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Concurrent delivery of tumor antigens and activation signals to
dendritic cells by irradiated CD40 ligand-transfected tumor cells
resulted in efficient activation of specific CD8
þ
T cells
Ko-Jiunn Liu, Li-Fan Lu, Hui-Ting Cheng, Yi-Mei Hung, Sheng-Ru Shiou, Jacqueline
Whang-Peng and Shin-Hun Juang
Cancer Research Cooperative Laboratory at National Taiwan University Hospital, Division of Cancer
Research, National Health Research Institutes, Taipei, Taiwan.
To improve the efficacy of tumor cell-based and dendritic cell (DC)-based cancer vaccines, this study explored the potential of a
new cancer vaccine strategy, that is, the use of CD40 ligand-transfected tumor (CD40L-tumor) cells to simultaneously deliver both
tumor-derived antigens (Ag) and maturation stimuli to DCs. Materials from frozen/thawed or irradiated human tumor cells, with or
without surface CD40L, were internalized efficiently by immature DCs after coincubation. However, during the internalization
process, only coculturing with irradiated CD40L-tumor cells resulted in concurrent, optimal DC maturation and production of
proinflammatory chemokines and pro-Th1 cytokines, such as IL-6, IL-8, IL-12, IFN-g, and TNF-a. These activated DCs were the
most potent cells to support the growth of CD8
þ
, IFN-g-producing T cells, and to process tumor Ag for the generation of specific
cytotoxic T cells in vitro. Animals vaccinated with irradiated CD40L-tumor cell-pulsed DCs were better protected against
subsequent challenge of a weakly immunogenic tumor cell line than animals vaccinated with irradiated CD40L-tumor cells alone.
Thus, our results strongly support the future clinical application of using DCs pulsed with irradiated CD40L-tumor cells as a cancer
vaccine.
Cancer Gene Therapy (2004) 11, 135–147. doi:10.1038/sj.cgt.7700663
Published online 28 November 2003
Keywords: dendritic cells; antigen processing; CD40 ligand
D
endritic cells (DCs) are ‘‘professional’’ antigen
presenting cells (APCs) involved in the initiation
and maintenance of both the innate and the adaptive
immune responses.
1
Protective cellular immunity was
induced by vaccination of DCs loaded with various forms
of tumor antigens (Ag) in several animal models.
2–4
Early
clinical trials using tumor Ag-pulsed DCs as vaccines for
the treatment of various advanced malignancies have been
reported.
5–7
Several DC-based clinical trials employed
preidentified tumor-associated Ag (TAA) or synthetic
peptides containing cytotoxic T lymphocyte (CTL)
epitopes of known TAA as the source of Ag. Alternative
approaches have been explored to expand DC-based
therapy to cancers for which appropriate TAA or relevant
CTL epitopes have not been determined. These ap-
proaches include pulsing DCs with tumor cell ly-
sates,
3,4,6,8,9
tumor-derived RNA,
10
necrotic or apoptotic
tumor cells,
11–15
and fusing DCs with tumor cells.
16,17
Gene-modified tumor cell vaccines, which also use
undefined tumor material as the source of tumor Ag,
represent another promising new approach for cancer
therapy. Immunization of animals with tumor cells
transfected with genes coding for costimulatory mole-
cules, cytokines, or chemokines can induce tumor-specific
CTL responses and inhibit the growth of parental tumor
cells.
18–21
One possible mechanism underlying the anti-
tumor effect of gene-modified tumor cell vaccine is that
tumor materials deposited at the injection site are
captured and subsequently presented to specific CTLs
by infiltrating or resident DCs.
22
Thus, tumor cells
transfected with genes that can promote such in vivo Ag
presentation processes are likely to serve as good tumor
vaccines. It has been shown that immunization with CD40
ligand (CD40L)-transfected tumor (CD40L-tumor) cells
can induce a protective immunity against tumor challenge
in animal models.
22–24
CD40L induces DC maturation
and triggers DCs to secrete various cytokines, including
IL-12, which are important in the induction of Th1 and
CTL responses.
25–27
Signaling through CD40 on the
surface of DCs plays an important role in DC migration
from the epidermal tissue to the lymph nodes,
28
and can
enable DCs to activate CTL in the absence of Th cells.
29
Received June 2, 2003.
Address correspondence and reprint requests to: Dr Ko-Jiunn Liu,
Cancer Research Cooperative Laboratory at National Taiwan
University Hospital, Division of Cancer Research, National Health
Research Institutes, 7, Chung-Shan South Road, 100 Taipei, Taiwan.
E-mail: kojiunn@nhri.org.tw
This work was supported by intramural grants from the National
Health Research Institutes, Taipei, Taiwan.
Cancer Gene Therapy (2004) 11, 135–147
r
2004 Nature Publishing Group All rights reserved 0929-1903/04 $25.00
www.nature.com
/
cgt
Vaccination with CD40L-tumor cells could potentially
result in the uptake of tumor Ag by infiltrating DCs, and
the concurrent activation of DCs. Thus, the antitumor
immune response to CD40L-tumor cells could be more
potent than the one to unmodified tumor cells. However,
with this approach, it is difficult to optimally control and
monitor the efficiency of Ag loading and activation of
DCs in vivo. To overcome this drawback and utilize the
benefit of CD40L signaling, we incorporated the gene-
modified tumor cells with the DCs prior to vaccination.
In this study, we have used an in vitro system to
investigate the effect of coculturing human immature DCs
with different forms of human CD40L-tumor cells. By
using CD40L-tumor cells to deliver both tumor Ag and
stimulating signals to DCs during in vitro vaccine
preparation, we had better control of Ag loading and
activation of DCs. DCs cultured with irradiated CD40L-
tumor cells were able to process and present tumor Ag,
secrete substantial amounts of various pro-Th1 and
proinflammatory cytokines, and promote the growth
and generation of specific CD8
þ
T cells. When CD40L-
tumor cell-vaccinated animals were compared to animals
receiving DCs pulsed with irradiated CD40L-tumor cells,
the latter were found to be more resistant to subsequent
challenge with a weakly immunogenic B16-F1 tumor line.
