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or Cytidine-Phosphate-Guanosine DNA Drives T Cell Activation In Vitro and Therapeutic Anti-Tumor Immune Responses In Vivo1
Divisions of
*
Clinical Pharmacology and
Neuroendocrinology,
Departments of Medicine and Radiation Therapy, Ludwig-Maximilians-University of Munich, Munich, Germany;
Institute of Pathology, University of Mainz, Mainz, Germany;
¶ Department of Internal Medicine, University of Iowa, Iowa City, IA 52242;
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Coley Pharmaceutical Group, Wellesley, MA 02481; and
#
Veteran Affairs Medical Center, Iowa City, IA 52246
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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or a
cytidine-phosphate-guanosine (CpG)-containing oligonucleotide
(ODN-1826), which mimics the immunostimulatory effect of bacterial DNA.
Flow cytometric analysis of costimulatory molecules and MHC class II
showed that DC maturation was stimulated most by ODN-1826, whereas
TNF- had an intermediate effect. The extent of maturation correlated
with the secretion of IL-12 and the induction of alloreactive T cell
proliferation. In BALB/c mice, s.c. injection of colon carcinoma cells
resulted in rapidly growing tumors. In this model, CpG-ODN-stimulated
DC cocultured with irradiated tumor cells also induced prophylactic
protection most effectively and were therapeutically effective when
administered 3 days after tumor challenge. Thus, CpG-ODN-enhanced DC
maturation may represent an efficient means to improve clinical tumor
vaccination. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,
IL-1, or CD40 ligand expressed by activated T cells leads to DC
maturation (5, 6) and IL-12 secretion (7, 8, 9).
IL-12 plays a central role in the tumor-directed immune response,
stimulating NK, cytotoxic CD8+ T, and
IFN-
-producing CD4+ Th1 cells. Through its
stimulatory activity on undifferentiated CD4+
precursors to develop into Th1 cells, IL-12 antagonizes the action of
IL-4, which promotes Th2 development (10, 11, 12). Synthetic oligodeoxynucleotides containing unmethylated cytidine-phosphate-guanosine (CpG) dinucleotides (CpG-ODN) in specific sequence contexts mimic the immunostimulatory qualities of bacterial DNA (13). In vitro, they up-regulate the expression of costimulatory and Ag-presenting molecules and the secretion of IL-12 by monocytes and DC (7, 9, 14, 15, 16). In vivo, CpG-ODN act as an adjuvant, promoting Th1 immune responses (17, 18) that can enhance protection from a subsequent tumor challenge when coadministered with tumor Ag (19).
In murine tumor models, DC act as potent inducers of tumor immunity. Tumor development was induced by injection of established tumor cell lines of various tissue origins. Following interaction with tumor cells (20, 21) or selected tumor Ags (22, 23, 24, 25), DC are effective as prophylactic tumor vaccines against subsequent tumor challenges. In a few cases, even therapeutic efficacy of DC-based tumor vaccines was reported, leading to rejection of established tumors and lung metastases. These vaccines were based on fusion (20) or coculture (21) of DC and tumor cells, on tumor peptide- (26) or tumor lysate-pulsed DC (27). In the two studies reporting complete remission of established tumors, DC were generated in the presence of GM-CSF (20) or of GM-CSF plus IL-4 (21) without additional DC-activating agents. Both research teams worked with the C57BL/6 mouse strain, which is prone to raise proinflammatory, Th1-mediated immune responses (28) and injected low to moderate aggressively growing tumor cells. However, in man, aggressive tumor growth is often met by a severely compromised immune response. First clinical trials applying DC (29, 30, 31) have demonstrated therapeutic benefit (partial or complete regressions) that was, however, limited to a small proportion of patients. As inflammatory stimuli induce maturation of DC (5, 6), characterized by increased expression of MHC and costimulatory molecules, we reasoned that they may improve the therapeutic potential of DC-based vaccines.
