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Blood, Vol. 95 No. 1 (January 1), 2000:
pp. 138-146
HEMATOPOIESIS
Development of dendritic cells in vitro from murine fetal
liver-derived lineage phenotype-negative c-kit+
hematopoietic progenitor cells
Yanyun Zhang,
Yi Zhang,
Yong Wang,
Masafumi Ogata,
Shin-ichi Hashimoto,
Nobuyuki Onai, and
Kouji Matsushima
From the Department of Molecular Preventive Medicine and CREST,
School of Medicine, The University of Tokyo, Japan.
 |
Abstract |
We describe here that lineage phenotype- negative
(Lin) c-kit+ hematopoietic
progenitor cells (HPCs) from day 13 postcoitus (dpc) murine fetal liver
(FL) can generate dendritic cell (DC) precursors when cultured in vitro
in the presence of PA6 stromal cells plus granulocyte/macrophage
colony-stimulating factor (GM-CSF) + stem cell factor (SCF) + Flt3
ligand (Flt3L) for 12 to 14 days, and develop into mature DCs when
stimulated with GM-CSF plus mouse tumor necrosis factor  (mTNF)
for an additional 3 to 5 days. A transwell culture system showed
that the generation of DC precursors depended on the support of PA6
cell-secreted soluble factor(s). The mature DCs derived from 13 dpc FL
Linc-kit+ HPCs showed
characteristic morphology and function of DCs and expressed high levels
of Ia, CD86, and CD40 molecules, low levels of DEC205, E-cadherin, and
F4/80 molecules, but barely detectable CD11c antigen. Once FL-derived
HPCs were cultured without GM-CSF, NK1.1+ cells developed
in the presence of PA6 cells + SCF + Flt3L. These NK1.1+ cells could develop into DC precursors at an
earlier stage of differentiation by reculturing with PA6 cells + SCF + Flt3L + GM-CSF, but they would be irreversibly
committed to NK cell precursors without GM-CSF after 3 days,
suggesting that GM-CSF plays a critical role in controlling the
transition of DC and NK cell precursors from 13 dpc FL-derived
Linc-kit+ HPCs. This study
represents the first success in generating mature DCs in vitro from
murine FL HPCs. (Blood. 2000;95:138-146)
© 2000 by The American Society of Hematology.
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Introduction |
Dendritic cells (DCs) are professional
antigen-presenting cells that are distributed throughout tissues and
organs, where they show functional and phenotypic
heterogeneity.1-4 DCs can uptake, process, and present
antigens on major histocompatibility complex (MHC) class II to induce T
cell immune responses, in particular to initiate primary
antigen-specific immune reaction.5,6 They also participate
in B cell-mediated immune responses,7-9 constituting an
integral part of the immune system.10 Recent studies also
suggest an important role for DCs in the induction of T cell tolerance.
When antigen-bearing DCs are directly injected into the developing
thymus or fetal thymic organ cultures, reactive T cells were
selectively deleted.11 Moreover, if MHC class II molecules
were only expressed by cortical epithelium, but not by DCs in thymic
medulla, the propensity to autoimmunity increases.12 All
these data suggest that DCs may be involved in the induction of
tolerance by deleting autoreactive T cells and the tolerance of T cells
to self-antigens during the fetal development.
During embryonic development of hematopoiesis, hematopoietic progenitor
cells (HPCs) sequentially appear in yolk sac, paraaortic splanchnopleura, aorta-gonad-mesonephros region (AGM), fetal liver (FL), and finally in the bone marrow (BM).13-18 The FL is
considered to be the principle hematopoietic organ during the murine
fetal stage until the early neonatal stage, and serves as a reservoir of founder hematopoietic cells generated at early hematopoietic sites
within the conceptus.13,17-19 It has been reported that the
progenitor cells derived from FL can differentiate in vitro into T
lymphocytes,20 natural killer (NK) cells, B lymphocytes, macrophages and mast cells in mouse,21 and T lymphocytes
and thymic NK cells in human.22 However, it remains
elusive whether murine FL-derived HPCs can differentiate into mature DCs.
It is well established now that DCs can be generated from blood
monocytes,23 BM-derived HPCs24-26 and cord
blood HPCs27 stimulated with granulocyte/macrophage
colony-stimulating factor (GM-CSF), interleukin-4 (IL-4), and tumor
necrosis factor  (TNF). In addition, CD4lo thymic
precursors obtained from adult thymus can develop into T cells and
thymic DCs bearing CD8 molecule after intrathymic transplantation
into irradiated congeneic mice,28,29 indicating the
existence of lymphoid-committed DC progenitors. In the fetus, thymic
DCs start to develop in the rat embryonic thymus as early as day
17.30 All these observations imply that the development of
different DC subsets from embryonic HPCs may be regulated under distinct mechanisms from those of adult BM HPCs.
Because the thymus and spleen form relatively late in gestation and are
involved in the production of highly differentiated more mature
hematopoietic cells, HPCs presumably seed these organs from other
embryonic hematopoietic sites, mostly FL.18,31 To explore
the capacity of generating DC from embryonic HPCs, lineage phenotype-negative (Lin)c-kit+
HPCs were isolated from day 13 postcoitus (dpc) FL, and cultured in the
presence of growth factors and stromal cell line PA6. We found that
these FL HPCs possess the capacity to generate DC precursors, which can
finally differentiate into mature DCs, in the presence of GM-CSF + stem
cell factor (SCF) + Flt3 ligand (Flt3L) and PA6 cells. We also describe
that DC precursors in FL-derived HPCs may share progenitor cells in
common with NK cells.
