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Prepublished online as a Blood First Edition Paper on October 24, 2002; DOI 10.1182/blood-2002-07-2254.
IMMUNOBIOLOGY
From the Division of Immunogenetics, Department of
Neuroscience and Immunology, Kumamoto University Graduate School of
Medical Sciences, Kumamoto, Japan; and the Division of
Developmental Genetics, Department of Organogenesis, Institute of
Molecular Embryology and Genetics, Kumamoto University,
Japan.
We developed a method to generate dendritic cells (DCs) from mouse
embryonic stem (ES) cells. We cultured ES cells for 10 days on feeder
cell layers of OP9, in the presence of granulocyte-macrophage colony-stimulating factor in the latter 5 days. The resultant ES
cell-derived cells were transferred to bacteriologic Petri dishes
without feeder cells and further cultured. In about 7 days, irregularly
shaped floating cells with protrusions appeared and these expressed
major histocompatibility complex class II, CD11c, CD80, and
CD86, with the capacity to stimulate primary mixed lymphocyte reaction
(MLR) and to process and present protein antigen to T cells. We
designated them ES-DCs (ES cell-derived dendritic cells), and
the functions of ES-DCs were comparable with those of DCs generated
from bone marrow cells. Upon transfer to new dishes and stimulation
with interleukin-4 plus tumor necrosis factor Dendritic cells (DCs) are the most potent
antigen-presenting cells (APCs) responsible for priming of naive T
cells in the immune response. DCs are involved in the maintenance of
immunologic self-tolerance in the periphery, inducing regulatory T
cells or anergy of autoreactive T cells.1-3 It has been
reported that distinct subpopulations of DCs preferentially induce
differentiation of either T helper 1 (Th1) or Th2
cells.4-6 Therefore, DCs physiologically play a central
role in immunoregulation. Manipulation of functions of DCs by genetic
modification and in vivo transfer of DCs with modified property is
considered a promising means to control immune responses in an
antigen-specific manner.7 As for the methods for gene
transfer to DCs, electroporation, lipofection, and virus vector-mediated transfection have been developed. However, there are
several problems related to presently used means (ie, efficiency of
gene transfer, stability of gene expression, potential risk accompanying the use of virus vectors, and the immunogenicity of virus
vectors). Although improvements have been made in these methods,
development of more efficient and safer means is
desirable.8,9
Embryonic stem (ES) cells are characterized by pluripotency and
infinite propagation capacity. Non-virus-mediated methods for gene
transfer, including targeted gene integration and procedures for
isolation of appropriate recombinant cell clones, have been established
for ES cells. Recently, a novel Cre-lox-mediated exchangeable gene-trap system has been developed using TT2 ES cells.10
The method enables efficient gene-trap, plasmid rescue for the analysis of trapped genes, and targeted integration of replacement vectors to
the gene-trapped sites. Genetic modification of ES cells and their
subsequent in vitro differentiation to DCs would be an attractive strategy for genetic manipulation of DCs and for analysis of gene functions in DCs.
For hematopoietic differentiation of ES cells in vitro, embryoid
body-mediated methods and the OP9-coculture method have been established.11,12 OP9 is a bone marrow stromal cell line
that originated from macrophage colony-stimulating factor
(M-CSF)-defective op/op mouse,13 and generation of
various hematopoietic cells from ES cells using OP9 cells as
feeder cells has been reported, including granulocytes, erythrocytes, B
lymphocytes, and osteoclasts.11-15 The method has been
applied to several molecular and cellular analyses for investigations
of hematopoiesis.16-19
In the current study, we attempted to establish a method to develop DCs
from ES cells in vitro. We adapted the OP9-coculture method for
hematopoietic differentiation of ES cells. For induction of
differentiation to DCs, we used granulocyte-macrophage
colony-stimulating factor (GM-CSF), the cytokine essential for in vitro
generation of DCs from hematopoietic cells.20-22 The
generated ES cell-derived DCs were morphologically and functionally
comparable with those differentiated in vitro from bone marrow cells.