Toward this goal, we studied potential improvements and limits of DC tumor vaccines. We tested whether tumor vaccination was effective against aggressively growing, syngeneic colon carcinoma cells in BALB/c mice, which have an impaired Th1 response (32). We analyzed how enhanced maturation of DC compared with basic stimulation by GM-CSF and IL-4 alone influences their potential to induce tumor immunity, both prophylactically and therapeutically. In addition, systemic administration of CpG-ODN as an adjuvant with or without tumor cells was investigated. The concomitant in vitro characterization of differentially stimulated DC allowed correlation of the surface expression of DC marker proteins, the IL-12 synthesis and the T cell activation potential with the anti-tumor efficacy observed in the mouse model.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female BALB/c mice, 6–8 wk old, were purchased from Harlan Winkelmann (Borchen, Germany). Animal studies were approved by the local regulatory agency (Regierung von Oberbayern, Munich, Germany). The murine, BALB/c-derived Colon-26 carcinoma line as well as the murine Renca cells (Cell Lines Service, Heidelberg, Germany) were maintained in DMEM supplemented with 10% FCS, 1% L-glutamine, and antibiotics.
Preparation of DC
DC were prepared as described (33) with minor
modifications. Bone marrow leukocytes from mouse tibia and femur were
depleted of T and B lymphocytes and granulocytes by incubation (30 min,
4°C) with rat anti-mouse CD4, CD8, Ly6G, and CD45R mAbs (Serotec,
Oxford, U.K.) followed by incubation (15 min, 4°C) with goat
anti-rat IgG conjugated to magnetic beads (Dynal, Oslo, Sweden) and
separation in a magnetic field. Unbound cells were cultured in DC
medium consisting of RPMI 1640 medium containing 10% FCS, 1%
L-glutamine, and antibiotics supplemented with rGM-CSF (200
U/ml; Roche, Mannheim, Germany) and IL-4 (PeproTech, London, U.K.).
After 7 days, loosely adherent cells were harvested, and expression of
MHC class II (MHC II), CD40, CD54, CD80, and CD86 was quantified by
flow cytometry. In some experiments at day 7 or 9, DC were additionally
stimulated with 5–80 ng/ml murine TNF- (R&D Systems, Minneapolis,
MN) or with 6 µg/ml phosphorothioate-modified oligonucleotide
ODN-1826 described by Yi (34) 5'-TCC ATG ACG TTC CTG ACG
TT-3' (provided by Coley Pharmaceutical Group, Wellesley, MA). To
assess the specificity of ODN-1826, stimulation of DC with 6 µg/ml
phosphorothioate-modified oligonucleotide ODN-1982 5'-TCC AGG ACT TCT
CTC AGG TT-3' served as control.
Flow cytometry
Cells were incubated for 30 min at 4°C with 5 µg/2 x 105 cells rat anti-mouse MHC II, CD40, CD54, CD80, and CD86 mAbs (PharMingen, San Diego, CA and Serotec, Oxford, U.K.). After washing with PBS containing 2% horse serum, FITC-conjugated mouse anti-rat IgG (Dianova, Hamburg, Germany) was added for 20 min at 4°C. After washing in PBS, samples were analyzed using an EPICS Profile II flow cytometer (Coulter, Miami, FL).
Quantification of IL-12
The concentration of IL-12p40-subunit in culture supernatants was determined by ELISA according to the manufacturer’s protocol (R&D Systems).
Allogeneic T cell proliferation
T cells were isolated by passing spleen from C57BL/6 mice through a 70-µm cell strainer (Falcon, Heidelberg, Germany) followed by lysis of erythrocytes (Ortho Diagnostic Systems, Neckargemünd, Germany) and magnetic bead conjugate-mediated depletion of B lymphocytes and granulocytes with rat anti-mouse CD45R and Ly6G mAbs (Serotec). T cells were 60–80% pure as determined by flow cytometry.
