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Immunology |
Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 [R. T. R., M. B. C. G., A. M. E., J-P. H. M., C. E. K., F. I. O., E. M. J.]; Genetic Therapy, Inc., Gaithersburg, Maryland 20878 [K. H. D.]; and Institute for Molecular Biology and Biotechnology, McMaster University, Hamilton, Ontario, Canada L8S 4K1 [W. J. M.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MART-1, gp-100, tyrosinase, and neu3 are among an increasing number of naturally occurring human TAs that have been demonstrated to be T-cell targets (8, 9, 10, 11, 12) . These antigens have the potential to serve as tumor rejection antigens in vivo. Interestingly, these antigens are tumor-associated or differentiation antigens rather than antigens that are only expressed by the tumor. Thus, these antigens are self-antigens against which natural mechanisms of T-cell deletion or peripheral tolerance are expected to occur. These findings provide strong evidence that T cells that target antigens expressed by spontaneously arising tumors may be susceptible to tolerizing mechanisms in the host.
TA transgenic mouse models are being developed to specifically dissect the mechanisms of TA-directed tolerance and to identify more potent vaccine strategies that have the potential to overcome these mechanisms of tolerance. TA transgenic mice, such as those that express carcinoembryonic antigen (13, 14, 15) , prostate-specific antigen (16) , HA (17) , Friend murine leukemia virus envelope protein (18) , and MUC-1 (19) seem to be more clinically relevant models because antigen-specific tolerance has been shown to occur against the protein encoded by the transgene. However, these mice fail to demonstrate antigen-associated spontaneous tumor development. In contrast, TA transgenic mice that express the v-Ha-ras oncogene develop spontaneous breast cancers. However, these spontaneous breast cancers are highly immunogenic, and the T-cell response is not directed against the transgene-encoded ras oncogene (20) . Spontaneous tumor development is also seen in the TRAMP mouse model for prostate cancer (21 , 22) , however, the immunological response to tumor in this model has not been fully characterized.
neu is an attractive target for enhancing antitumor immunity because
neu-specific antibody (23
, 24)
and T-cell
(25, 26, 27, 28, 29)
responses have been demonstrated in patients with
neu-expressing mammary and ovarian cancers. Yet, neu-expressing tumors
in these same patients continue to grow and metastasize, indicating
that immune tolerance exists to the proto-oncogene. Two types of neu
transgenic mice have also been developed. neu-N transgenic
mice developed by Guy et al. (30)
, which
were derived from the FVB/N strain, overexpress the nontransforming rat
neu cDNA under the control of a mammary-specific promoter.
As a consequence, these mice develop spontaneous focal mammary
adenocarcinomas beginning at approximately 125 days, with the majority
of mice developing spontaneous tumors by 300 days. In a similar
transgenic strain expressing the activated neu oncogene
neu-T (31
, 32)
, in which a point mutation
renders the neu gene product constitutively active, animals
rapidly develop spontaneous mammary tumors (100% of animals develop
tumors by 
30 weeks of age) with total glandular involvement,
indicating that neu-T overexpression alone is sufficient for
mammary carcinogenesis. These mice do not seem to exhibit tolerance to
neu (33
, 34)
.
The stochastic appearance of spontaneous mammary tumors in the neu-N mice suggests that, as in the clinical setting, mammary carcinogenesis occurs through a multistep process in which neu-N overexpression is an early and necessary event (30 , 35 , 36) . Because neu is expressed as a transgene in neu-N mice, these animals would, likewise, be expected to demonstrate similar tolerance to neu. We describe here the immunological characterization of the neu-N transgenic mouse model of breast cancer. Our findings demonstrate that tolerance to neu exists in these mice relative to nontransgenic mice. In addition, it is possible to induce neu-targeted protective immunity in the neu-N mice potent enough to overcome tolerance and significantly delay transplantable and spontaneous neu-expressing tumor development.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Lines and Media.
Five neu-expressing mammary tumor cell lines, designated NT1–NT5, were
derived from spontaneous mammary tumors in female neu-N
mice. In vitro cell lines were established by digestion of
spontaneous tumors with dispase and collagenase (Roche Molecular
Biochemicals, Indianapolis, IN), followed by differential
trypsinization to remove fibroblasts (37)
. NT lines were
grown in our defined Breast Media, which consisted of RPMI (Life
Technologies, Inc., Grand Island, NY) with 20% fetal bovine serum
(Hyclone Laboratories, Logan, UT), 1%
L-glutamine, 1% non-essential amino
acids, 1% Na pyruvate, 0.5% penicillin/streptomycin, 0.02%
gentamicin (JRH Biosciences, Lenexa, KS), and 0.2% insulin (Eli Lilly
and Co., Indianapolis, IN), and maintained at 37°C in 5%
CO2. NIH-3T3 cells (ATCC, Manassas, VA)
were grown in 3T3-media: DMEM (Life Technologies, Inc.) with 10%
bovine calf serum, 1% L-glutamine, 1% Na
pyruvate, 1% non-essential amino acids, and 0.5%
penicillin/streptomycin at 37°C and 10% CO2.
The 3T3-neu cell line ATCC CRL-1915 (ATCC), which expresses rat neu
(38)
, was grown in 3T3 media + 0.3
µM methotrexate at 37°C in 10%
CO2. The 3T3 and 3T3-neu lines were genetically
modified to express mGM-CSF by retroviral transduction with the MFG
murine GM-CSF-encoding retrovirus, using previously described methods
(39)
to produce the 3T3/GM and 3T3-neu/GM cell
lines. This method of mGM-CSF gene transfer results in high-efficiency
gene delivery (100% of cells) and, therefore, does not require
subsequent cell selection (39
, 40)
. mGM-CSF production was
assessed with a commercially available ELISA kit (Endogen, Woburn, MA)
and was determined to be 200–250 ng/106 cells/24
h for 3T3/GM and 3T3-neu/GM cells. mGM-CSF production by the parental
and tumor cell lines (3T3, 3T3-neu, NT2, and NT5) was not detectable by
ELISA. Functional assessment of mGM-CSF production was performed using
the mGM-CSF-dependent cell line NSF-60 (39)
. The NT2 and
NT5 tumor lines were expanded to large numbers to produce master cell
banks of each line to avoid extensive in vitro passage and
to insure the reproducibility of each in vivo study.
