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Experimental Therapeutics |
Department of Dermatology [L. J., J-F. A., J-H. S., C. H.] and Division of Immunology and Allergy [J-F. A., C. H.], University Hospital Geneva, 1211 Genève 14, Switzerland, and the Department of Dermatology, University Hospital Mainz, Mainz, Germany [H. J.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, and prostaglandin
E2, DCs containing apoptotic MEL-397 cell material matured
normally [cross-presenting DCs (cp-DCs)]. Autologous CD8+
CTL lines generated with cp-DCs produced tumor necrosis factor when
stimulated with HLA-A2-binding immunodominant peptides from
MelanA/MART1 and MAGE-3 (expressed by MEL-397 cells) but not tyrosinase
(absent in MEL-397). T2 target cells loaded with the respective
peptides were lysed by these cell lines, although to a lesser extent
than by CTL lines generated in the presence of mature DCs and peptides
from melanoma-associated antigens. In contrast, lines generated with
cp-DCs lysed HLA-A2+ MEL-526 melanoma cells or allogenic
HLA-A2+ cp-DCs efficiently, whereas the CTL generated with
DCs and peptides had little lytic activity. Mature DCs containing
apoptotic tumor cells may thus represent an alternative approach for
the therapy of malignant tumors. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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DCs3 play a pivotal role in the initiation of T cell-dependent immune responses (6) and can be obtained by culturing peripheral blood monocytes in the presence of GM-CSF and IL-4 (7 , 8) . Antigen loading can be performed by pulsing DCs with synthetic immunodominant peptides from identified antigens, as recently reported for prostate cancer (9) , carcinoembryonic antigen expressing tumors (10) , and cutaneous malignant melanoma (11) . Disadvantages of this approach include the uncertainty regarding the longevity of antigen expression (12) , the need to determine the patient’s HLA haplotype, the unavailability of peptides for all HLA haplotypes, and the lack of CD4 helper cell-related epitopes for most antigens. In addition, the CTLs resulting from such protocols have a good in vitro capacity for killing peptide-pulsed target cells but only a modest capacity for killing tumor cells (13) .
Other methods for antigen loading include whole tumor cell
preparations, i.e., tumor lysates. Exogenous antigen is not
presented solely by MHC class II antigens but also can gain access to
the antigen processing pathway for presentation by MHC class I
molecules (cross-presentation; for review see (14)
.
Recently, the capacity of DCs to take up apoptotic cell material using
either the vitronectin receptor vß3 (15)
or vß5
and CD36 (16)
was demonstrated. Albert et al.
(17)
showed efficient presentation of influenza antigen by
DCs that had phagocytosed infected monocytes that were apoptotic. They
showed influenza antigen presentation to T cells in the context of MHC
class I (cross-priming). Furthermore, it was also shown that MHC class
II restricted presentation of antigen from apoptotic cells by DCs is
efficient (18)
.
We performed this study to determine whether DCs may prime for tumor-specific CTL responses after uptake of apoptotic melanoma cells. To this end, we first studied the uptake of apoptotic melanoma cells by immature monocyte-derived DCs. DCs were then matured (cp-DCs) and cultured with autologous CD8+ T cells and IL-2 to generate T cell lines. In parallel, we generated T cell lines using DCs loaded with immunodominant peptides from melanoma-associated antigens. We compared the ability of the CTL lines generated by both methods to release TNF in response to the immunodominant TAA peptides and to kill target cells loaded with peptide. We further compared their ability to kill melanoma cells and allogenic cp-DCs. We demonstrate the generation of TAA-specific T cell lines by DCs loaded with apoptotic melanoma cells. Furthermore, CTL lines generated by this method are more potent in killing melanoma cells than CTL lines generated by peptide-loaded DCs.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Antibodies and Reagents.
The following mAbs were used. FITC-labeled murine CD80 (BB1), CD40
(5C3), HLA-DR (G46–6), and HLA-ABC (G46–2.6) mAbs were purchased from
PharMingen (Hamburg, Germany), and CD8 (DK25) mAb was purchased from
DAKO Diagnostika GmbH (Hamburg, Germany). PE-conjugated murine
CD86 (IT2.2) mAb was from PharMingen; CD83 (Hb15a) mAb was from
Immunotech (Marseilles, France); CD4 (MT310) and CD16 (DJ130c) mAbs
were from DAKO Diagnostika GmbH; and CD14 (MO-P9), CD19 (Leu-12), and
CD3 (SK7) mAbs were from Becton Dickinson (Heidelberg, Germany).