For gene transfer to ES cells to generate genetically modified DCs, we
adopted 2 means: an expression vector containing Mice
Peptides, cell lines, and cytokines
Hematopoietic differentiation of TT2 ES cells The induction of hematopoietic differentiation of TT2 ES cells was done as described.11 After 15 days of culture on feeder cell layers of OP9 without exogenous cytokines, ES cell-derived cells were harvested by pipetting and were then subjected to cytospin preparation and May-Giemsa staining.Induction of differentiation of ES cells into DCs The procedure for induction of differentiation of ES cells into DCs is shown in Figure 1. ES cells were suspended in -MEM supplemented with 20% FCS and seeded
(1.5 × 104/2 mL medium/well) onto OP9 cell layers in
6-well plates. On day 3, half of the medium was removed and 2 mL fresh
medium was added to each well. On day 5, cells were harvested using
phosphate-buffered saline (PBS)/0.25% trypsin/1 mM EDTA
(ethylenediaminetetraacetic acid), reseeded onto fresh OP9
cell layers, and cultured in -MEM supplemented with 20% FCS and
GM-CSF (1000 U/mL). At this step, cells recovered from 3 wells of
6-well culture plates were suspended in 20 mL medium and seeded into
one 150-mm dish. On day 10 (5 days after the transfer), floating cells
were recovered by pipetting. On average, 4 to 8 × 106
cells were recovered from one 150-mm dish, thus indicating 100 to 200 times increase in cell number from undifferentiated ES cells. The
recovered cells were transferred to bacteriologic Petri dishes
(2.5 × 105 cells/90-mm dish) without feeder cells, and
cultured in RPMI-1640 medium supplemented with 10% FCS, GM-CSF (500 U/mL), and 2-mercaptoethanol. After days 17 to 19, 1.5 to
2 × 105 floating or loosely adherent cells were
recovered per dish (ES cell-derived dendritic cells
[ES-DCs]), the number of cells increasing about 100 times
over the number of undifferentiated ES cells. When over half the number
of cells became adherent after day 12, the transfer of floating cells
to fresh dishes on around day 15 improved the purity and yield of
ES-DCs. To induce a complete maturation of ES-DCs, cells cultured
for longer than 10 days in Petri dishes were transferred to fresh Petri
dishes and cultured in RPMI/10% FCS without GM-CSF. The next day, IL-4
(10 ng/mL), TNF- (5 ng/mL), plus anti-CD40 mAb (10 µg/mL, clone
3/23), or IL-4, TNF-, plus lipopolysaccharide (LPS; 1 µg/mL) were added. After 2 or 3 days, cells were harvested by
pipetting and used for functional and flow cytometric analysis.
In most experiments, some cells harvested on days 5 and 10 were freeze-stocked for future use.
Generation of DCs from mouse bone marrow cells Generation of dendritic cells from mouse bone marrow cells was done according to the reported procedures20,27 with some modifications. In brief, bone marrow cells were isolated from (C57BL/6 × CBA) F1 mice and cultured in bacteriologic Petri dishes (1.5 × 106/90-mm dish) in RPMI-1640 medium supplemented with 10% FCS, GM-CSF (500 U/mL), and 2-ME (50 µM). Culture medium was changed by half on days 5 and 10, and floating cells harvested by pipetting between 9 to 12 days of the culture were used as bone marrow-derived DCs (BM-DCs) in functional experiments. For the purpose of maturation, TNF- (5 ng/mL) was added on the day
before analysis.
Flow cytometric analysis Staining of cells and analysis on a flow cytometer (FACScan, Becton Dickinson, San Jose, CA) was done as described previously.28 The procedure for intracellular staining with anti-human CD74 mAb was also described previously.29 Antibodies used for staining were as follows: fluorescein isothiocyanate (FITC)-conjugated anti-H-2Kb (clone CTKb, mouse IgG2a; Caltag, Burlingame, CA), anti-I-Ab (clone 3JP, mouse IgG2a), anti-I-Ek (clone 8705-A, mouse IgG2a; Cedarlane, Hornby, Canada), anti-mouse CD11c (clone N148, hamster IgG; Chemicon, Temecula, CA), R-PE-conjugated anti-mouse CD80 (clone RMMP-1, rat IgG2a; Caltag), R-PE-conjugated anti-mouse CD86 (clone RMMP-2, rat IgG2a; Caltag), anti-mouse CD40 (clone 3/23, rat IgG2a; Serotec, Oxford, United Kingdom), R-PE-conjugated anti-F4/80 (A3-1, rat IgG2b; Serotec), anti-mouse CD205 (clone NLDC-145, rat IgG2a; Serotec), FITC-conjugated anti-mouse CD8 (clone 53-6.7, rat IgG2a; Pharmingen, San Diego, CA),
FITC-conjugated anti-human CD74 (clone M-B741, mouse IgG2a;
Pharmingen), FITC-conjugated goat anti-mouse Ig (Pharmingen),
FITC-conjugated goat anti-hamster IgG (Caltag), FITC-conjugated goat
anti-rat Ig (Pharmingen), mouse IgG2a control (clone G155-178;
Pharmingen), FITC-conjugated mouse IgG2a control (clone G155-178;
Pharmingen), R-PE-conjugated rat IgG2a control (clone LO-DNP-16;
Caltag), FITC-conjugated rat IgG2a control (clone LODNP-57;
Beckman-Coulter, Tokyo, Japan), hamster IgG control (clone 530-6;
Caltag), and rat IgG2a control (clone LO-DNP-16; Caltag).