Our results support DCs pulsed with irradiated CD40L-
transfected tumor cells as a candidate vaccine strategy in
human clinical studies and may provide the foundation
for the design of a better DC-based cancer vaccine.
Materials and methods
Human tumor transfectant expressing CD40L
A lung cancer cell line was previously established in our
laboratory from the pleural effusion of a lung cancer
patient. The lung cancer cells were transfected with either
an empty pcDNA3.1/Zeo() vector or a vector carrying
the human CD40L sequence and selected with 200 mg/ml
zeocin (Invitrogen, San Diego, CA). The expression of
CD40L by transfected tumor cells was determined by
staining with a PE-anti-CD154 mAb (BD PharMingen,
San Diego, CA). Cells expressing surface CD40L were
sorted using a FACS Vantage cell sorter (Becton
Dickinson, Mountain View, CA) and cloned. All cell
lines used in this study were negative for mycoplasma
contamination as determined by a nested PCR-based
mycoplasma detection kit (ATCC, Rockville, MD).
Preparation of human DCs
Peripheral blood mononuclear cells (PBMCs) from
healthy donors (obtained from the Chinese Blood
Foundation) were enriched by density-gradient centrifu-
gation with Ficoll-Hypaque (Amersham Pharmacia Bio-
tech, Uppsala, Sweden). Cells at the interface were
collected, washed and suspended in serum-free AIM-V
medium (Life Technologies, Rockville, MD). Cells were
plated into plastic tissue culture dishes (25 10
6
cells/
dish) and incubated at 371C for 2 hours. Adherent
mononuclear cells were cultured with RPMI-1640 med-
ium with 10% fetal bovine serum (FBS) in the presence of
1000 U/ml recombinant human IL-4 (Peprotech, Rock
Hill, NJ) and 500 U/ml granulocyte–macrophage colony-
stimulating factor (GM-CSF) (a gift from Kirin Brewery
Co., Japan). After 7 days, nonadherent and loosely
adherent cells were collected and used as the source of
immature DCs in the subsequent experiments.
Flow cytometric analysis
Cells to be analyzed for the expression of surface markers
were stained with different fluorescence-labeled mAb and
then analyzed using a flow cytometer (EPICS XL-MCL,
Beckman Coulter, Fullerton, CA). The mAbs used in this
study were as follows: flourescein isothiocyanate (FITC)-
anti-HLA-DR (Immunotech, Marseille Cedex, France),
phycoerythrin (PE)-anti-CD86 (Immunotech), FITC-
anti-CD80 (Immunotech), PE-anti-CD83 (BD PharMin-
gen), and PE-anti-CD11c (Immunotech). Isotype-
matched control mAb were obtained from BD PharMin-
gen and Caltag (Burlingame, CA).
Internalization of tumor materials by DCs
Tumor cells were labeled with a green fluorescent dye,
PKH67 (Sigma, St. Louis, MO) according to the
manufacturer’s instruction, and treated with one cycle
of freezing/thawing to induce necrosis or irradiation
(12,000 rad) to initiate apoptosis. One cycle of freezing/
thawing resulted in mostly intact cells that were stained
positive with trypan blue. Irradiation (12,000 rad) in-
itiated a slowly progressing apoptosis, as 10–20% cells
were stained positive with FITC-annexin V (Caltag) after
16 hours. DCs (1 10
6
cells) were cultured with equal
numbers of frozen/thawed or irradiated dye-labeled
tumor cells at 371C for 16 or 40 hours. The mixture of
cells was collected, and DCs were counterstained with a
PE-anti-CD11c mAb (Immunotech) or a PE-anti-
CD83 mAb (BD PharMingen). The internalization of
green fluorescent tumor materials by DCs was evaluated
with a fluorescent microscope (BX50, Olympus, Tokyo,
Japan) or a flow cytometer (EPICS XL-MCL, Beckman
Coulter).
Induction of maturation and activation of DCs
Immature DCs (2.5 10
5
cells/well) were cultured in a 24-
well plate with equal numbers of frozen/thawed or
irradiated tumor cells with or without CD40L expression.
Cells were collected after 16 or 40 hours and analyzed for
surface expressions of CD11c, CD83, CD80, CD86, and
HLA-DR by flow cytometry, or for the induction of an
allogeneic mixed leukocyte response (MLR). In some
experiments, the culture supernatants were harvested and
assayed for cytokine production.
Detection of cytokine production
The production of IL-12p70, IL-1b, IL-6, IL-8, IL-10,
and TNF-a in the culture supernatant was detected by a
Human Inflammation Cytometric Bead Array (CBA) Kit
Antigen loading and concurrent activation of DC
K-J Liu et al
136
Cancer Gene Therapy
(BD PharMingen). Briefly, beads of different FL-3
fluorescent intensities were preconjugated with mAb
against various cytokines by the manufacturer. These
beads were incubated with 50 ml of culture supernatants
(or cytokine standards) and PE-labeled mAb against
various cytokines at room temperature for 3 hours. Beads
were then subjected to flow cytometric analysis. The
amount of a given cytokine in the culture supernatant was
calculated by converting the intensity of PE fluorescence
to concentration after comparing to results obtained with
the cytokine standards. The production of IFN-g in the
culture supernatant was detected by a sandwich-ELISA
using an IFN-g-OptEIA set (BD PharMingen).
Allogeneic MLR
Serial diluted DCs, after coculturing with tumor cells,
were irradiated (3000 rad) and cultured with allogeneic,
nonadherent PBMCs (10
5
cells/well) in 96-well plates for 6
days. In all, 1 mCi of
3
H-TdR (Amersham Pharmacia
Biotech) was added to the culture for the last 16 hours.
The uptake of
3
H-TdR by the proliferating cells was then
determined by a liquid scintillation counter (Packard
BioScience, Downers Grove, IL).
T-cell surface marker, secreted and intracellular
cytokine analysis
DCs were cultured with different forms of tumor cells for
40 hours. After washing, tumor material-pulsed DCs
(2.5 10
5
cells/well) were cultured with allogeneic, non-
adherent PBMCs (2.5 10
6
cells/well) for 6 days.
