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Mature Dendritic Cells Derived from Human
Monocytes Within 48 Hours: A Novel
Strategy for Dendritic Cell Differentiation
from Blood Precursors
Marc Dauer, Bianca Obermaier, Jan Herten, Carola Haerle,
Katrin Pohl, Simon Rothenfusser, Max Schnurr, Stefan
Endres and Andreas Eigler
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2003 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2003; 170:4069-4076; ;
doi: 10.4049/jimmunol.170.8.4069
http://www.jimmunol.org/content/170/8/4069
The Journal of Immunology
Mature Dendritic Cells Derived from Human Monocytes
Within 48 Hours: A Novel Strategy for Dendritic Cell
Differentiation from Blood Precursors1
Marc Dauer,2 Bianca Obermaier,2 Jan Herten, Carola Haerle, Katrin Pohl,
Simon Rothenfusser, Max Schnurr, Stefan Endres, and Andreas Eigler3
D
endritic cells (DCs)4 are highly specialized APC with
the unique capacity to establish and control primary immune responses. DCs reside in peripheral tissues in an
immature state where they capture and process Ag for presentation
in the context of MHC molecules (1). Ligation of receptors for
inflammatory chemokines recruits immature DCs and their blood
precursors to sites of inflammation or infection (2, 3). Upon encounter with microbial, proinflammatory, or T cell-derived stimuli,
characteristic phenotypic and functional changes are induced, a
process referred to as maturation of DCs. Mature DCs exhibit reduced phagocytic activity and increased expression of MHC and
costimulatory molecules, and secrete large amounts of immunostimulatory cytokines (4 – 6). Maturing DCs also change their pattern of chemokine receptor expression, rendering them sensitive to
lymphoid chemokines. Thereby, mature DCs acquire the capacity
Division of Clinical Pharmacology and Section of Gastroenterology, Medizinische
Klinik Innenstadt, University of Munich, Munich, Germany
Received for publication November 8, 2002. Accepted for publication February
4, 2003.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work is part of the doctoral thesis of B.O. at the Ludwig-Maximilians-University (Munich, Germany). M.D. is supported by a grant from the University of Munich
(FöFoLe 271) and the Friedrich-Baur-Stiftung (Munich, Germany).
2
M.D. and B.O. contributed equally to this manuscript.
3
Address correspondence and reprint requests to Dr. Andreas Eigler, Abteilung fuer
Klinische Pharmakologie, Medizinische Klinik Innenstadt, University of Munich,
Ziemssenstrasse 1, 80336 Munich, Germany. E-mail address: andreas.eigler@
med.uni-muenchen.de
4
Abbreviations used in this paper: DC, dendritic cell; CCR, chemokine receptor;
CD40L, soluble CD40 ligand trimer; MIP-1␣, macrophage inflammatory protein-1␣;
MoDC, monocyte-derived DC; SDF-1, stromal cell-derived factor-1; TT, tetanus
toxoid.
Copyright © 2003 by The American Association of Immunologists, Inc.
to migrate to the T cell areas of draining secondary lymphoid organs, where they encounter naive T cells and initiate an adaptive
immune response (7, 8).
Human DCs originating from pluripotent hemopoietic stem cells
can be divided into at least three subpopulations: interstitial DCs
residing in the skin and lymphoid organs and two subsets of blood
DCs, the CD11c⫹ myeloid and CD11c⫺ plasmacytoid DC (9). The
two subsets of blood DCs not only differentially express CD11c
and CD123 (IL-3R ␣-chain), but also show distinct patterns of
Toll-like receptor expression and regulation by cytokines (10 –12).
Circulating blood DCs are rare (they account for ⬍1% of human
PBMC) and are difficult to maintain in culture. Although in vivo
expansion of blood DCs by administration of the hemopoietic
growth factors Flt-3 ligand and GM-CSF can be used to increase
the yield of isolated cells (13), most experimental and clinical studies currently rely on the in vitro development of DC-like cells from
CD34⫹ progenitor cells or blood monocytes (14 –16). Commonly,
monocytes are cultured for 5–7 days with GM-CSF and IL-4 to
generate immature DCs that have to be activated for another 2–3
days with microbial, proinflammatory, or T cell-derived stimuli to
obtain mature DCs with full T stimulatory capacity.
There is increasing evidence that maturation of DCs from monocyte precursors may also be relevant in vivo, although the conditions under which it occurs and the exact time span required for the
differentiation process are not known. However, experimental data
indicate that the kinetics of DC differentiation from monocytes
under physiologic conditions may not be reflected by current protocols for the in vitro development of DCs. A subpopulation of
monocytes differentiates into DCs within 48 h in a model simulating transendothelial migration into lymphatic vessels (17). More
recently, spontaneous maturation of CD11c⫹ myeloid DCs from a
subset of CD14⫹CD16⫹ monocytes after overnight culture of
PBMCs has been described (18). Monocytes may give rise to a
0022-1767/03/$02.00
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It is widely believed that generation of mature dendritic cells (DCs) with full T cell stimulatory capacity from human monocytes
in vitro requires 5–7 days of differentiation with GM-CSF and IL-4, followed by 2–3 days of activation. Here, we report a new
strategy for differentiation and maturation of monocyte-derived DCs within only 48 h of in vitro culture. Monocytes acquire
immature DC characteristics by day 2 of culture with GM-CSF and IL-4; they down-regulate CD14, increase dextran uptake, and
respond to the inflammatory chemokine macrophage inflammatory protein-1␣. To accelerate DC development and maturation,
monocytes were incubated for 24 h with GM-CSF and IL-4, followed by activation with proinflammatory mediators for another
24 h (FastDC). FastDC expressed mature DC surface markers as well as chemokine receptor 7 and secreted IL-12 (p70) upon CD40
ligation in the presence of IFN-␥. The increase in intracellular calcium in response to 6Ckine showed that chemokine receptor 7
expression was functional. When FastDC were compared with mature monocyte-derived DCs generated by a standard 7-day
protocol, they were equally potent in inducing Ag-specific T cell proliferation and IFN-␥ production as well as in priming
autologous naive T cells using tetanus toxoid as a model Ag. These findings indicate that FastDC are as effective as monocytederived DCs in stimulating primary, Ag-specific, Th 1-type immune responses. Generation of FastDC not only reduces labor, cost,
and time required for in vitro DC development, but may also represent a model more closely resembling DC differentiation from
monocytes in vivo. The Journal of Immunology, 2003, 170: 4069 – 4076.
