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Publication of the International Union Against Cancer
Publication de l’Union Internationale Contre le Cancer
Int. J. Cancer: 70, 706–715 (1997)
r 1997 Wiley-Liss, Inc.
REGRESSION OF TUMORS IN MICE VACCINATED WITH PROFESSIONAL
ANTIGEN-PRESENTING CELLS PULSED WITH TUMOR EXTRACTS
Smita K. NAIR1, David SNYDER1, Barry T. ROUSE2 and Eli GILBOA1*
1Department of Surgery, Duke University Medical Center, Durham, NC
2Department of Microbiology, University of Tennessee, Knoxville, TN
Vaccination with tumor extracts circumvents the need to
identify specific tumor rejection antigens and extends the use
of active immunotherapy to the vast majority of cancers, in
which specific tumor antigens have not yet been identified. In
this study we examined the efficacy of tumor vaccines comprised of unfractionated tumor material presented by professional antigen-presenting cells (APC): dendritic cells (DC) or
macrophages (Mø). To enhance the relevance of these studies for human patients we used 2 poorly immunogenic murine
tumor models and evaluated the effectiveness of the vaccination protocols in tumor-bearing animals. APC (in particular
DC) pulsed with unfractionated extracts from these ‘‘poorly
immunogenic’’ tumors were highly effective in eliciting tumorspecific cytotoxic T lymphocytes. A measurable CTL response could be detected after even a single immunization
with tumor extract-pulsed DC. DC or Mø pulsed with tumor
extract were also effective vaccines in tumor-bearing animals. In the murine bladder tumor (MBT-2) model a modest
extension of survival and 40% cure rate was seen in the animal
groups immunized with DC or Mø pulsed with MBT-2 tumor
extract. DC or Mø pulsed with B16/F10.9 tumor extract were
also remarkably effective in the B16 melanoma lung metastasis model, as shown by the observation that treatment with
APC caused a significant reduction in lung metastases. Cumulatively, the CTL and immunotherapy data from the two
murine tumor systems suggest that APC (in particular DC)
pulsed with unfractionated cell extracts as a source of tumor
antigen may be equally or more effective than genetically
modified tumor vaccines. Int. J. Cancer 70:706–715, 1997.
r 1997 Wiley-Liss, Inc.
Animal studies support the notion that, with a few exceptions,
tumor-specific CD81 CTL constitute an important effector arm of
the antitumor immune response (Greenberg, 1991). Hence, antigens recognized by CD81 CTL cells are likely to function as tumor
rejection antigens capable of eliciting protective immunity in vivo.
The existence of specialized, or professional, antigen-presenting
cells (APC) that are responsible for the presentation of Ag to naive
CD81 T cells was based on observations that host MHC-restricted
CTL can be primed in vivo to Ag that was introduced on
MHC-disparate cells (Matzinger and Bevan, 1977). Additional
evidence stems from transplantation studies suggesting that only a
subset of allo-MHC donor cells, called passenger leukocytes, were
responsible for the induction of an immune response and rejection
of the mismatched tissue (Lafferty et al., 1983). Studies exploring
the mechanism of action of interleukin-2 (IL-2) or granulocyte/
macrophage colony-stimulating factor (GM-CSF)-secreting tumor
vaccines have also suggested that priming of an MHC class
I-restricted antitumor response required the transfer of antigens
from the tumor cell to a host-derived cell for presentation to CD81
CTL (Huang et al., 1994; Bannerji et al., 1994). As recently
discussed by Bevan (1995), a number of possible pathways exist by
which extracellular antigens can translocate into the cytosolic class
I presentation pathway of host-derived professional APC.
The main candidates for professional APC are macrophages
(Mø) and the bone marrow-derived dendritic cells (DC). Several
studies have documented the exceptional ability of DC to stimulate
naive T cells, both in vitro and in vivo. DC pulsed with protein or
peptide in the presence of lipid (Nair et al., 1993) or transfected
with DNA (Rouse et al., 1994) are capable of eliciting primary CTL
responses in vitro, and inoculation of mice with small numbers of
allogeneic DC (McKinney and Streilein, 1989) or with peptide-
pulsed DC (Takahashi et al., 1993) induces a potent CTL response
in vivo. In general, Mø are less effective than DC at inducing T-cell
responses in vitro or in vivo (Steinman, 1991). However, presentation of exogenous soluble antigens to CD81 T cells, a defining
feature of a professional APC, is carried out by cells with Mø
characteristics (Rock et al., 1993).