Mixed lymphocyte reaction (MLR) Splenic mononuclear cells were prepared from unprimed female Balb/c mice, and T cells were isolated from the splenic mononuclear cells by magnetic cell sorting using anti-CD90 (Thy1.2) supermagnetic MicroBeads (Miltenyi Biotec, Bergisch-Gladbach, Germany), and then used as responders. Graded numbers of stimulator cells were x-ray irradiated (35 Gy) and cocultured with responder cells (1.5 × 105) in wells of 96-well round-bottomed culture plates and cultured for 4 days. [3H]-thymidine (6.7 Ci/mmol [247.9 GBq/mmol]) was added to the culture (1 µCi/well [0.037 MBq/well]) in the last 16 hours. At the end of the culture, cells were harvested onto glass fiber filters (Wallac, Turku, Finland), and the incorporation of [3H]-thymidine was measured by scintillation counting.Antigen presentation assay DCs were seeded onto 96-well flat-bottomed culture plates with or without PCC protein (50 µg/mL; Sigma, St Louis, MO) or peptide (10 µM). After 6 hours, 2B4 hybridoma cells were added (5 × 104/well). After 24 hours of culture, the supernatant (50 µL/well) was collected and added to cultures of the IL-2-dependent cell line, CTLL-20 (5 × 103/100 µL/well), in 96-well flat-bottomed culture plates. After 16 hours, [3H]-thymidine was added and cells were incubated for a further 8 hours. The incorporation of [3H]-thymidine by CTLL-20 was measured by scintillation counting. In some experiments, ES-DCs were fixed as follows. Cells were washed with PBS and suspended in PBS at 106 cells/mL, and diluted glutaraldehyde was added to the final concentration of 0.002%. After incubation for 30 seconds at room temperature, equal volume of 0.2 M L-lysine/PBS was added and mixed gently, and cells were incubated for 1 minute at room temperature. Cells were sequentially washed with PBS and with culture medium and used in the experiments. In experiments using BM-DCs pulsed with peptide, OVA or PCC peptide was added to the final concentration of 1 or 10 µM, incubated for 4 hours, washed twice, and used as stimulator cells.Plasmid construction To obtain pCI-PCC, the expression vector presenting PCC epitope on major histocompatibility complex (MHC) class II molecules and driven by the SR promoter, double-stranded oligo DNA encoding the PCC epitope,
5'-AAGGCAGAAAGGGCAGACCTAATAGCTTATCTTAAACAAGCTACTGCCAAG-3', was
inserted into the previously reported human invariant chain-based epitope-presenting vector, pCI.30 The coding region of
this construct was transferred to pCAG-IP,31 a mammalian
expression vector containing the chicken  -actin promoter and
IRES-puromycin N-acetyltransferase gene cassette, to
generate pCAG-PCC-IP. The replacement vector, p6SEFPPF, and the Cre
expression vector, pCAGGS-Cre, have been described
elsewhere.10,32-35 A cDNA fragment coding for OVA protein
was inserted into a mammalian expression vector pCAGGS to make
pCAGGS-OV. The DNA fragment containing -actin promoter-cDNA for OVA-rabbit -globin poly (A) signal was excised from this construct and inserted into p6SEFPPF replacing the enhanced green fluorescent protein-coding sequence to obtain p6AOVP.
Quantitative analysis of -geo
(-galactosidase/neomycin resistance fusion gene)-lox
cassette was introduced as a reporter gene, have been reported
elsewhere.10 ES cell clones were differentiated to DCs
according to the procedure described above. After
differentiation, ES-DCs were suspended in lysis buffer (150 mM NaCl/20
mM tris(hydroxymethyl)aminomethane [Tris-HCl], pH 8.0), and
cell lysates were prepared by 2 cycles of freezing and thawing. For
each cell lysate sample, the protein concentration was measured using a
protein assay kit (MIRCO BCA assay kit; Pierce, Rockford, IL)
and the -galactosidase (-gal) activity was quantified using a
-gal assay kit (Gene Therapy Systems, San Diego, CA) using
chlorophenol red--D-galactopyranoside as substrate. Relative -gal
activity (-gal activity/protein concentration) was calculated for
each sample, and the gene-trapped ES cell clone showing the highest
-gal activity after DC differentiation was selected for transfection
with the replacement vector.