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Materials and methods |
Cytokines, antibodies, and cell lines
Recombinant murine SCF and anti-c-kit antibody (ACK-2) were
kindly provided by Dr T. Sudo, Basic Research Institute of Toray Co
(Kanagawa, Japan), murine GM-CSF by Kirin Brewery Co (Tokyo, Japan),
murine macrophage colony-stimulating factor (M-CSF) by Morinaga Co
(Tokyo, Japan), and Flt3L by Immunex Co (Seattle, WA). Mouse TNF
(mTNF) was produced in house.32 These cytokines were
used at the optimal concentrations as previously
described.26,33 DEC-205 (NLDC145), a rat monoclonal
antibody (MoAb) to murine DCs, was a generous gift of Dr R. M. Steinman (Rockefeller University, New York,
NY).34,35 The MoAb to mouse E-cadherin was purchased from
Dainipon Pharmaceutical Co (Osaka, Japan). Other MoAbs and reagents
used for immunostaining were obtained from PharMingen (San Diego, CA)
unless otherwise indicated.
BM-derived stromal cell line, PA6 and M-CSF defective stromal cell
line, OP9 were kindly provided by Dr S. Nishikawa (Kyoto University,
Kyoto, Japan) and Dr T. Nakano (Osaka University, Osaka, Japan), respectively.
Mice
C57BL/6 and BALB/c mice were obtained from CLEA Animal Co (Tokyo,
Japan) and maintained under specific pathogen-free conditions in the
Animal Facility of the Department of Molecular Preventive Medicine,
School of Medicine, The University of Tokyo, Tokyo, Japan. FL were
obtained from 13 dpc fetuses of female BALB/c or C57BL/6 mice mated
with male C57BL/6 mice. The presence of a postcoital plug was used to
determine day 0 of the embryonic age. All animal experiments complied
with the standards set out in the Guideline for Care and Use of
Laboratory Animals of The University of Tokyo.
Suspension culture of Linc-kit+
HPCs
BM Linc-kit+ HPCs were
obtained and cultured to induce DCs as previously
described.26,33 FL cells were obtained by aspirating FL
from 13 dpc murine embryos.
Linc-kit+ HPCs were isolated
from FL mononuclear cells (MNCs) using a cell sorter (EPICS ELITE,
Coulter Electronics, Hialeah, FL) as previously described.26,33 In brief, FL MNCs were subjected to
indirect staining using a biotin-conjugated anti-c-kit MoAb and
phycoerythrin (PE)-labeled streptavidin, followed by a set of
fluorescein isothiocyanate (FITC)-labeled MoAbs to
CD3 (145-2C11), CD4 (H129.19),
CD8 (53-6.7), B220 (RA3-6B2), Gr-1 (Ly-6G),
CD11a(2D7), and CD11b (M1/70). The degree of contamination
by other types of cells in these preparations was consistently < 0.5% as revealed by an immunofluorescence analysis.
Purified FL-derived Linc-kit+
HPCs were incubated at a cell concentration of
3 × 104 cells/mL in Iscove's modified Dulbecco's
medium (IMDM; GiBCO, Rockville, MD), supplemented with 20% fetal calf
serum (FCS), penicillin G (100 U/mL), and streptomycin (100 µg/mL) in
the presence of GM-CSF (4 ng/mL) + SCF (10 ng/mL) + Flt3L (10 ng/mL) + mTNF (50 ng/mL). Optimal conditions were maintained by splitting
these cultures at day 7 and every 2 to 3 days replacing 50% of the
medium with a new medium containing fresh cytokines. At the indicated time intervals, morphologic observation with an inverted microscope and
immunophenotypical analyses were performed on the cultured cells.
FL-derived Linc-kit+ HPCs were
cocultured with PA6 or OP9 stromal cell line as shown in Figure
1. In some experiments, FL-derived HPCs
were cultured with or without stromal cells in the presence or absence
of M-CSF (100 ng/mL) under the above described conditions. In other
experiments, the NK 1.1+ cell subset was sorted at day 3, 5, and 7 from Linc-kit+ HPCs,
which had been cocultured with PA6 cells in the upper compartments of
transwell plates in the presence of SCF and Flt3L. The purity of the
sorted cells was more than 99%. The sorted NK1.1+ cell
subset was recultured for 10 to 12 days, supplemented with GM-CSF.
These cells were collected for analyses after restimulation by GM-CSF + mTNF for an additional 3 to 5 days. Meanwhile, the sorted
NK1.1+ cell subset was also cultured continuously in the
absence of GM-CSF for an additional 10 to 12 days. All the staining and
sorting procedures were performed in the presence of 1 mM EDTA to avoid cell aggregation.
Immunofluorescence analysis
Immunofluorescence analyses were performed as previously
described.26,33 In 2-color analyses,
4 × 105 cells were sequentially incubated with
optimal concentrations of biotinylated hamster anti-CD86 or anti-CD11c,
and rat anti-DEC-205, anti-E-cadherin or anti-F4/80 MoAbs, followed by
FITC-labeled streptavidin and by FITC-conjugated goat antirat IgG
(Fab')2 antibodies (Caltag, Camarillo, CA),
respectively, or directly with FITC-labeled anti-CD8 or NK1.1 MoAbs.
