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Allogeneic monocyte-derived cells loaded with tumor antigens as a
combined antigen-delivery vehicle and adjuvant in cancer
immunotherapy
John Alder1 , Stefan Lange2, Bengt Andersson1 , AnnaCarin Wallgren1 ,Karin
Gustavsson3, Eva Jennische4, Luigi Pelletieri3, Vincenzo d´Angelo5, Peter S. Eriksson3 and
Alex Karlsson-Parra1
Short title for running head: Allogeneic APC as antigen-carrier and adjuvant
1
Department of Clinical Immunology, Gothenburg University, Sweden, 2Department of
Neuroscience, Gotheburg Universtiy, Sweden, 3Department of Clinical Bacteriology .
Gothenburg University, Sweden, 4Department of Anatomi and Cell Biology, Gothenburg
University, Sweden, 5Ospedal Casa Sollievo, San Giovanni, Italy.
Supported by grants from LUA-SAM, Gothenburg University, Regional FOU, Västra
Götaland, Magnus Bergwall Foundation, Assar Gabrielssons Foundation and the JKFoundation, Sahlgrenska University Hospital.
Correspondence:
Dr. Alex Karlsson-Parra
Department of Clinical Immunology
Sahlgrenska University Hospital, Guldhedsgatan 10A
413 46 Göteborg, Sweden
Tel +46 31 342 47617 ; fax: +46 31 826 791, E-mail address: [email protected]
Abstract
We have recently shown that addition of antigen-presenting cells (APCs) into an allogeneic
immune compartment in vitro elicits an inflammatory reaction that promotes maturation of
bystander dendritic cells with T helper 1 (Th1)-inducing capacity. We therefore proposed that
vaccination with allogeneic, tumor-antigen-loaded, APCs would lead to efficient
immunization against viable tumor cells. Fisher344 female rats were challenged
subcutaneously with the highly malignant breast cancer cell line MAT B III. The rats were
vaccinated in both prophylactic and therapeutic settings. In initial experiments, the vaccine
cells consisted of allogeneic monocytes cultivated for 2 days in GM-CSF. After 1 day the
monocytes were pulsed with apoptotic tumor cells with or without addition of Vibrio cholerae
neuraminidase (NAS), an enzyme known to induce inflammatory chemokine production in
monocytes and to enhance their allogenicity. Prophylactic vaccinations reduced tumor take
from 90% in non-vaccinated rats to 10% in vaccinated rats. This immunity was long-lasting
since re-challenge of tumor-rejecting rats with tumor cells 6 weeks later failed to induce any
tumor growth. In the therapeutic setting all rats developed tumors but tumor growth was
significantly reduced in rats given NAS-treated and tumor-loaded monocytes or monocytederived dendritic cells as compared to non-vaccinated controls. Our results indicate that NAStreated allogeneic monocyte-derived cells may act as antigen carrier as well as a potent
adjuvant in cancer vaccination and immunotherapy.
Introduction
The pivotal role of dendritic cells (DCs) in the activation of naïve T lymphocytes is now
being increasingly explored in clinical trials in humans. There are two main approaches to
harnessing DC´s antitumor response. The most common approach to date has been the
generation ex vivo of autologous antigen-loaded monocyte-derived DCs, followed by injection
of these DCs for the in vivo generation or boosting of antigen-specific T cell mediated
immunity [1, 2]. Effective induction of antitumor cytotoxic T lymphocyte (CTL) responses
requires fully mature DCs that express high levels of costimulatory molecules [3] and that can
migrate in response to lymph-node-derived CCR7 ligands [4]. In addition, high interleukine12p70 (IL-12 p70) secretion dramatically enhances the ability of DCs to induce tumorspecific Th1 cells and CTLs, and promotes tumor rejection in therapeutic mouse models [5,
6]. Unfortunately, the maturation stage of DCs obtained by current ex vivo protocols inversely
correlates with their ability to produce IL-12p70 and most likely explains the failure of
current DC-based cancer vaccines to induce consistently meaningful clinical responses [7].