Bone marrow-derived day 7 DC were exposed to ionizing radiation (50 Gy). Varying numbers of irradiated DC (600, 3,000, or 15,000 cells) were cocultured with 1.5 x 105 allogenic T cells in 96-well culture plates for 48 h. [3H]thymidine (1 µCi; Amersham Buchler, Braunschweig, Germany) per well was added for 24 h, cells were harvested, and [3H]thymidine incorporation was counted in a Betaplate scintillation counter (Wallac, Turku, Finland).
DC tumor cell coculture and immunization
DC (5 x 106) were cocultured with
1 x 106 irradiated tumor cells (100 Gy) for
4 days in culture medium supplemented with GM-CSF (200 U/ml) and IL-4
(20 ng/ml). For additional DC stimulation, CpG 1826 (6 µg/ml) or
TNF- (20 ng/ml) was added for the last 2 days. At day 11, loosely
adherent cells were harvested, washed 3 times, and resuspended in HBSS
(Life Technologies, Karlsruhe, Germany). For prophylactic immunization,
1 x 106 cells were injected s.c. into the
right lower flank (200 µl). Seven days later, mice were challenged by
ipsilateral injection of Colon-26 cells (5 x
105 in 200 µl HBSS). To assess tumor
specificity, mice were challenged by injection of Renca cells (5
x 105 in 200 µl HBSS) 7 days after
immunization with 1 x 106 DC cocultured
with irradiated Colon-26 cells. For therapeutic immunization, 1 x
106 coculture cells were injected at the
indicated days after tumor challenge (1 x
105 or 5 x 105
Colon-26 cells in 200 µl HBSS). For vaccination with oligonucleotide
as adjuvant, 50 µg CpG-ODN 1826 was injected s.c. alone or in
combination with 5 x 105 irradiated tumor
cells (100 Gy) in 200 µl HBSS. Seven days later, mice were
challenged ipsilaterally with 5 x 105 tumor
cells. According to the guidelines for the proper use of laboratory
animals, mice were killed when severely suffering (e.g.,
shivering and showing decreased mobility). The decision was made
by an investigator blinded toward the treatment allocation.
Statistics
Student’s t test was applied to reveal significant differences in tumor protection by differently stimulated DC. We compared the mean tumor size of controls and all treated animals (tumor-free and tumor-positive animals). In addition, we compared the mean of controls with the mean of only tumor-positive animals (tumor-free animals in treatment group excluded). A value of p < 0.05 was accepted as the level of significance.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The pronounced expression of MHC and costimulatory molecules
contributes to the ability of DC to potently activate T cells. As
surface expression of these proteins increases during differentiation,
induction of maturation may enhance the immunostimulatory capacity of
DC. Fig. 1
depicts the effect of
different culture conditions on the maturation of DC. Basal stimulation
with GM-CSF and IL-4 alone (Fig. 1, upper panel) generates
DC that express moderate levels of CD80 (mean fluorescence intensity
(MFI) 2.8), CD86 (MFI 10.1), and MHC II (MFI 3.9). The cells also
expressed CD40 and CD54 and were identified morphologically as DC by
phase contrast microscopy of May-Grünwald-stained cytospins and
by raster electron microscopy (data not shown). TNF- enhanced the
surface density of CD80 (MFI 8.2), CD86 (MFI 14.3), and MHC II (MFI
5.1, Fig. 1, middle panel). An even stronger increase of the
surface expression of costimulatory and Ag-presenting molecules was
induced by ODN-1826 (Fig. 1, lower panel). On average, CD80
was increased 4-fold (MFI 9.5), CD86 2-fold (MFI 24.1), and MHC II
6-fold (MFI 23.2) compared with DC stimulated with GM-CSF and IL-4
alone.