Production was performed at the NIH cGMP facility (Frederick, MD). neu
and MHC I levels were tested by FACS and confirmed to be stable before
freezing and storage in liquid nitrogen.
Flow Cytometry Analysis.
NT cell lines were assayed by flow cytometry using antibodies against
murine MHC I (28-14-8), human MHC I (W632), and the neu protein (neu
Ab4; Oncogene Science, Cambride, MA). A fluorescein-conjugated goat
antimouse IgG2a secondary antibody was used to detect expression of
surface molecules. To determine neu-specific serum IgG titers, 3T3
cells, and 3T3-neu cells were stained using mouse serum as
the primary antibody. A fluorescein-conjugated goat antimouse 
-chain
pan IgG secondary antibody was used to detect bound serum IgG (Vector
Laboratories, Inc., Burlingame, CA). A FACScan Flow Cytometer and
CellQuest Software (Becton Dickinson, San Diego, CA) were used to
acquire and analyze data.
PCR Primers and RT-PCR.
The thymus was removed from neu-N mice at gestational day
18, age 7 days, age 10 weeks, or age 15 weeks (1 week after weaning
from pups); and the thymocytes were separated from the thymic stroma
using a cell strainer. RNA was then isolated using RNAzol, and reverse
transcription was performed using DNase I, RNase Inhibitor, and AMV
Reverse Transcriptase (Life Technologies, Inc.) according to the
manufacturer’s specifications. The 5'- and 3'-neu-specific primers
5'-ATTCATCATTGCAACTGTAGA-3' and 5'-AAGCACCTTCACCCTTCCTTA-3',
respectively, amplify a 222-bp region between bases 2140 and 2362 of
the rat neu-N cDNA. Primers that amplify a 260-bp fragment
of ß-2 microglobulin (41)
were used as standard and
positive control in these assays.
Whole-Cell Vaccinations.
On the day of vaccination, cells grown in vitro were
trypsinized, washed three times in HBSS (pH 7.4), and counted. The
cells were resuspended in HBSS at 107 cells/ml
and irradiated with 5,000 rad from a 137Cs source
discharging 1400 rad/min. neu-N or FVB/N mice, 8 weeks of
age, were given three 100-µl s.c. injections (right and left
forelimb, left hind limb) using a 1-ml tuberculin syringe with a
27-gauge needle.
rVV Vaccinations.
To generate recombinant vaccinia expressing neu-N
(neu rVV), the 5-kb HindIII/SalI
fragment from pSV2-neu-N was cloned into pSC11–1, and rVV
was prepared and amplified as described (42)
. neu
expression was verified by FACS analysis of neu rVV-infected 3T3 cells.
neu-N mice, 8 weeks of age, received four weekly injections
of 3 x 107 pfu/animal given i.p.
using a 1-ml tuberculin syringe with a 27-gauge needle. rVV expressing
the influenza HA protein HA rVV, used as a negative control, was a gift
from Dr. Hyam Levitsky (Department of Oncology, The Johns Hopkins
School of Medicine, Baltimore, MD).
Tumor Challenge.
To titrate NT cell lines in vivo, neu-N or FVB/N
mice, 8 weeks of age, were given s.c. injections of NT cells in HBSS at
doses ranging from 1 x 103 cells
to 5 x 105 cells for
neu-N mice and 5 x 105
to 1 x 107 cells for FVB/N mice.
Animals were then monitored twice weekly for the development of
palpable (>5 mm diameter) tumors at the challenge site. For tumor
challenge experiments, neu-N mice were given 5 x 104 NT cells s.c. in the right hind limb
14 days after receiving a whole-cell vaccine or 7 days after the final
rVV vaccination.
T-Cell Assays.
ELISPOT analysis for neu-specific T cells was performed using
splenocytes isolated from neu-N mice. Mice were given a s.c.
NT2 challenge, followed 3 days later by vaccination with
3T3-neu/GM or 3T3/GM. On day 12 after the vaccine,
splenocytes were isolated by Ficoll separation and passed through a
nylon wool column to remove B-cell and macrophage contaminants.
neu-specific IFN- production was determined by a standard ELISPOT
protocol (42)
following a 24-h incubation of 5 x 105 NT2 target cells/well with serial
dilutions of T cells (1 x 105
cells/well to 1 x 103 cell/well).
Reagents used in the assay were: 10 µg/ml rat antimouse IFN-
(PharMingen, San Diego, CA), 2 µg/ml biotinylated rat antimouse
IFN- (PharMingen), 2 µg/ml avidin-alkaline phosphatase (Sigma
Chemical Co., St. Louis, MO), and 5-bromo-4-chloro-3-indolyl
phosphate/Nitro Blue Tetrazolium (Sigma Chemical Co.).
neu-specific lysis was determined as described previously
(42)
. Briefly, spleens were excised from mice vaccinated 2
weeks before assay. Splenocytes were isolated by Ficoll separation and
incubated for 5 days in the presence of murine IL-2 and IFN- and
mitomycin-C-treated NT2 cells. Lytic function was determined against
3T3 and 3T3-neu cells in a 4-h chromium-51 release assay
with and without the CD8-blocking rat monoclonal antibody 2.43 and the
CD4-blocking rat monoclonal antibody GK1.5. The percentage of
neu-specific lysis was determined by the following formula: %
neu-specific lysis = (% lysis against
3T3-neu targets) - (% lysis against 3T3
targets).
T-Cell Depletion.