Proliferating T cells were expanded with 20 U/ml IL-2
(Chiron, Amsterdam, The Netherlands) and cultured for
another 6 days. Cells were then collected and stained with
an FITC-anti-CD8 mAb and a PE-anti-CD3 mAb (Im-
munotech). The forward scatter (FSC) and the side scatter
(SSC) were used to gate on small lymphocytes for
subsequent analysis. For intracellular cytokine staining,
proliferating T cells were collected, washed and stimu-
lated with 20 ng/ml phorbol 12-myristate-13-acetate
(PMA) and 1000 ng/ml ionomycin (Sigma) in the presence
of GolgiStop (BD PharMingen) for 6 hours. Cells were
stained with an FITC-anti-CD8 mAb. After washing, cells
were fixed, permeabilized with Cytofix/Cytoperm (BD
PharMingen), and then stained with a PE-anti-IFN-g
mAb (BD PharMingen). The expression of intracellular
IFN-g by CD8
þ
T cells was analyzed with a flow
cytometer. Supernatants from day 6 and day 12 cultures
were collected and analyzed for cytokine secretion with a
human Th1/Th2 CBA kit (BD PharMingen).
Evaluation of T-cell responses
Immature DCs were generated from PBMCs of normal
donors with the HLA-A*0201 genotype and cultured with
equal numbers of tumor cells for 16 hours. After washing,
tumor cell material-pulsed DCs (1.5 10
5
cells/well) were
cultured with autologous CD8-enriched T cells (1.5 10
6
cells/well) in RPMI medium supplemented with 10%
human AB-type serum (BioWhittaker, Walkersville, MD)
in a 24-well plate. Autologous CD8-enriched T cells (80%
pure) were prepared by removing adherent cells and
CD4
þ
T cells with anti-CD4-coated magnetic beads and
a Magnetic Particle Concentrator (Dynal Biotech, Oslo,
Norway). IL-2 (20 U/ml) was added into the culture on
day 1, 3, and 6. On day 10, the T cells were restimulated
with irradiated autologous PBMCs pulsed with 50 mg/ml
of an HLA-A*0201-restricted HER2 peptide (HER2, 369-
377:KIFGSLAFL, Research Genetics, Huntsville, AL).
30
IL-2 (20 U/ml) was added into the culture on days 11 and
14. On day 17, T cells (1 10
5
cells/well) were stimulated
with irradiated (8000 rad) T2 cells (5 10
4
cells/well)
pulsed in advance for 3 hours with 50 mg/ml of the HER2
peptide or a control HLA-A*0201-restricted, HPV-16 E6
peptide (E6, 18-26:KLPQLCTEL, Research Genetics).
31
The secretion of IFN-g in the culture supernatant was
determined after 48 hours using a human Th1/Th2 CBA
kit (BD PharMingen). The number of viable cells at the
beginning (day 0) and the end of each stimulation
procedure (days 10 and 17) was determined by the trypan
blue exclusion assay. The cytotoxicity was determined by
a europium-ligand release assay (Wallac, Turku, Fin-
land). The counts of culture with unlabeled T2 cells were
subtracted from those of cultures with peptide-pulsed T2
cells before calculating the specific lysis.
Animal experiments
Murine DCs were generated by culturing bone marrow
cells with RPMI-1640 medium supplemented with 10%
FBS, 500 U/ml of murine GM-CSF, and IL-4 (Peprotech)
for 7 days as described.
26
CT-26 or B16-F1 tumor cells
(ATCC, Rockville, MD) were transfected with either a
control vector or a vector carrying the murine CD40L
gene. Transfected cells were selected and cloned similarly
to methods described above. Day-7 DCs were cocultured
with irradiated (20,000 rad) CD40L-tumor cells for 48
hours at a one-to-one ratio. Mice (n ¼ 5) were immunized
(s.c.) weekly with PBS, DC alone (1 10
6
cells), irradiated
CD40L-tumor alone (1 10
6
cells), or DCs (1 10
6
cells)
pulsed with irradiated CD40L-tumor cells. At 2 weeks
after the third immunization, C57BL/6 or BALB/c mice
were challenged (s.c.) with wild-type B16-F1 cells (1 10
4
cells) or CT-26 cells (2 10
4
cells), respectively. B16-F1
cells were treated with IFN-g 16 hours before injection to
induce the expression of MHC class I molecules. The
growth of tumor was monitored every 2–3 days. The size
of tumor was determined as follows: short diameter
2
long diameter 1/2. Mice were killed when the tumor
volume exceeds 2 cm
3
. This experiment was repeated once
using the same conditions.
Results
DCs can efficiently internalize tumor materials from
frozen/thawed or irradiated tumor cells transfected with
CD40L
Human cells (because they are most relevant to future
clinical applications) were investigated initially in an in
Antigen loading and concurrent activation of DC
K-J Liu et al
137
Cancer Gene Therapy
vitro system. The cDNA for human CD40L was
transfected into a human lung cancer cell line (Fig 1a)
to generate tumor cells that can both provide tumor
antigenic materials and DC stimulatory signals. Immature
DCs were generated by culturing adherent PBMCs with
GM-CSF and IL-4 for 7 days. They expressed a typical
surface staining phenotype of immature DCs (HLA-
DR
þ
, CD3
, CD11c
þ
, CD14
, CD20
, CD80
þ
,
CD83
, and CD86
þ
) and can be induced to express a
mature DC phenotype (CD86
high
and CD83
þ
) after
incubation with 1000 U/ml TNF-a for 40 hours (Fig 1b
and data not shown). To determine the capacity of
immature DCs to internalize tumor materials after
different treatments, tumor cells, with or without surface
CD40L (CD40L-tumor or vector-tumor cells, respec-
tively), were labeled with a green fluorescent dye (PKH67)
and treated with one cycle of freezing/thawing or
irradiation (12,000 rad). Irradiation at this dose initiated
only a slowly progressing apoptosis in the tumor cells, as
about 13 and 41% of cells were stained positive with
FITC-annexin V after 16 and 40 hours, respectively (Fig
1c). These treated tumor cells were then cultured with
equal numbers of immature DCs. After 16 hours, a
significant amount of green fluorescent material was
observed in CD11c
þ
DCs cultured with various forms of
tumor cells (Fig 2). These results indicate that immature
DCs were capable of internalizing antigenic materials
from tumor cells with or without CD40L expression on
the surface. Similar results were observed after cocultur-
ing for 40 hours (data not shown). Flow cytometric
analysis revealed that 65% (64/99) of all CD11c
þ
DCs
contained green fluorescent tumor materials (Fig 3a, left
panel) after coculturing with frozen/thawed CD40L-
tumor cells for 40 hours. Only a few intact tumor cells
(CD11c
PHK67
þ
) remained in the culture (the small
debris was not gated for analysis). In contrast, after
coculturing DCs with irradiated CD40L-tumor cells for
40 hours, 40% (28/70) of CD11c
þ
DCs contained green
fluorescent tumor materials, while a significant percentage
(about 29% of all gated cells) of tumor cells
(CD11c
PKH67
þ
) was still present in the culture (Fig
3a, right panel). These results indicate that immature DCs
appeared to engulf tumor materials more efficiently when
CD40L-tumor cells were treated with freezing/thawing. It
is likely that coculture with irradiated CD40L-tumor cells
led to maturation of immature DCs and thus down-
regulated their capacity to internalize tumor materials.