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Effect of IL-4, TNF-, and ODN-1826 on DC IL-12 synthesis
DC secrete IL-12 (7, 8, 9), a key inducer of
proinflammatory Th1 responses. We investigated to what extent
stimulation of DC influenced IL-12 secretion. As IL-4 has antagonistic
effects, promoting anti-inflammatory Th2 cytokine profiles, we
tested whether IL-4 (which is frequently added to DC cultures to
increase DC survival and differentiation) inhibits IL-12 synthesis by
DC. DC were cultured in the presence of GM-CSF and different
concentrations of IL-4 (0, 20, and 80 ng/ml) for 7 days. IL-4 had no
inhibitory effect on baseline IL-12 synthesis (Fig. 2a). However, IL-4 addition
increased DC yield. TNF- increased the IL-12 secretion induced by
GM-CSF and IL-4 alone up to 4-fold (Fig. 2b). ODN-1826
induced an extremely high IL-12 production, 19-fold higher than after
basic stimulation with GM-CSF and IL-4 alone. The extent of IL-12
secretion induced by TNF- and ODN-1826 correlated with enhancement
of surface expression of CD80, CD86, and MHC II (Fig. 1
).
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A major objective of tumor vaccination is enhancement of
tumor-specific T cell responses. To determine the effect of DC
maturation on T cell activation in vitro, we compared DC generated with
GM-CSF and IL-4 alone to TNF-- or ODN-1826-stimulated DC in their
capacity to induce allogeneic T cell proliferation (Fig. 3). Even at a relatively high DC number
(DC-T cell ratio, 1:10), DC grown in the presence of GM-CSF and IL-4
alone marginally activated T cells. T cell proliferation induced by
spleen cells was even lower (data not shown). In contrast, TNF-- or
ODN-1826-activated DC induced high T cell proliferation and there was a
1.3-fold increase between these latter preparations. However, at lower
DC numbers (DC-T cell ratios, 1:50 and 1:250) we observed marked
differences in the capacity of the respective DC to induce T cell
proliferation. At a DC-T cell ratio of 1:50, TNF--activated DC
induced a 2-fold (5 ng/ml TNF-), a 4-fold (20 ng/ml TNF-), and a
9-fold (80 ng/ml TNF-) higher T cell proliferation compared with
GM-CSF- and IL-4-stimulated DC, but were only marginally more effective
at low DC numbers (DC-T cell ratio, 1:250). In contrast,
ODN-1826-activated DC were potent T cell stimulators also at low
numbers, inducing a 15-fold (1:50) and 12-fold (1:250) higher T cell
proliferation than GM-CSF- and IL-4-stimulated DC. Thus, at low
numbers, only highly matured DC were able to activate T cells. The
effect of TNF-- or ODN-1826-induced DC activation on T cell
proliferation again correlated with the respective effects observed on
expression of DC marker proteins and on IL-12 secretion.
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DC maturation and physical contact to tumor cells in vitro improves protective tumor immunity in vivo
We evaluated whether enhanced maturation of DC and coculture with
tumor cells improves the induction of protective tumor immunity. DC
stimulated with GM-CSF and IL-4 for 7 days were cocultured with
irradiated tumor cells at a ratio of 5:1 for 4 days. For the last
48 h of coculture, DC were activated with either TNF- or
ODN-1826. Mice were vaccinated s.c. with 1 x
106 cocultured cells and, 7 days later, mice were
challenged with 5 x 105 viable tumor cells.