Transfection of ES cells TT2 ES cells maintained on layers of primary embryonic fibroblasts (PEFs) were harvested and suspended in Dulbecco modified Eagle medium at a concentration of 2.5 × 107/mL, and 1 × 107 cells were electroporated in a 4-mm gap cuvette under the condition of 200 V and 950 µF. For transfection with pCI-PCC or pCAG-PCC-IP, 40 µg linearized plasmid DNA was used. For Cre-mediated targeted integration of the replacement vector into a gene-trap ES clone, 20 µg each of p6AOVP and pCAGGS-Cre in circular form were used for transfection. Site-specific integration into ES cells using a pair of mutant lox, lox71 and lox66, has been described previously.33 Transfected ES cells were cultured on PEF feeder layers in 90-mm culture dishes and selected with puromycin (2 µg/mL) on days 3, 5, and 7 for 24 hours each, and drug-resistant colonies were picked up on day 9 into 24-well culture plates with PEFs.Transfer of ES-DCs into mice and cytotoxity assay ES-DCs were stimulated with IL-4, TNF-, and anti-CD40 mAb and
injected intraperitoneally into mice (5 × 105
cells/mouse). Injections were given twice at a 7-day interval, and 7 days after the second injection, mice were killed and spleen cells
isolated. Spleen cells were treated with hemolysis buffer (140 mM
NH4Cl, 10 mM HEPES
[N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid], pH 7.4) for one minute, washed, and cultured in
24-well culture plates (2.5 × 106/well) in 45%
RPMI/45% AIMV/10% horse serum supplemented with recombinant
human IL-2 (100 U/mL) and OVA257-264 peptide (0.1 µM).
After 5 days, cells were recovered, and viable cells isolated with
lympholyte-M (Cedarlane) were used as effector cells. As target cells,
EL-4, a thymoma cell line which originated from a C57BL/6 mouse, was
labeled with sodium [51Cr]-chromate for one hour at
37°C and washed twice with RPMI/10% FCS. Subsequently, target cells
were incubated in 24-well culture plates (1 × 106
cells/well) with or without 10 µM OVA peptide for 3 hours,
harvested, washed with RPMI/10% FCS, and seeded onto 96-well
round-bottomed culture plates (5 × 103 cells/well).
Effector cells were added (5 × 104cells/well) to the
target cells and incubated for 4 hours at 37°C. At the end of the
incubation, the plates were centrifuged, and supernatants (50 µL/well) were harvested and counted on a gamma counter. The
percentage of specific lysis was calculated as:
100 × [(experimental release  spontaneous release)/(maximal
release spontaneous release)]. Spontaneous release and maximal
release were determined in the presence of either medium or 1% Triton X-100, respectively.
Application of OP9-coculture method to generate hematopoietic cells from TT2 ES cells For induction of hematopoietic differentiation of ES cells, we used OP9 cells, a bone marrow stromal cell line defective in the M-CSF gene,13 as feeder cells. Successful hematopoietic differentiation by the OP9-coculture method of ES cell lines derived from 129 strain of mice, such as D3 and E14, has been reported.11,12,14,17 In the current study, we used another line of ES cell, TT2, established from (CBA × C57BL/6) F1 blastocysts,23 which has been used in gene targeting to generate many lines of mutant mice and also for a large-scale gene-trap project.10,24To determine if the OP9-coculture method can be applied to TT2,
we cultured TT2 ES cells following the reported culture
procedure.11 TT2 ES cells, maintained on PEFs in the
presence of leukemia inhibitory factor (LIF), were transferred
onto the OP9 cell layer and cultured without exogenous cytokines
(Figure 2A). Most of the ES cell colonies showed a differentiated morphology in 4 to 5 days (Figure 2B). After 5 days of culture on OP9 feeder layers, ES cell-derived cells were
transferred onto freshly prepared OP9 feeder layers and cultured for
another 10 days. Cells of various morphologies floating or loosely
adherent to the feeder cells appeared. We harvested the differentiated
cells and examined them after May-Giemsa staining. As shown in Figure
2C-E, we observed hematopoietic cells of at least erythroid, myeloid,
and megakaryocytic lineage. Therefore, this method is applicable also
to TT2 ES cells.