A number of genes that encode tumor antigens recognized by
CD81 T cells have been characterized (Boon et al., 1994). There
are at least 3 advantages to using defined tumor antigens in cancer
immunotherapy: 1) use of defined tumor antigen obviates the need
for tumor tissue and therefore will benefit patients with low tumor
burden; 2) the purity of the antigenic preparation is likely to
enhance the effectiveness of the vaccines; and 3) the absence of
irrelevant tumor material will minimize possible autoimmune
reactions against ‘‘self antigen.’’ There are, however, 3 possible
drawbacks in using defined CTL antigens: 1) it is unclear whether
or which of the identified human tumor-specific antigens are the
best choice to mount an effective anti-tumor immune response in
vivo; This potential concern was underscored in a report by
Anichini et al. (1996), who showed that the majority of CTL
present in HLA-A2.1 melanoma patients were not directed to the
tumor antigens, Melan-A/Mart-1, tyrosinase, gp100, or MAGE-3;
2) the use of vaccines consisting of a single antigen or a few tumor
antigens carry the risk of generating escape mutants; and 3) human
CTL antigens have been identified and isolated only from a small
number of cancers.
An alternative strategy, not encumbered by these limitations, is
to use unfractionated tumor-derived antigens obtained from tumors, such as whole tumor cells, total peptide extracts or total
protein extracts. The main advantages of using unfractionated
tumor material as a source of tumor antigen are: 1) the identity of
the effective tumor antigen(s) need not be known, a fact that
expands significantly the type of cancer that can be treated; and 2)
the (likely) presence of multiple tumor antigens reduces the risk of
escape mutants. There are, however, three potential drawbacks in
vaccinating cancer patients with unfractionated tumor-derived
peptides or proteins, compared with use of purified antigens: 1) use
of unfractionated tumor material as a source of tumor antigen will
depend on the availability of substantial amounts of tumor tissue
from the patient; 2) vaccination with unfractionated tumor-derived
antigens could induce autoimmune responses directed against
‘‘self’’ antigens; and 3) immunization with unfractionated tumor
material may be less effective due to the low concentration of
effective tumor antigens in the mixture.
Several studies have shown that immunization of mice with
professional APC pulsed with unfractionated tumor proteins induced protective immunity in mice against a subsequent challenge
with live tumor cells (Grabbe et al., 1991; Shimizu et al., 1989;
Flamand et al., 1994; Cohen et al., 1994). Approximating the
conditions prevailing in cancer patient more closely, genetically
engineered whole cell tumor vaccines were capable of causing the
regression of tumors devoid of intrinsic immunogenicity (Gilboa
and Lyerly, 1994).
*Correspondence to: Department of Surgery, Duke University Medical
Center, Box 2601, Durham, NC 27710, USA. Fax: 919 681 7970.
Received 26 August 1996; revised 21 November 1996
TUMOR EXTRACT-PULSED APC AND TUMOR IMMUNITY
In this study we examined the efficacy of tumor vaccines
comprised of unfractionated tumor material presented by professional APC, DC or Mø. Using 2 poorly immunogenic murine tumor
models, we show that immunization with APC pulsed with
unfractionated tumor cell extracts induce potent CTL responses in
vivo and cause the regression of pre-existing tumors in tumorbearing animals.
MATERIAL AND METHODS
Mice
Retired breeder female C57BL/6 mice (H-2b ), 7–8 weeks old and
C3H/He mice (H-2k ) were obtained from the Jackson Laboratory
(Bar Harbor, ME). In conducting the research described below, the
investigators adhered to the ‘‘Guide for the Care and Use of
Laboratory Animals’’ as proposed by the committee on care of
Laboratory Animal Resources Commission on Life Sciences,
National Research Council. The facilities are fully accredited by
the American Association for Accreditation of Laboratory Animal
Care.
Cell lines
The murine MBT-2 cell line, derived from a carcinogen-induced
bladder tumor in C3H mice, was obtained from Dr. T. Ratliff
(Washington University, St. Louis, MO). The MBT-2/IL-2 cell line
is derived by transfecting MBT-2 cell with human IL-2 cDNA
(Connor et al., 1993). The F10.9 clone of the B16 melanoma of
C57BL/6 origin is a highly metastatic, poorly immunogenic and a
low class I-expressing cell line. F10.9/K1 is a poorly metastatic and
highly immunogenic cell line derived by transfecting F10.9 cells
707
H-2Kb
cDNA (Porgador et al., 1995). Other
with a class I molecule,
cell lines used were EL4 (C57BL/6, H-2b, thymoma), E.G7-OVA
(EL4 cells transfected with the cDNA of chicken ovalbumin (OVA)
(Moore et al., 1988), A20 (H-2d, B cell lymphoma) and L929 (H-2k
fibroblasts). Cells were maintained in DMEM supplemented with
10% FCS, 25 mM Hepes, 2 mM L-glutamine and 1 mM sodium
pyruvate. E.G7-OVA cells and MBT-2/IL-2 cells were maintained
in medium supplemented with 400 µg/ml G418 (GIBCO, Grand
Island, NY), and F10.9/K1 cells were maintained in medium
containing 800 µg/ml G418.