4070
subset of DCs during infection or inflammation, when high levels
of proinflammatory mediators such as TNF-␣, IL-1␤, PGE2, and
IFN-␣ are produced. IFN-␣ not only induces the development of
mature DCs from monocytes in vitro within 3 days (19), but high
serum levels of IFN-␣ have been shown to be associated with the
acquisition of DC characteristics by monocytes isolated from peripheral blood in patients with systemic lupus erythematosus (20).
In the present study we describe a novel strategy for the development of mature DCs from monocytes within only 48 h of in vitro
culture. DCs obtained by this new strategy were compared with
monocyte-derived DCs (moDCs) generated by a standard 7-day
protocol for the expression of DC activation markers, sensitivity to
proinflammatory and lymph node-directing chemokines, and their
ability to induce proliferation and Ag-specific IFN-␥ production in
autologous T cells as well as to prime autologous naive T
lymphocytes.
Materials and Methods
Reagents and ELISA kits
Media
All cultures of human PBMC were maintained in RPMI 1640 medium
(Biochrom, Berlin, Germany) supplemented with 2% human AB serum
(BioWhittaker, Walkersville, MD), 2 mM L-glutamine (Life Technologies,
Paisley, Scotland), 100 U/ml penicillin, and 100 ␮g/ml streptomycin (Sigma-Aldrich, Munich, Germany), hereafter referred to as complete medium.
Isolation and culture of cells
PBMC were isolated from peripheral blood of healthy donors by FicollHypaque gradient centrifugation. Monocytes were purified using the
MACS CD14 isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany)
and were subsequently cultured in six-well plates (0.5–1.5 ⫻ 106 cells/ml)
in fresh complete medium supplemented with 1000 U/ml GM-CSF and 500
U/ml IL-4. To generate standard moDCs, cells were cultured for 6 days and
subsequently incubated with a combination of proinflammatory mediators
for 24 h (1000 U/ml TNF-␣, 10 ng/ml IL-1␤, 10 ng/ml IL-6, and 1 ␮M
PGE2). Alternatively, monocytes were cultured for 24 h with GM-CSF
(1000 U/ml) and IL-4 (500 U/ml), followed by incubation with the same
proinflammatory mediators for another 24 h (FastDC). In some experiments the different DCs were additionally stimulated with CD40L (500
ng/ml) or CD40L plus IFN-␥ (1000 U/ml). For cocultures, a fraction of
DCs was cryopreserved and thawed for restimulation. T cells were purified
from monocyte-depleted PBMC by negative selection using the Pan T Cell
Isolation kit (Miltenyi Biotec). To obtain CD45RA⫹ naive T cells,
CD45RO expressing cells were additionally depleted using CD45RO Micro Beads (Miltenyi Biotec).
Flow cytometry and mAbs
The following mAbs (all from BD PharMingen) were used for FACS analysis: TU39 (anti-HLA-DR, -DP, and -DQ, FITC-conjugated), L307.4 (antiCD80, PE-conjugated), 2331/FUN-1 (anti-CD86, allophycocyanin-conjugated), HB15e (anti-CD83, FITC-conjugated), M5E2 (anti-CD14, allophycocyanin-conjugated), 5C3 (anti-CD40, PE-conjugated), HIT3A (antiCD3,
FITC-conjugated), RPA-T4 (anti-CD4, PE-conjugated), RPA-T8 (anti-CD8,
allophycocyanin-conjugated), and HI100 (anti-CD45RA, PE-conjugated).
Chemokine receptor 7 (CCR7) expression was determined by incubation with
rat anti-CCR7 mAb (clone 3D12; provided by R. Förster, Hannover, Germany), followed by incubation with anti-rat IgG2a-biotinylated mAb (clone RG7/
1.30; BD PharMingen) and incubation with streptavidin-allophycocyanin
(BD PharMingen). Cells were analyzed on a FACSCalibur flow cytometer
(BD Biosciences, Heidelberg, Germany). Data were analyzed using
CellQuest (BD Biosciences; version 3.2.1) and FlowJo (Tree Star, San
Carlos, CA; version 2.7.8) software.
Scanning electron microscopy
Scanning electron microscopy was performed in cooperation with Prof. Dr.
U. Welsch (Institute for Anatomy, University of Munich, Munich, Germany). Cells were spun onto standard microscopy glass slides and fixed with
2.5% glutaraldehyde in PBS (pH 7.4). Following dehydration through
graded ethanols, cells were critical point-dried in CO2 and gold-coated by
sputtering. Samples were examined using a JSM-35-CF scanning electron
microscope (JEOL, Tokyo, Japan). After electron microscopy, prints were
scanned using Adobe Photoshop software (Adobe Systems, Mountain
View, CA; version 3.0).
Measurement of FITC-dextran uptake
Endocytic activity was assessed by incubating cells for 2 h with FITCdextran (0.5 mg/ml) at 37°C. Cells were washed extensively with PBS, and
FITC-dextran uptake was quantified as the mean fluorescence intensity
(unspecific FITC-dextran binding to the cell surface was assessed by incubating cells on ice).
Cytometric measurement of changes in intracellular ionized
calcium concentrations
MoDCs or FastDC were loaded for 40 min with the Ca2⫹-sensitive dyes
Fluo-3 (3 ␮g/ml) and Fura Red (10 ␮g/ml) in the presence of 0.02% of the
detergent Pluronic F-127 (all from Molecular Probes, Leiden, The Netherlands) as previously described (21). Cells were washed twice, and the
chemokines macrophage inflammatory protein-1␣ (MIP-1␣), 6Ckine (500
ng/ml; both from R&D Systems, Wiesbaden, Germany), and stromal cellderived factor-1 (SDF-1; 250 ng/ml; R&D Systems) were added to activate
chemokine receptors. Changes in intracellular calcium concentrations over
time (time resolution, 200 ms) were determined by flow cytometry using
the ratio of the fluorescence intensity of fluo-3 (increased intensity in the
presence of Ca2⫹) and Fura Red (reduced intensity in the presence of
Ca2⫹).