There were only a few necrotic cells (o5%) in the culture
Figure 1 (a) The expression of surface CD40L on transfected tumor cells was analyzed by flow cytometry. Upper panel: vector-tumor cells.
Lower panel: CD40L-tumor cells. Dotted line: cells stained with a PE-isotype-matched control mAb. Solid line: cells stained with a PE-anti-CD40L
mAb. (b) Immature DCs were generated by culturing adherent PBMCs with GM-CSF and IL-4 for 7 days. Cells were further cultured in the
presence (lower four panels) or absence (upper four panels) of TNF-a for 48 hours. The cell surface expression of HLA-DR, CD86, CD80, and
CD83 was determined by flow cytometry with specific mAb (solid line). Dotted line: isotype-matched control mAb. (c) CD40L-tumor cells were
irradiated (12,000 rad) and then cultured for 16 or 40 hours. Untreated tumor cells or irradiated cells after culture were collected and stained with
FITC-annexin V and PI, and analyzed by flow cytometry.
Antigen loading and concurrent activation of DC
K-J Liu et al
138
Cancer Gene Therapy
Figure 2 Vector-tumor (left four panels) or CD40L-tumor (right four
panels) cells were labeled with a green fluorescent dye, PKH67,
washed extensively and then treated with either one cycle of
freezing/thawing (frozen/thawed) or irradiation (irradiated). Dye-
labeled tumor cells were then cocultured with equal numbers of
immature DCs for 16 hours. The mixture of cells was collected and
DCs were counterstained with a PE-anti-CD11c mAb. The inter-
nalization of green fluorescent tumor materials by CD11c
þ
DCs was
examined using a fluorescent microscope. Image from the same cell
was displayed with filters for green (a, c, e, and g) or red (b, d, f, and
h) fluorescence. One representative result from three separate
experiments is shown.
Figure 3 (a) Immature DCs were cocultured with equal numbers of
PKH67-labeled, frozen/thawed (left panel) or irradiated (right panel)
CD40L-tumor cells for 40 hours. The mixture of cells was collected
and DCs were counterstained with a PE-anti-CD11c mAb. The
numbers in each quadrant represent the percentage of cells after
gating. One representative result from three separate experiments is
shown. (b) Immature DCs were cocultured with equal number of
frozen/thawed or irradiated vector-tumor (closed bars) or CD40L-
tumor (open bars) cells for 16 or 40 hours. The mixture of cells was
then collected and stained for the expression of HLA-DR and CD83.
The percentage of CD83
þ
cells in all cells positive for HLA-DR is
shown. (c, d) Immature DCs were cocultured with equal numbers of
irradiated, PKH67-labeled vector-tumor (closed bars) or CD40L-
tumor (open bars) cells for 4, 16, or 40 hours. The mixture of cells
was collected and DCs were counterstained with a PE-anti-CD11c
mAb or a PE-anti-CD83 mAb. The percentage of CD11c
þ
(c) or
CD83
þ
(d) cells from all cells associated with PHK67 green
fluorescence was determined. One representative result from two
separate experiments is shown.
Antigen loading and concurrent activation of DC
K-J Liu et al
139
Cancer Gene Therapy
up to 40 hours after irradiation (Fig 1c). Therefore, under
this condition, necrotic cells should not have a major role
in the uptake of tumor materials by DCs and the
induction of DC maturation.
Only irradiated CD40L-tumor cells can efficiently
induce the concurrent expression of CD83 on DCs
during the internalization of tumor materials
Ligation of surface CD40 on DCs can trigger their
maturation and activation. As shown in Figure 3b, less
than 25% of HLA-DR
þ
DCs expressed CD83 after
coculturing with frozen/thawed vector-tumor or CD40L-
tumor cells. Coculturing immature DCs with irradiated
CD40L-tumor cells, but not vector-tumor cells, resulted
in a dramatic induction of CD83 expression. Around 40%
of DR
þ
cells were CD83
þ
after 16 hours, while more
than 90% DR
þ
cells were CD83
þ
after 40 hours. These
results suggest that only coculturing immature DCs with
irradiated CD40L-tumor cells can lead to concurrent
internalization of tumor materials and maturation of
DCs. To simultaneously monitor these two events,
immature DCs were cocultured with irradiated, green
fluorescent dye-labeled tumor cells for 4, 16, or 40 hours.
The mixture of cells was collected and the distribution of
internalized green tumor material in DCs expressing
CD11c (all DCs) or CD83 (mature DCs) was evaluated.
Around 20% of green fluorescent material was associated
with CD11
þ
DC after 4 hours of coculture with
irradiated vector-tumor or CD40L-tumor cells, while
60–70% of green fluorescent tumor material was asso-
ciated with CD11c
þ
DC after 16 and 40 hours (Fig 3c).