Alternatively, components of the inflammatory response can be used to target
DC in vivo [8]. The orchestration of immune responses to natural pathogens is a complex
process involving cross-talk between elements of non-specific, innate and antigen-specific,
adaptive immunity. This cross-talk is controlled by expression of proinflammatory factors
including cytokines and chemokines [9, 10]. Approaches to circumvent the injection of
potentially harmful microbes or microbial components, such as BCG and Freunds complete
adjuvant, have included vaccination with DNA vaccines encoding tumor antigens in
conjunction with inflammatory mediators [11-14] or gamma-irradiated tumor cells
(autologous or allogeneic) transfected with genes encoding inflammatory mediators [15, 16].
Accumulating evidence suggests that immune priming induced by these DNA or tumor cell
vaccines is initiated by endogenous dendritic cells (DCs) rather than by DNA-transfected
myocytes [17-19] or injected tumor cells [15, 16]. Since DCs are not typically found in
normal tissue they presumably migrate to the site of antigen inoculation in response to
inflammatory or chemotactic signals following vaccination [12, 20]. Cross-priming may then
occur, in which CD4+ and CD8+ T cell responses are primed by exogenous peptides derived
from injected tumor cells or produced by DNA-transfected myocytes that are acquired,
processed and presented by recruited endogenous DCs. By trafficking antigens from the site
of injection to draining lymph nodes, these DCs thus serve to efficiently present antigen to
naïve T cells [4]. In particular, plasmid GM-CSF has received considerable attention given its
ability to enhance immune responses elicited by DNA vaccines [12] or cellular vaccines
consisting of irradiated tumor cells producing GM-CSF [16]. Co-delivery of plasmids
encoding GM-CSF and MIP-1 alpha is further associated with enhanced tumor rejection and
more efficient tumor-specific CTL priming [12]. Other C-C chemokines such as RANTES
have also been shown to potentiate DNA-based vaccine efficiency presumably by the same
mechanisms [21]. Finally, the use of plasmids that code for MIP-1 alpha in combination with
the maturation/activation factors fms-like tyrosine kinase 3 ligand (Flt3L), has been shown to
strongly enhance DNA vaccine potency [13].
One of the strongest known inflammatory responses is that generated against
MHC alloantigens expressed on allogeneic antigen-presenting cells (APCs) . T cells
recognizing allogeneic MHC molecules by the direct pathway of allorecognition are present at
very high frequencies in the T-cell repertoire, approximately 1–10% of an individual's T
lymphocytes will respond to intact foreign MHC molecules expressed on APCs from another,
allogeneic, individual [22]. By performing conventional allogeneic mixed leukocyte reactions
(MLRs) in vitro we recently showed that primary, and particularly secondary MLRsupernatants, contain high levels of monocyte/immature DC-recruiting CC-chemokines and
pro-inflammatory cytokines [23]. Exposure of immature DCs to primary or secondary MLRsupernatants was found to upregulate CD40-expression and further enhanced LPS-induced
interleukin-12
p70
production.
Secondary
MLR-supernatants
additionally
induced
upregulation of CD86 and deviated allogeneic T cells-responses towards Th1. Taken together,
these previous findings thus predict that the inflammatory process induced during direct
allorecognition in vivo may have the potential to provide a milieu rich in DC-recruiting, DCmaturating and Th1-deviating cytokines and chemokines.
In the present study, we therefore explored the ability of subcutaneously injected
antigen-pulsed and activated allogeneic monocyte-derived cells to induce prophylactic and
therapeutic anti-tumor immune responses in a rat-cancer model.
Materials and methods
Animal model
Inbred Fisher F-344 female and male Spraque-Dawley rats that were purchased from ALAB (
Sollentuna, Sweden). The animals were housed in an environmental room at 22 C and 58 to
65% relative humidity, with a controlled 12-hour light-dark cycle and with free access to
standard pellet diet and tap water. For at least 7 days before the experiments, the rats were
kept in cages containing up to 5 animals. All experiments were performed with approval from
the local research ethical committee (135-2005/Alex Karlsson-Parra).
