dendritic cells (dcs) are characterized by an irregu

Induction of Dendritic Cell Differentiation by Granulocyte-MacrophageColony-Stimulating Factor, Stem Cell Factor, and Tumor Necrosis Factor a

In Vitro From Lineage Phenotypes-Negative c-kit" MurineHematopoietic Progenitor Cells

By Yi Zhang, Naofumi Mukaida, Jian-bin Wang, Akihisa Harada, Mariko Akiyama, and Kouji Matsushima

To elucidate the capacity of murine early hematopoietic pro- whereas freshly isolated splenic DCs did not. These resultssuggested that DCs generated in our culture system pheno-genitor cells (HPCs) to differentiate into dendritic cells (DCs),

lineage phenotypes (Lin)Ïc-kit" HPCs were highly purified typically resemble thymic ones. In contrast, mouse TNFafailed to induce TNF-Rp55-deficient mice-derived LinÏc-kit"from either wild-type or tumor necrosis factor (TNF) receptor

p55 (TNF-Rp55)-deficient mice. Upon culture with granulo- HPCs to generate DCs with characteristic morphology, im-munophenotype, and accessory function for T cells undercyte-macrophage colony-stimulating factor (GM-CSF) and

stem cell factor (SCF) for 14 days, wild-type mouse LinÏc- the same culture conditions, suggesting a crucial role ofTNF-Rp55 in TNFa-mediated DC differentiation from HPCs.kit" HPCs did not exhibit characteristic features of DC such

as sheet-like projections and veil processes. Moreover, these Interestingly, human TNFa, which can bind to mouse TNF-Rp55 but not TNF-Rp75, was incapable to augment DC gen-cells expressed a marginal level of DC markers such as DEC-

205, CD86, and barely supported allogenic MLR. However, eration from wild-type mouse LinÏc-kit" HPCs. Collectively,these results suggest that TNFa has a pivotal role in DCthe addition of mouse TNFa generated a large number of

cells with typical DC morphology, expression of high levels generation from murine early HPCs in collaboration withGM-CSF and SCF through the interaction of TNF-Rp55 andof Ia, DEC-205, CD86, and function of stimulating allogenic

MLR. Moreover, a proportion of these mature DCs and thy- TNF-Rp75.q 1997 by The American Society of Hematology.mic DCs expressed Thy-1 mRNA as well as Thy-1 antigen,

D responding to the stimulation of several combined cytokines,ENDRITIC CELLS (DCs) are characterized by an irregu-lar shape with numerous projections in their surface and including granulocyte-macrophage colony-stimulating factor

expression of high levels of major histocompatibility complex (GM-CSF), interleukin-4 (IL-4), and tumor necrosis factor a(MHC) class II antigens and other DC markers.1-6 These cells (TNFa).3,9-11 DCs, which can differentiate from the commonare specialized to present antigens in an MHC-restricted man- precursors shared by granulocyte- and macrophage-lineagener and initiate a primary immune response more efficiently cells,12 were also generated in vitro from murine MHC classthan other types of antigen-presenting cells such as B cells II-negative bone marrow hematopoietic cells in a suspensionand macrophages.1,2 DCs comprise heterogeneous populations culture in response to GM-CSF.13 However, a relative highand are classified as follows depending on their location: thy- cell concentration was required for the induction of DCs inmic DC in thymus; interdigitating DC (IDC) in lymph node, the system, suggesting that an additional factor(s) may bethymus, and spleen; veiled cells in afferent lymph; interstitial necessary for the generation of DCs. This notion is supportedDC in heart, lung, kidney, and intestine; Langerhans’ cells by the observation that, in collaboration with GM-CSF, hu-(LC) in skin; and blood DC in peripheral blood.1-8 The various man TNFa is essentially required for DC differentiation andtissue distributions of DC suggest the presence of different maturation from human CD34/ hematopoietic progenitorDC precursors in vivo with differential requirements for their cells.14-21 Moreover, TNFa can also induce the terminal dif-differentiation into mature DC.1-3

ferentiation of blood monocyte-derived DCs that are gener-The ontogeny of DCs has been widely investigated using ated by the stimulation with GM-CSF and IL-4.10,11 These

various DC precursors. DCs can be generated in a large results implicate TNFa as an important regulator of humannumber from human peripheral blood mononuclear cells in DC differentiation and maturation. However, until now, it

remains to be established whether highly purified murineearly hematopoietic progenitor cells (HPCs) could generate

From the Department of Pharmacology, Cancer Research Insti- DC in vitro in response to TNFa.tute, and the Department of Hygiene, School of Medicine, Kanazawa Mice deficient in TNFa, lymphotoxin a (LTa), or TNF-University, Kanazawa, Japan; and the Department of Molecular receptor p55 (TNF-Rp55) gene lack germinal centers andPreventive Medicine, School of Medicine, University of Tokyo,

exhibit dysregulated antibody generation.22,23 Moreover,Tokyo, Japan.

these mice lack one type of DCs, follicular dendritic cellsSubmitted March 18, 1997; accepted August 14, 1997.(FDCs) within germinal centers, suggesting that the TNFa-Supported in part by Grants-in-Aid from the Ministry of Educa-TNF-Rp55 system is involved in the development of FDC,tion, Culture, Science, and Sports of the Japanese Government andthereby regulating antibody response.22,23 Although FDC isfrom CREST, Japan Technology Corp.