APC and responder T cells
Splenocytes obtained from naive C57BL/6 female retired breeders were treated with ammonium chloride Tris buffer for 3 min at
37°C to deplete red blood cells. Splenocytes (3 ml) at 2 3 107
cells/ml were layered over 2 ml metrizamide gradient column
(Nycomed Pharma, Oslo, Norway; analytical grade, 14.5 g added
to 100 ml PBS, pH 7.0) and centrifuged at 600g for 10 min. The
DC-enriched low-density fraction from the interface was further
enriched by adherence for 90 min to remove contaminating T and B
cells. Adherent cells (mostly DC and a few contaminating Mø)
were retrieved by gentle scraping and subjected to a second round
of adherence at 37°C for 90 min to deplete the contaminating Mø.
After the second round of adherence, non-adherent cells were
pooled as splenic DC and FACS analysis showed approximately
80–85% DC (MAb 33D1), 1–2% Mø (mAb F4/80), 5–10% T cells
and ,5% B cells (data not shown, Nair et al., 1993).
The pellet was resuspended and enriched for Mø by 2 rounds of
adherence at 37°C for 90 min each. More than 80% of the adherent
FIGURE 1 – Induction of OVA-specific CTL responses in mice immunized with DC pulsed with OVA protein. Mature splenic DC were pulsed
with OVA protein in the presence or absence of the cationic lipid, DOTAP, as described in Material and Methods. C57BL/6 mice were immunized
once i.p., and after 7 days splenocytes were harvested and restimulated in vitro. CTL assay was done on day 5 with E.G7-OVA, EL4 and
BALB/3T3 cells as targets. Control targets EL4 and BALB/3T3 showed insignificant lysis (data not shown).
708
NAIR ET AL.
population was identified as Mø by FACS analysis with 5%
lymphocytes and ,5% DC.
Pulsing of APC
APC were washed twice in Opti-MEM medium (GIBCO). Cells
were resuspended in Opti-MEM medium at 5–10 3 106 cells/ml
and added to 50 ml polypropylene tubes (Falcon, Oxnard, CA). The
cationic lipid DOTAP (Boehringer Mannheim, Indianapolis, IN)
was used to deliver protein or tumor extracts into cells. Tumor
extracts were obtained by sonicating tumor cells in Opti-MEM (107
cells/500 µl) using a Special Ultrasonic Cleaner (Laboratory
Supplies, Hicksville, NY). Cell sonicates were used without any
further manipulation as tumor extracts. Tumor cell sonicates or
tumor extracts (500 µl) and DOTAP (125 µg in 500 µl Opti-MEM
medium) were mixed in 12 3 75 mm polystyrene tubes at room
temperature (RT) for 20 min. The complex was added to the APC
(107 cells) in a total volume of 2–5 ml and incubated at 37°C in a
water bath with occasional agitation for 2 hr. The cells were
washed, irradiated at 3,000 rads and resuspended in PBS (2 3 106
DC pulsed with 2 3 106 tumor cell extract and 25 µg DOTAP in
500 µl PBS/mouse) for intraperitoneal immunizations. Purified
OVA protein was used at a concentration of 50 µg/mouse.
Induction of antigen-specific CTL in vivo
Cells were pulsed with tumor extracts or OVA protein as
described above. Naive, syngeneic mice were immunized intraperitoneally with 2 3 106 APC (irradiated at 3,000 rads)/mouse in 500
µl PBS. Irradiated tumor cells were used at a concentration of 5 3
106 cells per mouse.
Splenocytes were harvested after 7–10 days and depleted of red
blood cells with ammonium chloride Tris buffer. Splenocytes
(1.5 3 107 ) were cultured with 7.5–10 3 105 irradiated stimulator
cells (E.G7-OVA cells irradiated at 20,000 rads, MBT-2/IL-2 cells
irradiated at 7,500 rads and F10.9/K1 cells irradiated at 7,500 rads)
in 5 ml of IMDM with 10% FCS, 1 mM sodium pyruvate, 100
IU/ml penicillin, 100 mg/ml streptomycin and 5 3 1025 M
b-mercaptoethanol/well in a 6-well tissue culture plate. Cells were
cultured for 5 days at 37°C and 5% CO2. Effectors were harvested
on day 5 on Histopaque 1083 gradient prior to use in a CTL assay.
Alternatively, human recombinant IL-2 (5 units/ml, Schiapparelli
Biosystems, Columbia, MD) was used to restimulate effector cells
instead of irradiated tumor cells.