Coculture of DCs with autologous T cells
Coculture of DCs with autologous T cells was performed with minor modifications as previously described (22). To load the different DCs with Ag,
immature moDCs or monocytes cultured with GM-CSF and IL-4 were
incubated with TT (5 ␮g/ml) on day 5 or 1, respectively. After 24 h of
incubation with TT, cells were stimulated to induce DC maturation. The
different DC preparations were harvested on day 6 or 2, respectively,
washed, and cocultured with autologous T cells at a ratio of 1/10 in the
presence of soluble CD40L (500 ng/ml). Half the medium was replaced
every second day by fresh culture medium containing IL-2 (25 U/ml) and
IL-7 (10 ng/ml). On day 10, T cells were restimulated with DCs at a ratio
of 10/1 in the presence of IL-7 (10 ng/ml), IL-2 (25 U/ml), and soluble
CD40L (500 ng/ml). After 4 h of restimulation, culture supernatants were
harvested for cytokine measurements.
Proliferative T cell response
Proliferative T cell responses were measured in cooperation with Prof. Dr.
R. Wank (Institute of Immunology, University of Munich). T cell proliferation assay was performed with minor modifications as previously described (23). The different DC preparations were harvested and cocultured
in complete medium supplemented with CD40L (500 ng/ml) with a constant number of autologous T cells or naive CD45RA⫹ T cells (2 ⫻ 105/
200 ␮l) in 96-well, round-bottom microtiter plates at ratios ranging from
1/20 to 1/320 in triplicate. On day 5 the cells were pulsed with [3H]thymidine (1 ␮Ci/well) and harvested after 18 h onto a Filtermate (Wallac,
Turku, Finland). The amount of incorporated [3H]thymidine was analyzed
in a liquid scintillation counter (Wallac).
Statistical analysis
Data are expressed as the mean ⫾ SEM. Statistical significance was determined using paired two-tailed Student’s t test. Differences were considered statistically significant for p ⬍ 0.05. Significance is presented for
individual experiments (asterisks in figures). Statistical analysis was performed using StatView 4.51 software (Abacus Concepts, Calabasas, CA).
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Recombinant cytokines were obtained from the indicated sources. GMCSF was purchased from Novartis (Basel, Switzerland), IL-4 from Promega (Madison, WI), TNF-␣ from R&D Systems (Wiesbaden, Germany),
and IL-6 from Amersham International (Little Chalfont, U.K.); IFN-␥, IL1␤, IL-2, and IL-7 were obtained from Strathmann Biotech (Hannover,
Germany). PGE2 and FITC-dextran were purchased from Sigma-Aldrich
(Steinheim, Germany), [3H]thymidine from Amersham Buchler (Freiburg,
Germany), and tetanus toxoid (TT) from Statens Serum Institute (Copenhagen, Denmark). Soluble CD40 ligand trimer (CD40L) was a gift from
Immunex (Seattle, WA). Total IL-12 was determined using an assay that
detects both IL-12 (p40) and IL-12 (p70) (Bender Med Systems, Vienna,
Austria). IL-12 (p70) was measured using the OptEIA human IL-12 (p70)
set (from BD PharMingen, San Diego, CA). IL-4 and IFN-␥ were quantified using ELISA kits from BD PharMingen.
RAPID DC DIFFERENTIATION FROM MONOCYTES
The Journal of Immunology
Results
Within 48 h, monocytes incubated with GM-CSF and IL-4 plus
proinflammatory mediators differentiate into mature DCs with
distinct morphology (FastDC)
FIGURE 1. Monocytes incubated with GM-CSF and IL-4 plus proinflammatory mediators develop into mature DCs within 48 h (FastDC).
Monocytes were either cultured for 48 h with GM-CSF and IL-4 alone or
additionally stimulated with IL-6, IL-1␤, TNF-␣, and PGE2 for the last
24 h. The expression of DC activation markers and CD14 was determined
by FACS (A), and cell morphology was assessed by scanning electron
microscopy (B). The results of a representative FACS analysis of five independent experiments are shown.
FastDC are sensitive to the CCR7 ligand 6Ckine and to the
CXCR4 ligand SDF-1
DC maturation is associated with a coordinated switch of chemokine receptor expression (2). While receptors for inflammatory
chemokines are down-regulated, increased expression of CCR7
facilitates migration toward secondary lymphoid organs. CD14⫹
cells were cultured with GM-CSF and IL-4 alone for 48 h or were
activated with proinflammatory mediators during the last 24 h.
While monocytes cultured with GM-CSF and IL-4 alone remained
CCR7⫺ after 48 h, FastDC homogeneously expressed CCR7 (see
Fig. 1) to levels comparable to mature moDCs (data not shown).
The functional activity of chemokine receptor expression was determined by measuring intracellular calcium mobilization in response to agonistic chemokines. GM-CSF/IL-4-cultured monocytes responded to the inflammatory chemokine MIP-1␣, a CCR5
ligand, but were insensitive to the CCR7 ligand 6Ckine (Fig. 2). In
contrast, FastDC lost sensitivity to MIP-1␣, but responded to
6Ckine, correlating with phenotypic DC maturation (Fig. 2).
FastDC, but not GM-CSF/IL-4-cultured monocytes, also mobilized calcium in response to the CXCR4 ligand SDF-1 (Fig. 2,
bottom). The difference in the pattern of chemokine response of
GM-CSF/IL-4-cultured monocytes vs FastDC was similar to that
of immature vs mature moDCs (both cultured for 7 days; data not
shown).
PGE2, TNF-␣, and IL-1␤ are key mediators required for
phenotypic maturation of FastDC and synergize to induce IL-12
production
To identify which of the proinflammatory mediators are required
for maturation of FastDC, monocytes were cultured for 24 h with
GM-CSF and IL-4 and subsequently activated for the following
24 h using different stimuli and combinations of stimuli. Only
combinations of stimuli containing both PGE2 and TNF-␣ were
effective in inducing complete maturation of FastDC (⬎ 50%
FIGURE 2. FastDC matured with proinflammatory mediators are sensitive to CCR7 and CXCR4 ligands. Monocytes were cultured for 48 h with
GM-CSF and IL-4 alone or were additionally stimulated with IL-6, IL-1␤,
TNF-␣, and PGE2 for the last 24 h. Cells were loaded with Ca2⫹-sensitive
dyes in the presence of Pluronic F-127 (0.02%), and sensitivity to the
chemokines MIP-1␣, 6Ckine, and SDF-1 was determined by Ca2⫹ flux
analysis. Results are shown as the kinetics of the fluorescence ratio between Fluo-3 and Fura Red.