These cells were finally stained with PE-conjugated mouse antimouse Ia
MoAb (AF6-120.1). In other experiments, the cells were first incubated
with the appropriate concentration of biotinylated anti-Ly49c MoAb and
subsequently labeled by PE-conjugated streptavidin. Then the cells were
stained with FITC-conjugated anti-NK1.1 MoAb. In tri-color analysis,
the cells were sequentially incubated with rabbit antimouse asialo-GM1
antibody (Wako Co, Osaka, Japan) and PE-conjugated goat antirabbit IgG
(Fab')2 antibody, followed by the incubation with
FITC-labeled anti-CD3 or CD4 and biotinylated anti-NK1.1 MoAbs. The
cells were finally stained with Cy-chrome-conjugated streptavidin. The
instrument compensation was set in each experiment using single-color
or 2-color stained samples. In some experiments, the corresponding cell
subpopulations were isolated using a cell sorter.
Endocytosis
The endocytosis experiment was performed as previously
described.36,37 Briefly, in the endocytosis test the cells
were incubated with 0.1 mg/mL FITC-Dextran (FITC-DX; 4,000 daltons; Sigma Chemicol Co, St Louis, MO) at 0°C or 37°C for 60 minutes. The reaction was stopped by adding ice-cold phosphate-buffered saline
(PBS) containing 5% bovine serum albumin and 0.02% sodium azide, and
the cells were washed 3 times with 2.5% FCS-0.02% sodium azide-PBS,
respectively. Finally, the percentage and density of FITC-positive
cells were examined with a cell sorter as previously described.33
Mixed leukocyte reaction (MLR)
Splenic MNCs were prepared from allogeneic mice as previously
described.33 The adherent cells were first removed by
incubating them at 37°C for 60 minutes in IMDM medium containing
10% FCS. To obtain highly purified T cells, the nonadherent splenic
MNCs were incubated with superparamagnetic MicroBeads conjugated with hamster anti-mouse CD4 MoAb (Miltenyi Biotec, Germany), thereby isolating the CD4+ T cells by magnetic cell sorting. After
treatment with mitomycin C (MMC; 15 µg/mL),38 the
indicated stimulator cells from FL Linc-kit+ cell culture, BM
Linc-kit+ cell culture or
peritoneal macrophages (from 100 to 3 × 104 cells)
were added to the T cells (3 × 105) in wells of
96-well round-bottomed microtest tissue-culture plates (Nunc, Roskilde,
Denmark). After incubating at 37°C for 4 to 5 days, cell
proliferation was determined by using
3-(4,5-dimethylthiazolyl-2yl-2,5-diphenyltetrazolium bromide (MTT;
Sigma Chemical Co). In brief, 15 µL of MTT (5 mg/mL in PBS) was added
into each well and the plates were incubated at 37°C for an
additional 4 hours. The resultant absorbance at 550 nm was read by a
microplate immunoreader.
Nonspecific esterase (NSE) staining
Cells were cytocentrifuged for 5 minutes at 500 rpm on a microscope
slide and used for NSE staining (a-naphthyl acetate esterase staining
kit; Sigma Chemical Co), according to the instructions of the manufacturer.
Statistical analysis
Significant differences were evaluated with the Student's t
test. P < .05 were considered to be statistically significant.
| |
Results |
Culture conditions for generating DCs from 13 dpc FL-derived
Linc-kit+ HPCs
We first determined whether culture conditions that lead to the
differentiation of adult murine BM HPCs into DCs would also be
applicable in FL-derived progenitor cells. The capacity to generate DCs
from 13 dpc FL-derived Linc-kit+
HPCs in the presence of GM-CSF + SCF + Flt3L and mTNF was examined after culturing for 7, 14, and 21 days. As shown in Figure
2A, there was no typical DC aggregate
formation in the cultures. The immunofluorescence analysis showed that < 1.5% cells expressed Ia and CD86 antigens, characteristics of
mature DCs (Figure 2B). These results indicated that the combination of
GM-CSF + SCF + Flt3L + mTNF could not induce the
generation of mature DCs from 13 dpc FL-derived
Linc-kit+ HPCs, whereas
combination of these growth factors could induce mature DC
differentiation from adult murine BM
Linc-kit+ HPCs as reported
previously by us.26
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| Fig 2.
The culture of murine 13 dpc FL-derived
Linc-kit+ HPCs.
(A) Using a phase contrast microscope, morphologic analyses were
performed on cultured murine 13 dpc FL-derived
Linc-kit+ HPCs stimulated with
GM-CSF + SCF + Flt3L + mTNF at day 7, 14, and 21. Original
magnifications: × 200. (B) 2-color immunofluorescence analysis
was performed on the cultured cells at the indicated time points. The
cells were sequentially stained with biotinylated CD86 MoAb and
PE-labeled Ia MoAb. CD86 was revealed by FITC-streptavidin. The quads
were set up on the isotype-matched control dot plot. These results are
representative of 3 independent experiments.