Three independent experiments (n = 45) were performed,
and one representative result with five mice per group is shown in Fig. 4. Tumor growth in unvaccinated control
mice was rapid, leading to death (by natural causes or by tumor
burden-based euthanasia) by day 18, demonstrating the aggressive tumor
formation by Colon-26 cells in BALB/c mice (Fig. 4, 
). As a mean of
all three experiments, 31% of mice immunized with DC that were
cocultured with tumor cells in the presence of GM-CSF and IL-4 were
completely protected against tumor formation (no development of tumors
until day 21 and for at least 4 wk beyond). In the remaining 69% of
mice, tumor growth was slightly delayed with survival up to day 21
(Fig. 4a, p < 0.001). Injection of 5
x 105 Colon-26 cells 7 days after vaccination
with ODN-1826-stimulated DC that had not been exposed to tumor cells
led to s.c. tumor formation in all mice (n = 10, data
not shown). Vaccination with TNF--stimulated DC cocultured with
tumor cells rendered half of the mice (50%) resistant to tumor
challenge while delaying tumor progression in the remaining 50% of
mice and prolonging their survival to day 23 (Fig. 4b,
p < 0.001). Immunization with ODN-1826-stimulated DC
cocultured with tumor cells completely protected 59% of mice and
delayed tumor growth in the remaining mice, increasing their survival
up to day 25 (Fig. 4c, p < 0.001). In a
preliminary study, we observed that the tumor vaccination potential can
be further increased by two prophylactic immunizations before tumor
challenge (data not shown).
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To evaluate the tumor specificity of DC vaccination we also
performed control experiments with a different BALB/c-derived tumor
line (Renca). Two independent experiments at n = 30
each were performed, and one representative with five mice per group is
shown in Fig. 5. Tumor growth after
injection of 5 x 105 Renca cells in
unvaccinated mice (control group) was also rapid, leading to death by
day 23 in all mice. Renca cells (5 x 105)
were injected s. c. 7 days after vaccination with ODN-1826-stimulated
DC cocultured with Colon-26 cells; all mice in the group
(n = 10) finally succumbed to tumors (Fig. 5a, p = 0.290). However, 6 of 10 mice
challenged with Renca cells after prophylactic vaccination with Renca
cells cocultured with DC did not show any tumor formation (three of
five mice for the experiment shown in Fig. 5b,
p < 0.001). Injection of 5 x
105 Colon-26 cells 7 days after vaccination with
DC cocultured with Renca cells led to lethal tumor development in all
mice (Fig. 5c, p = 0.250). In contrast, 6 of
10 mice challenged with Colon-26 cells after prophylactic vaccination
(Colon-26) remained tumor free (three of five mice for the experiment
shown in Fig. 5d, p < 0.001). In addition,
injection of 5 x 105 Renca cells 10 wk
after complete rejection of Colon-26 tumors (following prophylactic
vaccination with ODN-1826-stimulated DC cocultured with Colon-26 cells)
led to s.c. formation of tumors in all mice (n = 10,
data not shown). This indicates maintained specificity even after
immunization-based rejection has occurred.
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As ODN-1826-stimulated DC cocultured with tumor cells were the
most effective prophylactic tumor vaccine, we next evaluated their
therapeutic potential in three independent experiments
(n = 40). Tumors were induced by s.c. injection of 1 or
5 x 105 Colon-26 cells followed by
ipsilateral injection of 106 cocultured cells 3,
7, or 10 days later. One representative result with five mice per group
is shown in Fig. 6. In the untreated
control mice, no tumors were visible 3 days after tumor challenge but
became visible (0.3–0.5 cm diameter) at day 7 (see also Figs. 4 and 8a). These mice die or have to be euthanized around day 18.
As a mean of all experiments, vaccination 3 days after injection of
1 x 105 tumor cells completely prevented
tumor growth in 40% (two of five mice in Fig. 6a,
p < 0.05). In the remaining three mice, tumor size at
day 18 was reduced, concomitant with a prolonged survival up to day 29
(n = 2) and beyond 32 days (n = 1).
Vaccination 3 days after s.c. injection of 5 x
105 tumor cells (Fig. 6b,
p < 0.05) prevented tumor growth in one of five mice,
and reduced tumor growth in the remaining mice prolonging survival up
to day 23. Even when mice were vaccinated 10 days after injection of
1 x 105 tumor cells, reduced tumor growth
and prolonged survival was observed (Fig. 6c,
p < 0.05). Thus, the time window for inducing tumor
immunity was 10 days after tumor cell injection. For the high tumor
challenge (5 x 105 cells), no prevention of
tumor growth and only a trend toward prolonged survival up to day 20
(in three of five mice) following tumor vaccination 7 days after tumor
challenge was observed (Fig. 6d, p =
0.371).