Purified control IgG1-PE was purchased from DAKO Diagnostika GmbH, and
IgG2b-PE, IgG1-FITC, and IgG2b-FITC mAbs were from PharMingen.
Flow Cytometric Analysis.
Cultured cells were washed; suspended at 3 x 105/ml in 50 µl of cold PBS, 0.1% sodium
azide, 10 mg/ml BSA, and 200 µg/ml mouse IgG (Sigma); and
incubated for 10 min on ice. Subsequent staining with labeled mAb or
appropriate isotypic controls was performed for 30 min. Cells were then
washed and resuspended in 300 µl of cold PBS, 1% human serum albumin
containing 10 µg/ml 7-AAD (Sigma). Stained cells were analyzed for
three-color immunofluorescence with a FACScalibur cell analyzer (Becton
Dickinson, Mountain View, CA). Cell debris was eliminated from the
analysis using a gate on forward and side scatter. A life gate was set
using 7-AAD. At least 104 cells were analyzed for
each sample. Results were processed using Cellquest software (Becton
Dickinson).
DC Generation from Buffy Coats.
Buffy coats of HLA-A2+ healthy donors were
obtained according to institutional guidelines. PBMCs were prepared by
density centrifugation using Ficoll-Paque (Amersham Pharmacia
Biotech, Uppsala, Sweden). PBMCs were resuspended (15 x 106 cells/well) in 6-well plates (Nunc,
Roskilde, Denmark) and incubated for 1 h at 37°C. Nonadherent
cells were removed and the remaining cells were fed with 3 ml of X-VIVO
15 medium (Bio-Whittaker, Walkersville, MD) containing 1% of
heat-inactivated autologous plasma, 103
IU of
GM-CSF/ml, and 103
IU of IL-4/ml
(Stratagen, Hannover, Germany). Cells were refed with 0.5 ml of
fresh medium containing 103
IU of GM-CSF and
103
IU of IL-4 per ml on days 2, 4, and 6. On day
7, the nonadherent cells were transferred to a new well with fresh
medium. DC maturation was induced with a cocktail of cytokines as
recently published (8)
. The following cytokines were
added: IL-4, 1000 units/ml; IL-1ß, 10 ng/ml; IL-6, 1000 units/ml (all
from Stratagene); GM-CSF, 1000 units/ml (Leukomax, Novartis, Basel,
Switzerland, kindly provided by Dr. P-Y. Dietrich, University of
Geneva); PGE2, 1 µg/ml (Prostin, Amersham
Pharmacia Biotech); and TNF-, 10 ng/ml (kindly provided by
Dr. J-M. Dayer, University of Geneva). Cells were harvested after 2
days and used for flow cytometric analysis and/or culture with T cells.
To have fresh autologous DCs at each time point of restimulation of
cultured CD8+ T cells, PBMCs were frozen in 10%
DMSO (Fluka, Buchs, Switzerland) and 90% human serum albumin
(Blutspendedienst SRK, Bern, Switzerland).
Induction of Apoptosis in MEL-397 Cells and Uptake of Apoptotic
Cell Material by DCs.
To induce apoptosis, MEL-397 cells were irradiated with UV-B (Philips
UV, Philips, the Netherlands). After irradiation, MEL-397 cells were
kept for 8 h in culture to allow apoptosis to occur. Apoptosis was
measured using an annexin-V kit (PharMingen) and 7-AAD staining
(22)
. The UV-B dose necessary to induce apoptosis in 70%
of the melanoma cells 8 h after irradiation was calculated to be 6
J/cm2. The uptake of apoptotic MEL-397 cell
material by immature DCs was measured as described recently
(16)
. Briefly, MEL-397 cells were labeled with PKH-26 dye
(Sigma-Aldrich, Steinheim, Germany). Apoptotic cell material was
then incubated with immature (day 7) DCs stained with PKH-67 (Sigma) at
a 1:1 ratio for various periods of time. Flow cytometric analysis of
samples from these cultures was performed using FL-1 (green
fluorescence, PKH-67) for detection of DCs and FL-2 (red, PKH-26) for
the detection of tumor cells.
Bioassay for TNF Quantification.