Cells and cell culture
The rat mammary adenocarcinoma cell line Mat B-II was obtained from the American Type
Culture Collection (Rockville, MD). Cells were maintained in culture in vitro in McCoy’s
modified medium supplemented with 10% fetal calf serum (FCS) and 1% gentamycin (Sigma,
St. Louis, MO). Before inoculation, tumor cells were washed in culture media and trypsinized
for 5 min. Cell viability of > 80% was ensured by the trypane blue exclusion test. Cells were
then collected in culture medium and centrifuged at 1500 rpm for 5 min. Cell pellets (1x
106cells) were resuspended in 0.25 ml PBS and subcutaneously injected in the left upper
flank.
Vaccine cell generation
About 10 ml of peripheral heparinized blood from each allogeneic Sprague-Dawley rat was
collected by cardiac puncture under Isofluorhane anesthesia. Peripheral blood mononuclear
cells (PBMCs) were obtained by gradient centrifugation using lymphocyte separation medium
(Lymphoprep; Nycomed, Oslo, Norway). PBMCs were then plated at 2.5 x 106 cells/ml in 6well culture plates (Nunc; Roskilde, Denmark). Cells were incubated for 2 h at 37C, and 5 %
CO2, in culture medium. After 2 h non-adherent PBMCs were removed by repeated washes
with culture medium. The remaining adherent cells were incubated at 37C in culture medium
in the presence of 5 ng/ml recombinant rat GM-CSF (R&D Systems, Abingdon, UK) with or
without addition of 5 ng/ml recombinant rat interleukin (IL)-4 (R&D Systems, Abingdon,
UK). Mat BIII tumor cells were induced to become apoptotic with 10.000 Joule/m2 UVBirradiation were added to the monocyte culture at a ratio of 1:1 after 24 h (for monocytes
cultured in GM-CSF) or after 5 days (for monocytes cultured in GM-CSF + IL-4). The UVB
light source was a bank of two unfiltered fluorescent tubes (Derma light 80, Scan-Med. a/s,
Drammen; Norway) placed 2 cm over the target cells. Emission was of broad spectrum with a
peak at 306 nm. At the same time GM-CSF-monocytes were treated with 25 mU/mL Vibrio
cholerae neuraminidase (NAS) (Sigma-Aldrich, Stenheim, Germany) and harvested after a
further 24 h incubation. Monocyte-derive DCs (MoDCs) were pulsed with apoptotic tumor
cells at day 4 and NAS-treated for 3 h at day 5 before harvest.
The vaccine cells (GM-CSF-monocytes or monocyte-derived DCs) were collected in culture
medium and centrifuged at 1500 rpm for 5 min. Cell pellets were resuspended in PBS
(4x106m cells/ml) and resuspended in 250µl PBS for subsequent subcutaneous vaccination.
Vaccination and tumor challenge
In the prophylactic setting, vaccine cells (GM-CSF-monocytes) were injected subcutaneously
in the lower right abdominal region day 14 and day 7 before tumor challenge. The therapeutic
vaccination (GM-CSF-monocytes or monocyte-derived DCs) was given at the day of tumor
challenge (day 0) and at day 7 after tumor challenge.
Breast adenocarcinoma tumors were induced ectopically by injection with 1 x 10 6 MAT BIII
cells in 0.25 ml PBS subcutaneously into the left flank of each Fischer344 rat. Rats rejecting
the first tumor challgenge were rechallenged 6 weeks later with new tumor cells ( 2x10 6 cells)
injected subcutaneously. Tumor growth was assessed every day from day 4 by inspection and
palpation. Groups of 5 rats were used and rats were sacrificed 12 days after tumor challenge.
The tumors were removed, weight and subsequently fixed in 4 % buffered formaldehyde.
Immunohistochemistry
The fixed tumors were embedded in paraffin, and sectioned at 4 um. Antigen retrieval was
performed by heating the sections in a microwave oven in 1 mmol EDTA buffer (pH 9.0).