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To study the persistence of tumor immunity, mice that had not
developed tumors after prophylactic or therapeutic vaccination were
rechallenged 6 wk after the first vaccination by s.c. injection of
5 x 105 tumor cells. As shown in the
representative experiment of Fig. 7
independent of the initial vaccination protocol used, no animal (of a
total of 20 animals rechallenged) developed a tumor. This suggests that
vaccination with DC cocultured with tumor cells mediates tumor
protection lasting for at least 6 wk. In addition, no tumors formed
even after repeated tumor challenges (data not shown).
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Our findings demonstrated that ODN-1826 enhanced the T
cell-activating capacity of DC in vitro. This prompted us to examine
whether in vivo the prophylactic vaccination using ODN-1826 (without
DC) could influence subsequent tumor development. ODN-1826 was injected
s.c. with or without irradiated tumor cells and, after 7 days, mice
were challenged s.c. with 5 x 105 vital
Colon-26 cells. Fig. 8 shows one
representative experiment of three (n = 30). Control
mice developed rapidly growing tumors with no mice surviving day 18
(Fig. 8a). Prophylactic injection of ODN-1826 without
irradiated tumor cells resulted in a delayed tumor growth but no
survival was observed beyond day 20 (Fig. 8b,
p = 0.173). ODN-1826 coinjected with irradiated tumor
cells protected a small proportion of mice (25% for all experiments)
and reduced tumor progression in most of the remaining animals,
prolonging survival up to day 25 (Fig. 8c, p
< 0.05). Injection of irradiated tumor cells alone resulted in no
protection (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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or with the CpG
motif-containing oligonucleotide ODN-1826 (34). We
demonstrate that the extent of DC maturation, as reflected by surface
density of costimulatory and MHC II molecules, correlated with the
secretion of IL-12, with the T cell-activating potential in vitro, and
with the induction of tumor immunity in vivo. ODN-1826 was identified
as a very potent DC activator, clearly superior to the well-established
DC stimulator TNF- (5, 27, 35).
For enrichment of DC from bone marrow, we modified the protocol
published by Inaba (33) by additionally supplementing the
culture medium with IL-4. In line with published data, this gave
consistently better yields in DC and suppressed the development of
monocytes (36). As IL-4 is the key inducer of Th2
responses and antagonizes proinflammatory IL-12, we ruled out that IL-4
inhibits IL-12 production by DC. DC secreted stable baseline amounts of
IL-12 independent of the IL-4 concentration used (Fig. 2). This is
concordant with the notion that the antagonism of IL-12 and IL-4 is not
due to a direct suppressive effect on each other’s synthesis
(37, 38, 39).
Analyzing the effect of differential DC stimulation on IL-12
production, we found that ODN-1826 was the most potent stimulator. Its
effect exceeded that of TNF- by a factor of 5, which by itself was
4-fold more potent than baseline stimulation. DC cultured in the
presence of GM-CSF and IL-4 alone expressed moderate levels of the
costimulatory molecules CD80 and CD86, of the DC activation marker
CD40, of the adhesion integrin CD54 (ICAM-1), and of the Ag-presenting
proteins MHC class I and MHC II. TNF- at its optimal concentration
up-regulated DC markers MHC II, CD86, and CD80 1.3- to 3-fold. Compared
with baseline stimulation, ODN-1826 induced an increase in mean
expression of CD80, CD86, and MHC II by a factor of 4, 2, and 6,
respectively. Examining the effect of DC maturation on in vitro T cell
proliferation, we found a maturation-dependent T cell-activating
potential of the respective DC preparations. Especially at low numbers,
ODN-1826-activated DC induced the highest T cell proliferation, whereas
TNF--stimulated DC again showed an intermediate effect. This finding
may be of particular relevance in vivo, where DC are scarce in
peripheral blood, tissue, and lymph nodes. Our data further imply that
DC have to be strongly stimulated to raise a productive T cell
response. We propose that this stimulation can be caused either by
strong signals like bacterial DNA or by combinations of weaker signals
such as TNF- plus CD40 ligation.
