzed by confocal microscopy. To initiate the differentiation to DCs, the shake-offs of mixed glial cultures were cultured in the presence of 10 ng/ml GM-CSF and IL-4. Seven to thirteen days later, these cultures consisted of large adherent, macrophage-like cells, and of freely floating, smaller cells with processes suggestive of DCs. Since there was no good antibody marker available that unequivocally identifies rat DCs, we characterized the floating cells according to their morphology, surface marker expression, response to LPS, and ability to activate naive T cells. All of these cells had cellular processes of variable length (Figure 1A) and were CD11c+, CD11b+, ICAM-1+, B7.1+, and B7.2+ (Figure 1B). The expression of the OX62 antigen was inconsistent, ranging from a complete or near-complete absence (in 56%) to moderate (in 13%) to high levels (in 31% of all cultures, data not shown). Even in the absence of additional maturation stimuli such as LPS, these cells expressed large amounts of MHC class II products and costimulatory molecules on the cell surface (Figure 1B) and were able to activate naive T cells (Figure 1C). They efficiently took up FITC-dextrane and FITC-BSA, but showed only surface-binding of FITC-K12 (Figure 1D), while undifferentiated microglial cells readily took up FITC-dextrane, FITC-BSA, and FITC-K12 (Figure 1E). Rat cells with such a phenotype have been called DCs previously.25Talmor M Mirza A Turley S Mellmann I Hoffman LA Steinman RM Generation or large numbers of immature and mature dendritic cells from rat bone marrow cultures.Eur J Immunol. 1998; 28: 811-817Crossref PubMed Scopus (117) Google Scholar However, since there is currently no marker available that unequivocally shows whether such differentiated cells are really of the DC lineage, or whether they provide an independent myeloid lineage, we rather prefer to term these cells "DC-like." Different subsets of murine26Shortman K Liu Y-J Mouse and human dendritic cell subsets.Nat Rev Immunol. 2002; 2: 151-161Crossref PubMed Scopus (1921) Google Scholar and rat27de la Mata M Riera CM Iribarren P Identification of a CD8a+ dendritic cell subpopulation in rat spleen and evaluation of its OX-62 expression.Clin Immunol. 2001; 101: 371-378Crossref PubMed Scopus (7) Google Scholar DCs can be readily distinguished by their expression of CD8α. We therefore characterized the expression of CD8α molecules on the surface of micDCs by FACS analysis. Twenty-four of 28 independent cultures gave rise to CD8α− micDCs. In 4/28 cultures, pure populations of CD8α+ micDCs were obtained. The differentiation of microglial cells to CD8α− and CD8α+ micDCs reflects the normal differentiation pattern of myeloid DCs in mice and rats. In these species, both CD8α− and CD8α+ myeloid DCs can derive from the same precursor cell,26Shortman K Liu Y-J Mouse and human dendritic cell subsets.Nat Rev Immunol. 2002; 2: 151-161Crossref PubMed Scopus (1921) Google Scholar, 28León B Martinez del Hoyo G Parrillas V Vargas HH Sánchez-Mateos P Longo N López-Bravo M Ardavin C Dendritic cell differentiation potential of mouse monocytes: monocytes represent immediate precursors of CD8- and CD8+ splenic dendritic cells.Blood. 2004; 103: 2668-2676Crossref PubMed Scopus (108) Google Scholar and do not change their CD8α status in vitro.29Vremec D Shortman K Dendritic cell subtypes in mouse lymphoid organs. Cross-correlation of surface markers, changes with incubation and differences among thymus, spleen and lymph nodes.J Immunol. 1997; 159: 565-573PubMed Google Scholar To further characterize the micDCs, we determined their transcription factor profile by PCR analysis. We concentrated on the transcription factors PU.1 (typically used by myeloid DCs30Anderson KL Perkin H Surh CD Venturini S Maki RA Torbett BE Transcription factor PU. 1 is necessary for development of thymic and myeloid progenitor-derived dendritic cells.J Immunol. 2000; 164: 1855-1861PubMed Google Scholar, 31Guerriero A Langmuir PB Spain LM Scott EW PU. 1 is required for myeloid-derived but not lymphoid-derived dendritic cells.Blood. 2000; 95: 879-885Crossref PubMed Google Scholar), and Spi-B (used by plasmacytoid DCs32Schotte R Nagasawa M Weijer K Spits H Blom B The ETS transcription factor Spi-B is required for human plasmacytoid dendritic cell development.J Exp Med. 2004; 200: 1503-1509Crossref PubMed Scopus (144) Google Scholar, 33Schotte R Rissoan MC Bendriss-Vermare N Bridon JM Duhen T Weije
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