A subclone (WEHI 1.14) of the TNF-sensitive WEHI 164 clone was used as
described (23)
with the following modifications. Fifty
µl of graded dilutions from culture supernatant were added to 50 µl
(2 x 104) WEHI 1.14 cells in
flat-bottomed 96-well plates (Nunc) in duplicates and incubated for
24 h at 37°C. Twenty µl of MTS (333 µg/ml) (Promega,
Madison, WI) and 1 µl of the electron coupling reagent phenazine
methosulfate (25 µM; Sigma) were subsequently
added to each well. After 2 h of incubation, the resulting
intensity of the coloration was measured at 490 nm in a Thermomax
microplate reader (Molecular Devices, Menlo Park, CA) and analyzed
using Softmax software from the same company. Recombinant human TNF-
(kindly provided by Dr. J-M. Dayer, University of Geneva) was used as a
standard. The sensitivity of this assay was 0.1 pg/ml. This assay does
not distinguish TNF- from lymphotoxin. Thus, the resulting activity
is referred to as TNF.
RNase Protection Assay.
Total RNA was extracted from DCs or MEL-397 melanoma cells using
TRizol reagent (Life Technologies, Inc.). Multiprobe template set hCK2
(containing DNA templates for IL-12 p35, IL-12 p40, IL-10, IL-1,
IL-1ß, IL-1RA, IL-6, IFN-
, L32, and GAPDH) was purchased from
PharMingen. The pSP64-hTNF plasmid (kindly provided by Dr. V.
Jongeneel, Ludwig Institute of Cancer Research) was linearized with
EcoRI and used to synthesize with SP6 RNA polymerase (Roche
Molecular Biochemicals, Mannheim, Germany), an antisense
riboprobe encoding for human TNF-. hCK2 DNA templates were used to
synthesize the [-32P]UTP (800 Ci/mmol, 5
mCi/ml, Hartmann Analytic, Braunschweig, Germany)-labeled riboprobes in
the presence of rNTPs using T7 RNA polymerase (Promega). Hybridization
with 2.5 µg of each target RNA was performed overnight at 56°C
followed by digestion with RNases A and T1 at 37°C for 30 min. The
samples were then treated with a proteinase K/SDS mixture, extracted
with phenol:chloroform:isoamylalcohol (25:24:1), and precipitated in
the presence of carrier Escherichia coli MRE 600 tRNA (Roche
Molecular Biochemicals). The samples were loaded onto an
acrylamide-urea sequencing gel next to the labeled control probes,
which had been treated with RNases A and T1 or left untreated, and run
at 50 W for 1.5 h. The gel was absorbed to filter paper, dried
under vacuum, and exposed on Kodak X-AR film with intensifying screens
at –80°C.
The relative amounts of proinflammatory cytokine encoding mRNA were measured by scanning densitometry (Molecular Dynamics, Sunnyvale, CA) on subexposed autoradiograms, and further normalized using both housekeeping gene values (L32 and GAPDH).
T Cell Isolation and Cultures.
PBMCs from allogenic buffy coats were allowed to adhere to
plastic for 1 h at 37°C and then passed through a nylon wool
column (Biotest, Dreieich, Germany). T cells (1 x 105 cells/well) were cultured in the presence of
graded numbers of irradiated (3000 rad, 137Cs
source) DCs. Cells were cultured for 5 days in round-bottomed 96-well
plates in 200 µl of RPMI 1640 supplemented with
L-glutamine and penicillin and containing 5%
heat-inactivated human AB+ serum (Blood
Transfusion Center, Annemasse, France). Proliferation was assessed by
tritiated thymidine incorporation in the last 8–12 h of culture. All
conditions were set up in triplicate.
Autologous CD8+ T cells were isolated from nonadherent PBMCs using magnetic bead-conjugated mouse anti-human CD8+ mAb (Miltenyi, Bergisch Gladbach, Germany), a MACS column for positive selection (VS), and a vario-MACS magnet according to the manufacturer’s instructions. The purified cells contained 96–99% CD8+ cells as assessed by flow cytometry.
Autologous CD8+ cells (1.5 x 106) were added to 5 x 104 mature autologous DCs or cp-DCs and cultured in 1.5 ml/well X-VIVO 15 medium supplemented with 1% autologous plasma in 24-well plates. IL-2 was added in a concentration of 40 IU/ml at days 1, 4, and 7. The three peptides described above were added at 10–5 M when indicated. Nine days later, CD8+ cells were restimulated under identical conditions using fresh generated autologous DCs. CTL lines were used after two cycles of stimulation for TNF assays and after three or four cycles of restimulation for CTL assays.
CTL Assay.
MEL-526 cells, cp-DCs or T2 cells (106) were
labeled with 60–100 µCi of
Na51CrO4 (Amersham
Pharmacia Biotech, Buckinghamshire, England) in 200 µl of serum-free
RPMI for 1 h at 37°C. When T2 cells were used, a 1 x 10–5 M concentration of
peptide was added 1 h prior to the labeling. After three washes,
labeled cells were incubated with graded numbers of effector cells in
round-bottomed 96-well plates. After 4 h, the supernatants were
harvested and counted in an Isomedic gamma counter (Wallac, Turku,
Finland). The mean of triplicate samples was calculated, and the
percentage of specific 51Cr release was
determined according to the following equation: % specific
51Cr release = 100 x (experimental 51Cr release – spontaneous
release)/(maximum 51Cr release – spontaneous
release).