Cytotoxicity assay
Target cells (5–10 3 106 ) were labeled with europium diethylenetriamine penta-acetate for 20 min at 4°C. After several washes, 104
europium-labeled targets and serial dilutions of effector cells at
an effector/targer ratio of 80:1 to 5:1 were incubated in 200 µl
of RPMI 1640 with 10% heat-inactivated FCS in 96-well
V-bottomed plates. The plates were centrifuged at 500g for 3 min
and incubated at 37°C and 5% CO2 for 4 hr; 50 µl of the supernatant
were harvested, and europium release was measured by timeresolved fluorescence (Delta fluorometer, Wallac, Gaithersburg,
MD) (Saito et al., 1994). Spontaneous release was less than 25%.
Standard errors (SE) of the means of triplicate cultures were less
than 5%.
FIGURE 2 – Priming of OVA-specific CTL in mice immunized with DC pulsed with cellular extracts from OVA expressing tumor cells. Mature
splenic DC were pulsed with OVA protein, EG7, EL4 or A20 extracts in the presence of the cationic lipid, DOTAP, EG7 extract/DOTAP alone or
DC 1 EG7 extract without DOTAP as described in Material and Methods. C57BL/6 mice were immunized once i.p., and after 10 days splenocytes
were harvested and restimulated in vitro. CTL assay was done on day 5 with E.G7-OVA, EL4, RMA-S cell pulsed with OVA peptide and A20 cells
as targets. Control targets A20 showed insignificant lysis (data not shown).
TUMOR EXTRACT-PULSED APC AND TUMOR IMMUNITY
Immunotherapy
MBT-2 murine model.Orthotopic implantation of MBT-2 cells
into the bladder of C3H mice was performed as described
previously (Connor et al., 1993). Briefly, under magnification, a 0.8
cm incision was made transversely in the abdomen just above the
pubis. The anterior abdominal wall muscles were incised, and the
bladder was delivered into the surgical field. Using a 1 ml
tuberculin syringe, MBT-2 cells (2 3 104 ) in 50 µl PBS were
injected into the bladder wall. The incision was closed in one layer
using a 5.0 prolene suture. The procedure was well tolerated, and
postoperative mortality was less than 5%. Mice were vaccinated on
days 4, 8 and 12 following implantation of MBT-2 cells. Mice were
evaluated on a daily basis and were sacrificed when moribund.
Each treatment group consisted of 5–10 mice.
F10.9-B16 melanoma model.Mice were injected intrafootpad
with 2 3 105 F10.9 cells. The postsurgical protocol was used as
described previously with a few modifications (Porgador et al.,
1995). Mice were amputated when the local tumor in the footpad
was 5.5–7.5 mm in diameter. Postamputation mortality was less
than 5%. Two days postamputation mice were immunized intraperitoneally followed by weekly vaccinations twice, for a total of 3
vaccinations. Mice were sacrificed based on the metastatic death in
the non-immunized or control groups (28–32 days postamputation). Metastatic loads were assayed by weighing the lungs and by
counting the number of metastatic nodules.
Statistical methods
For the MBT-2 model, overall significance of the study was
calculated using the Kaplan-Meier method, and the significance of
709
the differences between the survival rates was calculated using the
log-rank test. In the B16 melanoma model, the different experimental groups within the study were compared using the KruskalWallis test. Comparisons of significance for differences in lung
weights between specific pairs of groups were then compared by
the Mann-Whitney U-test. A probability of less than 0.05 ( p , 0.05)
was used for statistical significance.
RESULTS
Dendritic cells pulsed with OVA protein in the presence of the
cationic lipid DOTAP induce OVA-specific CTL responses in vivo
To test whether DC pulsed with protein are capable of inducing
CTL in vivo, mice were immunized once with DC pulsed with OVA
protein in the presence or absence of DOTAP. The OVA protein
encodes an H-2Kb epitope (aa 257–264, SIINFEKL) that is
recognized by CD81 CTL in C57BL/6 mice. As controls, DC were
incubated with DOTAP with no antigen, or with antigen in the
absence of DOTAP. Splenocytes were restimulated in vitro with
E.G7-OVA cells, which are EL4 (H-2b ) thymoma cells transfected
with and expressing the chicken OVA protein (Moore et al., 1988).
Cytotoxic activity of the splenocytes was tested on E.G7-OVA,
EL4 and BALB/3T3 cells as targets. As shown in Figure 1, only DC
pulsed with OVA protein in the presence of DOTAP were able to
induce a strong and specific CTL response in the treated mice.