Address reprint requests to Kouji Matsushima, MD, PhD, Depart- one type of DC that presents native antigens as immunement of Molecular Preventive Medicine, School of Medicine, Univer- complexes to B cells,2,24 we addressed whether the TNFa-sity of Tokyo, 7-3-1, Hongo, Bunkyoku, Tokyo 113, Japan; email: TNF-Rp55 system might be involved in regulating the [email protected]. entiation of other types of DC in vitro.

The publication costs of this article were defrayed in part by pageAccumulating evidence indicates that murine bone mar-

charge payment. This article must therefore be hereby markedrow Lin0c-kit/ cells represent uncommitted HPCs with the‘‘advertisement’’ in accordance with 18 U.S.C. section 1734 solely tocapability of reconstitution of lymphoid and myeloid hema-indicate this fact.topoiesis in vivo.25,26 However, it is not clarified whetherq 1997 by The American Society of Hematology.

0006-4971/97/9012-0033$3.00/0 this cell population can differentiate in vitro into DC. We

4842 Blood, Vol 90, No 12 (December 15), 1997: pp 4842-4853

AID Blood 0024 / 5h42$$$461 11-10-97 10:41:15 blda WBS: Blood

For personal use only.on November 19, 2018. by guest www.bloodjournal.orgFrom

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MURINE DENDRITIC CELL DIFFERENTIATION 4843

provide evidence here that TNFa, in concert with GM-CSFand stem cell factor (SCF), has a crucial role in inducinghighly purified murine Lin0c-kit/ HPCs to differentiate intofunctionally competent DCs and that the interaction of TNFawith both TNF-Rp55 and TNF-Rp75 is indispensable forthis process.

MATERIALS AND METHODS

Cytokines and antibodies. Recombinant murine GM-CSF andmurine SCF were generous gifts from Kirin Brewery Co (Tokyo,Japan). Human TNFa was a kind gift from Dainippon Pharmaceuti-cal Co (Osaka, Japan). Mouse TNFa was produced as describedpreviously.27 Endotoxin was not detected in these cytokine prepara-tions using a Toxicolor assay kit (Seikagaku-Kogyo, Tokyo, Japan).These cytokines were used at the optimal concentrations determinedin the preliminary experiments as follows: GM-CSF, 4 ng/mL; SCF,20 ng/mL; mouse TNFa, 25 ng/mL; and human TNFa, 50 ng/mL. Fig 1. Schematic representation of culture conditions of highlyAn anti-c-kit antibody (ACK-2) was kindly provided by Dr T. Sudo purified murine LinÏc-kit" HPCs.(Toray, Kamakura, Japan)28 and conjugated with biotin by using aNHS-Biotin kit (Pharmacia-Biotech, Uppsala, Sweden) according tothe manufacturer’s instructions. A rat monoclonal antibody (MoAb) inverted microscope (Nikon, Tokyo, Japan) and on cytocentrifugedto murine dendritic cell marker, DEC-205 (NLDC145),5,29 was a cells that were stained with Giemsa solution.generous gift by Dr R.M. Steinman (Rockefeller University, New Immunofluorescence analysis. Immunofluorescence analysesYork, NY). Other MoAbs and reagents used for immunostaining were performed as previously described.31,32 In two-color immuno-were obtained from PharMingen (San Diego, CA), unless otherwise fluorescence analysis, 2 to 4 1 105 cells were incubated with theindicated. optimal concentrations of anti-CD86 (GL1) or anti-DEC-205 MoAb,

Mice. TNF-Rp55-deficient mice (designated thereafter as TNF- followed by FITC-labeled goat antirat IgG(Fab*)2 antibody (Caltag,Rp550/0 mice) were obtained from Dr H. Bluethmann (Hofmann- Camarillo, CA). The cells were then stained with PE-conjugatedLa Roche Ltd, Basel, Switzerland)30 and maintained by mating with mouse antimouse Ia MoAb (AF6-120.1). In three-color immunoflu-C57BL/6 mice under pathogen-free conditions in the Animal Re- orescence analysis, the cells were sequentially incubated with ansearch Center of Kanazawa University (Kanazawa, Japan). F1 mice anti-DEC-205 MoAb and FITC-labeled goat antirat IgG(Fab*)2 anti-between C57BL/6 and 129 (Japan SLC, Hamamatsu, Japan) were body, followed by the incubation with the optimal concentrations ofused as wild-type mice (designated as TNF-Rp55///). All animal PE-labeled anti-Ia and biotinylated anti-CD86 MoAbs. Then the cellsexperiments complied with the standards set out in the Guideline were finally stained with CY-conjugated streptavidin. In anotherfor Care and Use of Laboratory Animals of Takara-machi Campus series of three-color immunofluorescence analyses, the cells wereof Kanazawa University. sequentially incubated with either a rat anti-DEC-205 or anti-CD86