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To explore what conditions might be essential for inducing DCs from
FL-derived Linc-kit+ HPCs, we
first cocultured them with BM-derived stromal cell line PA6 or M-CSF
defective stromal cell line OP9 in the presence of GM-CSF + SCF + Flt3L
for 12 to 14 days. Because addition of mTNF significantly inhibited
the proliferation of FL
Linc-kit+ HPCs (data not shown),
this was excluded from the cultures. On the coculture with the PA6 or
OP9 cells in the presence of GM-CSF + SCF + Flt3L, FL-derived HPCs did
not develop DC aggregates (Figure 3A), and
only a few cells were Ia+CD86+ cells (Figure
3B). Because our previous findings showed that mTNF plays a critical
role in stimulating the differentiation of DCs from adult murine
BM-derived Linc-kit+
HPCs,26 the nonadherent cells from FL-derived HPCs
cocultured with PA6 stromal cells in the presence of GM-CSF + SCF + Flt3L were stimulated with GM-CSF + mTNF for an additional 3 to 5 days. As shown in Figure 3A, this resulted in the development of
DC-like aggregates surrounded by cells with large sheet-like processes in the periphery. Phenotypically, such cells expressed high levels of
Ia, CD86, and CD40, and low levels of F4/80, DEC205, and E-cadherin antigens. However, CD11c molecule was barely detected, and CD8 and
NK1.1 antigens were negative (Figures 3B and
4A). Giemsa-Wright staining showed that the
aggregated cells were morphologically DCs with eccentric nuclei
and polarized lamellipodia. They were negative for nonspecific
esterase activity (Figure 4B).39 Functionally, these cells
could enhance the proliferation of allogeneic T lymphocytes in an MLR assay at a comparable level with DCs generated from adult BM
Linc-kit+ HPCs (Figure
5). On the contrary, cells derived from OP9
cells + GM-CSF + SCF + Flt3L-induced FL-derived HPCs did not
differentiate into mature DCs when restimulated with GM-CSF + mTNF (Figure 3). These results suggested that DC precursors were
generated from murine FL-derived
Linc-kit+ HPCs cocultured with
PA6 stromal cells in the presence of GM-CSF + SCF + Flt3L,
whereas mTNF and GM-CSF were essential for the final maturation of
DCs from these precursors.
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| Fig 3.
Generation of DCs from 13dpc FL-derived
Linc-kit+ HPCs.
13 dpc FL-derived Linc-kit+ HPCs
were cocultured with stromal cell lines PA6 or OP9 cells in the
presence of GM-CSF + SCF + Flt3L for 12 to 14 days, and restimulated by
GM-CSF + mTNF for an additional 3 to 5 days. (A) A phase contrast
microscopic observation was performed on cocultured FL-derived HPCs
with PA6 or OP9 cells in the presence of GM-CSF + SCF + Flt3L for 12 to
14 days before and after replanted into new plates and restimulated by
GM-CSF + mTNF for an additional 3 to 5 days. Original
magnification: × 200. (B) Immunophenotypic detection was
performed on nonadherent cells from FL-derived HPC cultures at the
indicated time points and culture conditions. These cells were
sequentially stained with biotinylated CD86 MoAb and PE-labeled Ia
MoAb, whereas CD86 was revealed by FITC-streptavidin. The quads were
set up on the isotype-matched control dot plot. These results are
representative of 3 independent experiments.
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| Fig 4.
Immunophenotypic and morphologic analyses of DC-like
cells.
13dpc FL-derived Linc-kit+ HPCs
were first supplemented with PA6 cells + GM-CSF + SCF + Flt3L for 12 to
14 days and then stimulated with GM-SCF + mTNF for an additional 3 to 5 days. (A) The phenotype of the nonadherent cells was analyzed by
2-color immunofluorescence staining as described in the Materials and
Methods. The indicated FITC-labeled MoAbs (CD8, NK1.1, F4/80, CD40,
CD11c, DEC205, and E-cadherin) were used to demonstrate the phenotypic
characteristics of the generated DCs. The quads were set up on the
isotype-matched control dot plot. (B) Giemsa staining and nonspecific
esterase activity analyses were performed on GM-CSF + mTNF-stimulated DC precursors at day 3 to day 5 that were derived
from FL-derived HPCs cocultured with PA6 cells + GM-CSF + SCF + Flt3L
for 12 to 14 days. Original magnification: × 400. These results
are representative of 3 independent experiments.
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| Fig 5.
The capacity of the cultured cells to enhance allogeneic
MLR.
Allogeneic MLR was performed using purified T cells
(3 × 105 cells/well in 96-round-well plate) as
responder cells. The unfractionated nonadherent cells, which were
generated from FL-derived HPCs cocultured with PA6 cells + GM-CSF + SCF + Flt3L for 12 to 14 days before and after restimulation by GM-CSF + mTNF for an additional 3 to 5 days, were treated by MMC and used as
stimulator cells at the indicated cell numbers. DCs derived from
BM-derived HPCs stimulated with SCF + Flt3L + GM-CSF + mTNF, and
peritoneal macrophages were used as controls. The proliferation of T
cells was measured using MTT after 5 days of culture. Results are
expressed as the mean ± 1 SD of triplicate cultures and are
representative of 3 independent experiments. Black squares indicate
macrophages; red diamonds, DCs from BM HPCs; green circles, DCs from FL
HPCs; and purple triangles, FL-derived DC precursors.