The proportion of green fluorescent tumor material
associated with CD83
þ
DCs after culturing with irra-
diated CD40L-tumor cells for 4, 16, or 40 hours was
similar to that associated with CD11c
þ
DCs (Fig 3c and
d, open bars). However, less than 20% of green
fluorescent tumor material from irradiated vector-tumor
cells was associated with CD83
þ
DC even after 40 hours
(Fig 3d, closed bars). These results demonstrate that after
coculturing immature DCs with irradiated CD40L-tumor
cells, a significant amount of tumor material was
internalized by CD11c
þ
DCs, and that most of these
DCs also became CD83
þ
within 16 hours. Coculturing
immature DCs with irradiated vector-tumor cells also
resulted in efficient internalization of tumor materials into
CD11c
þ
DCs, but the majority of them remained CD83
even after 40 hours.
Only irradiated CD40L-tumor cells induce a significant
secretion of pro-Th1 and pro-inflammatory cytokines by
DCs
DCs are known to dictate and modulate immune
responses by releasing various cytokines and chemokines.
A significant level of IL-12p70, in addition to substantial
amounts of IFN-g and several proinflammatory cytokines
including IL-6 and TNF-a, was detected only in the
culture containing DCs and irradiated CD40L-tumor cells
after 40 hours (Fig 4). Significant levels of IL-8 were
present under all four culture conditions, and the highest
level was detected in the culture with irradiated CD40L-
tumor cells. A low level of IL-10 and IL-1b was detected
in the culture containing DCs stimulated with irradiated
CD40L-tumor cells. These results indicate that cocultur-
ing immature DCs with irradiated CD40L-tumor cells
drives the activation of DCs and the production of several
pro-Th1 and proinflammatory cytokines. This cytokine
and chemokine profile should foster the generation of an
effective antitumor CTL response.
DCs cultured with irradiated CD40L-tumor cells induce
a much stronger primary allogeneic MLR
We then evaluated the T-cell stimulatory function of these
DCs. DCs cultured with irradiated CD40L-tumor cells
clearly stimulated a much stronger allogeneic MLR than
those cultured with frozen/thawed CD40L-tumor cells or
vector-tumor cells (Fig 5a and c). This result further
demonstrated that irradiated CD40L-tumor cells were the
most effective stimulus of DC maturation in our
experimental setting. Nevertheless, DCs cultured for 16
or 40 hours with frozen/thawed or irradiated tumor cells,
with or without CD40L expression, stimulated allogeneic
MLR equally well in the presence of LPS (Fig 5b and d).
These results suggest that frozen/thawed or irradiated
vector-tumor cells and frozen/thawed CD40L-tumor cells
failed to provide necessary maturation stimuli for
immature DCs, but they did not interfere with the ability
of immature DCs to undergo maturation in the presence
of a valid DC maturation signal such as LPS.
DCs cultured with irradiated CD40L-tumor cells
preferentially promote the growth of CD8
þ
T cells
To further investigate the influence of tumor material-
pulsed DCs on the growth of T cells, DCs were cultured
with tumor cells after different treatments as described
above. After 40 hours, DCs were collected, washed and
cultured with allogeneic T cells. T cells were harvested and
examined on day 12, whereas the culture supernatant was
Figure 4 Immature DCs were cocultured with equal numbers of
frozen/thawed vector-tumor (vector/F), CD40L-tumor (CD40L/F),
irradiated vector-tumor (vector/R), or CD40L-tumor (CD40L/R) cells
for 40 hours. The culture supernatants from each culture were
collected and assayed for the production of IL-1b, IL-6, IL-8, IL-10,
IL-12p70, and TNF-a with a CBA kit. The production of IFN-g was
detected by an ELISA. The detection limits (pg/ml) are: IFN-g, 3.6;
TNF-a, 3.7; IL-1b, 7.2; IL-6, 2.5; IL-8, 3.6; IL-10, 3.3, and IL-12p70,
1.9. The symbol (*) indicates data exceeding the upper range of the
standard (5000 pg/ml). One representative result from three separate
experiments is shown.
Antigen loading and concurrent activation of DC
K-J Liu et al
140
Cancer Gene Therapy
collected on day 6 and day 12 for cytokine analysis. The
highest number of T cells was recovered from the culture
stimulated by DCs pulsed with irradiated CD40L-tumor
cells, and more than 40% (37/92) of all CD3
þ
T cells in
the culture were CD8
þ
(Fig 6a). The majority (478%,
29/37) of these CD8
þ
T cells expressed IFN-g after
stimulation with PMA and ionomycin, indicating that
they are in a rather activated state. The percentage of
CD8
þ
T cells of all CD3
þ
T cells in the other three
groups was lower than 13% (8/96, 5/94, and 12/96). These
results strongly suggest that DCs cultured with irradiated
CD40L-tumor cells can efficiently promote the growth of
CD8
þ
, IFN-g producing T cells. Similar results were also
observed in the experiments using CD45RO-depleted
autologous or allogeneic T cells (data not shown). Some
cells expressed lower levels of CD3 and CD8. These cells
were probably the CD56
þ
cells and thus were not gated
for analysis. Examination of the cytokine environment
during T-cell stimulation indicated that the culture
supernatant of T cells plus DCs pulsed with irradiated
CD40L-tumor cells contained the highest level of IFN-g
and TNF-a, and the lowest level of IL-5 (Fig 6b). This
cytokine profile should promote the development of Th1
and CTL responses. In contrast, the cytokine environ-
ment of the other three groups is more likely to favor a
Th2 response (Fig 6b).
DCs cultured with irradiated CD40L-tumor cells
generate more Ag-specific T cells in vitro
It has been reported that the proto-oncogene HER2 was
overexpressed in many cancers including the non-small-
cell lung cancer.
32
The parental and transfected human
lung cancer cell lines used in this study also expressed
similar levels of HER2 on the surface (data not shown).