Suspension culture of Lin0c-kit/ HPCs. Bone marrow cells were MoAb and PE-labeled goat antirat IgG(Fab*)2 antibody, followedobtained by aspirating femurs and tibiae of 8- to 10-week-old female by the incubation with biotinylated anti-Ia MoAb (25-9-17). TheTNF-Rp55/// or TNF-Rp550/0 mice. Lin0c-kit/ HPCs were isolated cells were then finally incubated sequentially with CY-conjugatedfrom nonadherent bone marrow mononuclear cells with the use of streptavidin and FITC-labeled MoAbs to CD8, Thy-1.2 (53-2.1),an EPICS ELITE cell sorter (Coulter Electronics, Hialeah, FL) as CD11a, CD11b, and CD11c (HL3). All incubations were performedpreviously described, with some modifications.31,32 In brief, nonad- for 30 minutes on ice, followed by two washes, and instrumentherent cells were stained with an indirect staining composed of bio- compensation was set in each experiment using single-color and/ortin-labeled anti-c-kit MoAb and phycoerythrin (PE)-conjugated two-color stained samples. In some experiments, the correspondingstreptavidin followed by a set of fluorescein isothiocyanate (FITC)- cell populations were isolated by using an EPICS ELITE cell sorterlabeled MoAbs to CD3 (145-2C11), CD4 (H129.19), CD8 (53-6.7), (Coulter Electronics, Hialeah, FL) according to the manufacturer’sB220 (RA3-6B2), Gr-1 (Ly-6G), CD11a (2D7), and CD11b (M1/ instructions.70). The contamination of other types of cells in this preparation Separation of DCs from thymus and spleen. DCs were separatedwas consistently less than 0.5%, as shown by an immunofluorescence from thymuses and spleens of wild-type mice according to the methodanalysis. Purified Lin0c-kit/ HPCs were incubated at a cell concen- previously described by Vremec et al.6 Briefly, single-cell suspensionstration of 1 1 104 cells/mL in Iscove’s modified Dulbecco’s medium were prepared by digesting thymus and spleen with 1 mg/mL of colla-supplemented with 10% fetal bovine serum (FBS), 5 1 1005 mol/L genase type IV (Sigma Chemical Co, St Louis, MO) at 377C for 302-mercaptoethanol, penicillin G (100 U/mL), and streptomycin (100 minutes in Hank’s buffer. Low-density cells were separated from amg/mL) in the presence of GM-CSF, SCF, and TNFa until 7 days single-cell preparation suspended in phosphate-buffered saline (PBS)after the initiation of culture (Fig 1). The cells were thereafter cul- containing 5% FBS and 10 mmol/L EDTA by a sequential centrifuga-tured in the presence of TNFa and GM-CSF. Because this condition tion on Histopaque 1077 and 14.5% metrizamid medium (Sigmaincreased cell yields without changing the morphology and pheno- Chemical Co). For allogenic MLR, nonadherent cells were furthertype of the obtained cells,16,18 it was used in the present study unless depleted from the resultant cell preparations according to the methodotherwise stated. The cells were split at day 5 or 7 after the initiation as previously described.33 The purity of DCs prepared by this methodof the culture and half of the medium was exchanged every 3 to 4 was usually greater than 60%, as shown by two-color immunostainingdays. At the indicated time intervals, the cells were collected for with anti-Ia and CD11c MoAbs. In some experiments, Ia/CD11c/

immunofluorescence staining and allogenic MLR analysis. Morpho- CD30CD40B2200Gr-10NK1.10TER-1190 cells were enriched fromlow-density cells by a cell sorter after three-color immunofluorescence,logic analyses were performed directly on cultured cells with an





ZHANG ET AL4844

because these phenotypes are presumed to represent mature marrow Lin0c-kit/ HPCs in the cultures (1 1 104 at day 0,DCs.1,2,6-8,34 A highly homogenous DC population with a purity of 51.1 { 7.7 1 104 cells on day 7, and 26.0 { 2.5 1 104

greater than 99.6% was consistently obtained by repeatedly sorting cells at day 14, n Å 7). On a phase contrast microscopicthree times and was used for extraction of total RNA. observation, at day 5 of culture, GM-CSF induced cell clus-

Reverse transcription-polymerase chain reaction (RT-PCR). To- ter formation without sheet-like cells in its periphery (Figtal RNAs were extracted from 1 1 105 thymocytes, splenocytes,