Presumably, the function of DOTAP is to facilitate cytoplasmic
incorporation of the exogenous antigen for MHC class I presentation to CD81 T cells. Control targets, EL4 and BALB/3T3 cells
(data not shown) were not lysed by the CTL. Mice immunized
FIGURE 3 – Induction of MBT-2 tumor-specific CTL by immunization with tumor extract pulsed APC. Splenic DC and Mø were pulsed with
MBT-2 tumor cell sonicates in the presence or absence of DOTAP as described in Material and Methods. C3H mice were immunized i.p. 3 times at
weekly intervals with 2 3 106 APC or with 5 3 106 tumor cells. Splenocytes were harvested and restimulated with recombinant IL-2, and a CTL
assay was done after 5 days. EL4 cells were used as targets for MHC restriction and showed insignificant lysis. This experiment was repeated 3
times with similar results.
710
NAIR ET AL.
intraperitoneally with OVA 1 DOTAP without DC were much less
effective at in vivo CTL induction as shown in Figure 1. Comparison between intraperitoneal and intravenous routes of immunization did not show any significant differences in the levels of
cytotoxicity generated (data not shown).
Figure 1 demonstrates that it is possible to induce an OVAspecific CTL in vivo following a single immunization with DC
pulsed with OVA protein and DOTAP. The aim of our study was to
determine if we could generate CTL responses using crude tumor
extracts instead of using purified protein antigens as a practical
alternative for active immunotherapy of cancer patients when
tumor antigens have not been identified. To determine if we could
generate OVA-specific and tumor-specific CTL using unfractionated tumor-derived antigen, we pulsed DC with tumor cell sonicates from EL4 thymoma cells and E.G7-OVA in the presence of
DOTAP. Splenocytes were harvested from immunized mice to
determine CTL activity (refer to Material and Methods). E.G7OVA, EL4, A20 and RMA-S pulsed with OVA peptide were used as
targets.
As shown in Figure 2, DC pulsed with OVA protein/DOTAP
were more effective at generating OVA-specific CTL responses
compared with DC 1 E.G7-OVA extract/DOTAP (70% lysis of
RMA-S cells pulsed with OVA peptide compared with 30% lysis),
whereas both groups were comparable in their ability to lyse
E.G7-OVA targets. Similarly, DC pulsed with EL4 or E.G7-OVA
cell sonicates plus DOTAP were equally effective at generating
EL4-specific CTL responses in vivo. This is also evident in Figure 2
as shown by the lysis of E.G7-OVA targets by effectors generated
from mice immunized with DC 1 EL4 extract/DOTAP. Thus a
single immunization with DC pulsed with unfractionated E.G7OVA cell sonicates primed OVA-specific CTL that were capable of
lysing E.G7-OVA (55% specific lysis), RMA-S pulsed with OVA
peptide (30% specific lysis) and EL4 cells (17% specific lysis).
Mice immunized with DC pulsed with E.G7-OVA extract in the
absence of DOTAP or with E.G7-OVA extract/DOTAP complexes
in the absence of DC generated insignificant E.G7-OVA-specific
CTL responses in vivo. Control targets A20 showed insignificant
lysis (data not shown). This experiment demonstrates that immunization with DC pulsed with extracts from OVA-expressing EL4
tumors is capable of eliciting CTL responses against OVA antigen
as well as undefined EL4 tumor antigens.
In vivo priming of tumor-specific CTL responses using DC
or Mø pulsed with unfractionated tumor material
To test whether unfractionated proteins from tumors can elicit
CTL responses in vivo, we used tumor cell extracts from MBT-2
(H-2k ) murine bladder tumor cells. The MBT-2 tumor cell line is a
poorly immunogenic tumor, as indicated by the fact that repeated
immunizations with irradiated MBT-2 cells fail to elicit a measurable CTL response (Connor et al., 1993; Saito et al., 1994; Fig. 3).
However, as we have previously shown, C3H (H-2k ) mice immunized 3 consecutive times at weekly intervals with genetically
modified MBT-2 cells expressing the human IL-2 gene (MBT-2/
IL-2) elicit a strong tumor-specific CTL response (Connor et al.,
1993; Saito et al., 1994; Fig. 3). As shown in Figure 3, under the
same experimental conditions, DC or Mø pulsed with MBT-2
FIGURE 4 – A single immunization with DC pulsed with MBT-2 or with L929 tumor cell extracts elicits antigen-specific CTL responses in vivo.
C3H mice were immunized with DC or Mø pulsed with MBT-2 or with L929 tumor extracts as described in Material and Methods. Ten days later
splenocytes were harvested and cultured in the presence of IL-2. CTL assay was done on day 5 with MBT-2, L929 and EL4 cells as targets. EL4
targets showed insignificant lysis (data not shown).
TUMOR EXTRACT-PULSED APC AND TUMOR IMMUNITY
extracts in the presence of the lipid DOTAP were also capable of
eliciting a strong CTL response, although DC were reproducibly
more effective than Mø. DC or Mø pulsed with tumor extracts in
the absence of DOTAP also elicited CTL, albeit, a low level. EL4
tumor cells used as control targets (H-2b ) showed no specific lysis.