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Monocytes were enriched from PBMC by CD14-positive selection
using MACS and were subsequently cultured with GM-CSF and
IL-4 for 48 h. Morphology, determined by light microscopy, and
forward/side scatter intensity of the cells were similar to those in
the initial population of monocytes (data not shown). However,
cells already showed down-regulation of CD14 and high levels of
MHC II expression consistent with early immature DC development (Fig. 1A, top row). When proinflammatory mediators
(TNF-␣, IL-1␤, IL-6, PGE2) were added after 24 h of culture with
GM-CSF and IL-4 to accelerate DC maturation, cells displayed
a fully mature DC immunophenotype (CD83⫹CD40⫹CCR7⫹
CD14⫺CD80⫹CD86highMHC IIhigh) after only 48 h of total culture period (Fig. 1A, bottom row). Although a moderate increase in
the forward/side scatter intensity of these stimulated cells compared with monocytes cultured for 48 h with GM-CSF and IL-4
alone could be observed, they were considerably smaller and less
granular than moDCs (cultured for 7 days; data not shown). Scanning electron microscopy was performed to assess the morphology
of cells more closely; while monocytes cultured with GM-CSF and
IL-4 alone for 48 h maintained monocyte-like morphology, cells
that were activated with proinflammatory mediators following 24 h
of culture with GM-CSF and IL-4 formed long, fine cytoplasmatic
protrusions typical of mature DC development (Fig. 1B). Using
this two-step differentiation strategy, high numbers of mature and
viable DCs (⬃30 – 40% of the initial population of monocytes)
could be recovered after 48 h of culture. These cells will further be
referred to as FastDC. Rapid DC maturation from monocytes
could also be induced by adding the proinflammatory mediators
together with GM-CSF and IL-4 at the initiation of culture. However, the expression of DC activation markers was less pronounced
compared with the two-step differentiation and activation sequence
(⬍60% CD83⫹ cells; data not shown). Monocytes cultured with
GM-CSF alone or GM-CSF plus proinflammatory mediators in the
absence of IL-4 for 48 h did not develop DC characteristics (data
not shown).
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4072
RAPID DC DIFFERENTIATION FROM MONOCYTES
CD83⫹ cells). Removal of PGE2 from the combination of proinflammatory mediators used in prior experiments almost completely
abolished FastDC development (Fig. 3, top; p ⬍ 0.05; n ⫽ 6).
PGE2 and TNF-␣ were not only required for maturation, but also
synergized to induce production of total IL-12 by FastDC (Fig. 3,
bottom; p ⬍ 0.05; n ⫽ 6). While IL-6 was completely dispensable
in the combination of proinflammatory mediators, addition of
IL-1␤ significantly enhanced IL-12 secretion of FastDC induced
by PGE2 and TNF-␣ and synergized with PGE2 to induce DC
maturation in the absence of TNF-␣ (Fig. 3). Neither PGE2 nor any
other stimulus used as a single agent (CD40L, TNF-␣, IL-1␤,
IL-6) could induce DC maturation or IL-12 production (data not
shown).
CD40L synergizes with proinflammatory mediators to maximize
IL-12 production by FastDC, but a second signal is required for
secretion of functional IL-12 (p70)
FIGURE 4. CD40 ligation enhances IL-12 production by FastDC, but
requires IFN-␥ to induce IL-12 (p70). Monocytes were cultured for 48 h
with GM-CSF and IL-4 alone or were additionally stimulated with IL-6,
IL-1␤, TNF-␣, and PGE2 for the last 24 h. Soluble CD40L or CD40L plus
IFN-␥ were added 12 h after addition of the proinflammatory mediators as
indicated. After 48 h culture supernatants were harvested for measurement
of total IL-12 (top; one representative experiment of six is shown) and
IL-12 (p70) (bottom). Data are the mean ⫾ SEM of six experiments with
different donors.
48 h or were activated with proinflammatory mediators during the
last 24 h, and culture supernatants were harvested for measurement
of total IL-12 and IL-12 (p70). While GM-CSF/IL-4-cultured
monocytes did not secrete IL-12, FastDC produced moderate
amounts of total IL-12, but failed to secrete IL-12 (p70) (Fig. 4).
Soluble CD40 ligand, a stimulus known to induce IL-12 (p70)
production in moDCs, was added after 12 h of proinflammatory
stimulation to simulate encounter of CD40L-expressing activated
T cells with CD40-expressing FastDC after migration to secondary lymphoid organs. CD40 ligation following proinflammatory
stimulation enhanced production of total IL-12 by FastDC, but left
secretion of IL-12 (p70) unaffected (Fig. 4, top). Although an ⬃3fold increase in IL-12-production by FastDC after additional
CD40 ligation could be observed in each individual experiment
performed (n ⫽ 6), this trend did not reach statistical significance
due to the large differences in maximal levels of IL-12 detected. It
is known that CD40 ligation requires the presence of T cell-derived cosignals, such as IFN-␥ or IL-4, for maximal induction of
IL-12 (p70) in moDCs (25, 26). If CD40 ligation of FastDC occurred in the additional presence of IFN-␥, the production of IL-12
(p70) could be induced (Fig. 4, bottom; p ⬍ 0.05; n ⫽ 6).
FastDC can be loaded with Ag: monocytes increase endocytosis
of soluble dextran within 24 h of culture with GM-CSF and IL-4
FIGURE 3. PGE2, TNF-␣, and IL-1␤ are key mediators for maturation
and IL-12 production of FastDC. Monocytes were cultured for 48 h with
GM-CSF and IL-4 alone or were stimulated with different combinations of
proinflammatory mediators for the last 24 h. The expression of mature DC
surface markers was determined by FACS (top), and culture supernatants
were harvested for measurement of total IL-12 (IL-12 (p40) and IL-12
(p70) (bottom). Data are the mean ⫾ SEM of six experiments with different
donors.