The depletion of CD4+ and
CD8+ T-cell subsets was accomplished by i.p.
injection of 500 µg GK1.5 (anti-CD4) or 2.43 (anti-CD8) antibody,
respectively, given every other day for 3 days. Depletion of
CD4+ and CD8+ T cells was
verified by FACS analysis of splenocytes 1 day after the three
injections and maintained by continuing the antibody injections twice
weekly for the duration of the tumor challenge experiment. Spleens were
excised from additional animals in each group throughout the experiment
to confirm the depletion of lymphocyte subsets. The anti-NK cell
antibody pk136 was used as a negative control.
Prevention of Spontaneous Tumor Formation.
Because lactation hastens spontaneous tumor formation in
neu-N
mice,4
females, 8–10 weeks of age, were allowed to breed and the pups were
weaned 3 weeks after birth. The parous neu-N females (20
weeks of age, 3 weeks after weaning from pups) were given a series of
five weekly injections of 1 x 107
pfu neu rVV (vaccine group) or no treatment (control group) and were
monitored for the development of spontaneous mammary tumors.
Statistical Analyses.
Statistical analyses were performed using the Statview software
program. Kaplan-Meier nonparametric regression analyses for
tumor-protection and tumor-prevention experiments were performed, and
significance was determined using the Mantel-Cox log rank test.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). NT2 and NT5 cells were each expanded to generate a certified master
cell bank of each line at the National Cancer Institute cGMP laboratory
(Frederick, MD). Each lot demonstrated stable neu and MHC I expression
when grown in vitro for up to 3 months (data not shown).
Frozen aliquots of each lot were thawed, expanded for 1 week in
vitro, and used in tumor challenge experiments. Both cell lines
were tested at least twice in each of the experiments described below.
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. In contrast, neu-N mice demonstrated significant tolerance
to the neu-expressing tumor line; the minimum dose required for tumor
growth in 100% of animals was at least 100-fold lower for the
transgenic mice compared with FVB/N mice. Data obtained using the NT2
cell line were virtually identical to what was seen with an NT5 tumor
challenge (data not shown).
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, the data indicated that neu expression is seen in parous adult thymi
but not in samples from fetal or virgin adult mice. In addition,
low-level neu expression was also found in newborn mice. The apparent
discrepancy in thymic expression of neu in newborn and virgin adult
mice may be explained by the exposure of the newborns to hormones in
their milk that activated the MMTV promoter driving the rat
neu-N transgene. Control samples taken from parental FVB/N
animals showed no detectable neu expression.
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, as well as the generation of neu-specific CTLs (data not shown).
Because the transgenic mice demonstrated profound tolerance to
neu-expressing tumors in vivo, we sought to determine
whether or not a neu-specific immune response analogous to that
observed in the parental animals could be induced by vaccination. As
with the FVB/N animals, neu-N mice were vaccinated with
either 3T3/GM or 3T3-neu/GM, and serum samples were obtained
14 days after vaccination. Titers of neu-specific serum IgG were
determined by FACS analysis of serial dilutions of mouse serum. In
contrast to FVB/N mice, transgenic mice demonstrate low basal levels of
neu-specific IgG (Fig. 4)
. Vaccination with a GM-secreting
3T3-neu vaccine resulted in an increase in neu-specific IgG
relative to mock-vaccinated animals, although the level of induction
was much less than that seen in FVB/N mice.
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production by ELISPOT assay. As shown in Fig. 5A
, mice receiving a neu-specific vaccine showed a 3–4-fold
increase in the number of T cells producing IFN- in response to NT2
cells relative to mock-vaccinated mice. Further evidence of
neu-specific effector function was demonstrated by chromium-51-release
assay using 3T3-neu cell targets. Animals were vaccinated
with 1 x 106 irradiated 3T3 or
3T3-neu/GM cells, and splenic T cells were isolated 14 days
later. T cells, cultured for 5 days in the presence of interleukin 2
and IFN- and mitomycin-C-treated NT2 cells, were assayed for the
lysis of 3T3 or 3T3-neu target cells in a 4-h
chromium-release assay. As shown in Fig. 5B
, splenocytes
from 3T3-neu/GM-vaccinated mice demonstrated
CD8+ T cell-dependent lysis that is not seen with
splenocytes from 3T3/GM vaccinated control mice. This neu-specific
lysis is almost completely abrogated by the addition of the CD
8-blocking antibody 2.43.
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, neu-N mice given a 3T3-neu/GM
vaccine demonstrated a significant delay in NT2 tumor development
relative to mice treated with the control 3T3/GM vaccine
(P < 0.0005). Although unlikely, 3T3 cells
may express antigens other than neu in common with the mammary tumors.
However, this delay in transplantable tumor development was confirmed
to be neu-specific by vaccinating neu-N mice with
rVV-expressing neu. Mice vaccinated with one i.p. injection of
rVV-expressing neu demonstrated as significant delay in NT2 tumor
growth compared with mice vaccinated with a control rVV expressing the
irrelevant HA antigen (P < 0.01). Similar
results were obtained in two additional experiments in which animals
received an NT5 challenge (data not shown). In contrast, FVB/N mice,
which do not exhibit tolerance to neu, were completely protected from
tumor challenge after identical vaccine protocols (Fig. 6B)
.
These data clearly demonstrate that, although profound tolerance to neu
exists in neu-N mice, neu-specific vaccination can generate
immune responses that are potent enough to significantly delay the
growth of transplantable neu-expressing tumors.
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, show that mice depleted of CD4+ T cells develop
tumors with kinetics that are similar to that seen in unvaccinated,
undepleted controls. The deletion of CD8+ T cells
has a less dramatic effect on tumor growth, although NT2 growth is
statistically distinct from vaccinated, undepleted animals. The
tumor-free survival of animals given the NK cell-depleting antibody
pk136 and vaccine were identical to that of undepleted, vaccinated
mice. These data establish the importance of both
CD4+ and CD8+ T cells in
inducing neu-specific immunity.