To study the cross-presentation and T-cell stimulation
function of tumor material-pulsed DCs, we have exam-
ined the ability of DCs to process tumor materials and
Figure 5 Immature DCs were cultured with equal numbers of frozen/
thawed (open symbols) or irradiated (closed symbols), vector-tumor
(triangles), or CD40L-tumor (circles) cells for 16 hours (a, b) or 40
hours (c, d), with (b, d) or without (a, c)1mg/ml LPS. Cells were then
collected, washed and irradiated (3000 rad). Two-fold serial diluted
cells were cultured with allogeneic PBMCs (10
5
cells/well) in 96-well
plates for 6 days. The proliferation of allogeneic PBMCs was
determined in a thymidine incorporation assay.
Figure 6 DCs were cocultured with equal numbers of frozen/thawed
vector-tumor (vector/F), CD40L-tumor (CD40L/F), irradiated vector-
tumor (vector/R), or CD40L-tumor (CD40L/R) cells for 40 hours.
After washing, DCs were cultured with allogeneic nonadherent
PBMCs for 6 days. Proliferating T cells were expanded by adding IL-
2 and cultured for another 6 days. Cells were then collected, counted
and stained with an FITC-anti-CD8 mAb and a PE-anti-CD3 mAb.
The number of cells obtained from each culture is: vector/F, 2 10
6
vector/R, 2.2 10
6
; CD40L/F, 2.6 10
6
, and CD40L/R, 3.5 10
6
.
For intracellular cytokine staining, proliferating T cells were collected
and stimulated with PMA and ionomycin in the presence of GolgiStop
for 6 hours. Cells were stained with an FITC-anti-CD8 mAb. After
washing, cells were fixed, permeabilized, and then stained with a PE-
anti-IFN-g mAb. Expression of intracellular IFN-g by CD8
þ
T cell was
analyzed with a flow cytometer (a). Supernatants from day 6 and day
12 cultures were collected and analyzed for cytokine secretion (b).
Antigen loading and concurrent activation of DC
K-J Liu et al
141
Cancer Gene Therapy
generate HER2-specific T cells in vitro. DCs were
obtained from normal donors with the HLA-A*0201
genotype and cultured with tumor cells after different
treatments for 16 hours. These DCs were washed,
irradiated, and then cultured with autologous (HLA-
A*0201) CD8-enriched T cells in the presence of human
AB-type serum. After 10 days, T cells were restimulated
with irradiated autologous PBMCs pulsed with an HLA-
A*0201-restricted HER2/369-377 peptide to enrich for
HER2-specific T cells. Surviving T cells on day 17 were
collected and stimulated with T2 cells pulsed with the
HER2/369-377 peptide or a control HPV E6 peptide. We
have found that only T cells initially stimulated with DCs
cultured with either irradiated vector-tumor or CD40L-
tumor cells were able to secrete a significant amount of
IFN-g in response to T2 cells pulsed with the HER2/369-
377 peptide (Fig 7a). These results indicate that DCs
cultured with irradiated tumor cells can process inter-
nalized tumor materials and present the HER2/369-377
epitope to autologous T cells. T cells initially stimulated
with DCs pulsed with frozen/thawed tumor cells secreted
little IFN-g. Therefore, it is not likely that the second
stimulation with peptide-pulsed autologous PBMCs was
priming, instead of boosting, the HER2-specific response.
The cell numbers from all four groups decreased in the
first 10 days of stimulation probably due to the death of
nonspecific autologous T cells. However, after two in vitro
stimulations, a much higher number (44-fold) of T cells,
initially primed by DCs pulsed with irradiated CD40L-
tumor cells, were recovered (Fig 7b). In addition, after
four in vitro stimulations, significant cytotoxicity of these
CD8
þ
T cells against T2 cells pulsed with the HER2/369-
377 peptide could be detected (Fig 7c). In contrast, the
number of T cells from the other three groups declined
rapidly during the first two in vitro stimulations and these
T cells died out during the subsequent stimulations. These
results are consistent with our previous observations and
indicate that DCs pulsed with irradiated CD40L-tumor
cells are superior in supporting the growth of specific
CD8
þ
T cells during in vitro culture.
Animals vaccinated with DCs pulsed with irradiated
CD40L-tumor cells were more resistant to subsequent
challenge of a weakly immunogenic tumor
Our in vitro results with human cells suggested that DCs
pulsed with irradiated CD40L-tumor cells can serve as a
potent vaccine to generate tumor-specific T-cell responses.
An animal model was then established to further compare
the efficacy of tumor cell-based vaccine with cancer
vaccine consisting of DCs pulsed with irradiated CD40L-
tumor cells. Similar to results described above, upregula-
tion of CD86 and secretion of IFN-g and IL-12 were
Figure 7 Immature DCs were generated from an HLA-A*0201 donor
and cultured with equal numbers of frozen/thawed vector-tumor
(vector/F), CD40L-tumor (CD40L/F), irradiated vector-tumor (vector/
R), or CD40L-tumor (CD40L/R) cells for 16 hours. Tumor material-
pulsed DCs were collected and incubated with autologous CD8-
enriched T cells. On day 10, T cells were restimulated with
autologous PBMC pulsed with 50 mg/ml of an HLA-A*0201-restricted
HER2/369-377 peptide. On day 17, cells were collected and
counted. T cells were then incubated with irradiated T2 cells pulsed
with 50 mg/ml of the HER2/369-377 peptide (closed bars) or an HLA-
A*0201-restricted, HPV-16 E6 peptide (open bars). (a) The secretion
of IFN-g in the culture supernatant by T cells on day 19 was
determined. (b) T-cell numbers were determined at the beginning
(day 0) and the end of the two stimulation cycles (days 10 and 17).
(c) T2 cells were labeled with a fluorescence enhancing ligand and
then pulsed at 371C for 2 hours without peptides, with 50 mg/ml of the
HER2 peptide, or two control CEA and MET peptides. Different
numbers of effector T cells were incubated with labeled T2 cells (10
4
cells/well) for 4 hours at 371C. The counts of culture with unlabeled
T2 cells were subtracted from those of cultures with peptide-pulsed
T2 cells before calculating the specific release. One representative
result from two separate experiments is shown.