Statistical Analysis.
Statistical analysis was done with Wilcoxon’s signed rank test.
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RESULTS |
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). Very few double-positive cells were detected immediately after adding
apoptotic MEL-397 cells to DCs. Most of the DCs and MEL-397 cells
stained double-positive after 18 h. Additional incubation for a
total of 48 h only slightly increased the percentage of
double-positive cells. Internalization of MEL-397 cells by DCs was
confirmed microscopically (not shown). No double-positive cells were
observed in cultures kept on ice. Together, these results indicate that
immature DCs can efficiently internalize cell material from apoptotic
MEL-397 cells, presumably by phagocytosis as shown previously with
other cells (16)
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, IL-1, IL-1ß, IL-1RA, and IL-6) were measured
by RNase protection assay 1 and 2 h after culturing immature DCs
with or without apoptotic MEL-397 cells (Fig. 2a
). The relative amount of proinflammatory cytokine
mRNA induced by apoptotic cells was lower than by stimulation of
immature DCs with exogenous TNF-. To see whether the small induction
of TNF- mRNA induced by apoptotic MEL-397 cells was accompanied by
release of protein, we measured bioactive TNF in the 48 h
supernatant of these cultures. Induction of TNF protein induced by
apoptotic MEL-397 cells was measurable and significant (Fig. 2b
).
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We next tested the effect of apoptotic cell material on the capacity of DCs to stimulate allogenic T cells. DCs cultured with apoptotic MEL-397 cells for 2 days induced T cell proliferation comparable to that of immature control DCs (data not shown).
Together, these data suggest that the presence or the uptake of apoptotic MEL-397 cells does not induce features of DC maturation, with the exception of moderate induction of some inflammatory cytokines.
Characterization of cp-DCs.
Because apoptotic MEL-397 cells did not induce maturation in DCs, we
attempted to achieve this by adding a mixture of maturation factors. We
exposed DCs containing or not containing apoptotic MEL-397 cells for 2
days to IL-1ß, TNF-, IL-6, and PGE2, a
mixture previously shown to induce full maturation in DCs
(8)
. DCs incubated with apoptotic MEL-397 cells and
subsequently matured are herein referred to as cp-DCs. In three
independent experiments, we found no significant difference in the
expression of maturation markers, costimulatory molecules, and MHC
molecules in cp-DCs as compared to control mature DCs. Mean
values were as follows: for CD83, 83.3% (SD, 12.5%) in cp-DCs
versus 82.7% (SD, 10%) in normal DCs; for CD80, 69.7%
(SD, 1.5%) versus 73% (SD, 4.6%); for CD86, 90.3% (SD,
6.7%) versus 94.5% (SD, 2.3%); for CD40, 54% (SD, 26%)
versus 70% (SD, 19.3%); for MHC class I, 96% (SD, 2.8%)
versus 94.2% (SD, 7.4%); and for MHC class II, 97.5% (SD,
1.3%) versus 98.6% (SD, 1%).
Furthermore, DCs matured alone or after the uptake of apoptotic MEL-397
cells had a similar capacity to stimulate the proliferation of
allogenic T cells, as shown in Fig. 3
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To assess antigen-dependent responses, we incubated the T cell lines
after two restimulations with immunodominant peptides from
MelanA/MART1, MAGE-3, or tyrosinase. It is important to mention that
MEL-397 cells used to prepare cp-DCs express MelanA/MART1 and MAGE-3
but not tyrosinase (19)
. After 48 h, we measured TNF
in the supernatant. As shown in Fig. 4
, T cells generated in the presence of peptide plus DCs only produced
TNF when the same peptide used for primary culture was added for
restimulation but not when unrelated peptides or no peptide was added.
The CD8+ cell lines generated in the presence of
cp-DCs produced TNF only in presence of MelanA/MART1 and MAGE-3,
whereas no TNF was released in the presence of tyrosinase peptide or in
the absence of peptide. CD8+ cells primed with
DCs in the absence of exogenous peptide or apoptotic MEL-397 cells did
not produce TNF. Together, these results demonstrate that
CD8+ T cell lines generated with either cp-DCs or
DCs plus peptide from healthy individuals release TNF in an antigen
specific manner.