Development of DCs from ES cells To induce differentiation to DCs, the above-described mesodermally differentiated ES cell-derived cells harvested from a 5-day culture on OP9 feeder layers were cultured on fresh OP9 cell layers in the presence of exogenous GM-CSF (Figure 1). In comparison with the culture without exogenous GM-CSF, addition of this cytokine resulted in appearance of a larger number of floating cells. On day 8 (3 days after the transfer), we observed many round and relatively homogenous floating cells (Figure 3A) and most expressed CD11b, suggesting their commitment to myeloid cell lineage. On day 10, cells floating or loosely adherent to feeder cells were recovered by pipetting and transferred to bacteriologic Petri dishes without feeder cells (Figure 3B-C). After this passage, some (< 30%) of the transferred cells adhered to the dish surface and resembled macrophages. On days 15 to 18, floating cells could be divided roughly into 2 types, 1 with a round shape and of a larger size, the other smaller and irregularly shaped with protrusions (Figure 3D). In addition, clusters of floating cells (Figure 3E) of the latter type were observed after days 17 to 19, and the cell clusters gradually increased. Floating cells were positive for MHC class I, MHC class II, CD80, CD86, DEC205, and CD11c (Figure 4A-B). Based on the morphology, surface phenotype, and function (described below), we referred to cells with protrusions as ES-DCs. ES-DCs were positive for F4/80 and CD11b and negative for CD8, suggesting that they were of
myeloid lineage. However, further analysis may be necessary to
definitely determine the lineage of ES-DCs. Further culture in Petri
dishes in the presence of GM-CSF resulted in appearance of cells with
longer protrusions showing morphology of mature DCs (Figure
3F).
Induction of maturation of ES-DCs The induction of maturation by separating floating cells from adherent cells was noted in culture of bone marrow- and skin-derived DCs.20,27,36,37 To induce further maturation of ES-DCs, we recovered floating cells from 14- to 16-day cultures in Petri dishes (on days 24-26 in Figure 1) and transferred these to new Petri dishes. Some transferred cells became typical mature DCs in 2 to 3 days, bearing large veils or long dendritic protrusions, along with dead cells and adherent cells. We tested several factors reported to have effects on maturation of DCs (IL-4, TNF-, IL-1, anti-CD40 mAb,
and LPS) in single use or in combinations. As a result, we found that
the combination of IL-4, TNF-, plus anti-CD40, or IL-4, TNF-,
plus LPS drastically increased the number of typical mature DCs. Most
of the mature ES-DCs formed clusters if stimulated with IL-4, TNF-,
plus anti-CD40 (Figure 3G). On the other hand, most floating cells did
not cluster if stimulated with IL-4, TNF-, plus LPS (Figure 3H). The
stimulated ES-DCs expressed higher levels of surface MHC class I, MHC
class II, CD80, and CD86 than did cells before the treatment (Figure
4A-C). In addition, cells stimulated with IL-4, TNF-, plus LPS
expressed CD40, for which ES-DCs before stimulation were negative. On
the other hand, expression of CD11b and F4/80 decreased slightly and
that of CD11c did not change significantly. We compared the surface
phenotype of ES-DCs with that of BM-DCs generated by culture
in the presence of GM-CSF for 10 days (Figure 4D) and those further
stimulated with TNF- (Figure 4E). Levels of expression of MHC class
II, CD40, CD80, and CD86 were almost comparable between mature ES-DCs
and BM-DCs.
Stimulation of primary MLR by ES-DCs To test ES-DCs for the capacity to activate naive T cells, we did primary MLR assays using ES-DCs as stimulators. Because H-2 of TT2 cells is of the k/b haplotype, allogenic splenic T cells purified from unprimed Balb/c mice (H-2d) were used as responders. ES-DCs prepared from 7-day culture in Petri dishes (day 17 in Figure 1) had a capacity to stimulate MLR comparable with that of BM-DCs cultured for 10 days27 (Figure 5A), although it is possible that BM-DCs generated by different culture protocols have stronger stimulation capacity. In contrast, ES cell-derived cells harvested from a 10-day culture on OP9 feeder cell layers (day 10 in Figure 1) did not stimulate T cells. We also tested DCs given maturation stimuli (Figure 5B). We tested the T-cell stimulatory capacity of ES-DCs treated with 3 different stimulation cocktails: IL-4/TNF-/anti-CD40 mAb,
IL-4/TNF-/LPS, or IL-4/TNF-/anti-CD40 mAb/LPS. Consistent with
the elevation of surface MHC class II and costimulatory molecules, the
maturation stimuli enhanced the capacity to stimulate T cells.
Antigen processing and presentation by ES-DCs To examine the APC function of ES-DCs, presentation of PCC epitope to a T-cell hybridoma, 2B4, recognizing PCC88-104 in the context of I-Ek, was tested. ES-DCs cultured for 17 days in Petri dishes (day 27 in Figure 1) and bone marrow-derived DCs cultured for 10 days were harvested and incubated with PCC protein, to allow for capture of the antigen. After 6 hours, 2B4 cells were added and presentation of the PCC epitope by DCs was measured by quantifying the IL-2 produced by 2B4. We compared functions of these 2 kinds of DCs under the conditions of different numbers of APCs and concentrations of antigenic protein. As shown in Figure 6A-B, APC activity of ES-DCs was even stronger than BM-DCs. To rule out the possibility that the response of T cells was induced by direct binding of peptide fragments contaminated in the PCC protein to I-E molecules, we fixed ES-DCs with glutaraldehyde. As shown in Figure 6C, fixation of ES-DCs before addition of the protein abrogated the response of T cells, whereas ES-DCs fixed under the same condition and added with PCC88-104 synthetic peptide induced the response of 2B4. These results indicate that ES-DCs can capture and process soluble protein antigens and present the resultant peptides in the context of MHC class II molecules.