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Mediators required for generating DC precursors from FL-derived
Linc-kit+ HPCs
To elucidate whether the cell-cell contact is required for the
generation of DC precursors, transwell plates with 0.4-µm pore size
were used in the coculture system. The direct interaction of HPCs and
stromal cells could be blocked in the transwell system, while soluble
factors could diffuse into the cell cultures. After coculturing with
PA6 cells + GM-CSF + SCF + Flt3L for 12 to 14 days, the nonadherent
cells in the upper compartments of transwell plates from FL-derived
HPCs were transferred into a new 6-well plate and restimulated with
GM-CSF + mTNF. As shown in Figure 6, DC
precursors could be generated in the transwell culture system, and
these cells could subsequently differentiate into mature DCs by
restimulation with GM-CSF + mTNF.
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| Fig 6.
The role of stromal cells in the induction of DCs from
FL-derived Linc-kit+ HPCs.
13 dpc FL-derived HPCs were cultured in the presence of cytokines,
GM-CSF + SCF + Flt3L, with or without stromal cell line OP9 or PA6
cells in a transwell culture system. M-CSF was added into these
cultures as indicated. After 12 to 14 days of culture, the nonadherent
cells were replanted in a new 6-well plate, restimulated with GM-CSF + mTNF for an additional 3 to 5 days and then collected for
immunofluorescence analysis. Ia+CD86+ cells
were examined by staining with PE-labeled Ia MoAb and biotinylated CD86
MoAb, and revealed by FITC-streptavidin. Results are expressed as the
mean ± SD of 3 independent experiments. *P < .05
significance compared with cultures lacking stromal cells or M-CSF
addition, or between different stromal cells. Gray bars represent
cultures without M-CSF; colored bars, with M-CSF.
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In contrast, coculture with OP9 cells in the same condition could not
generate DC precursors from FL-derived
Linc-kit+ HPCs. Because OP9
cells are deficient in the production of functional M-CSF, we asked
whether M-CSF might account for the generation of DC precursors in
PA6-stimulated culture. Addition of M-CSF to OP9 cells + GM-CSF + SCF + Flt3L resulted in significant induction of FL HPCs-derived DC
precursors that subsequently differentiated into mature DCs by
restimulation with GM-CSF + mTNF. However, the effect of M-CSF on
the generation of DC precursors was significantly less than that of PA6
cells + GM-CSF + SCF + Flt3L-stimulated culture. Moreover, addition of
M-CSF alone to the combination of cytokines, GM-CSF + SCF + Flt3L,
could not induce the generation of DC precursors from FL-derived HPCs.
Furthermore, addition of M-CSF to PA6 cells + GM-CSF + SCF + Flt3L-induced culture did not enhance the generation of DC precursors
(Figure 6).
Further characterization of DC precursors derived from 13 dpc FL
Linc-kit+ HPCs
To better understand the cellular basis for mature DC development
from 13 dpc FL-derived Linc-kit+
HPCs, we analyzed the features of the cells generated at day 12 to
day14 in culture with PA6 cells + SCF + Flt3L + GM-CSF. Morphologically, these cells showed monocyte-like cells.
Phenotypic analysis demonstrated that these cells expressed high level
of CD11b (data not shown). When these nonadherent cells were further examined with a panel of MoAbs, the analysis revealed that these cells
expressed high levels of CD40 and E-cadherin and low levels of
NK1.1 and Gr-1, but lacked certain DC-associated markers Ia, CD86, and
DEC205, whereas CD11c and F4/80 molecules were barely detected.
However, CD8 antigen was negative (Figure
7). The above evidence indicates that
culture with PA6 cells + GM-CSF + SCF + Flt3L is not enough to support
the generation of mature DCs from 13 dpc FL-derived
Linc-kit+ HPCs.
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| Fig 7.
Immunofluorescence analysis on DC precursors generated
from murine FL-derived Linc-kit+
HPCs.
Immunofluorescence analysis was performed on the nonadherent cells from
FL-derived HPCs cocultured with PA6 cells + GM-CSF + SCF + Flt3L for 12 to 14 days. The indicated markers of FITC-labeled MoAbs were used to
demonstrate the phenotypic characteristics of the DC precursors. Solid
and dotted lines indicated the immunofluoresence intensity of cells as
a control and the test of MoAbs, respectively. Representative results
from 3 independent experiments are shown.
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Functionally, these DC precursors were active in endocytic
uptake of FITC-DX at 37°C, but not at 0°C (Figure
8). These cells were not effectively able
to stimulate allogeneic T-cell proliferation in MLR (Figure 5). When
these DC precursors were further stimulated by GM-CSF and mTNF to
differentiate into mature DCs, such endocytic capacity was
significantly reduced (Figure 8), and the capacity to
stimulate T cell proliferation in allogeneic MLR was considerably enlarged (Figure 5).
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| Fig 8.
Endocytic activity of FL HPC-derived DC precursors and
mature DCs.
The capacity of FITC-DX uptake was analyzed by a cell sorter as
described in "Materials and Methods." The FITC intensity of cells
as a control or the test of FITC-DX uptake was indicated. The results
are representative of 3 independent experiments.
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DCs derived from FL Linc-kit+
HPCs share progenitor cells in common with NK cells
Previous reports demonstrated that
Sca-1+c-kit+ FL cells could
differentiate into lymphocytes.20,21 In our studies,
culture of FL-derived Linc-kit+
HPCs with PA6 alone for 7 days led to the generation of
NK1.1+ cells accounted for 75% of the suspended cells.