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). The killing of MelanA/MART1 and MAGE-3 peptide-loaded T2
cells was significantly higher compared to tyrosinase-loaded and
unpulsed T2 cells (P 
0.01, Wilcoxon’s
test). When the natural killer-sensitive cell line K562 was used as
target cell, no killing was observed (data not shown). CTL lines
generated in the presence of peptide from MelanA/MART1, MAGE-3, or
tyrosinase and autologous mature DCs killed T2 cells pulsed with the
relevant peptide but not those pulsed with irrelevant peptide (Fig. 5
)
or no peptide (data not shown). In addition, these T cell lines killed
peptide-loaded target cells more efficiently than CTLs primed with
cp-DCs in three independent experiments (Fig. 5 and d
). T cell lines generated with DCs in the absence of
exogenous antigen did not kill peptide-loaded or unloaded T2 cells
(Fig. 5 and d
). These results demonstrate as a
novel finding that priming of CD8+ T cells with
cp-DCs from normal donors can generate CTL lines specific for peptides
from melanoma-associated antigens expressed by the MEL-397 cells. The
results also confirm that peptide from MelanA/MART1, MAGE-3, and
tyrosinase can be used to generate CTL lines that lyse peptide-loaded
target cells.
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shows a representative experiment. The average percentage of MEL-526
cell lysis was 31.7% (SE, 8.2%) at an E:T cell ratio of 60:1. The
average lysis of MEL-397 cells at the 60:1 E:T cell ratio was 6.4%
(SE, 2.5%). The difference in the lysis of MEL-526 and MEL-397 cells
was statistically significant (P < 0.01,
Wilcoxon’s test). CTL lines generated with DCs and peptide from
MelanA/MART1 or MAGE-3 were significantly less efficient in killing
MEL-526 cells (6.9 ± 2.3%) (P < 0.01, Wilcoxon’ s test). The T cell lines generated with DCs
alone lysed neither MEL-526 nor MEL-397 cells.
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) but not HLA-A2+ DCs without
apoptotic MEL-397 cells (Fig. 7b
). Furthermore,
HLA-A2- DCs, containing MEL-397 cells or not,
were not significantly lysed (data not shown). As observed with MEL
target cells, CTLs generated in the presence of DCs plus peptide were
not capable of exhibiting significant killing toward cp-DCs (Fig. 7a
). These results confirm the higher killing capacity of
CD8+ T cell lines generated with cp-DCs than with
peptide-loaded DCs when antigen is processed and presented naturally in
target cells. Furthermore, these results suggest that cp-DCs are
capable of inducing CTLs against antigen presented in a MHC
class I-restricted manner.
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DISCUSSION |
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, IL-6, and PGE2.
Maturation in cp-DCs is supported by the surface marker profile and the
enhanced stimulation of allogenic T cell proliferation. Exogenous antigen can be presented by MHC class I molecules, a process termed cross-presentation (14) . It was suggested by Bevan (27) that damaged cells could gain access to cross-presentation pathways. In addition, it has been demonstrated that particulate antigens prime more efficiently than soluble molecules for class I-restricted CTL responses (28) . It was only recently that the highly efficient presentation of viral antigen after the phagocytosis of influenza-infected, apoptotic macrophages was demonstrated (17) . These authors (17) induced CTL recall responses using cp-DCs. Presentation of antigen (ovalbumin) from apoptotic cells in the context of MHC class II has been demonstrated in a mouse model (18) . To study whether the uptake of apoptotic cells by DCs can be used for in vitro priming of T cells against melanoma we cultured autologous CD8+ cells with HLA-A2+ DCs that were first allowed to take up apoptotic MEL-397 melanoma cells and then matured with a mixture of maturation factors (cp-DCs). CD8+ cells cultured in the presence of peptide (10-5 M) and DCs served as control. CD4+ T cells were eliminated to prevent any suppressor activity and overgrowth by this subset. T cell help was substituted by IL-2. Under other experimental conditions, help from CD4+ T cells may be required for the successful induction of CD8-dependent immunity to exogenous antigen (29) . As our mature DCs and cp-DCs expressed high levels of accessory molecules, including CD40, helper factors other than IL-2 from CD4+ T cells were not required.