2A), and thereafter many round cells appeared in the cul-highly purified thymic and splenic DCs, or DCs generated fromtures. Giemsa staining on these cells demonstrated that theyLin0c-kit/ HPC culture by using RNAzol B (Biotex Laboratorieswere mostly macrophages but not DCs after 14 days ofInc, Houston, TX), according to the manufacturer’s instructions.culture (Fig 2E and G). Addition of SCF during the firstIa/CD11c/ cells were enriched from Lin0c-kit/ HPC-derived DCs

and, in some experiments, they were further sorted into Thy-1/ and week of the culture remarkably increased the number ofThy-10 populations. First-strand cDNA was synthesized in a 25-mL nonadherent cells (268 { 8.0 1 104 cells, n Å 8) withoutreaction volume using an RT-PCR kit (Takara Shuzo, Kyoto, Japan) affecting the phenotype of the recovered cells (data notwith random primers. Thereafter, cDNA was amplified for 35 cycles shown), consistent with the previous reports.16,18 The addi-consisting of 947C for 30 seconds, 607C for 1 minute, and 727C for tion of mouse TNFa to the cultures increased the yields of1.5 minutes with the Thy-1-specific oligonucleotide primers (5*- nonadherent cells to 78.1 { 15.1 1 104 (n Å 3) and 42.7 {AACCCAGCCATCAGCGTCGC-3* and 5*-CAGAGAAATGAA-

7.5 1 104 cells at day 14 (n Å 8) in the presence and theGTCCAGGG-3*), which will give rise specifically to a 480 bp-absence of SCF, respectively. After culturing Lin0c-kit/cDNA encoding Thy-1 glycoprotein.35 As a control, mouse b-actinHPCs for 5 days in the presence of mouse TNFa combinedtranscript was amplified in parallel as previous described.32 The PCRwith GM-CSF, cell aggregates appeared with the profilesproducts were fractionated on a 1.5% agarose gel and visualized by

ethidium bromide staining. of typical sheet-like projections and veil processes on itsMLR. T cells were obtained from spleens of allogenic mice periphery, irrespective of the presence of SCF (Fig 2B). The

(BALB/c) by depleting nylon wool column-retained cells from aggregates grew thereafter, adjoining the adherent cells thatspleen-derived mononuclear cells.13,33 The T-cell preparations were also progressively grew. When the cell density was keptfurther incubated with a mouse anti-Ia MoAb and followed by goat relatively low by splitting the cells at day 7, the number ofantimouse IgG-conjugated magnetic beads (Dynal, Oslo, Norway) aggregates with apparent sheet-like cells in the peripheryto remove residual antigen-presenting cells. After being exposed to

continued to increase with generating DC-like cells until 1430 Gy of x-ray irradiation, the indicated numbers of the cells fromdays of the culture (Fig 2C). The aggregates were easilyLin0c-kit/ cell cultures, freshly isolated splenic DCs, or peritonealdislodged from the adherent layers and the cells with veil-macrophages were added to 3 1 105 allogeneic T cells per well inor sheet-like processes were clearly observed after replating96-well, round-bottom culture plates. In some experiments, the cells

from Lin0c-kit/ HPC cultures were further sorted based on their into a new well (Fig 2D), consistent with the previous re-immunofluorescence intensities of Ia, CD86, and DEC-205 and used port.13 On Giemsa staining, these cells exhibited an irregularfor MLR. After the incubation at 377C for 5 days, cell proliferation shape with an eccentric nuclear and polarized lamellipodiawas determined using 3-(4,5-dimethyl thiazolyl-2-)-2,5-diphenyl- (Fig 2F and H), suggesting that these were DCs on morpho-tetrazolium bromide (MTT; Sigma Chemical Co). In brief, 15 mL logic criteria.of MTT (5 mg/mL in PBS) was added into each well and the plates The effects of GM-CSF and TNFa on immunophenotypeswere incubated at 377C for an additional 4 hours. After removing

of cultured HPCs. Because murine mature DCs expressthe supernatant, 150 mL of isopropanol containing 0.04 N HCl washigh levels of MHC class II antigen, DEC-205, and costimu-added to each well and mixed thoroughly. The resultant absorbancelatory molecule, CD86,1,2,4-8 we then performed immunoflu-at 550 nm was read by a microplate immunoreader.orescence analyses on the cells using MoAbs to these anti-Statistical analysis. Differences were evaluated using the Stu-

dent’s t-test. P values of less than .05 were considered to be statisti- gens. GM-CSF and SCF marginally increased the proportioncally significant. of both Ia/DEC-205/ and Ia/CD86/ cells at days 7 and

14 (Fig 3A). The addition of mouse TNFa synergisticallyRESULTS increased the proportion of Ia/DEC-205/ and Ia/CD86/

cells at days 7 and 14, although Ia/CD86/ cells started toEnhanced DC-like cell generation from TNF-Rp55///

increase later than that of Ia/DEC-205/ cells (Fig 3A).mice-derived Lin0c-kit/ HPCs by mouse TNFa. HPCsThree-color immunofluorescence analyses detected a sig-were extinguished by 3 days after the start of the culture innificant proportion of Ia/DEC-205/CD86/ cells in HPCsthe absence of any cytokines or in the presence of mousecultured for 14 days (Fig 3B). These cells expressed highTNFa alone (data not shown). GM-CSF increased the num-

ber of nonadherent cells from highly purified murine bone levels of CD11b and DC marker, CD11c (Fig 4), but not

rFig 2. Generation of mature DCs by culturing murine LinÏc-kit" HPCs in the presence of mouse TNFa, GM-CSF, and SCF. Morphologic