Addition of SCF + Flt3L further increased the proliferation of
NK1.1+ cells. However, GM-CSF significantly inhibited the
generation of NK1.1+ cells in PA6 cells + SCF + Flt3L
cocultures (Figure 9). The cultured cells
derived from 13 dpc FL Linc-kit+
HPCs in the presence of PA6 cells + SCF + Flt3L at day 14 were immunophenotypically analyzed. These NK1.1+ cells were
CD3 and CD4 negative, and expressed similar level of asialo-GM1
antigen (Figure 10A) to that of adult
murine spleen NK cells (data not shown). They also partly expressed a
mature NK cell marker, Ly49c antigen (Figure 10B). The results suggest that these cultured cells possess characteristics of NK cells, but not
of NK T cells.
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| Fig 9.
Generation of NK1.1+ cells from FL-derived
Linc-kit+ HPCs.
13 dpc FL-derived Linc-kit+ HPCs
were cocultured with PA6 cells in the presence or absence of SCF + Flt3L with or without GM-CSF in a transwell culture system. The
nonadherent cells were collected at the indicated time points and
stained with FITC-conjugated anti-NK1.1 MoAb. The data represent mean
value ± SD of NK1.1+ cell percentage in the cultures
from 5 independent experiments. Black squares represent PA6 cells; red
diamonds, PA6 cells with SCF and Flt3L; and green circles, PA6 cells
with GM-CSF, SCF, and Flt3L.
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| Fig 10.
Immunophenotypic analysis on NK1.1+ cells
generated from FL-derived
Linc-kit+ HPCs.
Linc-kit+ HPCs from 13 dpc FL
were cultured in the presence of PA6 cells + SCF + Flt3L for 14 days.
The phenotypes of the nonadherent cells were analyzed by 2- and
tri-color immunofluorescence staining as described in "Materials and
Methods." (A) In the tri-color analyses, expression of asialo-GM1
and CD3 or CD4 antigens on NK1.1+ cells was analyzed.
(B) The 2-color analysis was performed on expression of NK1.1 and Ly49c
antigens from the cultured cells. The quads were set up on the
isotype-matched control dot plot and the results are representative of
3 independent experiments.
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To clarify whether DCs share a common progenitor with NK cells, we
sorted NK1.1+ cells from PA6 cells + SCF + Flt3L-stimulated
FL Linc-kit+ HPCs cultures at
the indicated time points, cultured them again in the presence of the
PA6 cells + SCF + Flt3L + GM-CSF for an additional 10 to 12 days, and restimulated them with GM-CSF + mTNF. Mature DCs (> 26%) could be generated from the NK1.1+ cells sorted at
day 3 of original culture, but not from a prolonged identical culture
of more than 5 days duration (Figure 11).
In contrast, flow cytometry analysis revealed that more than 92% of
the cells expressed NK1.1 antigen from the continuous culture of the
sorted NK1.1+ cells in the absence of GM-CSF for an
additional 10 to 12 days. These results suggest that FL
Linc-kit+ HPC-derived DCs may
share a common progenitor with NK cells.
View larger version (38K):
[in this window]
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| Fig 11.
Capacity to generate DC precursors from
NK1.1+ cells.
13 dpc FL-derived HPCs were co-cultured with PA6 cells + SCF + Flt3L in
transwell plates. At the indicated time points, the nonadherent cells
were collected and NK1.1+ cells were sorted and recultured
with PA6 cells + GM-CSF + SCF + Flt3L. After being recultured for 10 to
12 days, the nonadherent cells were restimulated with GM-CSF + mTNF
for an additional 3 to 5 days. The expression of Ia and CD86 antigens
on these cells was examined as described in "Materials and
Methods." Solid and dotted lines indicated the immunofluoresence
intensity of cells stained with a control and the test MoAbs,
respectively. The quads were set up on the isotype-matched control dot
plot. Representative results from 3 independent experiments are
shown.
|
|
| |
Discussion |
Identification of the progenitors of DCs in the early stages of
fetal hematopoiesis is of particular interest in understanding the
origin and development of DCs in the establishment of the immune
system.1,10-12,40,41 We have described here that purified murine 13 dpc FL Linc-kit+ HPCs
can differentiate into DC precursors in vitro after coculture with PA6
stromal cells for 12 to 14 days in the presence of GM-CSF + SCF + Flt3L. These DC precursors can subsequently be induced to develop into
mature DCs by further stimulation with GM-CSF + mTNF for an
additional 3 to 5 days. The resultant DCs not only show typical
morphologic characteristics and immunophenotype of DCs, but also
markedly stimulate T cell proliferation in allogeneic MLR as
effectively as adult murine BM
Linc-kit+ HPC-derived DCs
do,26 suggesting that these cells are mature DCs with
antigen-presenting function.