The sections were incubated with a monoclonal antibody against rat CD3 (W3/13), CD8 (Ox8) and CD69 (ED1). All anti-rat antibodies were purchased from Serotc Ltd. (Oxford, United
Kingdom). The PAP procedure (Dako, Glostrum, Denmark) was used as secondary reagents,
and the sections were developed with diaminobenzidine as substrate. The sections were
lightly counterastined with hematoxylin, dehydrated, and mounted.
Results
Prophylactic vaccination
The profylactic vaccinations were conducted using GM-CSF-monocytes as vaccine cells.
Nine out of 10 (90%) non-vaccinated rats and 2 out 5 (40%) of rats given prophylactic
vaccinations with tumor-loaded allogeneic GM-CSF-monocytes developed subcutaneous
tumors within 3 weeks. Only 1 out or 5 (10%) of rats given prophylactic vaccination with
NAS-treated and tumor-loaded vaccine cells developed a tumor. (Fig.1). This immunity was
long-lasting since re-challenge of tumor-rejecting rats with new tumor cells (2x106 cells) 6
weeks later failed to induce any tumor growth (data not shown).
Therapeutic vaccination
In the therapeutic setting all rats developed tumors but tumor growth was significantly
reduced (p< 0,05) in rats given NAS-treated and tumor-loaded allogeneic monocytes
compared to controls (Fig. 2 and 3). Vaccination with NAS-treated but unloaded allogeneic
monocytes or vaccination with apoptotic tumor cells gave no significant reduction of tumor
growth. Vaccination with antigen-loaded and NAS-treated GM-CSF-monocytes or monocytederived DCs induced a significant reduction of tumor growth (p < 0.05 and
> 0.001,
respectively)
Histology and immunohistology
Immunohistological examinations in tumors taken at day 12 were performed in control rats
and rats receiving therapeutic vaccination with NAS-treated and antigen-loaded GM-CSFmonocytes.
Tumors taken from control rats usually exhibited a more “invasive” behaviour in that they
usually were more adherent to adjacent normal tissues. In contrast, tumors from the
vaccinated group usually were relative free of attachments to the overlying skin and
underlying musculature. Some tumors from the control group further became sacs filled with
turbid, amber fluid, a phenomenon not observed in tumors from vaccinated rats.
Microscopically, tumors from both sources where characterized by a surrounding capsular
structure and a subcapsular rim of infiltrating mononuclear cells. Control tumors exhibited
well-defined acinic structures (composed of epithelial tumor cells) in more superficial parts of
the tumors while large central areas of the tumor exhibited prominent cellular necrosis. In
contrast, tumors taken from vaccinated animals exhibited necrotic areas more superficially
while central areas were characterized by scattered acinar structures with intact epithelial cells
mixed with a variable number of infiltrating mononuclear cells. Only minor areas with
fulminant cellular necrosis where seen in the central parts. Immunohistological examinations
revealed a prominent infiltration of ED1+ macrophages as well as W3/13-positive T cells
within the peripheral rim of inflammatory cells in both tumor-types (Fig.4) . In control
tumors, a moderate infiltration of T cell and macrophages was seen within some peripheral
acinar structures, often those that were in close vicinity to the peripheral rim of inflammatory
cells (Fig.4). More centrally located acini usually exhibited very few infiltrating T cells or
macrophages (Fig.4). In contrast, a substantial T cell and macrophage infiltration in was
usually seen in both central and peripheral acinar structures in tumors from vaccinated rats
(Fig.4). Staining for CD8+ cells showed that lymphocyte-like cells as well as macrophagelike cells were positively stained in tumors from both groups. Interestingly, the main
proportion of CD8 positive cells within acinar structures in tumors from vaccinated rats was
macrophage-like (Fig.5b). Careful examination of sequential sections revealed that these cells
were also positively stained for ED1 (Fig.5c).