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20 weeks of age). The animals were then monitored weekly for the
development of spontaneous tumors. As shown in Fig. 7
, there was a significant delay in the onset of tumor formation in the
vaccinated mice by 7 weeks after the initial vaccination, relative to
unvaccinated controls (P = 0.02). Nearly
identical results were obtained using whole-cell vaccination
(3T3-neu/GM; data not shown), demonstrating that the
neu-specific vaccines can delay tumor onset and suggesting that more
complete protection may be possible.
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DISCUSSION |
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It is clear from our data that neu tumors are highly immunogenic in nontransgenic mice in which tolerance to neu is absent. This is consistent with the findings of Bernards et al. (43) and Chen et al. (44) , who induced protective immunity in nontransgenic NFS and FVB/N mice, respectively, that was sufficient to give protection from a neu-expressing tumor challenge. Similarly, Cefai et al. (34) and Amici et al. (33) demonstrated potent antitumor immunity using the FVB/N neu-T mammary tumor model, in which mice are not tolerant to the transgene-encoded neu-T oncoprotein. In separate studies using neu-specific whole-cell or plasmid DNA vaccinations, it was shown that neu-T mice were completely protected from neu-expressing tumor challenge (34) , and spontaneous tumor development could also be prevented (33 , 34) . Thus, our data confirm that neu is a relevant tumor rejection target in nontransgenic immunocompetent mice. However, our data also demonstrate that neu is a relevant rejection target recognized by T cells in tolerized transgenic mice.
The presence of tolerance to a TA in vivo represents a significant challenge to successful immunotherapy of human cancers. For example, patients with breast cancer show neu-specific responses in the form of antibody and CTL, but these responses are not sufficient to prevent tumor progression (23 , 24 , 29) . We have made a similar observation in the neu-N mice. Despite the fact that we were unable to detect neu expression in the thymus of fetal neu-N mice, the central deletion of high-avidity neu-specific T-cell populations is implicit in the qualitative and quantitative differences in neu-specific immune responses seen in FVB/N mice versus neu-N mice. It is possible that neu expression in the thymus, although below the detection level of RT-PCR, was sufficient to cause the deletion of high-avidity neu-specific T-cell precursors. Alternatively, because neu expression is seen in a number of tissues in neu-N mice (30) , it is conceivable that neu antigen was acquired by antigen-presenting cells in the periphery and trafficked to the thymus for presentation during T-cell development. neu expression is demonstrated in the thymus at times when the mice have higher levels of peripheral neu expression (i.e., when prolactin levels, an inducer of the MMTV promoter, are high during nursing or pregnancy). Therefore, it is probable that higher-avidity T cells that are present in the FVB/N mice and are responsible for the tumor immunogenicity observed have undergone deletion in the transgenic mice (45) . The fact that antibody and T-cell responses are induced in neu-N mice after vaccination but are not fully protective strongly suggests that the T cells present in neu-N mice may well be of lower avidity and have more stringent requirements for activation and effector function. Consistent with this interpretation is our recent finding that there is a predominant oligoclonal TCR Vß usage among the vaccine-induced FVB/N-derived T cells that is very different from the predominant oligoclonal TCR Vß usage observed among the vaccine-induced transgenic-derived T cells.5 It is also possible that the lower-avidity T cells undergo peripheral anergy induction in the transgenic mice.
We have shown that two neu-specific vaccines can generate antitumor
immunity in neu-N mice capable of significantly delaying
tumor growth. These results clearly implicate neu as the in
vivo rejection antigen. The fact that FVB/N mice are fully
protected from a 100-fold greater tumor burden after an identical
vaccination is indicative of the profound tolerance to neu in the
neu-N mice. Depletion experiments carried out in
neu-N mice demonstrated that the significant delay in tumor
development is both CD4+ and
CD8+ T cell mediated. It is unlikely that
CD4+ T cells play a direct role in tumor cell
lysis because significant MHC II levels cannot be induced on NT cells,
even after treatment with -IFN (data not shown). Thus, the role of
CD4+ T cells in tumor rejection seems to be at
the level of T-cell help. Likewise, CD8+ T-cell
depletion abrogates the tumor-protective effects of neu-specific
vaccination, although the delayed tumor growth observed in
CD8+ T cell-depleted mice is not as great as that
seen in the undepleted, vaccinated mice. This may reflect the fact that
the undepleted, vaccinated mice are capable of both neu-specific
antibody production and CTL induction. It is likely that the
vaccine-induced CD4+ T-cell response up-regulates
neu-specific humoral and CTL responses and that each contributes to the
retardation of tumor growth.
Our studies also show that it is possible to induce protective immunity capable of delaying the development of spontaneous mammary tumors in the transgenic mice. The modest delay in tumor onset seen in our prevention studies is consistent with the delays seen in the tumor challenge model using virgin neu-N mice, thus giving validation to the use of transplantable tumors for the initial evaluation of vaccine approaches for tumor prevention. It is unlikely that neu-specific immune responses differ greatly in virgin neu-N mice vaccinated before inoculation with transplantable tumors versus vaccinated postlactational neu-N mice in which spontaneous tumors develop. Our investigation of neu-specific T-cell and antibody responses in virgin neu-N females, 8–10 weeks of age, clearly indicates these mice are already profoundly tolerant to neu. The prevention of spontaneous tumor formation has been reported in the neu-T transgenic mouse model using i.p. injections of antibody against neu (46) as well as whole-cell and plasmid DNA immunization (33 , 34) , demonstrating the successful use of neu-specific immunotherapy for tumor prevention in the absence of tolerance to neu. The fact that we were able to delay tumor onset in neu-N mice is significant in that it demonstrates that we can generate a neu-specific response in neu-N transgenic mice that is capable of overcoming in vivo tolerance and is sufficient to significantly delay spontaneous tumor development. Esserman et al. (47) also demonstrated vaccine-mediated prevention of spontaneous tumors using neu-N mice. Vaccination of mice that were heterozygous for the neu-N proto-oncogene with the neu extracellular domain was sufficient to prevent spontaneous tumor development in 50% of transgenic mice. The improved vaccine efficacy in their tumor prevention studies could be due to a number of factors. First, in our studies, we used mice that were homozygous for the neu-N transgene. It is possible that differences in neu expression in heterozygous versus homozygous mice results in different levels of tolerance to neu. Second, our protection experiments used postlactational neu-N mice. Whereas tolerance to neu is unlikely to differ significantly in postlactational versus virgin females, the increase in hormones such as prolactin that are associated with milk production are known to increase expression by the MMTV promoter, which drives neu expression in these mice. neu expression in the thymus, which is not detected in fetal or virgin females, is clearly up-regulated in nursing and postlactational mice. Third, these studies use whole-cell and rVV vaccines expressing the entire neu protein as opposed to plasmid DNA vaccination with the neu extracellular domain. Fourth, the timing of neu-specific vaccination may be important for vaccine efficacy. Esserman et al. (47) vaccinated heterozygous virgin neu-N females very early, at 10 weeks of age, well before the onset of tumorigenesis, and boosted at 24 weeks of age. In our model, animals were not vaccinated until 20 weeks of age, possibly at a time when these animals have significant, although not macroscopic, tumors.