Generation of DC precursors from 13 dpc murine FL
Linc-kit+ HPCs in vitro depends
on the support of PA6 stromal cell line even in the presence of a
cytokine mixture of GM-CSF + SCF + Flt3L, which suffices to induce
adult BM Linc-kit+ HPCs to
develop into DC precursors.26 In transwell culture, we
showed that some soluble factors secreted by PA6 stromal cells together
with the cytokine cocktail could induce FL-derived HPCs to develop into
DC precursors. Previous studies have shown that the number of
Langerhans cells (LCs) is reduced in the skin of osteopetrotic (op/op)
mice which is defective in the production of functional M-CSF
protein.42 This implied that OP9 stromal cells might induce
13 dpc FL-derived Linc-kit+ HPCs
to develop into DCs by the addition of M-CSF. Addition of M-CSF to OP9
cells + GM-CSF + SCF + Flt3L generated DC precursors from FL-derived
HPCs to a much less extent than that of PA6 cells and the
cytokines-stimulated culture. This result indicates that M-CSF
partially compensates for the incapability of OP9 cells to induce the
differentiation of DC precursors from FL-derived HPCs. However, the
addition of M-CSF to the culture of PA6 cells + GM-CSF + SCF + Flt3L
did not enhance the yield of DCs generated from FL-derived
Linc-kit+ HPCs. Moreover, M-CSF
plus the cytokines GM-CSF + SCF + Flt3L without stromal cells could not
induce FL-derived HPCs into DC precursors, indicating that M-CSF cannot
substitute for the role of PA6-derived soluble factor(s) which play an
essential role in stimulating FL
Linc-kit+ HPCs to differentiate
into DC precursors.
Several other cytokines have been considered to be the candidates
accounting for the generation of DC precursors from FL-derived HPCs.
For example, IL-4 and TGF- have been shown to be essential factors
for DC differentiation.43,44 However, addition of IL-4 to
the combination of GM-CSF + SCF + Flt3L failed to generate DC
precursors from FL-derived HPCs, or anti-IL-4 MoAb did not block the
generation of FL-derived DC precursors in the PA6 cells and cytokine
cocktail culture system (data not shown). We have recently observed
that TGF- polarizes adult murine BM-derived HPCs to differentiate
into monocytes/macrophages that can eventually differentiate into
LC-like DCs expressing high level of E-cadherin.45 Interestingly, DC precursors generated in our cultures expressed E-cadherin, which is believed to be a specific marker expressed on LCs
and to be tightly regulated by TGF-.46 However, without PA6 cells, addition of TGF- to the combination of GM-CSF + SCF + Flt3L did not stimulate the generation of DC precursors from FL-derived
HPCs. Moreover, both OP9 and PA6 cells expressed IL-4 mRNA, whereas
TGF- mRNA was undetectable in either of them (data not shown). Taken
together, these observations indicate that neither IL-4 nor TGF-
substitutes for PA6 cells to induce the generation of DCs from
FL-derived HPCs. Thereafter, the collaborative role of IL-4 or
serum-derived TGF- with some PA6 cell-secreted factors may not be
ruled out.
Most recently, it has been demonstrated that generation of T cells, NK
cells, and DCs in vitro from human FL-derived
CD34+D38 cells required the fetal thymic
organ cultures,47 in- consistent with our findings that the
generation of DC precursors from murine FL HPCs needs the support of
PA6 cells. Obviously, the culture conditions reported here are
artificial and might not exactly reflect the physiological situation in
vivo. PA6 stromal cells were derived from BM.48 It has been
shown that PA6 cells can promote the proliferation of BM hematopoietic
cells through a short range cell-to-cell interaction by providing an in
vitro microenvironment similar to that for in vivo
hematopoiesis.49 It is anticipated that the unidentified
factor produced by PA6 cells may be available in bone marrow or thymus
environments that may skew development of HPCs toward the DC lineage.
Biochemical purification of the factor(s) that are responsive for the
generationof DC precursors from FL HPCs is in progress, but so far unsuccessful.
FL HPC-derived DCs in this study displayed low levels of DEC205 and
E-cadherin and barely detectable CD11c antigen. Previous studies
demonstrated that murine splenic DCs express high level of CD11c and
can be classified into at least 2 subpopulations CD8+DEC205+ and
CD8DEC205 cells. On culture in
vitro, these mature splenic DCs express high level of
DEC205.2 This phenotype of splenic DCs significantly differs from that of mature DCs or their precursors generated from
FL-derived Linc-kit+ HPCs in our
culture system. We have recently reported that the development of BM
Linc-kit+ HPC-derived DCs could
be mediated through CD11b/dullCD11c+ and
CD11b+/hi CD11c+ precursors.33 When
we evaluated FL Linc-kit+ HPCs,
it was found that the cytokerastic kinetics differ between BM- and
FL-derived Linc-kit+ HPCs.
FL-derived DCs were generated from the precursors on which CD11c
antigen was barely expressed. Except for Gr-1 marker, these cells
lacked the phenotypic characteristics of other hematopoietic cell
lineages, whereas BM Linc-kit+
HPC-derived ones expressed high levels of DEC205 and CD11c
molecules.26,33 These findings would be reminiscent of the
generation of CD11c DC from FL-derived
Linc-kit+ HPCs. In human blood,
CD11c+ and CD11c DC subsets have been
identified.50A recent study demonstrated that
CD11c+ and CD11c DC subsets possibly
localize in the B-cell and T-cell areas, respectively, and may exert
distinct roles in initiating cellular and humoral immune
responses.50 Further characterization of the phenotype of
DC precursors in vivo in murine fetus will be helpful for understanding
DC development and its function in educating the immune system during
fetal stage.