Discussion
The present study indicates that NAS-treated allogeneic monocyte-derived cells ( GM-CSFmonocytes or monocyte-derived DCs) may act as antigen carrier as well as adjuvant in cancer
vaccination and immunotherapy. A substantial protection against subsequent tumor growth
was thus obtained in rats given prophylactic vaccination with NAS-treated and tumor-loaded
vaccine cells. This protection/immunity was long lasting since challenge of tumor-rejecting
rats with new tumor cells 6 weeks later failed to induce any tumor growth. In the therapeutic
setting, tumor growth was significantly reduced in rats given NAS-treated and tumor-loaded
vaccine cells compared to controls.
The current vaccine approach is based on our recent in vitro findings that
alloreactivity (direct pathway of allorecognition) creates an inflammatory milieu with potent
impact on bystander (“endogenous”) DCs [23]. A hypothetical model could be that the
vaccine cells, recently activated by NAS-treatment, initially would produce inflammatory
molecules, including MIP-1 alpha [24], that recruit an initial set of CCR5-expressing mature
alloreactive T cells. Their subsequent interaction with the injected allogeneic APCs (in vivo
MLR) will likely escalate the production of DC (and mature T cell) recruiting chemokines
and induce production of DC-maturating cytokines [23]. Release of the tumor-antigen “cargo”
may be through NK-cell/CTL-induced apoptosis of the vaccine cells, or active release of
exosomes [25]. Tumor-derived peptides within apoptotic cells/bodies or in complex with heat
shock proteins within secreted exosomes may thus subsequently be acquired, processed and
presented by endogenous DCs. By trafficking antigens from the site of injection to draining
lymph nodes, these DCs thus serve to efficiently present antigen to naïve and self-restricted T
cells (CD8+ as well as CD4+ T cells). This model is thus fully in line with the proposed
models for immunization induced by vaccination with DNA vaccines encoding tumor
antigens in conjunction with inflammatory mediators [11-14] or gamma-irradiated tumor cells
transfected with genes encoding inflammatory mediators [16]. The exact nature of
immunological mechanisms that contributed to the prophylactic as well as therapeutic benefits
seen with our present vaccine approach is however still unknown. Tumors from both groups
exhibited a dense peripheral rim of ED1-positive macrophages and T cells beneath a capsular
structure. Control tumors were also characterized by macrophage and T cell infiltration in
superficial parts of the tumor while deeper parts usually exhibited extensive necrosis without
substantial infiltration of macrophages or T cells. These findings may indicate that nonimmunological factors, such as hypoxia within central parts of this rapidly growing tumor,
participate in necrosis-induction. Our observation that a substantial number of infiltrating
macrophages within acinar structures in tumors from vaccinated rats expressed CD8 is of
clear interest since CD8-expression on monocytes/ macrophages taken from rejecting rat
allografts has been shown to correlate with their ability to kill tumor target cells [26].
Moreover, cross-linking of membrane-expressed CD8 on activated rat macrophages by OX-8
antibodies has been shown to stimulate nitric oxide (NO) production [27]. The intense
infiltration of CD8-expressing macrophages and the close contact between such macrophages
and viable tumor cells may thus indicate an active contribution of macrophages in the tumorrejection process rather than representing secondary unspecific phagocytic functions.
The concept of harnessing alloreactivity in cancer treatment is not new. The
main hypothesis has been that alloreactive CD4+ cells may help cancer-specific CTLs by
providing helper factors such as IL-2 and IFN-gamma [28] and semiallogeneic DCs
consisting of allogeneic DCs fused with HLA-compatible tumor cells (from the patient) has
therefore been proposed as the optimal concept [28]. This follows the theory that foreign
MHC class II molecules can be directly recognized by a number of highly reactive allogeneic
CD4+ T cells, which would in turn, support the induction of CD8+ cytotoxic T cells specific
for antigen presented by autologous hybridized tumor cell MHC class I molecules. Indeed,
clinical trials have shown a substantial response [29, 30] but the underlying protective
mechanisms have not been fully explored. Theoretically, such cell hybrids must themselves
migrate to regional lymph nodes in order to get in contact with naïve T cells, thus possessing
similar “logistic” problems as antigen-loaded autologous DCs propagated ex vivo (see
introduction). Moreover, this approach poses important technical problems. First, a semiallogeneic vaccine depends on the availability of adequate number of tumor cells, which are
rarely available because of the reactive processes that are found infiltrating the tumor cells of
many common cancers [16]. Second, a semi-allogneic vaccine requires de novo production of
hybrids for treatment of each patient, which is labour intensive and may cause variable
numbers of intact hybrids. Third, there is significant expense and time required to certify each
patient’s lot of vaccine cells so that they meet local Food and Drug Administration guidelines.