Additional work is required to determine whether the level of transgene expression influences the extent of tolerance to neu, or whether lactational status has an effect on vaccine-mediated antitumor immunity. Furthermore, now that it is clear that neu-specific antitumor immunity can be induced in neu-N mice despite tolerance, the next step is to determine what factors are necessary for the improvement of vaccine efficacy.
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FOOTNOTES |
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1 Supported by Department of Defense and National
Cooperative Drug Discovery Group Grant DAMD17-96-6138 (to E. M. J.),
Grant U19CA72108 (to E. M. J.), NIH Grant T32A107247 (to R. T. R.),
the AACR–Cancer Research Foundation of America Fellowship in
Prevention Research (to R. T. R.), a grant from Belgian Fonds
National de la Recherche Scientifique-Televie (to J-P. H. M.), and a
grant from Oeuvre Belge du Cancer (to J-P. H. M.). W. J. M. is a
Medical Research Council of Canada Scientist. 
2 To whom requests for reprints should be
addressed, at Bunting-Blaustein Cancer Research Building, 1650 Orleans
Street, Room 4 M07, Baltimore, MD 21231.
3 The abbreviations used are: TA, tumor antigen;
neu, HER-2/neu; neu-N, wild-type
HER-2/neu; neu-T, mutated transforming
HER-2/neu; NK, natural killer; FACS,
fluorescence-activated cell sorter; ELISPOT, enzyme-linked immunospot
assay; rVV, recombinant vaccinia virus; pfu, plaque-forming unit;
mGM-CSF, murine granulocyte-macrophage colony-stimulating factor; HA,
hemagglutinin; 3T3, NIH-3T3; 3T3/GM, 3T3 cells producing mGM-CSF;
3T3-neu, NIH-3T3 cells expressing rat
neu-N; 3T3-neu/GM, 3T3-neu
cells producing mGM-CSF; ATCC, American Type Culture Collection;
RT-PCR, reverse transcription-PCR.
4 R. Reilly, unpublished observations.
5 A. Ercolini and E. Jaffee, personal
communication.
Received 9/ 9/99. Accepted 4/25/00.
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P. Nanni, G. Nicoletti, A. Palladini, S. Croci, A. Murgo, A. Antognoli, L. Landuzzi, M. Fabbi, S. Ferrini, P. Musiani, et al. Antimetastatic Activity of a Preventive Cancer Vaccine Cancer Res., November 15, 2007; 67(22): 11037 - 11044. [Abstract] [Full Text] [PDF]
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S. L. Kominsky, B. Tyler, J. Sosnowski, K. Brady, M. Doucet, D. Nell, J. G. Smedley III, B. McClane, H. Brem, and S. Sukumar Clostridium perfringens Enterotoxin as a Novel-Targeted Therapeutic for Brain Metastasis Cancer Res., September 1, 2007; 67(17): 7977 - 7982. [Abstract] [Full Text] [PDF]
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H.-J. Ko, Y.-J. Kim, Y.-S. Kim, W.-S. Chang, S.-Y. Ko, S.-Y. Chang, S. Sakaguchi, and C.-Y. Kang A Combination of Chemoimmunotherapies Can Efficiently Break Self-Tolerance and Induce Antitumor Immunity in a Tolerogenic Murine Tumor Model Cancer Res., August 1, 2007; 67(15): 7477 - 7486. [Abstract] [Full Text] [PDF]
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E. M. Wall, K. Milne, M. L. Martin, P. H. Watson, P. Theiss, and B. H. Nelson Spontaneous Mammary Tumors Differ Widely in Their Inherent Sensitivity to Adoptively Transferred T Cells Cancer Res., July 1, 2007; 67(13): 6442 - 6450. [Abstract] [Full Text] [PDF]
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S. D. Allen, J. T. Garrett, S. V. Rawale, A. L. Jones, G. Phillips, G. Forni, J. C. Morris, R. G. Oshima, and P. T. P. Kaumaya Peptide Vaccines of the HER-2/neu Dimerization Loop Are Effective in Inhibiting Mammary Tumor Growth In Vivo J. Immunol., July 1, 2007; 179(1): 472 - 482. [Abstract] [Full Text] [PDF]
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E. A. Manning, J. G.M. Ullman, J. M. Leatherman, J. M. Asquith, T. R. Hansen, T. D. Armstrong, D. J. Hicklin, E. M. Jaffee, and L. A. Emens A Vascular Endothelial Growth Factor Receptor-2 Inhibitor Enhances Antitumor Immunity through an Immune-Based Mechanism Clin. Cancer Res., July 1, 2007; 13(13): 3951 - 3959. [Abstract] [Full Text] [PDF]
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J. T. Garrett, S. Rawale, S. D. Allen, G. Phillips, G. Forni, J. C. Morris, and P. T. P. Kaumaya Novel Engineered Trastuzumab Conformational Epitopes Demonstrate In Vitro and In Vivo Antitumor Properties against HER-2/neu J. Immunol., June 1, 2007; 178(11): 7120 - 7131. [Abstract] [Full Text] [PDF]
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S. Rolla, C. Nicolo, S. Malinarich, M. Orsini, G. Forni, F. Cavallo, and F. Ria Distinct and Non-Overlapping T Cell Receptor Repertoires Expanded by DNA Vaccination in Wild-Type and HER-2 Transgenic BALB/c Mice J. Immunol., December 1, 2006; 177(11): 7626 - 7633. [Abstract] [Full Text] [PDF]