Evidence has accumulated indicating that CD40 molecule, a member of TNF
receptor family, could be expressed on tonsil DCs,51 blood
DCs,52 and LCs53 in human and some murine
DCs.33 Moreover, triggering of CD40 ligand and CD40 could
stimulate human CD34+ HPCs to differentiate into mature DCs
and activate the function of mature DCs.54 Interestingly,
our results show that CD40 molecule was highly expressed on DC
precursors generated from FL
Linc-kit+ HPCs in the presence
of PA6 cells + GM-CSF + SCF + Flt3L. Because CD40 and other
members of TNF receptor family can bind to their ligands expressed on
activated and memory T cells, it is presumed that the stimulatory
effects of DCs and T cells are mutual because DCs induce T cells into
immune or tolerance state by presenting antigens,1,11,12,54
and T cells in turn promote the development of precursors into
functionally mature DCs.10,54-56
It has been reported that NK cells are developmentally close to
lymphoid cells in mouse57-59 and in human.60
Murine FL-derived Sca-1+c-kit+ cells
generated in vitro mixed NK colonies with B220+
cells.21 A T cell precursor subset in murine thymus has
been shown to be tripotential with full potential to generate DCs and also NK cells although the ability to develop B cells has been lost.59,61,62 Further study has demonstrated that NK cells and DCs may branch off the T cell lineage from a common intermediate bipotential progenitor in human postnatal thymus.63
However, it remains to be elucidated which factors may determine the
branch-off of DCs from NK and T cells. A remarkable feature from our
findings is that murine FL-derived HPCs are able to differentiate into either DC or NK cell precursors, depending on the presence of GM-CSF.
When cocultured with PA6 cells, murine FL-derived HPCs could generate
DC precursors in the presence of GM-CSF, whereas the development of
NK1.1+ cells was drastically suppressed. In the absence of
GM-CSF, a large number of NK1.1+ cells proliferated in PA6
cells + SCF + Flt3L-stimulated cultures. Interestingly, the
generated NK1.1+ cells could revert to DC precursors at an
early stage of differentiation by reculturing with GM-CSF + PA6 cells + SCF + Flt3L. These observations reveal that unlike IL-2, which can
simultaneously induce bipotential differentiation of human thymic
progenitors into DCs and NK cells in similar proportion in
vitro,63 GM-CSF plays a critical role not only in promoting
the viability, proliferation, survival, function, and mobilization of
DC precursors and DCs, but also in the commitment into DC precursors at
the expense of NK cell precursors from FL-derived HPCs. The transition
between DC and NK cell precursors suggests that DCs and NK cells may
share a common progenitor of fetal hematopoietic origin. Thus, owing to the relationship of NK cells and DCs as well as their dependence on
stromal cells in differentiation, we speculate that 13 dpc murine FL
Linc-kit+ HPCs may be able to
generate thymic DCs under appropriate culture condition. It is believed
that CD8+ DCs represent murine lymphoid DCs that may
derive from lymphoid progenitor cells.64 However, it
remains to establish a culture system for generating
CD8+ lymphoid DCs in vitro.65 We therefore
could not directly compare the NK1.1+ precursor-derived DCs
with CD8+ lymphoid DCs. Further experiments will be
performed in vivo to address the relationship of NK1.1+
precursor-derived DCs with CD8+ lymphoid DCs; these
experiments are in progress in our laboratory.
It has been demonstrated that the primary stromal cultures from 16 dpc
rat embryonic thymus can differentiate into morphologically and
phenotypically mature DCs after several days of culture.30 In agreement with these results, our findings suggest that FL HPCs gain
the capacity to generate DCs as early as 13 dpc. Probably, the
embryonic DC precursors have developed in the early embryonic hematopoietic organs such as fetal liver and thymus possibly with the
phenotype different from that of the adult counterparts; for example,
embryonic DC precursors barely display CD11c molecule. However, the
differentiation of these DC precursors may require other factors during
the embryonic development. FL
Linc-kit+ HPCs may migrate into
various tissues where they produce essential factors required for DC
development and function in the fetal stage, to generate diverse DC
precursors. This study would provide a novel insight into the mechanism
of DC ontogeny and their role in the establishment of the immune system.
| |
Acknowledgments |
We express our gratitude to Dr R. M. Steinman (Rockefeller
University, New York, NY) for his kind gift of MoAb to DEC-205 (NLDC
145); and Dr Tohru Nakano (Osaka University, Osaka, Japan) and Dr
Shin-ichi Nishikawa (Kyoto University, Kyoto, Japan) for their generous
gifts of PA6 and OP9 stromal cell lines. We highly appreciate Dr J. J. Oppenheim (NCI-FCRDC, Frederick, MD) for his critical review of the
manuscript. We also thank Drs V. Christian, H. Iizasa, and Hongyan Dong
for their kind assistance.
| |
Footnotes |
Submitted December 30, 1998; accepted September 1, 1999.
Reprints: Kouji Matsushima, Department of Molecular Preventive
Medicine, School of Medicine, The University of Tokyo, 7-3-1, Hongo,
Bunkyo-ku, Tokyo 113-0033, Japan.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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C. Liu, S. Yu, J. Kappes, J. Wang, W. E. Grizzle, K. R. Zinn, and H.-G. Zhang
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Top
Abstract
Introduction