DC activating potential for ODN-1826 was described by Vogel and
coworkers who showed that this oligonucleotide was nearly as effective
as LPS in activating C57BL/6 fetal skin-derived DC to express MHC II,
costimulatory, and accessory molecules and to stimulate T cell
proliferation (9). In addition, it was about 10 times more
potent than LPS in stimulating IL-12 secretion. Vogel et al. did not
compare it to other stimuli such as TNF- or IL-4. Using the control
ODN-1982, we could not observe any stimulatory effects on the surface
expression of MHC II, CD86, and CD80, the IL-12 secretion or the T cell
activation of DC in vitro. This is completely in line with previous
reports of control ODN, in which the CpGs were methylated or, inverted,
lacked these stimulatory activities, confirming that the stimulatory
effects of ODN-1826 were dependent on its unmethylated CpG motifs
(Refs. 9, 15 ; and A. M. Krieg, unpublished
observations).
Of particular interest was the observation that the expression of DC
activation and maturation markers correlated with the IL-12 synthesis
and the capacity to stimulate T cell proliferation. For all parameters
tested, ODN-1826 was the most potent stimulus, TNF- had an
intermediate effect, and GM-CSF and IL-4 alone showed the weakest
effects. Therefore, a common intracellular pathway underlying DC
activation and regulating the cellular aspects required for T cell
activation seems to be triggered by different maturation-inducing
stimuli. Recently, it has been shown that Toll-like receptors are
evolutionarily conserved immune response-activating molecules and are
expressed by DC, monocytes, and various other mammalian cell types
(40). Toll-like receptor 2 was identified as the
signal-transducing receptor for LPS, triggering a signal cascade that
leads to activation of NF-
B (41) and c-Jun
NH2-kinase (42, 43). NF-B
activation is central to the process of DC maturation (44)
and NF-B is activated by CpG-ODN in lymphoma cells (34)
and macrophages (45, 46). It is not known whether
Toll-like receptors also participate in the activation of DC by
inflammatory signals other than LPS but it seems likely that the
efficacy by which NF-B is activated by the respective signal cascade
determines the degree of DC maturation.
A key finding of this work is that maturated DC after coculture with
tumor cells were able to confer tumor-specific protection even in the
BALB/c mouse strain, which is prone to raise a Th2 response
(32). Using the aggressively growing colon carcinoma line
Colon-26, which leads to euthanasia around day 18, allowed us to
investigate the limits of DC-based tumor vaccination and to assess
differences in the potency of the respective DC preparations.
Vaccination with ODN-1826-stimulated DC cocultured with tumor cells
prevented the development of tumors in 
60% of mice. Preliminary
findings of ongoing studies in our laboratory indicate that two
vaccinations with ODN-1826-stimulated DC are even more protective,
conferring tumor resistance to about 80% of mice. ODN-1826-stimulated
DC were also therapeutically effective in this model, preventing tumor
formation in up to 40% of mice (Fig. 5). Thus, even in a Th2-biased
mouse strain, a high level of protection against tumor challenge can be
induced. This extends a recent study comparing different CpG-containing
oligonucleotides in their ability to act as adjuvants in the induction
of a Th1 response. ODN-1826 was identified as the most potent one
(17).
In correlation to their maturation status, DC stimulated with
TNF- or GM-CSF plus IL-4 alone and cocultured with tumor cells
were less effective as prophylactic tumor vaccines and, also,
ODN-1826-stimulated DC did not induce complete tumor immunity. At first
glance, this seems to be at variance with studies published by the
groups of Kufe and Mayordomo, who reported complete protection even
though DC were stimulated only with GM-CSF (20) or with
GM-CSF and IL-4 (21). We propose that this discrepancy may
be due to the rather slow tumor growth induced by the cell lines used
in both studies (MC-38, B16, and 3LL led to death between 28 and >60
days) and/or due to the Th1 bias of C57BL/6 mice (28).