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P. Otahal, T. D. Schell, S. C. Hutchinson, B. B. Knowles, and S. S. Tevethia Early Immunization Induces Persistent Tumor-Infiltrating CD8+ T Cells against an Immunodominant Epitope and Promotes Lifelong Control of Pancreatic Tumor Progression in SV40 Tumor Antigen Transgenic Mice. J. Immunol., September 1, 2006; 177(5): 3089 - 3099. [Abstract] [Full Text] [PDF]
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R. Singh and Y. Paterson Vaccination Strategy Determines the Emergence and Dominance of CD8+ T-Cell Epitopes in a FVB/N Rat HER-2/neu Mouse Model of Breast Cancer. Cancer Res., August 1, 2006; 66(15): 7748 - 7757. [Abstract] [Full Text] [PDF]
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K. L. Knutson, H. Lu, B. Stone, J. M. Reiman, M. D. Behrens, C. M. Prosperi, E. A. Gad, A. Smorlesi, and M. L. Disis Immunoediting of Cancers May Lead to Epithelial to Mesenchymal Transition J. Immunol., August 1, 2006; 177(3): 1526 - 1533. [Abstract] [Full Text] [PDF]
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K. L. Knutson, Y. Dang, H. Lu, J. Lukas, B. Almand, E. Gad, E. Azeke, and M. L. Disis IL-2 Immunotoxin Therapy Modulates Tumor-Associated Regulatory T Cells and Leads to Lasting Immune-Mediated Rejection of Breast Cancers in neu-Transgenic Mice J. Immunol., July 1, 2006; 177(1): 84 - 91. [Abstract] [Full Text] [PDF]
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R. E. Nair, M. O. Kilinc, S. A. Jones, and N. K. Egilmez Chronic Immune Therapy Induces a Progressive Increase in Intratumoral T Suppressor Activity and a Concurrent Loss of Tumor-Specific CD8+ T Effectors in her-2/neu Transgenic Mice Bearing Advanced Spontaneous Tumors. J. Immunol., June 15, 2006; 176(12): 7325 - 7334. [Abstract] [Full Text] [PDF]
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T. Pannellini, M. Spadaro, E. Di Carlo, E. Ambrosino, M. Iezzi, A. Amici, P. L. Lollini, G. Forni, F. Cavallo, and P. Musiani Timely DNA Vaccine Combined with Systemic IL-12 Prevents Parotid Carcinomas before a Dominant-Negative p53 Makes Their Growth Independent of HER-2/neu Expression. J. Immunol., June 15, 2006; 176(12): 7695 - 7703. [Abstract] [Full Text] [PDF]
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S. Murata, B. H. Ladle, P. S. Kim, E. R. Lutz, M. E. Wolpoe, S. E. Ivie, H. M. Smith, T. D. Armstrong, L. A. Emens, E. M. Jaffee, et al. OX40 Costimulation Synergizes with GM-CSF Whole-Cell Vaccination to Overcome Established CD8+ T Cell Tolerance to an Endogenous Tumor Antigen J. Immunol., January 15, 2006; 176(2): 974 - 983. [Abstract] [Full Text] [PDF]
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A. I. Garbe, B. Vermeer, J. Gamrekelashvili, R. v. Wasielewski, F. R. Greten, A. M. Westendorf, J. Buer, R. M. Schmid, M. P. Manns, F. Korangy, et al. Genetically Induced Pancreatic Adenocarcinoma Is Highly Immunogenic and Causes Spontaneous Tumor-Specific Immune Responses Cancer Res., January 1, 2006; 66(1): 508 - 516. [Abstract] [Full Text] [PDF]
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 H. Zhang, K. L. Knutson, K. E. Hellstrom, M. L. Disis, and I. Hellstrom Antitumor efficacy of CD137 ligation is maximized by the use of a CD137 single-chain Fv-expressing whole-cell tumor vaccine compared with CD137-specific monoclonal antibody infusion Mol. Cancer Ther., January 1, 2006; 5(1): 149 - 155. [Abstract] [Full Text] [PDF]
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L. A. Emens and E. M. Jaffee Leveraging the Activity of Tumor Vaccines with Cytotoxic Chemotherapy Cancer Res., September 15, 2005; 65(18): 8059 - 8064. [Abstract] [Full Text] [PDF]
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R. Singh, M. E. Dominiecki, E. M. Jaffee, and Y. Paterson Fusion to Listeriolysin O and Delivery by Listeria monocytogenes Enhances the Immunogenicity of HER-2/neu and Reveals Subdominant Epitopes in the FVB/N Mouse J. Immunol., September 15, 2005; 175(6): 3663 - 3673. [Abstract] [Full Text] [PDF]
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 A. M. Ercolini, B. H. Ladle, E. A. Manning, L. W. Pfannenstiel, T. D. Armstrong, J.-P. H. Machiels, J. G. Bieler, L. A. Emens, R. T. Reilly, and E. M. Jaffee Recruitment of latent pools of high-avidity CD8+ T cells to the antitumor immune response J. Exp. Med., May 16, 2005; 201(10): 1591 - 1602. [Abstract] [Full Text] [PDF]
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F. Rohrbach, R. Weth, M. Kursar, A. Sloots, H.-W. Mittrucker, and W. S. Wels Targeted Delivery of the ErbB2/HER2 Tumor Antigen to Professional APCs Results in Effective Antitumor Immunity J. Immunol., May 1, 2005; 174(9): 5481 - 5489. [Abstract] [Full Text] [PDF]
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 L. D. Yee, D. C. Young, T. J. Rosol, A. M. VanBuskirk, and S. K. Clinton Dietary (n-3) Polyunsaturated Fatty Acids Inhibit HER-2/neu-Induced Breast Cancer in Mice Independently of the PPAR{gamma} Ligand Rosiglitazone J. Nutr., May 1, 2005; 135(5): 983 - 988. [Abstract] [Full Text] [PDF]