Antigen loading and concurrent activation of DC
K-J Liu et al
142
Cancer Gene Therapy
observed after culturing bone marrow-derived DCs with
irradiated B16-F1 or CT-26 tumor cells transfected with
murine CD40L (data not shown). Mice immunized with
PBS or DCs alone developed tumors quickly after
challenging with the wild-type tumor cells (Fig 8a,b,e,f).
For the more immunogenic CT-26 tumor, vaccination of
either irradiated CD40L-tumor or DCs pulsed with
irradiated CD40L-tumor both provided protection in
the majority of the mice (Fig 8g and h). However, for the
weakly immunogenic B16-F1 tumor, immunization with
DCs pulsed with irradiated CD40L-B16-F1 tumor cells
(Fig 8d) was better than vaccination with irradiated
CD40L-B16-F1 tumor cells alone (Fig 8c) in inhibiting or
delaying the growth of tumor. Vaccination with DCs
pulsed with irradiated vector-tumor cells failed to provide
such additional protection in both tumor models (data
not shown). These results suggested that for certain
weakly immunogenic tumors, the response to tumor
vaccine consisting of DCs pulsed with irradiated
CD40L-tumor cells might be more protective than that
to tumor vaccine consisting of just the irradiated CD40L-
tumor cells alone.
Discussion
DC-based clinical trials for advanced cancers with partial
effectiveness have been reported.
6,17,33,34
One aspect to
improve current DC-based vaccines could be the source
and format of tumor Ag. Both necrotic and apoptotic
tumor cells have been explored as the source of Ag.
However, the consequences of Ag presentation by DCs
after internalization of materials from necrotic or
apoptotic cells may be different. It has been proposed
that immature DCs that have taken up Ag from apoptotic
cells during normal tissue turnover may induce anergy in
T cells or generate regulatory T cells as a mechanism of
peripheral tolerance.
35
Nevertheless, some studies demon-
strated that DCs can acquire Ag from apoptotic cells and
induce Ag-specific T-cell responses.
12,14,15
Both apoptotic
cells and lysate or supernatant of necrotic cells have been
shown to induce DC maturation in some reports.
36,37
DCs
exposed to apoptotic cells mediated by virus infection or
in the presence of inflammatory stimuli may undergo
maturation.
38,39
Rovere et al have demonstrated that a
higher number of apoptotic cells may trigger DC
maturation and increase their ability to crossactivate T
cells.
13,40
It has been shown that the maturation of DCs is
better stimulated by necrotic tumor cells than by
apoptotic tumor cells or primary tissue cells, probably
due to the secretion of proinflammatory mediators or
heat-shock proteins during necrosis.
36,41
However, in
other reports, immature DCs were not matured after
coculturing with tumor cell lysate or supernatant of
necrotic cells alone.
42–44
To reconcile these contradictory
results, Salio et al
43
and Somersan et al
37
have suggested
Figure 8 C57BL/6 (a-d) or BALB/c (e-h) mice (n ¼ 5) were immunized weekly with PBS (a, e), DCs alone (b, f), irradiated CD40L-tumor alone
(c, g), or DCs pulsed with irradiated CD40L-tumor cells (d, h). At 2 weeks after the third immunization, mice were challenged with wild-type B16-
F1 (a-d) or CT-26 (e-h) tumor cells, respectively. The growth of tumor was monitored every 2–3 days. Mice were killed when the tumor volume
exceeded 2 cm
3
. Several mice (#) died when the tumors were still developing. The numbers in the parentheses indicate the ratio of tumor-free
animals. Results from two separate experiments are shown.
Antigen loading and concurrent activation of DC
K-J Liu et al
143
Cancer Gene Therapy
that DCs are triggered to maturation not simply by the
death of surrounding cells, but also by other coexisting
stimulatory signals. These factors include inflammatory
cytokines,
42
heat-shock proteins released by dead cells,
37
and microbial materials (e.g., mycoplasma) contained in
the cell lysate.
43
Feng et al
45
also demonstrated that only
stressed apoptotic cells (but not nonstressed apoptotic
cells) could efficiently stimulate DC maturation, possibly
through upregulation of surface heat-shock proteins. Our
results support the notion that exposure of immature DCs
to necrotic or apoptotic cells may not be sufficient to
induce DC activation. Additional stimulations are re-
quired to fully mature DCs. Since our tumor cell culture
was free from mycoplasma contamination, it is likely that
little (if any) heat-shock protein was released from the
tumor cells under the conditions of our experiment, and
hence DCs were not induced to maturation after
coculturing with necrotic or unmodified apoptotic tumor
cells. In our case, the concurrent signaling by functional
membrane-CD40L is required and sufficient for optimal
stimulation of DCs to secrete IL-12 and other proin-
flammatory cytokines, and to promote the growth of
CD8
þ
T cells. This is further supported by a recent study
indicating that both crosstolerance and crosspriming
require the presence of mature DCs and the determining
factor is whether DCs receive an additional activation
signal from CD4
þ
T cells, most likely through CD40–
CD40L interaction.