The capacity to take up and process Ag for presentation of MHC
molecules is a hallmark of immature DC function and is rapidly
lost upon maturation of DCs (16). To assess the uptake of soluble
Ag via endocytosis, CD14⫹ cells were cultured with GM-CSF and
IL-4 alone for 48 h or were activated with proinflammatory mediators during the last 24 h and incubated with FITC-conjugated
dextran at 37°C for 2 h at various time points. Dextran uptake was
determined by FACS analysis. Monocytes increased FITC-dextran
uptake by 12 h after initiation of culture with GM-CSF and IL-4
(Fig. 5). After 24 h, a ⬎10-fold increase in mean fluorescence
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DCs stimulated by infectious organisms, proinflammatory mediators, or CD40L produce IL-12, a key cytokine in the induction of
IFN-␥ production by activated T cells (24). However, many of the
stimuli known to induce DC maturation are not sufficient to induce
the production of functional IL-12 (p70) by moDCs. To determine
the capacity of FastDC to secrete the bioactive form of IL-12,
CD14⫹ cells were cultured with either GM-CSF and IL-4 alone for
The Journal of Immunology
4073
docytic activity, activation with proinflammatory mediators for the
last 24 h lead to a rapid reduction of dextran uptake as a result of
DC maturation (Fig. 5).
FastDC loaded with TT induce Ag-specific autologous T cell
proliferation and IFN-␥ production
intensity could be observed, indicating a markedly enhanced capacity to internalize soluble dextran. While monocytes that were
cultured with GM-CSF and IL-4 alone continued to increase en-
FIGURE 6. TT-loaded FastDC induce Ag-specific proliferation and IFN-␥ production by autologous T cells. Monocytes were cultured for 24 h
with GM-CSF and IL-4 in the presence of TT (5
␮g/ml) and were stimulated with IL-6, IL-1␤,
TNF-␣, and PGE2 for another 24 h to generate Agloaded FastDC. Alternatively, cells were cultured
for 5 days with GM-CSF and IL-4, loaded with TT,
and stimulated for 24 h with proinflammatory mediators to generate mature Ag-loaded moDCs.
Monocytes cultured with GM-CSF plus IL-4 alone
for 48 h, immature moDCs, unloaded FastDC, and
unloaded, stimulated moDCs were used as controls. DCs were cocultured with purified autologous T cells as described (see Materials and Methods). To determine T cell proliferation, cells were
pulsed with [3H]thymidine on day 5, and incorporation was measured after 18 h. T cell proliferation
alone (without DCs) measured as an additional
control accounted for only 0.4 ⫻ 103 cpm (data not
shown). To assess cytokine production of T cells,
supernatants were harvested for ELISA measurements after 4 h of restimulation with DCs on day
10 of coculture. One representative experiment of
three performed with different donors is shown for
T cell proliferation. The results of cytokine measurements are expressed as the mean ⫾ SEM of
these three experiments.
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FIGURE 5. Ag loading of FastDC. Monocytes show increased FITCdextran uptake after 24 h of culture with GM-CSF and IL-4. Monocytes
were cultured for 48 h with GM-CSF and IL-4 alone or were additionally
stimulated with IL-6, IL-1␤, TNF-␣, and PGE2 for the last 24 h. Cells were
incubated with FITC-conjugated dextran at 37°C at the indicated time
points of the differentiation process (controls on ice). After 2 h cells were
washed, and dextran uptake was quantified as the mean fluorescence intensity (MFI) using FACS.
Mature DCs are very effective stimulators of Ag-specific T cell
responses. The T cell stimulatory capacity of FastDC was assessed
and compared with that of moDCs using TT as a model Ag. To
load FastDC with Ag, cells were incubated with TT during the first
24 h of culture with GM-CSF and IL-4 before they were activated
with proinflammatory mediators for another 24 h. Immature
moDCs were loaded with TT on day 5 and were subsequently
stimulated with proinflammatory mediators for 24 h to induce DC
maturation. FastDC or moDCs, each loaded with TT or left unloaded, were cocultured with autologous T cells following activation. CD40L was added to induce maximal levels of functional
IL-12 secretion. Before the initiation of coculture, FastDC and
activated mature moDCs were compared for their expression of surface markers. No differences in the expression of CD14, CD83,
CD80, CD86, and MHC II were detected (data not shown). After 5
days of coculture, cells were pulsed with [3H]thymidine and harvested
after 18 h to measure proliferation. Ag-loaded FastDC were equally
effective in inducing the proliferation of autologous T cells compared
with Ag-loaded, activated moDCs (Fig. 6, top row). In an additional
set of experiments T cells were restimulated after 10 days of coculture
with the same DCs (FastDC or moDCs, respectively, left unloaded or
4074
RAPID DC DIFFERENTIATION FROM MONOCYTES
FIGURE 7. TT-loaded FastDC induce Agspecific proliferation of autologous CD45RA⫹ T
cells. TT-loaded FastDC and mature TT-loaded
moDCs were generated as described. Monocytes
cultured with GM-CSF plus IL-4 alone for 48 h,
immature moDCs, unloaded FastDC, and unloaded, stimulated moDCs were used as controls.
The different DCs were cocultured with purified
autologous naive CD45RA⫹ T cells as described
(see Materials and Methods). Cells were pulsed
with [3H]thymidine on day 5 and harvested after
18 h to determine proliferation. One representative experiment of four is shown.
Ag-loaded FastDC efficiently prime autologous naive T lymphocytes
Ag-bearing, mature DCs are the most potent inducers of primary
adaptive immune responses. To test the ability of FastDC to prime
naive T cells, FastDC or activated moDCs, each loaded with TT or
left unloaded, were cocultured with autologous, CD45RA-expressing T cells. T cell proliferation was determined after 5 days of
coculture in the presence of CD40L. Ag-loaded FastDC were
equally effective in inducing the proliferation of naive T cells compared with Ag-loaded, activated moDCs (Fig. 7).