analyses were performed on cultured cells (A through D) or Giemsa-stained cells after cytocentrifugation (E through H). (A through D) A phasecontrast microscopic observation was performed on murine LinÏc-kit" HPCs stimulated by the combination of GM-CSF " SCF (A) or that ofGM-CSF " SCF " mouse TNFa (B through D) for 5 (A and B) or 14 days (C) as described in Fig 1. In (D), the aggregates shown in (C) wereisolated and observed for 2 hours after replating into a new plate. Original magnifications: (A), (B), and (D), Ì 200; (C), Ì 100. (E through H)Giemsa staining was performed after cytocentrifuging nonadherent cells or aggregate cells (from D) from murine LinÏc-kit" HPCs cultured for14 days in the absence (E and G) or in the presence of mouse TNFa (F and H) combined with GM-CSF and SCF. Original magnifications: (E)and (F), Ì 160; (G) and (H), Ì 400.






AID Blood 0024 / 5h42$$0024 11-10-97 10:41:15 blda WBS: Blood




ZHANG ET AL4846

served in splenic DCs but not thymic DCs upon stimulationwith GM-CSF and TNFa for 3 days. Furthermore, RT-PCRshowed that Thy-1 mRNA was expressed by freshly isolated,purified thymic DCs and Thy-1/ DCs generated from Lin0c-kit/ HPCs, but neither splenic DCs nor Thy-10 DCs gener-ated from Lin0c-kit/ HPCs (Fig 5B). These results suggestthat Thy-1 on thymic DCs and HPC-derived DCs was, atleast in part, endogenously synthesized from Thy-1 tran-scripts, although it has been shown that Thy-1 expressionon the surface of thymic DCs may be partially due to passiveacquisition from surrounding cells.34,36,37 Moreover, HPC-derived DCs shared several characteristic surface markerswith freshly isolated thymic DCs.

Enhancement of allogenic MLR by DCs generated fromTNF-Rp55/// mice-derived Lin0c-kit/ HPC cultures. Wenext examined whether the nonadherent cells including ag-

Fig 3. Immunofluorescence analysis on nonadherent cells gener-ated in LinÏc-kit" HPCs cultures. (A) Using a two-color immunofluo-rescence analysis, the proportion of Ia"DEC-205" (

��) or Ia"CD86" cells

(j) was determined on nonadherent cells generated from LinÏc-kit"

HPCs cultured with or without mouse TNFa combined with GM-CSFand SCF for 7 and 14 days, respectively. The data are expressed asthe mean Ô SD of four independent experiments or more. Error barsindicate 1 SD. *P Ú .05 significance as compared with the cultureswithout mouse TNFa addition. (B) Three-color immunofluorescenceanalysis on nonadherent cells generated in LinÏc-kit" HPCs stimu-lated with mouse TNFa combined with GM-CSF and SCF for 14 days.These cells were sequentially stained with DEC-205 MoAb, biotinyl-ated CD86 MoAb, and PE-labeled Ia MoAb. DEC-205 and CD86 werethen shown by antirat IgG(Fab* )2-conjugated FITC and CY-streptavi-din, respectively. The cell population that expressed high levels ofIa-antigen was gated and the coexpression of DEC-205 and CD86 wasanalyzed. More than 20,000 events were acquired. Three independentexperiments were performed and representative results are shownhere. The figures in each quadrant represent the percentage of thecells.

Fig 4. Immunofluorescence analysis on murine LinÏc-kit" HPCscultured for 14 days in the presence of mouse TNFa, GM-CSF, andSCF. Three-color immunofluorescence analyses were performed asdescribed in the Materials and Methods on the cultured HPCs at day

CD4, B220, and Gr-1 (data not shown). The expression of 14. The indicated FITC-labeled MoAbs (CD8, Thy-1, CD11a, CD11b,and CD11c) were used to demonstrate the phenotype characteristicsCD8a was not detectable (Fig 4). Moreover, both Ia/DEC-of the generated dendritic cells by gating on Ia"DEC-205" or Ia"CD86"205/ and Ia/CD86/ cells expressed Thy-1 at similar levelscell population. Solid and dotted lines indicate the immunofluores-as CD11b (Fig 4). Thy-1 antigen was expressed by freshly cence intensity of cells stained with a control and the test antibodies,

isolated thymic DCs, but not those splenic ones (Fig 5A), respectively. Representative results from three independent experi-ments are shown.although marginal upregulation of Thy-1 expression was ob-






eration (Fig 6A). After incubation for 14 days with GM-CSF and SCF, a marginal level of T-cell proliferation wasobserved upon their addition. In contrast, the cells, whichwere incubated for 14 days with mouse TNFa and the combi-nation of GM-CSF and SCF, enhanced T-cell proliferationat a comparable level to splenic DCs (Fig 6A), suggestingthat mouse TNFa was indispensable for the generation offunctionally competent DCs from purified HPCs. Becausethe cultured cells were heterogeneous with respect to CD86and DEC-205 expression, they were further purified by acell sorter to determine the capacity of each subpopulation toaugment allogenic MLR. The four subpopulations obtained(Ia/CD86/, Ia/CD860, Ia/DEC-205/, and Ia/DEC-2050

cells) enhanced allogenic MLR as efficiently as splenic DC(Fig 6B). Under the same conditions, peritoneal macro-phages supported MLR marginally even at the highest cellconcentrations used (Fig 6B). Ia/CD860 and Ia/DEC-2050

cells in these experiments should represent Ia/CD860DEC-205/ and Ia/CD86/DEC-2050 cells, respectively (Fig 3B).Thus, these results suggest that the obtained highly Ia-anti-gen-positive cells represented functionally competent DCsirrespective of expression of CD86 and DEC-205.