To investigate whether the cell lines generated by repetitive stimulation can recognize immunodominant peptide epitopes from melanoma-associated antigens expressed by MEL-397 (MelanA/MART1 and MAGE-3), we stimulated the T cell lines with these peptides and measured the TNF response as the first readout. It was not necessary to add exogenous antigen-presenting cells to observe responses by the CD8+ T cell lines. This is not surprising, as activated CD8+ cells were recently described to present peptide efficiently to each other (30) . Both cell lines primed with cp-DCs and with DCs plus antigenic peptide exhibited specific responses. It is impressive that T cell lines generated by cp-DCs recognized single peptide epitopes from MelanA/Mart1 or MAGE-3 expressed by MEL-397 cells. From these experiments, it can be concluded that cp-DCs can prime for responses to MelanA/MART1 and MAGE-3. These data, together with the CTL experiments with cp-DCs as target cells, give evidence that DCs can cross-present melanoma antigens to CD8+ T cells. Furthermore, these results show that many healthy individuals have circulating precursors for these antigens in the blood. Their frequency should be >1.5 x 10-6 as we used 1.5 x 106 cells per condition in the starting cultures. This is in the order of magnitude reported for MAGE-3 specific T cells measured by ELISPOT assay (6) . Whether the specific cells within our T cell lines derive from naïve or memory cells remains to be established.
The differential killing capacity of CTL lines generated with the two different priming strategies when using peptide-loaded T2 target cells as opposed to MEL-526 cells and cp-DCs was striking. We did not measure the density of immunogenic peptide on the surface of DCs and the different target cells. It is very likely, however, that it was higher on T2 cells loaded with peptide and DCs to which peptide was added than on cp-DCs and MEL-526 cells. It has previously been shown with murine cells that priming with high levels of peptide selects for low-affinity/low-avidity T cells whereas low levels of peptide on antigen-presenting cells select for high-affinity/high-avidity T cells (31 , 32) . This may explain our results. The high density of peptide used for priming may have selected for low-affinity T cells that killed efficiently only when target cells with high peptide density (i.e., peptide-loaded T2 cells) are used but not when naturally processed and presented antigen is displayed in low density on target cells, such as MEL-526 cells and cp-DCs. Conversely, the low peptide density on cp-DCs may have selected for high-affinity T cells that killed MEL-526 cells and cp-DCs efficiently. CTL lines generated with cp-DCs may have been less efficient to kill T2 cells loaded with a single antigenic peptide than CTL lines generated with DCs and peptide because the former may contain cells specific for several antigens from MEL-397 cells, whereas the latter contain cells with a single relevant specificity. Cross-primed CD8+ T cell lines exhibited clear CTL activity (an average of 25% lysis above negative control) only at high E:T ratios (60: 1). This may be linked to the low density of antigen expressed on target cells (unlike in T2 cells loaded with peptide) and the relative low abundance of high avidity T cells in the effector population. Despite the differential killing activity of T cell lines generated by the two methods, the antigen presenting capacity of both peptide-exposed DCs and cp-DCs was high, because with both methods, lines with antigen specificity could be generated. Our cytotoxicity results therefore suggest that vaccination with mature DCs expressing naturally processed and presented antigen, such as cp-DCs, may be more efficient than vaccination with peptide-pulsed mature DCs. Our findings may thus contribute to the optimization of vaccination strategies using DCs. Transfection of RNA from tumor cells may represent an alternative approach to generate a low and natural density of T cell epitopes on DCs (33 , 34) .
Another potential advantage of cp-DCs is that determination of MHC class I haplotypes is not a prerequisite (as in the peptide approach) because melanoma-associated antigens may also be recognized in the context of haplotypes other than HLA-A2. Furthermore, presentation of apoptotic melanoma cells by DCs has the potential benefit that presentation via MHC class II may generate helper epitopes that support the development of specific CTLs that might be important for antitumoral immunity (35) . The induction of autoimmunity could be a potential disadvantage of cp-DCs, as with all whole cell preparations of tumor cells and RNA derived thereof (discussed in Ref. 36 ).
In conclusion, we have demonstrated that DCs containing apoptotic melanoma cells can efficiently prime autologous CD8+ T cells in vitro to give rise to CD8+ T cell lines specific for epitopes from the melanoma-associated antigens MelanA/MART1 and MAGE-3 and that these cell lines killed melanoma target cells more efficiently than cell lines generated with DCs and peptides.
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FOOTNOTES |
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1 This project was supported in part by the MEDIC
Foundation (Geneva, Switzerland). L. J. was supported by Deutsche
Forschungsgemeinschaft grant 282/6. 