Also shown is the fact that immunization with MBT-2 tumor
extract 1 DOTAP alone was consistently ineffective at inducing
CTL responses in vivo.
As shown in Figures 1 and 2, a single immunization with DC
pulsed with a defined CTL antigen, the chicken OVA protein, was
sufficient to induce a strong CTL response in vivo, while 3
immunizations with MBT-2 tumor extract-pulsed DC or Mø were
used to elicit the tumor-specific cytotoxic responses shown in
Figure 3. We therefore tested whether a single immunization with
unfractionated MBT-2 tumor extracts loaded onto APC would
suffice to generate CTL in vivo. As shown in Figure 4, a single
immunization with tumor extract-pulsed DC elicited a CTL response in vivo, whereas Mø pulsed with tumor extract were not
effective stimulators of CTL induction. Irradiated MBT-2/IL-2
cells were also incapable of CTL priming in vivo following a single
immunization. Similarly, a single immunization with DC, but not
Mø, pulsed with L929 (H-2k ) tumor extracts elicited an L929specific CTL response. These observations show that in this
experimental system DC pulsed with unfractionated tumor extracts
were superior to either Mø pulsed with tumor extracts or to
IL-2-secreting MBT-2 tumor cells in eliciting CTL in vivo. The
antigen specificity of the CTL response is illustrated by the fact that
DC pulsed with MBT-2 extracts generated CTL responses capable
711
of lysing only MBT-2 targets, and DC pulsed with L929 extracts
generated only CTL capable of lysing L929 cells. EL4 tumor cells
used as control targets (H-2b ) showed no specific lysis (data not
shown).
Treatment of MBT-2 tumor-bearing animals with APC
pulsed with tumor extracts
The mouse MBT-2 tumor is an excellent model for human
bladder cancer. Using experimental conditions that simulate as
closely as possible the conditions prevailing in the cancer patients,
we have previously shown that IL-2- or GM-CSF-secreting irradiated MBT-2 cell preparations were capable of curing animals of
orthotopically established tumors and also engendered protective
immunological memory in the cured animals (Connor et al., 1993;
Saito et al., 1994). In the experiment shown in Figure 5, tumors
were established orthotopically by implanting MBT-2 cells in the
bladder wall. Four days post-tumor implantation, mice were
vaccinated 3 times with APC pulsed with tumor extracts or with
irradiated tumor cells (MBT-2 and MBT-2/IL-2). All mice in the
group treated with irradiated MBT-2 cells or with DC pulsed with
L929 tumor extract died within 4 weeks. Mice treated with
irradiated IL-2-secreting MBT-2 cells had a modest survival
advantage, similar though less pronounced, than that observed in
previous studies (Connor et al., 1993; Saito et al., 1994).
The most pronounced therapeutic benefit was seen in mice
treated with DC or with Mø pulsed with MBT-2 extract. Two of 5
mice (40%) in each treatment group survived for over 60 days, after
which mice were challenged again with MBT-2 cells. Mice
FIGURE 5 – Treatment of tumor-bearing mice with APC pulsed with MBT-2 tumor extracts. Tumors were established in C3H mice by orthotopic
implantation of 2 3 104 MBT-2 cells into the bladder of the animal. Vaccinations consisting of 5 3 106 tumor cells or 2 3 106 APC pulsed with
tumor extracts were given as described in Material and Methods. Mice were vaccinated a total of 3 times and were evaluated on a daily basis and
sacrificed when moribund. Each treatment group consisted of 5 mice. Data are representative of 3 experiments performed with similar results.
Differences in survival for the groups MBT-2/IL-2, Mø 1 MBT-2 extract and DC 1 MBT-2 extract are significant compared with the control
MBT-2 group, based on log-rank analysis ( p 5 0.0017 for MBT-2/IL-2 and p 5 0.0013 for Mø 1 MBT-2 extract and DC 1 MBT-2 extract). p 5
0.395 for the control DC 1 L929 extract group compared with the MBT-2 group. The overall significance of this study is p 5 0.0001 based on the
Kaplan-Meier test.
712
NAIR ET AL.
immunized with DC or Mø pulsed with tumor extracts in the
presence of DOTAP survived the rechallenge and were sacrificed
on day 90. In a separate experiment, all survivors were tested for
CTL activity and demonstrated MHC-restricted, MBT-2-specific
cytotoxic responses (data not shown). This experiment therefore
suggests that vaccines based on DC or Mø pulsed with unfractionated tumor extracts are equally or more potent than IL-2-secreting
MBT-2 cells as vaccines.