46
A combination of DC-based cancer vaccines with
costimulatory molecules, cytokines, and chemokines
could provide potential activation signals and allow for
a greater control of DC functions particularly in the
microenvironment of vaccine injection site. Soluble forms
of these immune-regulatory mediators are convenient to
handle, but may have limitations due to a short half-life if
delivered locally, or undesired toxicities if administered
systemically. One strategy of enhancing the efficacy of
DC-based vaccine is to use gene-modified tumor cells as a
provider of both tumor Ag and activation stimulus for
DCs in vitro. In our study, coculturing immature DCs
with frozen/thawed or irradiated vector-tumor cells
resulted in uptake of tumor materials by DCs, but failed
to induce DC maturation and activation. In contrast,
irradiated, but not frozen/thawed, CD40L-tumor cells can
efficiently induce the maturation and activation of DCs in
the absence of additional cytokines. DCs cultured with
irradiated CD40L-tumor cells secreted not only IL-12 but
also a substantial amount of IL-6, IL-8, IFN-g, and TNF-
a. Thus, vaccination with such tumor material-loaded,
activated DCs might create an inflammatory microenvir-
onment at the injection site, and effectively promote the
generation of Th1 and CTL responses. Indeed, more IFN-
g-producing, CD8
þ
T cells were obtained after stimulat-
ing allogeneic or autologous T cells with DCs precultured
with irradiated CD40L-tumors. Although soluble CD40L
may provide a similar stimulus, results reported pre-
viously
47
and from our own experiments (data not shown)
indicate that its stimulatory effect might be lower than
that of membrane-bound CD40L. We have also observed
that DCs cultured with either irradiated vector- or
CD40L-tumor cells can both process internalized tumor
materials and generate HER2/369-377-specific T cells in
vitro. However, T cells survived for a longer period in vitro
if they are initially primed by DCs cultured with
irradiated CD40L-tumor cells. Since DCs stimulated with
irradiated vector-tumor cells secrete very little IL-12, it is
possible that a large amount of IL-12 may be more
important in the maintenance (in contrast to the priming)
of peptide-specific CD8
þ
T cells. Nevertheless, other
CD40L-mediated signals may also increase the ability of
DCs to support the growth of CD8
þ
T cells. DCs
cocultured with irradiated vector-tumor cells did not
mature efficiently, but could still induce HER2/369-377-
specific T cells (Fig 7a). One possible explanation is that
these immature DCs also secreted a substantial amount of
IL-6 (Fig 4). Several investigators have reported that IL-6
plays an important role in the induction of CTL in
vitro.
48–51
A combination of IL-6 and IL-12 is likely to
further enhance the survival of CTLs,
51
as occurs in
cultures containing DCs pulsed with irradiated CD40L-
tumor cells. Our results are in agreement with these
previous findings.
We have observed that a low but significant amount of
IL-10 was present in the culture containing DCs and
irradiated CD40L-tumor cells. IL-10 has been shown to
suppress immature DC development.
52–54
However, the
presence of IL-10 in our cultures did not seem to interfere
with the induction of CD83 expression on DCs, and the
ability of DCs to mediate a strong allogeneic MLR. It has
been shown that mature DCs are more resistant to the
inhibitory effect of IL-10.
52,55
Therefore, it is likely that
DCs cocultured with irradiated CD40L-tumor cells have
undergone maturation and activation before the IL-10
can act. Signaling through CD40L and the presence of
TNF-a may counteract the inhibitory effect of IL-10 as
well.
56
Alternatively, IL-10 in this setting may be
stimulatory, since IL-10 was shown to promote the
maintenance of tumor-specific CD8
þ
T-cell effector
function.
57
This is in agreement with our finding that
CD8
þ
T cells initially stimulated with DCs cultured with
irradiated CD40L-tumor cells survived longer during the
in vitro stimulation procedure. The failure of frozen/
thawed CD40L-tumor cells to induce DC maturation may
be due to the denaturation of surface CD40L during the
freezing/thawing treatment. Although most tumor cells
remain intact after only one cycle of freezing/thawing, it is
also possible that frozen/thawed CD40L-tumor cells are
internalized or break down early in the culture before
sustained contact with DCs needed for efficient induction
of maturation is achieved.
This study was initiated mainly to evaluate the
potential use of DCs pulsed with CD40L-tumor cells as
a cancer vaccine in future clinical studies. Therefore,
human tumor cells and monocyte-derived DCs were used
in our in vitro system. We have demonstrated that pulsing
DCs ex vivo with irradiated CD40L-tumor cells can result
in simultaneous internalization of tumor materials and
activation of DCs without adding exogenous cytokines.
Pulsing with irradiated CD40L-tumor cells proved to be a
better strategy for increasing the capacity of DCs to
Antigen loading and concurrent activation of DC
K-J Liu et al
144
Cancer Gene Therapy
stimulate CD8
þ
T cells than pulsing with unmodified
tumor cells. Also, the in vitro irradiated CD40L-tumor
cell-stimulated production of various pro-Th1 cytokines
and proinflammatory chemokines should increase the
efficacy of vaccination in vivo. It is likely that the secretion
of IL-6, IL-8, IFN-g, and TNF-a at the vaccination site by
these activated DCs might attract and/or activate cells of
the innate immune system such as NK cells and
eosinophils and lead to an amplification of the immune
responses. Thus, pulsing DCs with irradiated CD40L-
tumor cells in vitro may allow Ag loading and activation
of DCs to be better controlled, and vaccination with DCs
pulsed with irradiated CD40L-tumor cells may have a
greater therapeutic effect than vaccination with irradiated
CD40L-tumor cells alone. Our preliminary experiments
demonstrated that animals immunized with DCs pulsed
with irradiated CD40L-tumor cells were indeed better
protected against a weakly immunogenic tumor cell line.
It is possible that tumor cell-based vaccines and DC-based
vaccines may generate different immune responses or
involve different effector cells. We are currently conduct-
ing animal experiments to clarify the underlying mechan-
isms of these two different immunization strategies. Since
most human cancer cells are likely to be of low
immunogenicity, our results suggest that DCs pulsed with
irradiated CD40L-tumor cells could be useful clinically
and may provide the basis of cancer vaccines that could
be tested in future clinical studies. Since sufficient tumor
cells may not always be available and freshly isolated
tumor cells are difficult to transfect, such an approach
(using autologous gene-modified tumor cells) may be
inappropriate for the treatment of some cancer patients.
However, in several recent clinical studies of cancer
vaccine, unmodified or gene-modified allogeneic tumor
cells were chosen as the source of tumor Ag.
18,58
The basis
for this choice was the assumption and observation that
tumor cells from different patients express some of the
same TAA (e.g., CEA, MUC-1, and HER2/neu), and that
DCs are superior in crosspresentation.
59,60
Therefore, it is
possible that well-characterized, allogeneic tumor cell
lines transfected with CD40 ligand could be used as the
source of tumor Ag to pulse and stimulate DCs in a future
clinical application of our proposed vaccination strategy.
Acknowledgments
We would like to thank a former collaborator, Dr Kun-
Tai Lu, at the National Taiwan University Hospital, for
providing malignant pleural effusions and a former senior
technician, Mrs Den-Mei Yang, for her technical assis-
tance in establishing the human lung cancer cell line.
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