Genetic modification of ES-DCs using an expression vector containing IRES-puromycin N-acetyltransferase cassette For the genetic modification of ES-DCs, we planned to introduce expression vectors into ES cells and develop DCs from the ES cell transfectants. For this purpose, we first used the MHC class II-restricted epitope presentation vector, pCI,29,30 in which the class II-associated invariant chain peptide (CLIP) region of the human invariant chain (Ii) is substitutive with antigenic peptides and the expression in mammalian cells is driven by the SR promoter. We inserted a DNA fragment coding
PCC88-104 into pCI to make pCI-PCC and transfected ES cells
with this vector. We differentiated 48 transfected ES cell clones to
DCs, and examined their expression of human Ii (CD74), using flow
cytometry. To our disappointment, only 1 of 48 clones expressed human
Ii and the level of expression of the clone was very low.
To improve the efficiency of expression of the introduced genes, we
used a vector containing a
Cre-lox-mediated targeted gene introduction into gene-trapped ES cell clones As an alternative strategy to efficiently obtain ES cell transfectants, which expressed the introduced genes after differentiation to DCs, we used targeted gene integration into gene-trapped ES cell clones. Cell lineage-specific gene-expression patterns are regulated not only by transcription factors that bind specific nucleotide sequences but also by epigenetic mechanisms; that is, activation and inactivation of specific chromatin domains are controlled by the status of histone acetylation and DNA methylation. We supposed that, by integrating expression constructs into certain chromosomal region replacing some gene, which is actively transcribed in DCs, we could efficiently obtain ES cell transfectant clones expressing the introduced genes after differentiation to DCs.A library of gene-trapped TT2 ES cells has been recently generated, in
which a lox-
Priming of antigen-specific cytotoxic T cells with genetically modified ES-DCs The capacity of ES-DCs introduced with an OVA-expression vector as described above to prime OVA-specific T cells in vivo was analyzed. ES-DCs (5 × 105) with or without OVA expression vector were injected intraperitoneally into syngenic (CBA × C57BL/6) F1 mice twice with a 7-day interval. Splenocytes were isolated 7 days after the second injection and cultured in vitro in the presence of a suboptimal concentration (0.1 µM) of OVA257-264 peptide, the major Kb-binding epitope of OVA protein. After 5 days, viable cells were recovered and assayed for their capacity to kill EL-4 thymoma cells (H-2b) prepulsed with the OVA peptide. The results shown in Figure 8C indicated that cytotoxic T cells specific to the OVA epitope were primed in vivo with ES-DCs expressing OVA protein but not with ES-DC without OVA. These results demonstrate that ES-DCs genetically engineered to express an antigenic protein have the capacity to prime antigen-specific cytotoxic T cells in vivo.
We developed a method to generate DCs from mouse ES cells in vitro. The ES-DCs were comparable with bone marrow cell-derived DCs in morphology, surface phenotypes, and function. Several improvements have been made in the procedure for differentiation culture. For feeder cells used in days 5 to 10 of culture, we compared OP9 with other bone marrow stromal cell lines, PA6 and ST2, both producing M-CSF. Although DCs could differentiate when PA6 or ST2 cells were used as feeder cells, the number of generated DCs was fewer and the phenotype of the generated DCs differed. With PA6 or ST2, generated DCs did not express CD80 and CD205, and their activity to stimulate MLR was weaker than that of DCs produced with OP9. The use of tissue culture-grade dishes in the culture after transfer from the OP9 feeder cell layer (after day 10 in Figure 1) gave rise to a fewer number and a lower purity of ES-DCs than did the use of bacterial-quality Petri dishes. If we used dishes of tissue-culture grade, many cells firmly adhered to the dish surface, resembling macrophages or fibroblasts, and inhibited the generation of ES-DCs. The beneficial effect of bacterial-quality Petri dishes to DC development has been noted also for culture of BM-DCs.27 GM-CSF has been reported to be essential for in vitro generation of DCs from hematopoietic cells.20-22 GM-CSF is also necessary for generation of ES-DCs. We applied the current culture protocol to ES cell lines other than TT2. We tested 3 lines of ES cells, D3, R1, and CCE, and observed that all of these lines also differentiated to DCs, thereby suggesting that the method is applicable to most lines of mouse ES cells. Recently, another method to generate DCs in vitro from mouse ES cells