Involvement of TNF-Rp55 and TNF-Rp75 in generationof DC from HPCs. TNFa differentially uses two distinctreceptors, TNF-Rp55 and TNF-Rp75, to mediate intracellu-lar signals.38,39 We previously showed that TNF-Rp55 has apivotal role to regulate the differentiation and proliferationof myeloid cells from HPCs in vivo and in vitro.31 Hence,using TNF-Rp550/0 mice, we examined the role of TNF-Rp55 in TNFa-induced enhancement of DC generation fromLin0c-kit/ HPCs. Combined with GM-CSF and SCF, mouseTNFa could neither induce the DC aggregate formation norenhance the expression of DEC-205 and CD86 antigens onthose cultured cells from TNF-Rp550/0 mice-derived Lin0c-kit/ HPCs during the whole culture periods (Figs 7 and 8Aand B). Moreover, these cells were incapable to stimulatethe allogenic MLR as compared with that of TNF-Rp55///

mice-derived Lin0c-kit/ HPCs (Fig 9). To further dissectthe role of TNF-Rp55 and TNF-Rp75, the effects of humanTNFa were investigated on DC generation from Lin0c-kit/

HPCs. Interestingly, under the same culture conditions, hu-Fig 5. The expression of Thy-1 antigen and its mRNA in thymicDCs, splenic DCs, and DCs generated from LinÏc-kit" HPCs.I- man TNFa could not induce DC differentiation and func-a"CD11c"CD3ÏCDB220ÏGr-1ÏNK1.1ÏTER-119Ï thymic and splenic tional maturation from either TNF-Rp55/// or TNF-Rp550/0

DCs were highly purified by sorting them from low-density thymo- mice-derived Lin0c-kit/ HPCs, even in the presence GM-cytes and splenocytes after centrifuging on 14.5% metrizamid me-

CSF and SCF (Figs 7, 8, and 9). On the other hand, wedium. The expression of Thy-1 antigen was reanalyzed on eitherobserved that human TNFa was able to significantly inhibitfreshly isolated or cultured thymic and splenic DCs upon stimulation

with GM-CSF " mouse TNFa for 3 days. The percentage of Thy-1" the proliferation of Gr-1/ granulocytes from TNF-Rp55///

cells is indicated in the histograms (A). Thy-1 mRNA was examined mice-derived Lin0c-kit/ HPCs, but not from TNF-Rp550/0

in the indicated cells using RT-PCR (B). Total RNAs were extractedmice-derived ones, to the comparable levels as mouse TNFafrom 1 Ì 105 cells of splenocytes, thymocytes, splenic DCs, thymicdid (data not shown), indicating that human TNFa used inDCs, and LinÏc-kit" HPC-derived DCs. DCs generated from LinÏc-kit"

HPCs were further sorted into Thy-1" and Thy-1Ï subpopulations. our culture system retained biologic activities. Thus, theseThe b-actin transcripts were used as control (B). Similar experiments results strongly suggest that TNF-Rp75 may also be coopera-were performed three times and representative results are shown tively involved in TNFa-induced DC differentiation andhere.

maturation from HPCs in vitro.

DISCUSSIONgregated cells could exhibit a characteristic activity ofDCs1-4 to enhance allogenic MLR. When we cultured Lin0c- Several groups independently reported that functional

DCs could be generated from human CD34/ and mousekit/ HPCs with either combination of cytokines only for 7days, these HPC-derived cells failed to induce T-cell prolif- MHC class II-negative bone marrow cells.40-43 However, be-





ZHANG ET AL4848

Fig 6. The capacity of the cultured cells to en-hance allogenic MLR. Allogenic MLR was performedusing purified T cells (3 Ì 105 cells per well in 96-round-well plates) as responder cells. The unfraction-ated nonadherent cells, which were derived fromLinÏc-kit" HPC cultures in the presence of mouseTNFa combined with GM-CSF and SCF at days 7 and14, respectively, as described in the Materials andMethods, were irradiated and used as stimulators atthe indicated cell numbers (A). In (B), the indicatedcell populations were used as stimulator cells thatwere separated by using a cell sorter from LinÏc-kit" HPC cultures stimulated with mouse TNFa incombination with GM-CSF and SCF for 14 days. Theproliferation of T cells was measured by MTT assayafter 5 days of culture. Results are expressed as themean Ô 1 SD of triplicate cultures. Results of eachpanel are representative of three independent exper-iments.