Discussion
In the present study we report for the first time that mature DCs
with full T stimulatory capacity can be derived from human monocytes within only 48 h of in vitro culture. Monocytes cultured for
24 h with GM-CSF and IL-4 followed by stimulation with the
proinflammatory mediators TNF-␣, IL-1␤, IL-6, and PGE2 (27)
for another 24 h rapidly undergo all phases of DC differentiation;
cells transiently display immature DC characteristics (down-regulation of the monocytic marker CD14, increase in endocytotic uptake of soluble dextran and responsiveness to the inflammatory
chemokine MIP-1␣) before they develop into mature DCs expressing high levels of CD83, costimulatory, and MHC II molecules as
well as functional CCR7 (FastDC). FastDC loaded with TT induced equal rates of autologous T cell proliferation compared with
TT-loaded, stimulated moDCs generated according to a 7-day
standard protocol, indicating that they are capable of taking up and
processing proteins for presentation on MHC molecules. Strong
proliferative responses of autologous naive CD45RA⫹ cells indicated that Ag-loaded FastDC not only stimulate immune responses
to recall Ags, but are also effective in initiating primary immune
responses. Moreover, FastDC were equally capable of stimulating
a Th1-type immune response as Ag-loaded, activated moDCs, as
shown by IFN-␥ production of autologous T cells cocultured with
TT-loaded FastDC.
Our results confirm previous experimental evidence that circulating monocytes may represent a pool of stand-by precursor cells
capable of rapid differentiation into DCs. Randolph et al. (17)
showed that a subpopulation of monocytes, after migration across
an endothelial barrier into a collagen matrix, reverse direction and
migrate back. This reverse transmigration, simulating entry into
lymphatic vessels, was associated with down-regulation of the
monocytic marker CD14 and up-regulation of MHC II molecules.
If monocytes encountered yeast cell walls or other phagocytic particles in the collagen matrix, reverse transmigrated cells acquired
a mature DC phenotype and high allostimulatory capacity within
48 h. As we show here, monocytes efficiently take up and process
Ag, undergo DC maturation, up-regulate the lymph node-directing
chemokine receptor CCR7, and acquire T cell stimulatory within
48 h of incubation with GM-CSF and IL-4 plus proinflammatory
mediators. During inflammation or infection, the production of
GM-CSF by endothelial cells and the secretion of proinflammatory
mediators such as TNF-␣, IL-1␤, and PGE2 by tissue-residing immune cells may cooperate with microbial stimuli to induce rapid
differentiation and maturation of monocytes into DCs in vivo (9,
28, 29). Thus, monocytes may give rise to a subset of tissue-derived DCs capable of transit to secondary lymphoid tissues via lymphatic vessels to initiate or maintain adaptive immune responses.
The capacity of the proinflammatory mediators TNF-␣ and
PGE2 to induce complete DC maturation of monocytes by day 2 of
culture with GM-CSF and IL-4 is a novel finding of this study.
However, the effects of some of the proinflammatory mediators
used here on maturation, cytokine production, and T cell activation
by FastDC confirm previous results obtained in studies with standard moDCs. PGE2 and a number of other stimuli tested, including
CD40L, were not capable of inducing phenotypic maturation of
FastDC by themselves; cooperation of PGE2 with TNF-␣ or IL-1␤
was required for complete maturation of FastDC (30 –32). Stimulation with PGE2 rendered FastDC sensitive not only to the
CCR7 ligand 6Ckine, but also to the CXCR4 ligand SDF-1
(CXCL12), as Luft and co-workers have very recently shown for
moDCs (29). We also identified IL-1␤ as a key factor for optimal
activation of FastDC; IL-1␤ synergized with PGE2 to induce phenotypic maturation in the absence of TNF-␣, and removal of IL-1␤
from the proinflammatory mediators markedly reduced IL-12 production of FastDC. Failure of FastDC activated with proinflammatory cytokines plus PGE2 to secrete IL-12 (p70) is not surprising if recent reports on the effect of PGE2 on cytokine production
by moDCs are taken into account; it has repeatedly been shown
that PGE2 inhibits the secretion of IL-12 (p70) by moDCs even if
potent stimuli, such as CD40L, are used for activation (29, 33).
Nevertheless, activation with proinflammatory cytokines plus
PGE2 does not affect the Th1-inducing capacity of FastDC or
moDCs (29, 34). A marked increase in IL-12 production by
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loaded with TT) that had initially been added to the coculture. After
4 h of restimulation with DCs, supernatants were harvested for cytokine measurements. T cells stimulated with FastDC as well as with
activated moDCs loaded with Ag secreted IFN-␥ (Fig. 6, middle row).
In contrast, neither FastDC nor moDCs induced significant amounts
of IL-4 in autologous T cells independent of Ag loading and maturation status (Fig. 6, bottom row).
The Journal of Immunology
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References
1. Banchereau, J., F. Briere, C. Caux, J. Davoust, S. Lebecque, Y. J. Liu,
B. Pulendran, and K. Palucka. 2000. Immunobiology of dendritic cells. Annu.
Rev. Immunol. 18:767.
2. Sallusto, F., and A. Lanzavecchia. 2000. Understanding dendritic cell and Tlymphocyte traffic through the analysis of chemokine receptor expression. Immunol. Rev. 177:134.
3. Dieu, M. C., B. Vanbervliet, A. Vicari, J. M. Bridon, E. Oldham, S. Ait-Yahia,
F. Briere, A. Zlotnik, S. Lebecque, and C. Caux. 1998. Selective recruitment of
27.
28.
immature and mature dendritic cells by distinct chemokines expressed in different
anatomic sites. J. Exp. Med. 188:373.
Cella, M., D. Scheidegger, K. Palmer-Lehmann, P. Lane, A. Lanzavecchia, and
G. Alber. 1996. Ligation of CD40 on dendritic cells triggers production of high
levels of interleukin-12 and enhances T cell stimulatory capacity: T-T help via
APC activation. J. Exp. Med. 184:747.
Cella, M., A. Engering, V. Pinet, J. Pieters, and A. Lanzavecchia. 1997. Inflammatory stimuli induce accumulation of MHC class II complexes on dendritic
cells. Nature. 388:782.
Schnurr, M., F. Then, P. Galambos, C. Scholz, B. Siegmund, S. Endres, and
A. Eigler. 2000. Extracellular ATP and TNF-␣ synergize in the activation and
maturation of human dendritic cells. J. Immunol. 165:4704.