has been reported.38 In the procedure, embryoid bodies (EBs) made from ES cells were cultured for 14 days in the presence of
GM-CSF and IL-3. Resultant DCs, referred to as esDCs, lack expression
of CD8 For investigation of the physiologic function of genes, generation of mutant mice by gene targeting in ES cells is a potent and widely used means. However, it takes a relatively long time to develop mutant mice by gene targeting, and if the disrupted gene is essential for embryogenesis, homozygous mutant mice cannot be obtained because of embryonic lethality. ES cell clones homozygous for mutated allele can be obtained from single-allele mutant cell clones by selection with a high dose of a selection drug39 or sequential gene targeting with 2 targeting vectors bearing different selection markers.28 The method for in vitro generation of DCs from ES cells may prove useful for analyzing genes essential for both embryogenesis and function of DCs. Using the TT2 ES cell line, the generation of a large-scale library of gene-trap ES clones is now in progress. Using the method established in the current study, it is possible to efficiently screen large numbers of gene-trap ES cell clones to search for genes expressed in DCs. After selection of ES cell clones in which genes expressed in DCs were trapped, one can generate homozygous mutant ES cell clones by high-dose drug selection, differentiate them to DCs, and evaluate the functional significance of the identified genes in DCs. For genetic modification of ES-DCs, we found 2 means for gene transfer to ES cells useful: an expression vector containing IRES-puromycin N-acetyltransferase gene and Cre-lox-mediated targeted gene integration into gene-trapped ES cell clones. Both worked very efficiently to generate ES cell transfectant clones expressing the gene products after DC differentiation; the frequency of appropriate transfectant clones in picked-up drug-resistant clones was 85% and 90% when we used the vector with IRES-puromycin N-acetyltransferase gene and targeted gene integration, respectively. We are now preparing ES-DCs expressing both antigenic protein and immunomodulating molecules, such as immunostimulatory or inhibitory molecules, cytokines, or chemokines. One of our goals is to manipulate immune responses in an antigen-specific manner by in vivo transfer of such engineered DCs. We have already succeeded in introducing 2 different plasmid vectors with different selection markers, the puromycin- and neomycin-resistance gene, by sequential transfection, and confirmed that generation of DCs from double-transfected ES cells is also feasible. Possible future applications of this method may be treatment of autoimmune and allergic diseases, inhibition of graft rejection in transplantation medicine, antitumor immunotherapy, and vaccination against intractable infectious diseases. Recently, human ES cells have been generated.40,41 In the mouse system, a method was devised to generate ES cell lines with an appropriate genetic background by nuclear transfer from somatic cells to already established ES cells.42,43 With the advances in ES cell-related technology, immunomodulation therapy using DCs generated from genetically engineered ES cells may be considered.
We thank Drs T. Nakano, K. Inaba, K. Matsushima, K. Iwabuchi, and K. Matsuno for valuable suggestions; Dr S. Aizawa for TT2; Drs N. Takakura and T. Suda for OP9 and PA6; Dr K. Lock for RF33.70; Drs T. Koda and M. Bevan for a cDNA clone for OVA; Dr H. Niwa for pCAG-IP; and Kirin Brewery for rGM-CSF. M. Ohara provided helpful comments on the manuscript.
Submitted July 26, 2002; accepted October 1, 2002.
Prepublished online as Blood First Edition Paper, October 24, 2002; DOI 10.1182/blood-2002-07-2254.
Supported by Grants-in-Aid 11557027, 12213111, 14370115, 14657082, and 14570421 from the Ministry of Education, Science, Technology, Sports, and Culture of 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.
Reprints: Yasuharu Nishimura, Division of Immunogenetics, Department of Neuroscience and Immunology, Kumamoto University Graduate School of Medical Sciences, 2-2-1 Honjo, Kumamoto 860-0811, Japan; e-mail: mxnishim{at}gpo.kumamoto-u.ac.jp.
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© 2003 by The American Society of Hematology.