Irrespective of its reason, this discrepancy highlights the importance
of thoroughly investigating different models before generalized
conclusions on DC-based tumor vaccination for the application
in man can be drawn.
First clinical studies on DC-based tumor vaccination have been
performed in patients with B cell lymphoma (29), melanoma
(31), and prostate carcinoma (30). These
studies achieved some clinical benefit (partial or complete responses),
which was, however, limited to a rather small proportion of patients
(25%). The observations of this study suggest that one reason for
the limited success of the vaccination trials may reside in
insufficient means to induce DC maturation. Because even the strongest
stimulation of DC did not confer complete protection against
aggressively growing tumors, other strategies are required to
eventually maximize the clinical benefit of DC-based tumor
vaccination.
In a last set of in vivo experiments, we evaluated the injection of ODN-1826 as an adjuvant. When coadministered with inactivated tumor cells, it retarded the growth of subsequently injected colon carcinoma cells and in 25% of mice it led to complete protection from tumor growth. Other bacterial products are already approved or in clinical trials as adjuvants for tumor vaccines. The mycobacterial preparation bacillus Calmette-Guérin (which contains bacterial DNA with CpG motifs) has been approved for immunotherapy of urothelial cancer (47). Furthermore, it has been studied in patients who were tumor free following surgery for colon carcinoma. Bacillus Calmette-Guérin combined with autologous tumor cells reduced the recurrence rate by 44% in patients with stage II colon carcinoma resulting in a trend toward prolonged survival (48). For patients with stage III carcinoma, no clinical benefit was noted. Other adjuvants are currently in clinical trials, i.e., the saponin formulation QS21 and the LPS derivative monophosphonyl lipid A (MPL) (49, 50). Because IL-12 improves the efficacy of QS21 and MPL (51), and CpG-ODN induces high amounts of IL-12, we propose that a combination of QS21, MPL, and CpG-ODN may improve antitumor responses even further.
In summary, we have demonstrated that surface expression of DC markers,
IL-12 secretion, and T cell proliferation are coordinatedly enhanced
during DC maturation. ODN-1826 stimulated this process most potently,
whereas TNF- showed an intermediate effect compared with basal
stimulation with GM-CSF and IL-4 alone. In correlation with the in
vitro stimulation, we found that, likewise, the induction of tumor
immunity depends upon the extent of DC maturation. We conclude that in
vitro maturation of DC is an efficient strategy to improve tumor
vaccination protocols.
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Acknowledgments |
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Footnotes |
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2 C.B. and J.S. contributed equivalently to this work.
3 This work contains parts of the doctoral thesis of J.S. and that of A.S. at the Ludwig-Maximilians-University of Munich.
4 Address correspondence and reprint requests to Dr. Stefan Endres, Division of Clinical Pharmacology, Medizinische Klinik Innenstadt, Ziemssenstrasse 1, 80336 München, Germany.
5 Abbreviations used in this paper: DC, dendritic cell(s); CpG, cytidine-phosphate-guanosine; CpG-ODN, synthetic oligodeoxynucleotides containing unmethylated CpG dinucleotides; MFI, mean fluorescence intensity; MHC II, MHC class II; MPL, monophosphenyl lipid A.
Received for publication January 27, 2000. Accepted for publication September 6, 2000.
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) or additionally with
5 ng/ml TNF-
), 20 ng/ml TNF-
),
or with 6 µg/ml ODN-1826 (
) was rapid, leading to death at day 23. In addition, 5
x 105 Colon-26 cells were injected 7 days after
vaccination with ODN-1826-stimulated DC cocultured with Renca cells
(c, 