Sozzani, S., P. Allavena, G. D’Amico, W. Luini, G. Bianchi, M. Kataura, T. Imai,
O. Yoshie, R. Bonecchi and A. Mantovani. 1998. Differential regulation of chemokine receptors during dendritic cell maturation: a model for their trafficking
properties. J. Immunol. 161:1083.
Randolph, G. J. 2001. Dendritic cell migration to lymph nodes: cytokines, chemokines, and lipid mediators. Semin. Immunol. 13:267.
Shortman, K.. and Y. J. Liu. 2002. Mouse and human dendritic cell subtypes. Nat.
Rev. Immunol. 2:151.
Hornung, V., S. Rothenfusser, S. Britsch, A. Krug, B. Jahrsdorfer, T. Giese,
S. Endres, and G. Hartmann. 2002. Quantitative expression of Toll-like receptor
1–10 mRNA in cellular subsets of human peripheral blood mononuclear cells and
sensitivity to CpG oligodeoxynucleotides. J. Immunol. 168:4531.
Krug, A., A. Towarowski, S. Britsch, S. Rothenfusser, V. Hornung, R. Bals,
T. Giese, H. Engelmann, S. Endres, A. M. Krieg et al. 2001. Toll-like receptor
expression reveals CpG DNA as a unique microbial stimulus for plasmacytoid
dendritic cells which synergizes with CD40 ligand to induce high amounts of
IL-12. Eur. J. Immunol. 31:3026.
Liu, Y. J. 2001. Dendritic cell subsets and lineages, and their functions in innate
and adaptive immunity. Cell 106:259.
Pulendran, B., J. Banchereau, S. Burkeholder, E. Kraus, E. Guinet, C. Chalouni,
D. Caron, C. Maliszewski, J. Davoust, J. Fay et al. 2000. Flt3-ligand and granulocyte colony-stimulating factor mobilize distinct human dendritic cell subsets
in vivo. J. Immunol. 165:566.
Caux, C., C. Dezutter-Dambuyant, D. Schmitt, and J. Banchereau. 1992. GMCSF and TNF-alpha cooperate in the generation of dendritic Langerhans cells.
Nature 360:258.
Romani, N., S. Gruner, D. Brang, E. Kampgen, A. Lenz, B. Trockenbacher,
G. Konwalinka, P. O. Fritsch, R. M. Steinman, and G. Schuler. 1994. Proliferating dendritic cell progenitors in human blood. J. Exp. Med. 180:83.
Sallusto, F., and A. Lanzavecchia. 1994. Efficient presentation of soluble antigen
by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis
factor alpha. J. Exp. Med. 179:1109.
Randolph, G. J., S. Beaulieu, S. Lebecque, R. M. Steinman, and W. A. Muller.
1998. Differentiation of monocytes into dendritic cells in a model of transendothelial trafficking. Science 282:480.
Ho, C. S., D. Munster, C. M. Pyke, D. N. Hart, and J. A. Lopez. 2002. Spontaneous generation and survival of blood dendritic cells in mononuclear cell culture
without exogenous cytokines. Blood 99:2897.
Santini, S. M., C. Lapenta, M. Logozzi, S. Parlato, M. Spada, T. Di Pucchio, and
F. Belardelli. 2000. Type I interferon as a powerful adjuvant for monocyte-derived dendritic cell development and activity in vitro and in Hu-PBL-SCID mice.
J. Exp. Med. 191:1777.
Blanco, P., A. K. Palucka, M. Gill, V. Pascual, and J. Banchereau. 2001. Induction of dendritic cell differentiation by IFN-␣ in systemic lupus erythematosus.
Science 294:1540.
Novak, E. J., and P. S. Rabinovitch. 1994. Improved sensitivity in flow cytometric intracellular ionized calcium measurement using fluo-3/Fura Red fluorescence
ratios. Cytometry 17:135.
Schnurr, M., C. Scholz, S. Rothenfusser, P. Galambos, M. Dauer, J. Robe,
S. Endres, and A. Eigler. 2002. Apoptotic pancreatic tumor cells are superior to
cell lysates in promoting cross-priming of cytotoxic T cells and activate NK and
␥␦ T cells. Cancer Res. 62:2347.
Schnurr, M., P. Galambos, C. Scholz, F. Then, M. Dauer, S. Endres, and
A. Eigler. 2001. Tumor cell lysate-pulsed human dendritic cells induce a T-cell
response against pancreatic carcinoma cells: an in vitro model for the assessment
of tumor vaccines. Cancer Res. 61:6445.
Trinchieri, G. 1998. Interleukin-12: a cytokine at the interface of inflammation
and immunity. Adv. Immunol. 70:83.
Snijders, A., P. Kalinski, C. M. Hilkens, and M. L. Kapsenberg. 1998. High-level
IL-12 production by human dendritic cells requires two signals. Int. Immunol.
10:1593.
Hochrein, H., M. O’Keeffe, T. Luft, S. Vandenabeele, R. J. Grumont,
E. Maraskovsky, and K. Shortman. 2000. Interleukin (IL)-4 is a major regulatory
cytokine governing bioactive IL-12 production by mouse and human dendritic
cells. J. Exp. Med. 192:823.
Feuerstein, B., T. G. Berger, C. Maczek, C. Roder, D. Schreiner, U. Hirsch,
I. Haendle, W. Leisgang, A. Glaser, O. Kuss, et al. 2000. A method for the
production of cryopreserved aliquots of antigen-preloaded, mature dendritic cells
ready for clinical use. J. Immunol. Methods 245:15.
Sato, T., Y. Kirimura, and Y. Mori. 1997. The co-culture of dermal fibroblasts
with human epidermal keratinocytes induces increased prostaglandin E2 production and cyclooxygenase 2 activity in fibroblasts. J. Invest. Dermatol.
109:334.