2 To whom requests for reprints should be
addressed, at Allergy Unit, Hôpital Cantonal Universitaire de
Genève, 24 rue Micheli-du-Crest, 1211 Genève 14,
Switzerland. Phone: 0041-22-37-829-381; Fax: 0041-22-37-294-75; E-mail: Conrad.Hauser{at}medecine.unige.ch
3 The abbreviations used are: DC, dendritic cell;
cp-DC, cross-presenting DC; TNF, tumor necrosis factor; IL,
interleukin; PGE2, prostaglandin E2; GM-CSF,
granulocyte-macrophage colony-stimulating factor; mAb, monoclonal
antibody; 7-AAD, 7-amino-actinomycin D; PBMC, peripheral blood
mononuclear cell.
Received 10/27/99. Accepted 6/12/00.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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 S. N. Franki, K. K. Steward, D. J. Betting, K. Kafi, R. E. Yamada, and J. M. Timmerman Dendritic cells loaded with apoptotic antibody-coated tumor cells provide protective immunity against B-cell lymphoma in vivo Blood, February 1, 2008; 111(3): 1504 - 1511. [Abstract] [Full Text] [PDF]
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Y. Tanaka, S. Koido, M. Ohana, C. Liu, and J. Gong Induction of Impaired Antitumor Immunity by Fusion of MHC Class II-Deficient Dendritic Cells with Tumor Cells J. Immunol., February 1, 2005; 174(3): 1274 - 1280. [Abstract] [Full Text] [PDF]
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P. R. Hoffmann, J. A. Kench, A. Vondracek, E. Kruk, D. L. Daleke, M. Jordan, P. Marrack, P. M. Henson, and V. A. Fadok Interaction between Phosphatidylserine and the Phosphatidylserine Receptor Inhibits Immune Responses In Vivo J. Immunol., February 1, 2005; 174(3): 1393 - 1404. [Abstract] [Full Text] [PDF]
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S. J. Prasad, K. J. Farrand, S. A. Matthews, J. H. Chang, R. S. McHugh, and F. Ronchese Dendritic Cells Loaded with Stressed Tumor Cells Elicit Long-Lasting Protective Tumor Immunity in Mice Depleted of CD4+CD25+ Regulatory T Cells J. Immunol., January 1, 2005; 174(1): 90 - 98. [Abstract] [Full Text] [PDF]
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 A. Jalili, M. Makowski, T. Switaj, D. Nowis, G. M. Wilczynski, E. Wilczek, M. Chorazy-Massalska, A. Radzikowska, W. Maslinski, L. Bialy, et al. Effective Photoimmunotherapy of Murine Colon Carcinoma Induced by the Combination of Photodynamic Therapy and Dendritic Cells Clin. Cancer Res., July 1, 2004; 10(13): 4498 - 4508. [Abstract] [Full Text] [PDF]
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 F. Ebstein, C. Sapede, P.-J. Royer, M. Marcq, C. Ligeza-Poisson, I. Barbieux, L. Cellerin, G. Dabouis, and M. Gregoire Cytotoxic T Cell Responses against Mesothelioma by Apoptotic Cell-pulsed Dendritic Cells Am. J. Respir. Crit. Care Med., June 15, 2004; 169(12): 1322 - 1330. [Abstract] [Full Text] [PDF]
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D. Atanackovic, N. K. Altorki, E. Stockert, B. Williamson, A. A. Jungbluth, E. Ritter, D. Santiago, C. A. Ferrara, M. Matsuo, A. Selvakumar, et al. Vaccine-Induced CD4+ T Cell Responses to MAGE-3 Protein in Lung Cancer Patients J. Immunol., March 1, 2004; 172(5): 3289 - 3296. [Abstract] [Full Text] [PDF]
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V. S. Zimmermann, A. Bondanza, A. Monno, P. Rovere-Querini, A. Corti, and A. A. Manfredi TNF-{alpha} Coupled to Membrane of Apoptotic Cells Favors the Cross-Priming to Melanoma Antigens J. Immunol., February 15, 2004; 172(4): 2643 - 2650. [Abstract] [Full Text] [PDF]
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L. T. van den Broeke, E. Daschbach, E. K. Thomas, G. Andringa, and J. A. Berzofsky Dendritic Cell-Induced Activation of Adaptive and Innate Antitumor Immunity J. Immunol., December 1, 2003; 171(11): 5842 - 5852. [Abstract] [Full Text] [PDF]
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M. Crittenden, M. Gough, K. Harrington, K. Olivier, J. Thompson, and R. G. Vile Expression of Inflammatory Chemokines Combined with Local Tumor Destruction Enhances Tumor Regression and Long-term Immunity Cancer Res., September 1, 2003; 63(17): 5505 - 5512. [Abstract] [Full Text] [PDF]