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  S. Senju, M. Haruta, Y. Matsunaga, S. Fukushima, T. Ikeda, K. Takahashi, K. Okita, S. Yamanaka, and Y. Nishimura Characterization of Dendritic Cells and Macrophages Generated by Directed Differentiation from Mouse Induced Pluripotent Stem Cells Stem Cells, May 1, 2009; 27(5): 1021 - 1031. [Abstract] [Full Text] [PDF]
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K.-D. Choi, J. Yu, K. Smuga-Otto, G. Salvagiotto, W. Rehrauer, M. Vodyanik, J. Thomson, and I. Slukvin Hematopoietic and Endothelial Differentiation of Human Induced Pluripotent Stem Cells Stem Cells, March 1, 2009; 27(3): 559 - 567. [Abstract] [Full Text] [PDF]
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S. Senju, H. Suemori, H. Zembutsu, Y. Uemura, S. Hirata, D. Fukuma, H. Matsuyoshi, M. Shimomura, M. Haruta, S. Fukushima, et al. Genetically Manipulated Human Embryonic Stem Cell-Derived Dendritic Cells with Immune Regulatory Function Stem Cells, November 1, 2007; 25(11): 2720 - 2729. [Abstract] [Full Text] [PDF]
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 T. Okazaki and T. Honjo PD-1 and PD-1 ligands: from discovery to clinical application Int. Immunol., July 2, 2007; (2007) dxm057v1. [Abstract] [Full Text] [PDF]
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 S. Hirata, H. Matsuyoshi, D. Fukuma, A. Kurisaki, Y. Uemura, Y. Nishimura, and S. Senju Involvement of Regulatory T Cells in the Experimental Autoimmune Encephalomyelitis-Preventive Effect of Dendritic Cells Expressing Myelin Oligodendrocyte Glycoprotein plus TRAIL J. Immunol., January 15, 2007; 178(2): 918 - 925. [Abstract] [Full Text] [PDF]
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 H. Komori, T. Nakatsura, S. Senju, Y. Yoshitake, Y. Motomura, Y. Ikuta, D. Fukuma, K. Yokomine, M. Harao, T. Beppu, et al. Identification of HLA-A2- or HLA-A24-Restricted CTL Epitopes Possibly Useful for Glypican-3-Specific Immunotherapy of Hepatocellular Carcinoma. Clin. Cancer Res., May 1, 2006; 12(9): 2689 - 2697. [Abstract] [Full Text] [PDF]
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I. I. Slukvin, M. A. Vodyanik, J. A. Thomson, M. E. Gumenyuk, and K.-D. Choi Directed Differentiation of Human Embryonic Stem Cells into Functional Dendritic Cells through the Myeloid Pathway. J. Immunol., March 1, 2006; 176(5): 2924 - 2932. [Abstract] [Full Text] [PDF]
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 Y. Motomura, S. Senju, T. Nakatsura, H. Matsuyoshi, S. Hirata, M. Monji, H. Komori, D. Fukuma, H. Baba, and Y. Nishimura Embryonic Stem Cell-Derived Dendritic Cells Expressing Glypican-3, a Recently Identified Oncofetal Antigen, Induce Protective Immunity against Highly Metastatic Mouse Melanoma, B16-F10 Cancer Res., February 15, 2006; 66(4): 2414 - 2422. [Abstract] [Full Text] [PDF]
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 A. L. Olsen, D. L. Stachura, and M. J. Weiss Designer blood: creating hematopoietic lineages from embryonic stem cells Blood, February 15, 2006; 107(4): 1265 - 1275. [Abstract] [Full Text] [PDF]
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S. Hirata, S. Senju, H. Matsuyoshi, D. Fukuma, Y. Uemura, and Y. Nishimura Prevention of Experimental Autoimmune Encephalomyelitis by Transfer of Embryonic Stem Cell-Derived Dendritic Cells Expressing Myelin Oligodendrocyte Glycoprotein Peptide along with TRAIL or Programmed Death-1 Ligand J. Immunol., February 15, 2005; 174(4): 1888 - 1897. [Abstract] [Full Text] [PDF]
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M. M. Dikov, J. E. Ohm, N. Ray, E. E. Tchekneva, J. Burlison, D. Moghanaki, S. Nadaf, and D. P. Carbone Differential Roles of Vascular Endothelial Growth Factor Receptors 1 and 2 in Dendritic Cell Differentiation J. Immunol., January 1, 2005; 174(1): 215 - 222. [Abstract] [Full Text] [PDF]
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H. Matsuyoshi, S. Senju, S. Hirata, Y. Yoshitake, Y. Uemura, and Y. Nishimura Enhanced Priming of Antigen-Specific CTLs In Vivo by Embryonic Stem Cell-Derived Dendritic Cells Expressing Chemokine Along with Antigenic Protein: Application to Antitumor Vaccination J. Immunol., January 15, 2004; 172(2): 776 - 786. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
), ES-derived
cells on day 10 (
), and (CBA × C57BL/6) F1 BM-DCs cultured for
11 days (
) were irradiated and cocultured with splenic T cells
(1.5 × 105) isolated from Balb/c mice for 4 days, and
the proliferative response of T cells in the last 16 hours of the
culture was measured by [3H]-thymidine uptake. (B)
ES-DCs harvested on day 24 and stimulated with IL-4/TNF-
), or IL-4/TNF-
) were
used as stimulators. BM-DCs cultured for 10 days and stimulated with
TNF-
) and BM-DCs
(