By harnessing the phenomenon that alloreactivity (direct pathway of
allorecognition) creates an inflammatory milieu with potent impact on bystander
(“endogenous”) DCs it would be possible to circumvent the inherent technical obstacles in
strategies using semiallogeneic cellular vaccines. Instead of using autologous (or syngeneic as
in the current study) tumor cells as antigen-source, fully allogneic tumor cells could
potentially be used as antigen-source due to the frequent presence of shared or “universal”
tumor antigens [31]. The combination of allogeneic vaccine cells with an allogeneic antigensource would then make it possible to develop a fully allogeneic vaccine strategy based on a
panel of antigen-loaded vaccine cells that can be formulated and stored before the initiation of
clinical studies. This is a particularly attractive approach for the majority of common cancers
for which specific tumor antigens have not yet been identified.
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Figure legends
Figure 1. Prophylactic induction of antitumor protection by immunization with tumor cellallogeneic APC vaccine. The antitumor response was evaluated using tumor-pulsed vaccine
cells with or without NAS treatment. The rats were immunized with vaccine cells (1 × 106) on
days 0 and 14 and then challenged s.c. with 1.0 × 106 tumor cells (13762 MAT B III) on day
21. The percentage of tumor-free mice on day 12 after tumor injection is shown. Naive
unvaccinated mice were used as a negative control.
Figure 2. Immunization with tumor cell-allogeneic APC vaccine in a therapeutic setting. The
antitumor response was evaluated using tumor-pulsed and NAS-treated vaccine cells. The rats
were challenged s.c. with 1.0 × 106 tumor cells (13762 MAT B III) on day 0 and subsequently
immunized with vaccine cells (1 × 106) on days 0 and 7. Tumors were removed day 12. Naive
unvaccinated mice were used as a negative control.
Figure 3. Immunization with tumor cell-allogeneic APC vaccine in a therapeutic setting. The
antitumor response was evaluated using tumor-pulsed and NAS-treated vaccine cells. The rats
were challenged s.c. with 1.0 × 106 tumor cells (13762 MAT B III) on day 0 and subsequently
immunized with vaccine cells (1 × 106) on days 0 and 7. Tumors were removed day 12. Naive
unvaccinated mice were used as a negative control.
Figure 4. Immunohistological staining of consecutive sections from MAT B III tumors
removed 12 days after subcutaneous inoculation of 1 x106 tumor cells in otherwise untreated
rats (A, B, E, F) or rats vaccinated with tumor-pulsed allogeneic monocytes at day 0 and day
7 (C,D,G,H). Peripheral parts (A-D) and central parts (E-H) of the tumors are shown. Sections
were stained according to the labeling in each figure. * indicate necrotic areas. Original
magnification x 200.
Figure 5. Immunohistological staining of central parts of consecutive sections from MAT B
III tumors removed 12 days after subcutaneous inoculation of 1 x106 tumor cells rats
vaccinated with tumor-pulsed allogeneic monocytes at day 0 and day 7. Sections were stained
according to the labeling in each figure. Original magnification x 400.
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% Tumor-free rats (n=5)
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Figure 5
Bild 4 och 5 (färgbilder) tar för stor plats och gör därmed filen för stor.
Figure 6
1,4
Tumor weight (gram)
1,2
1
0,8
0,6
0,4
0,2
0
Control
Vaccine