cause these cell populations consist of heterogeneous HPCs cells.36-37 However, the expression of Thy-1 mRNA was de-tected in this study in freshly isolated thymic DCs and HPC-that are thought to be at different differentiation stages,40-43

it remains elusive whether purified early HPCs can differen- derived Thy-1/ DCs but not HPC-derived Thy-10 ones, indi-cating that a subclass of freshly isolated thymic DCs andtiate in vitro into DC. We have provided evidence here that

highly purified murine Lin0c-kit/ HPCs could differentiate HPC-derived DCs could produce Thy-1 antigen, at least inpart, from its specific transcript. In addition, Thy-1 expres-into DCs with characteristic morphology and surface mark-

ers after culture for 14 days at a relatively low cell density sion was not detectable in freshly isolated splenic DCs, eventhough marginal upregulation of its expression was observedin the presence of GM-CSF, SCF, and mouse TNFa. More-

over, the cells enhanced allogenic MLR as effectively as upon stimulating these DCs with GM-CSF and TNFa. Theseresults would suggest that a subpopulation of HPC-derivedspleen-derived DCs, suggesting that these cells also function

as an antigen-presenting cell. The results would imply that DCs phenotypically resembled those freshly isolated thymicDCs. The latter notion may be corroborated by the previousthe generated cells are DCs, concordant with the proposed

characteristics of DC.1-3 observation that intrathymic injection of murine Lin0Sca-1/

primitive hematopoietic stem cells generated DCs with DEC-Murine DC exhibits heterogeneity in surface markers andintracytoplasmic structures, depending on their locations.1-8 205 in thymus.7

Previously, Inaba et al13 reported that GM-CSF alone canDEC-205 is expressed by a majority of DCs in murine thy-mus, whereas only less than 20% of DCs in spleen express induce murine MHC class II-negative bone marrow cells to

differentiate into splenic-type DCs that expressed high levelsDEC-205.4,5,7,8,29,44 However, a major difference in splenicand thymic DC lies in the expression of T-cell mark- of Ia-antigens but low levels of DEC-205 and CD11c without

Thy-1 expression, in contrast to our present results. More-ers.6,34,37,45 Thymic DCs express moderate levels of Thy-1and CD2 with a low level of CD4, whereas splenic DCs over, functional DCs appeared at 6 to 7 days after the culture

in their system,13 whereas Lin0c-kit/ cells could not enhanceexpress little, if any, of these antigens. Hence, these T-cellmarkers, especially Thy-1 and CD2, can be useful to distin- allogenic MLR at 7 days after the culture under our experi-

mental conditions, even in the presence of mouse TNFa,guish between thymic and splenic DCs,6 although there issome controversy concerning the expression by thymic DCs GM-CSF, and SCF. These results suggest that the kinetics

of DC maturation from Lin0c-kit/ HPCs also differs fromof certain cell-surface markers.6,34,36,37,46 It has been reportedthat thymic DCs absorbed Thy-1 antigen from surrounding that of MHC class II-negative DC precursors. Moreover, in






Fig 7. Development of dendritic cell aggregates in cultured LinÏc-kit" HPCs-derived from TNF-Rp55"/" mice bone marrow. Morphologicobservation was performed under a phase contrast microscope on cultured LinÏc-kit" HPCs. LinÏc-kit" HPCs were isolated from TNF-Rp55"/"

or TNF-Rp55Ï/Ï mice bone marrow cells and incubated for 14 days (14 d) as described in the Materials and Methods. Similar experiments wererepeated three times and representative results are shown here. Original magnification Ì 100.

their system, a relatively high cell concentration was used of granulocytes, macrophages, and a few numbers of DC.Thus, GM-CSF may induce MHC class II-negative boneto induce DC differentiation.13 When we incubated these

HPCs at a low cell concentration, only in the presence of marrow cells to produce some unknown factor(s) that affectsthe differentiation of DCs. It has been reported that thereGM-CSF and/or SCF, most of the shown colonies consisted





ZHANG ET AL4850

Fig 8. Two-color immunofluorescence analysison the cultured LinÏc-kit" HPCs. LinÏc-kit" HPCs fromTNF-Rp55"/" wild-type or TNF-Rp55Ï/Ï mice bonemarrow were cultured in the presence or absence ofmouse or human TNFa combined with GM-CSF andSCF for 14 days, and two-color immunfluorescenceanalysis was performed to determine the expressionof Ia with DEC-205 (A) or CD86 (B) as described inthe Materials and Methods. Representative resultsfrom three independent experiments are shownhere.

exist at least two distinct types of DC precursors with inde- DCs contain abundant members of transcription factors, NF-kB, particularly p50, and relB.11,48 Moreover, targeted dis-pendent differentiation pathways in human CD34/ hemato-

poietic progenitors.17,21 Thus, the discrepancy may be also ruption of relB gene has resulted in an impairment of DCdevelopment in either spleen or thymus,49,50 suggesting thatexplained by the presence of an additional type(s) of DC

precursors in murine MHC class II-negative bone marrow members of the NF-kB family have an essential role inregulating differentiation and maturation of DC in vivo. Ac-cells, which possibly differ from those in Lin0c-kit/ HPCs.