Induction of tumor immunity in the B16/F10.9
melanoma metastasis model
The potency of APC pulsed with unfractionated tumor extracts
was further evaluated in the B16/F10.9 (H-2b ) melanoma metastasis model. The B16/F10.9 melanoma tumor is poorly immunogenic, expresses low levels of MHC class I molecules and is highly
metastatic in both experimental and spontaneous metastasis assay
systems (Porgador et al., 1995). Porgador et al. (1995) have shown
that when vaccinations are carried out after the removal of the
primary tumor implant, only irradiated tumor cells transduced with
both the IL-2 and the H-2Kb genes were capable of significantly
impacting the metastatic spread of B16/F10.9 tumor cells in the
lung. Thus, the B16/F10.9 melanoma model and the experimental
design used by Porgador et al. (1995) constitute a stringent and
highly informative experimental system to assess the efficacy of
adjuvant treatments for metastatic cancer.
We first tested whether a single immunization of C57BL/6
(H-2b ) mice with DC or Mø pulsed with extracts from B16/F10.9
cells elicits a CTL response against B16/F10.9 cells, or against
B16/F10.9 cells expressing H-2Kb (F10.9/K1 cells). As shown in
Figure 6, only DC pulsed with B16/F10.9 tumor extracts were able
to elicit a strong tumor-specific CTL response against either target,
while tumor extract-pulsed Mø elicited a barely detectable CTL
response. No CTL could be detected in mice immunized with
irradiated B16/F10.9 cells, or with DC or Mø pulsed with EL4
tumor extracts. In another experiment EL4 cells and BALB/3T3
cells were also used as control targets. No lysis of BALB/3T3 cells
and EL4 was observed when effectors were generated using DC
pulsed with F10.9 cells (data not shown). As shown in Figure 6,
F10.9 tumor extracts 1 DOTAP in the absence of DC or Mø were
not capable of priming F10.9-specific CTL responses in vivo, even
after 3 consecutive immunizations.
To test whether immunization with DC or Mø was capable of
causing the regression of pre-existing lung metastases, primary
tumors were induced by implantation of B16/F10.9 tumor cells in
the footpad. When the footpad reached 5.5–7.5 mm in diameter, the
tumors were surgically removed, and 2 days later mice were
immunized with irradiated B16/F10.9 cells, or with APC pulsed
with tumor extract (Fig. 7). The mice received a total of 3
vaccinations given at weekly intervals. The average lung weight of
a normal mouse is 0.18–0.22 g. Mice immunized with irradiated
B16/F10.9 cells were overwhelmed with metastases. The mean
lung weight of mice in this treatment group was 0.84 g, about
three-quarters of the weight was contributed by the metastases,
which were too many to count. A similar metastatic load was seen
in animals treated with PBS (data not shown), which confirms
numerous previous observations that treatment with irradiated
B16/F10.9 tumor cells alone has no therapeutic benefit in this
tumor model (Porgador et al., 1995). As also previously shown,
immunization with H-2Kb expressing B16/F10.9 cells (F10.9/K1)
had a modest therapeutic benefit, as indicated by a statistically
FIGURE 6 – Induction of tumor-specific CTL following a single immunization with DC pulsed with B16/F10.9 tumor extracts. DC or Mø were
pulsed with extracts derived from B16/F10.9 tumor cells. C57BL/6 mice were immunized once i.p.; splenocytes were then harvested 10 days later
and restimulated with irradiated K1 cells (B16/F10.9 cells expressing H-2Kb ). CTL assay was done 5 days later with F10.9 cells and F10.9/K1
cells as targets. DC and Mø pulsed with EL4 cell extract were used as controls for antigen specificity.
TUMOR EXTRACT-PULSED APC AND TUMOR IMMUNITY
713
FIGURE 7 – Regression of lung metastases in mice treated with APC pulsed with F10.9 extracts. B16/F10.9 tumors established in the footpads of
C57BL/6 mice were amputated when they reached 5.5–7.5 mm in diameter. Two days after the amputation, and weekly thereafter, mice were
vaccinated i.p. for a total of 3 vaccinations. Mice were sacrificed 28–32 days post-amputation (as determined by the metastatic death monitored for
the control F10.9 group), and lung weights and metastatic loads were determined. Columns represent mean lung weight, and dots represent
individual lung weights (6–7 mice/group). The results are representative of 3 different experiments. For more details see Material and Methods.
Relative to the control F10.9 immunized group, p values were 0.0379, 0.62, 0.0012 and 0.0023 for F10.9/K1, DC 1 EL4 extract, DC 1 F10.9
extract and Mø 1 F10.9 extract immunized mice, respectively. The overall significance of the study as determined by the Kruskal-Wallis test is
p 5 0.0002.
NAIR ET AL.
714
significant decrease in the average lung weight of the animals in
this treatment group. One of 7 animals in this group had no visible
metastasis, 4 of 7 animals had 20–35 nodules and 1 mouse did not
respond to treatment (. 100 nodules).