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 L A Emens, R T Reilly, and E M Jaffee Breast cancer vaccines: maximizing cancer treatment by tapping into host immunity Endocr. Relat. Cancer, March 1, 2005; 12(1): 1 - 17. [Abstract] [Full Text] [PDF]
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 N. K. Dakappagari, K. D. Lute, S. Rawale, J. T. Steele, S. D. Allen, G. Phillips, R. T. Reilly, and P. T. P. Kaumaya Conformational HER-2/neu B-cell Epitope Peptide Vaccine Designed to Incorporate Two Native Disulfide Bonds Enhances Tumor Cell Binding and Antitumor Activities J. Biol. Chem., January 7, 2005; 280(1): 54 - 63. [Abstract] [Full Text] [PDF]
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S. Croci, G. Nicoletti, L. Landuzzi, C. De Giovanni, A. Astolfi, C. Marini, E. Di Carlo, P. Musiani, G. Forni, P. Nanni, et al. Immunological Prevention of a Multigene Cancer Syndrome Cancer Res., November 15, 2004; 64(22): 8428 - 8434. [Abstract] [Full Text] [PDF]
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N. Takeuchi, S.-i. Hiraoka, X.-Y. Zhou, M. Nagafuku, S. Ono, T. Tsujimura, M. Nakazawa, Y. Yura, T. Hamaoka, and H. Fujiwara Anti-HER-2/neu Immune Responses Are Induced before the Development of Clinical Tumors but Declined following Tumorigenesis in HER-2/neu Transgenic Mice Cancer Res., October 15, 2004; 64(20): 7588 - 7595. [Abstract] [Full Text] [PDF]
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P. Nanni, L. Landuzzi, G. Nicoletti, C. De Giovanni, I. Rossi, S. Croci, A. Astolfi, M. Iezzi, E. Di Carlo, P. Musiani, et al. Immunoprevention of Mammary Carcinoma in HER-2/neu Transgenic Mice Is IFN-{gamma} and B Cell Dependent J. Immunol., August 15, 2004; 173(4): 2288 - 2296. [Abstract] [Full Text] [PDF]
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C. De Giovanni, G. Nicoletti, L. Landuzzi, A. Astolfi, S. Croci, A. Comes, S. Ferrini, R. Meazza, M. Iezzi, E. Di Carlo, et al. Immunoprevention of HER-2/neu Transgenic Mammary Carcinoma through an Interleukin 12-Engineered Allogeneic Cell Vaccine Cancer Res., June 1, 2004; 64(11): 4001 - 4009. [Abstract] [Full Text] [PDF]
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M. A. Mihalyo, A. D. H. Doody, J. P. McAleer, E. C. Nowak, M. Long, Y. Yang, and A. J. Adler In Vivo Cyclophosphamide and IL-2 Treatment Impedes Self-Antigen-Induced Effector CD4 Cell Tolerization: Implications for Adoptive Immunotherapy J. Immunol., May 1, 2004; 172(9): 5338 - 5345. [Abstract] [Full Text] [PDF]
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A. Allen, Y. Zheng, L. Gardner, M. Safford, M. R. Horton, and J. D. Powell The Novel Cyclophilin Binding Compound, Sanglifehrin A, Disassociates G1 Cell Cycle Arrest from Tolerance Induction J. Immunol., April 15, 2004; 172(8): 4797 - 4803. [Abstract] [Full Text] [PDF]
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M. P. Piechocki, Y.-S. Ho, S. Pilon, and W.-Z. Wei Human ErbB-2 (Her-2) Transgenic Mice: A Model System for Testing Her-2 Based Vaccines J. Immunol., December 1, 2003; 171(11): 5787 - 5794. [Abstract] [Full Text] [PDF]
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J. Li, W. Li, S. Liang, D. Cai, M. P. Kieny, L. Jacob, A. Linnenbach, J. W. Abramczuk, H. Bender, K. Sproesser, et al. Recombinant CD63/ME491/Neuroglandular/NKI/C-3 Antigen Inhibits Growth of Established Tumors in Transgenic Mice J. Immunol., September 15, 2003; 171(6): 2922 - 2929. [Abstract] [Full Text] [PDF]
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M. E. Wolpoe, E. R. Lutz, A. M. Ercolini, S. Murata, S. E. Ivie, E. S. Garrett, L. A. Emens, E. M. Jaffee, and R. T. Reilly HER-2/neu-Specific Monoclonal Antibodies Collaborate with HER-2/neu-Targeted Granulocyte Macrophage Colony-Stimulating Factor Secreting Whole Cell Vaccination to Augment CD8+ T Cell Effector Function and Tumor-Free Survival in Her-2/neu-Transgenic Mice J. Immunol., August 15, 2003; 171(4): 2161 - 2169. [Abstract] [Full Text] [PDF]
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V. Renard, L. Sonderbye, K. Ebbehoj, P. B. Rasmussen, K. Gregorius, T. Gottschalk, S. Mouritsen, A. Gautam, and D. R. Leach HER-2 DNA and Protein Vaccines Containing Potent Th Cell Epitopes Induce Distinct Protective and Therapeutic Antitumor Responses in HER-2 Transgenic Mice J. Immunol., August 1, 2003; 171(3): 1588 - 1595. [Abstract] [Full Text] [PDF]
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 Y. Luo, H. Zhou, M. Mizutani, N. Mizutani, R. A. Reisfeld, and R. Xiang Transcription factor Fos-related antigen 1 is an effective target for a breast cancer vaccine PNAS, July 22, 2003; 100(15): 8850 - 8855. [Abstract] [Full Text] [PDF]
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D. Artemov, N. Mori, R. Ravi, and Z. M. Bhujwalla Magnetic Resonance Molecular Imaging of the HER-2/neu Receptor Cancer Res., June 1, 2003; 63(11): 2723 - 2727. [Abstract] [Full Text] [PDF]