Nevertheless, these results would imply that different HPC cumulating evidence indicates that TNFa can induce thetranscription of several genes by activating NF-kB.51 Thus,subpopulations or those hematopoietic cells at different mat-

uration stages may generate distinct DC subsets, depending it is tempting to speculate that TNFa activates NF-kB, whichmay be involved in the expression of a gene(s) essentiallyon the added stimuli.

A growing number of experiments demonstrates that each required for DC differentiation. The identification of a targetgene(s) might provide a novel insight into the molecularstep of hematopoietic cell differentiation is under the strict

and cooperative control of transcription factors.47 Mature mechanisms of DC differentiation.






Fig 9. The capacity of the cultured HPCs fromTNF-Rp55"/" or TNF-Rp55Ï/Ï mice bone marrow tostimulate allogenic MLR. LinÏc-kit" HPCs were ob-tained from TNF-Rp55"/" or TNF-Rp55Ï/Ï mice bonemarrow cells and incubated for 14 days in the pres-ence of the indicated combinations of cytokines. Al-logenic MLR was performed using purified T cellsand cultured HPC progenies as responder and stimu-lator cells, respectively, as described in the Materialsand Methods.

Two related but distinct receptors, p55 and p75, exist for difference of target cells. However, the phenotypic changesof TNF-Rp550/0 were much milder in spleen as comparedTNF.38,39 Although these receptors show sequence similarity

with each other in the extracellular domains, those of intra- with those of TNFa0/0 and LTa0/0 mice.22,23,53,54 Thus, TNF-Rp75, an additional receptor for TNFa and LTa, may playcellular domains diverge, accounting for mediation of over-

lapping but distinct signals.38,39 We recently observed in- a role in FDC differentiation as well as in vitro DC differenti-ation. Additionally, many other cytokines have been showncreased HPC numbers in TNF-Rp550/0 mice, implicating

TNF-Rp55 as a physiologic regulator of HPC differentiation to be able to induce DC’s differentiation either in vivo or invitro.2,3,34,46,55,56 These may explain the failure to detect aber-and proliferation.32 Moreover, TNF-Rp550/0, TNFa0/0, or

LTa0/0 mice exhibit impaired FDC development, although rant distribution and phenotypic changes in DCs in thymus,where bone marrow-derived DCs have been recorded,7 fromno information has been available yet on other types of

DCs.22,23,50 Hence, we first evaluated here the role of TNF- TNF-Rp550/0 mice under normal conditions (data notshown).Rp55 in in vitro DC differentiation from HPCs. Our study

using TNF-Rp550/0 mice-derived Lin0c-kit/ HPCs showed We show here that murine HPCs could differentiate intoDC resembling freshly isolated thymic one with respect tothat these early HPCs cannot generate morphologic and func-

tional mature DCs in response to the stimulation of mouse surface marker expression. Thymic DCs have been proposedto have a pivotal role in negative and positive selection inTNFa combined with GM-CSF and SCF, implying a critical

role of TNF-Rp55 in mouse TNFa-induced in vitro DC dif- thymus.57,58 Hence, the generation of a large number of thy-mic DC-like cells using this system may facilitate to eluci-ferentiation. However, human TNFa, which can only bind

to mouse TNF-Rp55 but not to mouse TNF-Rp75, could not date the mechanisms of thymic selection in vitro. Moreover,DCs generated in our culture system may be a useful weaponinduce DC generation even from TNF-Rp55/// mice-derived

Lin0c-kit/ HPCs. Thus, these results suggested that both to control autoimmune diseases in which disturbed thymicselection is frequently observed.24,57,58types of TNF-Rs p55 and p75 are coordinately involved in

in vitro TNFa-induced DC differentiation from early HPCs.ACKNOWLEDGMENTThe recruitment of TNF by TNF-Rp75 for TNF-Rp55 has

been documented.52 Thus, endogenously produced TNFa The authors express their gratitude to Dr R.M. Steinman (Themay interact initially with TNF-Rp75 and be recruited to Rockefeller University) for his kind gifts of MoAbs to DEC-205

(NLDC-145) and 33D1. We also thank Dr H. Bluethmann for hisTNF-Rp55. The interaction between TNFa and TNF-Rp55generous present of TNF-Rp55 gene-targeted knockout mice. Wemay eventually prevent HPCs from proliferation, therebyhighly appreciate the productive discussion with Dr K. Inaba (De-inducing differentiation into more committed cells, includingpartment of Zoology, Faculty of Science, Kyoto University, Kyoto,DCs.Japan).Targeted disruption of TNF-Rp55 resulted in a lack of

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1997 90: 4842-4853

Yi Zhang, Naofumi Mukaida, Jian-bin Wang, Akihisa Harada, Mariko Akiyama and Kouji Matsushima Progenitor Cells

Murine Hematopoietic+In Vitro From Lineage Phenotypes-Negative c-kitαColony-Stimulating Factor, Stem Cell Factor, and Tumor Necrosis Factor

Induction of Dendritic Cell Differentiation by Granulocyte-Macrophage

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dendritic cells (dcs) are characterized by an irregu

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