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S. Mehrotra, R. Stevens, R. Zengou, N. G. Chakraborty, L. H. Butterfield, J. S. Economou, D. I. Dorsky, and B. Mukherji Regulation of Melanoma Epitope-specific Cytolytic T Lymphocyte Response by Immature and Activated Dendritic Cells, in Vitro Cancer Res., September 1, 2003; 63(17): 5607 - 5614. [Abstract] [Full Text] [PDF]
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 D. Messmer, B. Messmer, and N. Chiorazzi The global transcriptional maturation program and stimuli-specific gene expression profiles of human myeloid dendritic cells Int. Immunol., April 1, 2003; 15(4): 491 - 503. [Abstract] [Full Text] [PDF]
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K. Akiyama, S. Ebihara, A. Yada, K. Matsumura, S. Aiba, T. Nukiwa, and T. Takai Targeting Apoptotic Tumor Cells to Fc{gamma}R Provides Efficient and Versatile Vaccination Against Tumors by Dendritic Cells J. Immunol., February 15, 2003; 170(4): 1641 - 1648. [Abstract] [Full Text] [PDF]
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B. Yu, S. Kusmartsev, F. Cheng, M. Paolini, Y. Nefedova, E. Sotomayor, and D. Gabrilovich Effective Combination of Chemotherapy and Dendritic Cell Administration for the Treatment of Advanced-Stage Experimental Breast Cancer Clin. Cancer Res., January 1, 2003; 9(1): 285 - 294. [Abstract] [Full Text] [PDF]
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G. Miller, S. Lahrs, V. G. Pillarisetty, A. B. Shah, and R. P. DeMatteo Adenovirus Infection Enhances Dendritic Cell Immunostimulatory Properties and Induces Natural Killer and T-Cell-mediated Tumor Protection Cancer Res., September 15, 2002; 62(18): 5260 - 5266. [Abstract] [Full Text] [PDF]
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M. Schnurr, C. Scholz, S. Rothenfusser, P. Galambos, M. Dauer, J. Robe, S. Endres, and A. Eigler Apoptotic Pancreatic Tumor Cells Are Superior to Cell Lysates in Promoting Cross-Priming of Cytotoxic T Cells and Activate NK and {gamma}{delta} T Cells Cancer Res., April 1, 2002; 62(8): 2347 - 2352. [Abstract] [Full Text] [PDF]
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 X.-Q. Zhao, X.-L. Huang, P. Gupta, L. Borowski, Z. Fan, S. C. Watkins, E. K. Thomas, and C. R. Rinaldo Jr. Induction of Anti-Human Immunodeficiency Virus Type 1 (HIV-1) CD8+ and CD4+ T-Cell Reactivity by Dendritic Cells Loaded with HIV-1 X4-Infected Apoptotic Cells J. Virol., February 22, 2002; 76(6): 3007 - 3014. [Abstract] [Full Text] [PDF]
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 K. M. Dhodapkar, J. Krasovsky, B. Williamson, and M. V. Dhodapkar Antitumor Monoclonal Antibodies Enhance Cross-Presentation of Cellular Antigens and the Generation of Myeloma-specific Killer T Cells by Dendritic Cells J. Exp. Med., January 7, 2002; 195(1): 125 - 133. [Abstract] [Full Text] [PDF]
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G. Pietra, R. Mortarini, G. Parmiani, and A. Anichini Phases of Apoptosis of Melanoma Cells, but not of Normal Melanocytes, Differently Affect Maturation of Myeloid Dendritic Cells Cancer Res., November 1, 2001; 61(22): 8218 - 8226. [Abstract] [Full Text] [PDF]
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M. Salio, D. Shepherd, P. R. Dunbar, M. Palmowski, K. Murphy, L. Wu, and V. Cerundolo Mature Dendritic Cells Prime Functionally Superior Melan-A-Specific CD8+ Lymphocytes as Compared with Nonprofessional APC J. Immunol., August 1, 2001; 167(3): 1188 - 1197. [Abstract] [Full Text] [PDF]
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H. Feng, Y. Zeng, L. Whitesell, and E. Katsanis Stressed apoptotic tumor cells express heat shock proteins and elicit tumor-specific immunity Blood, June 1, 2001; 97(11): 3505 - 3512. [Abstract] [Full Text] [PDF]
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) or not containing (
) apoptotic MEL-397
cells were exposed to the maturation inducing cytokines TNF-
).
The data represent mean of triplicates ± SD. One
representative experiment of five with similar results is shown. In all
experiments, the proliferation of T cells without DCs was below 600
cpm.
) or with cross-primed CTLs (
),
CTL lines generated without peptide (
), and cross-primed CTL
lines (
), DCs and tyrosinase peptide (