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N. K. Dakappagari, J. Pyles, R. Parihar, W. E. Carson, D. C. Young, and P. T. P. Kaumaya A Chimeric Multi-Human Epidermal Growth Factor Receptor-2 B Cell Epitope Peptide Vaccine Mediates Superior Antitumor Responses J. Immunol., April 15, 2003; 170(8): 4242 - 4253. [Abstract] [Full Text] [PDF]
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A. M. Ercolini, J.-P. H. Machiels, Y. C. Chen, J. E. Slansky, M. Giedlen, R. T. Reilly, and E. M. Jaffee Identification and Characterization of the Immunodominant Rat HER-2/neu MHC Class I Epitope Presented by Spontaneous Mammary Tumors from HER-2/neu-Transgenic Mice J. Immunol., April 15, 2003; 170(8): 4273 - 4280. [Abstract] [Full Text] [PDF]
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 L. Luznik, J. E. Slansky, S. Jalla, I. Borrello, H. I. Levitsky, D. M. Pardoll, and E. J. Fuchs Successful therapy of metastatic cancer using tumor vaccines in mixed allogeneic bone marrow chimeras Blood, February 15, 2003; 101(4): 1645 - 1652. [Abstract] [Full Text] [PDF]
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 L. SFONDRINI, D. BESUSSO, C. RUMIO, M. RODOLFO, S. MENARD, and A. BALSARI Prevention of spontaneous mammary adenocarcinoma in HER-2/neu transgenic mice by foreign DNA FASEB J, November 1, 2002; 16(13): 1749 - 1754. [Abstract] [Full Text] [PDF]
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S. C. Hewitt, W. P. Bocchinfuso, J. Zhai, C. Harrell, L. Koonce, J. Clark, P. Myers, and K. S. Korach Lack of Ductal Development in the Absence of Functional Estrogen Receptor {alpha} Delays Mammary Tumor Formation Induced by Transgenic Expression of ErbB2/neu Cancer Res., May 1, 2002; 62(10): 2798 - 2805. [Abstract] [Full Text] [PDF]
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P. Nanni, G. Nicoletti, C. De Giovanni, L. Landuzzi, E. Di Carlo, F. Cavallo, S. M. Pupa, I. Rossi, M. P. Colombo, C. Ricci, et al. Combined Allogeneic Tumor Cell Vaccination and Systemic Interleukin 12 Prevents Mammary Carcinogenesis in HER-2/neu Transgenic Mice J. Exp. Med., October 29, 2001; 194(9): 1195 - 1206. [Abstract] [Full Text] [PDF]
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S. A. Pilon, M. P. Piechocki, and W.-Z. Wei Vaccination with Cytoplasmic ErbB-2 DNA Protects Mice from Mammary Tumor Growth Without Anti-ErbB-2 Antibody J. Immunol., September 15, 2001; 167(6): 3201 - 3206. [Abstract] [Full Text] [PDF]
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M. M. Soares, V. Mehta, and O. J. Finn Three Different Vaccines Based on the 140-Amino Acid MUC1 Peptide with Seven Tandemly Repeated Tumor-Specific Epitopes Elicit Distinct Immune Effector Mechanisms in Wild-Type Versus MUC1-Transgenic Mice with Different Potential for Tumor Rejection J. Immunol., June 1, 2001; 166(11): 6555 - 6563. [Abstract] [Full Text] [PDF]
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J.-P. H. Machiels, R. T. Reilly, L. A. Emens, A. M. Ercolini, R. Y. Lei, D. Weintraub, F. I. Okoye, and E. M. Jaffee Cyclophosphamide, Doxorubicin, and Paclitaxel Enhance the Antitumor Immune Response of Granulocyte/Macrophage-Colony Stimulating Factor-secreting Whole-Cell Vaccines in HER-2/neu Tolerized Mice Cancer Res., May 1, 2001; 61(9): 3689 - 3697. [Abstract] [Full Text]
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R. T. Reilly, J.-P. H. Machiels, L. A. Emens, A. M. Ercolini, F. I. Okoye, R. Y. Lei, D. Weintraub, and E. M. Jaffee The Collaboration of Both Humoral and Cellular HER-2/neu-targeted Immune Responses Is Required for the Complete Eradication of HER-2/neu-expressing Tumors Cancer Res., February 1, 2001; 61(3): 880 - 883. [Abstract] [Full Text]
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) and neu-N mice (
) 14
days after vaccination with irradiated 3T3/GM or
3T3-neu/GM cells. Serial dilutions of serum were then
used as the primary antibody solution in a FACS analysis of both 3T3
and 3T3-neu cells using a secondary antibody against
murine IgG. Neu-specific serum IgG titers were determined based on the
greatest dilution of serum that gave a significant shift when staining
3T3-neu cells relative to background staining of 3T3
cells.
) and control
(
) groups. 
, neu-specific lysis in the presence of the
CD8-blocking antibody 2.43. This experiment was repeated three times
with similar results.
) or HA rVV (•; n = 10
mice/group), with Ps of 0.005 and 0.01 for the
whole-cell and rVV groups, respectively. The data shown are combined
from two separate experiments. Similar results were obtained with an
NT5 challenge (data not shown). B, FVB/N mice vaccinated
with irradiated 3T3-neu/GM vaccine given s.c. (
;
n = 12 mice) on day 0 were protected from
a s.c. NT2 challenge given in the right hind limb on day 14. Control
mice received mock vaccinations with either s.c. 3T3/GM (