Downloaded from http://www.jimmunol.org/ by guest on April 28, 2017
FastDC was observed if CD40 ligation followed proinflammatory
signaling, indicating that CD40L synergizes with proinflammatory
mediators to induce IL-12 production by DCs, a mechanism that
has been described for IL-1␤ and LPS (35, 36). Increased levels of
total IL-12 production were associated with secretion of the bioactive IL-12 (p70) heterodimer if IFN-␥ was present during CD40
ligation. Thus, even after stimulation with PGE2, low levels of
IL-12 (p70) can be induced in FastDC if an additional signal is
provided by T cell-produced cytokines such as IFN-␥ (25, 26). We
therefore hypothesize that FastDC matured with proinflammatory
cytokines plus PGE2 become capable of migrating to secondary
lymphoid organs where encounter with activated T cells induces
the secretion of IL-12 (p70) and facilitates the stimulation of Th1type immune responses.
Historic aspects of the study of DC differentiation may help to
explain why more rapid development of DC-like cells from monocyte precursors has not been attempted previously; when DCs were
first derived in vitro from blood precursors, specific surface markers had yet to be discovered, and morphologic criteria had to be
applied to identify DCs (14). Ever since, the concept has persisted
that moDCs had to be of Langerhans cell-like morphology; increased cell size, development of granules, and formation of cytoplasmatic protrusions are some of the morphologic features attributed to the typical moDC. In vitro, culture of 5–7 days with
GM-CSF and IL-4 is required to induce these morphologic
changes and hence has become the standard protocol for generation of DCs from monocytes (15, 16). Although this regimen can
be used for the generation of DC-like cells that have been proven
to efficiently induce antitumoral immune responses in vivo in a
number of vaccination trials (37–39), it may not reflect the time
span required for DC differentiation from monocytes under physiologic conditions. Moreover, compared with the prolonged in
vitro culture required in standard protocols, generation of FastDC
not only saves time, but may also prove to be less sensitive to
external disruptive factors such as microbial contamination. In our
hands, generation of FastDC yielded high percentages of viable,
fully mature DCs more reliably than the standard 7-day protocol
used to generate moDCs.
In summary, we were able to demonstrate that the development
of mature DCs from monocyte precursors does not require ⬎2
days of in vitro culture. FastDC matured with proinflammatory
mediators were compared with moDCs generated by a standard
7-day protocol and were found to possess equal capacity to induce
Ag-specific proliferation of T cells as well as to prime autologous
naive T lymphocytes. Moreover, FastDC are sensitive to chemokines agonistic to the lymph node-directing chemokine receptor
CCR7 and induce a Th1 cytokine profile in autologous T cells, thus
demonstrating all the qualities currently thought to be necessary
for an effective DC-based, antitumor vaccine. This new protocol
markedly reduces the time, work load, and cost associated with in
vitro culture of DC precursors and thus may facilitate the use of
DCs in clinical trials of cellular immunotherapy. Moreover, our
findings support the hypothesis that monocytes represent a pool of
circulating precursor cells capable of rapid differentiation into mature DCs after transit into inflamed or infected tissues.
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4076
29. Luft, T., M. Jefford, P. Luetjens, T. Toy, H. Hochrein, K. A. Masterman,
C. Maliszewski, K. Shortman, J. Cebon, and E. Maraskovsky. 2002. Functionally
distinct dendritic cell (DC) populations induced by physiologic stimuli: prostaglandin E2 regulates the migratory capacity of specific DC subsets. Blood 100:
1362.
30. Rieser, C., G. Bock, H. Klocker, G. Bartsch, and M. Thurnher. 1997. Prostaglandin E2 and tumor necrosis factor ␣ cooperate to activate human dendritic cells:
synergistic activation of interleukin 12 production. J. Exp. Med. 186:1603.
31. Kalinski, P., J. H. Schuitemaker, C. M. Hilkens, and M. L. Kapsenberg. 1998.
Prostaglandin E2 induces the final maturation of IL-12-deficient CD1a⫹CD83⫹
dendritic cells: the levels of IL-12 are determined during the final dendritic cell
maturation and are resistant to further modulation. J. Immunol. 161:2804.
32. Scandella, E., Y. Men, S. Gillessen, R. Forster, and M. Groettrup. 2002. Prostaglandin E2 is a key factor for CCR7 surface expression and migration of monocyte-derived dendritic cells. Blood 100:1354.
33. Kalinski, P., P. L. Vieira, J. H. Schuitemaker, E. C. de Jong, and
M. L. Kapsenberg. 2001. Prostaglandin E2 is a selective inducer of interleukin-12
p40 (IL-12p40) production and an inhibitor of bioactive IL-12p70 heterodimer.
Blood 97:3466.
RAPID DC DIFFERENTIATION FROM MONOCYTES
34. Steinbrink, K., L. Paragnik, H. Jonuleit, T. Tuting, and J. Knop. 2000. Induction
of dendritic cell maturation and modulation of dendritic cell-induced immune
responses by prostaglandins. Arch. Dermatol. Res. 292:437.
35. Luft, T., M. Jefford, P. Luetjens, H. Hochrein, K. A. Masterman, C. Maliszewski,
K. Shortman, J. Cebon, and E. Maraskovsky. 2002. IL-1␤ enhances CD40 ligandmediated cytokine secretion by human dendritic cells (DC): a mechanism for T
cell-independent DC activation. J. Immunol. 168:713.
36. Wesa, A. K., and A. Galy. 2001. IL-1␤ induces dendritic cells to produce IL-12.
Int. Immunol. 13:1053.
37. Nestle, F. O., S. Alijagic, M. Gilliet, Y. Sun, S. Grabbe, R. Dummer, G. Burg, and
D. Schadendorf. 1998. Vaccination of melanoma patients with peptide- or tumor
lysate-pulsed dendritic cells. Nat. Med. 4:328.
38. Thurnher, M., H. Klocker, C. Papesh, R. Ramoner, C. Radmayr, A. Hobisch,
G. Gastl, N. Romani, S. Ebner, G. Bock, et al. 1997. Dendritic cells for the
immunotherapy of renal cell carcinoma. Urol. Int. 59:1.
39. Kugler, A., G. Stuhler, P. Walden, G. Zoller, A. Zobywalski, P. Brossart,
U. Trefzer, S. Ullrich, C. A. Muller, V. Becker, et al. 2000. Regression of human
metastatic renal cell carcinoma after vaccination with tumor cell-dendritic cell
hybrids. Nat. Med. 6:332.
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