A more marked response was seen in the animal groups treated
with APC pulsed with tumor extract. The mean lung weight of mice
treated with DC pulsed with B16/F10.9 tumor extract was 0.30 g,
only 33% above the normal lung weight. Four mice in this group
(n 5 7) had no visible nodules, 2 mice had less than 5 nodules and
1 mouse had 15 nodules. Mø were almost as effective as DC, the
average lung weight being 0.38 g, 80% above the normal lung
average. In this treatment group (n 5 7) 3 mice had no visible
metastasis, 3 mice had between 5 and 10 nodules and 1 mouse had
22 nodules. The antigen specificity of the therapeutic benefit seen
in this experiment is indicated by the fact that no decrease in
metastatic load was seen in mice treated with DC pulsed with EL4
tumor extract.
DISCUSSION
In this study we have shown that immunization with unfractionated tumor extracts presented by professional APC, in particular
DC, elicits potent anti-tumor immunity in mice. To enhance the
relevance of these results to human patients, we used two poorly
immunogenic murine tumor models and evaluated the effectiveness
of the vaccination protocols in tumor-bearing animals.
Neither B16/F10.9 nor MBT-2 tumor cells alone were capable of
eliciting CTL, a fact that reflects the poor intrinsic immunogenicity
of the tumors used here. On the other hand, APC pulsed with
unfractionated extracts from these ‘‘non-immunogenic’’ tumors
were highly effective in eliciting tumor-specific CTL. A measurable
CTL response could be detected after even a single immunization
with B16/F10.9- or MBT-2 extract-pulsed DC (Figs. 4, 6). The
observation that 3 consecutive immunizations with IL-2-secreting
MBT-2 cells were required to induce a measurable CTL response
suggests that MBT-2 tumor extract-pulsed DC are more potent
inducers of CTL than the genetically engineered, IL-2-secreting
tumor cells.
DC or Mø pulsed with tumor extract were also effective vaccines
in tumor-bearing animals. A modest extension of survival and 40%
cure rate were seen in the animal groups immunized with DC or Mø
pulsed with MBT-2 tumor extract, while immunization with
IL-2-secreting MBT-2 cells led to extended survival but no cures.
This observation, together with the CTL data presented in Figures 3
and 4, shows that DC pulsed with MBT-2 tumor proteins are more
potent inducers of tumor immunity than IL-2-secreting MBT-2
tumor cells (Connor et al., 1993; Saito et al., 1994).
DC or Mø pulsed with tumor extract were also remarkably
effective in a metastasis tumor model. The B16/F10.9 melanoma
tumor system used in this study is an excellent model for minimal
residual metastatic disease; it measures the efficacy of adjuvant
therapy in reducing the growth of pre-existing lung metastases in
animals in which the primary tumor is surgically removed. The
only treatment that has shown significant therapeutic benefit in this
disease model was vaccination with doubly transduced tumor cells,
B16/F10.9 cells transduced with both the IL-2 and H-2Kb genes
(Porgador et al., 1995). As shown above (Fig. 7), treatment of the
tumor-bearing mice with DC or Mø pulsed with tumor extract
caused a significant reduction in lung metastasis. The magnitude of
the effect, especially in the group treated with tumor extract-pulsed
DC, was similar to the effect reported with the doubly transduced
vaccine (Porgador et al., 1995).
Cumulatively, the CTL and immunotherapy data from the 2
murine tumor systems suggest that vaccines based on APC, in
particular DC, pulsed with unfractionated cell extracts as a source
of tumor antigen may be equally or more effective than genetically
modified tumor vaccines. However, additional studies will be
required to assess more definitively the comparative value of
APC-based versus genetically modified tumor cell-based vaccines,
or the use of DC vs. Mø as APC pulsed with tumor antigen. We are
currently characterizing the active components in the tumor
extracts and studying the cellular and immunological mechanisms
responsible for the induction of tumor immunity mediated by APC.
Vaccination with tumor extracts circumvents the need for
identifying specific tumor rejection antigens and hence extends the
use of active immunotherapy to the vast majority of cancers, in
which specific tumor antigens have not been identified. Zitvogel et
al. (1996) have shown that vaccination of mice with bone
marrow-derived DC pulsed with unfractionated acid-eluted tumor
peptides was capable of reducing the growth of subcutaneously
established, weakly immunogenic tumors. One potential drawback,
however, of immunizing with unfractionated tumor material,
compared with the use of defined tumor antigens, is the increased
risk of inducing autoimmune responses, with pathological consequences. While no evidence of autoimmunity was seen in the
current or previous animal vaccination studies employing unfractionated tumor material as a source of tumor antigens, the
development of increasingly potent vaccines may very well lead to
some autoimmune manifestations.
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