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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 1 (January 1), 1998:
pp. 222-230
Gene Immunotherapy in Murine Acute Myeloid Leukemia:
Granulocyte-Macrophage Colony-Stimulating Factor Tumor Cell Vaccines
Elicit More Potent Antitumor Immunity Compared With B7 Family
and Other Cytokine Vaccines
By
Kyriaki Dunussi-Joannopoulos,
Glenn Dranoff,
Howard J. Weinstein,
James L.M. Ferrara,
Barbara E. Bierer, and
James M. Croop
From the Dana-Farber Cancer Institute and Massachusetts General
Hospital, Harvard Medical School, Boston, MA.
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ABSTRACT |
In an attempt to explore novel treatment modalities in acute myeloid
leukemia (AML), we studied the role of costimulatory and cytokine gene
immunotherapy in murine AML. We have previously shown that leukemic
mice can be cured with CD80 transfected leukemic cells (B7.1-AML
vaccine) administered early in the course of the disease and that the
failure B7.1-AML vaccines administered late cannot be attributed to
immunosuppression induced by tumor growth. CD8+ T cells,
which are necessary for tumor rejection, are activated rather than
suppressed during the first half of the leukemic course in
nonvaccinated mice. In this report, we question whether CD86 (B7.2) or
the cytokines granulocyte-macrophage colony-stimulating factor
(GM-CSF), interleukin-4 (IL-4), or tumor necrosis factor- (TNF-)
can improve the vaccination potential of AML cells. The choice of
cytokines was based on their combined and alone as well ability to
direct the differentiation of CD34+ cells into potent
antigen-presenting dendritic cells in vitro. Our studies show that (1)
mice vaccinated with a leukemogenic number of AML cells engineered to
express B7.2 (B7.2-AML) or to secrete GM-CSF, IL-4, or TNF- (GM-,
IL-4-, TNF--AML) do not develop leukemia; (2) GM-AML cells are
tumorigenic in sublethally irradiated SJL/J mice but not in Swiss nu/nu
mice, indicating that killing of tumor cells is not T-cell-dependent;
(3) vaccines with irradiated GM-AML, but not B7.2-, IL-4-, or
TNF--AML cells, can elicit leukemia-specific protective and
therapeutic immunity; and (4) in head-to-head comparison experiments,
vaccination with irradiated GM-AML is more potent than B7.1-AML, curing
80% and providing 20% prolonged survival of the leukemic mice at week
2, as opposed to cures only up to 1 week with B7.1-AML vaccines. These
preclinical data emphasize that GM-CSF gene immunotherapy deserves
clinical evaluation in AML.
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INTRODUCTION |
IN RECENT YEARS, a tremendous amount of
information has emerged regarding biochemical and molecular mechanisms
that control the biology of acute myelogenous leukemia
cells.1-3 However, these advances have not yet translated
into novel therapeutic approaches. Despite the developments in new
regimens for induction of remission therapy and in supportive care,
long-term survival is usually only achieved in 25% to 30% of
patients.4 Factors predisposing to this unfavorable outcome
have still to be defined. Certain karyotypic abnormalities and the
multidrug resistance phenotype have been considered as principal
mechanisms affecting the rate and duration of complete remission in
acute myeloid leukemia (AML).4,5 At present, two new
treatment modalities represent hopeful prospects for improving the
outcome in AML: (1) the use of potent multidrug resistance reversal
agents that do not cause immunosuppression4,6 and (2) the
use of immunomodulatory compounds or tumor-cell vaccines as adjuvant
treatment.7,8 Both treatment modalities are novel
approaches that need careful clinical evaluation.
Intense research on animal tumor models has shown that tumor growth
does not eliminate immunity against nonself tumor-specific
antigens.9,10 Primary factors that have been implicated for
the escape of tumor cells from an effective cytolytic response are the
lack of expression of costimulatory molecules by most of the tumor
cells11 and the absence of an appropriate cytokine
microenvironment.12 Numerous studies have emphasized the
effectiveness of heightened expression of B7 costimulatory
molecules13,14 and immunoregulatory cytokines, such as
interleukin-2 (IL-2), IL-4, IL-6, interferon- , and
granulocyte-macrophage colony-stimulating factor (GM-CSF), in antitumor
immunity.15-19 Because few tumor-specific antigenic
determinants are currently known, most of these studies have used whole
tumor cell vaccines.
The mechanisms by which tumor cells that are engineered to secrete
cytokines induce tumor-specific immunity differ from vaccine to
vaccine.20,21 Variables influencing this outcome include
the immunogenicity of the tumor, the microenvironment surrounding the
tumor, the type of cytokine secreted, and, finally, the amount of
cytokine secreted.20 In recent reports comparing the
ability of different cytokines to enhance the immunogenicity of murine
tumor cells, GM-CSF was the most potent molecule for inducing antitumor
immunity.19,22 It has been speculated that this effect may
be due to the ability of GM-CSF to promote differentiation of dendritic
cells (DC),10 which are very potent antigen-presenting
cells (APC) for activating both class I- and class II-restricted T
cells.23 This idea is strengthened by studies on the ex
vivo generation of functionally mature DC from human CD34+
bone marrow precursors, showing that GM-CSF or the combination of
GM-CSF and IL-4 or tumor necrosis factor- (TNF-) promote the
differentiation of CD34+ bone marrow cells and their
acquisition of DC phenotypic and functional characteristics within 7 to
8 days.24,25
Murine acute leukemia cells that are genetically modified to express B7
costimulatory molecules can become immunogenic and be used effectively
as vaccines.26-29 We and others have shown that B7-1
vaccines eliminate only a relatively small leukemic burden; hence,
their efficacy is lost in later stages of the disease.26,27
The hypothesis that progressive tumor growth modulates the outcome of
immunotherapeutic strategies was not confirmed by our later studies,
showing that the CD8+ T cells necessary for tumor rejection
are activated rather than suppressed during the leukemic
course.30 In this respect, we investigated whether
transduction of AML cells with molecules other than B7.1 can enhance
immunogenicity and vaccine efficacy. First, we tested whether the
costimulatory ligand CD86 (B7.2) was more efficient than CD80 in this
model of AML vaccines. B7.2, the second member of the B7 family
costimulatory molecules, has been variously efficacious in a number of
murine tumor models.28,31-33 Second, we studied the role of
GM-CSF, IL-4, and TNF- as single cytokine vaccines in murine AML. In
this report, we show that vaccination with B7.2-, IL-4-, and
TNF--AML cells activates tumor-killing mechanisms resulting in
rejection of the inoculated leukemic burden, but does not elicit
leukemia-specific immunity. GM-AML vaccines, on the other hand, provide
potent, long-lasting antitumor immunity and can cure mice with a
considerably larger tumor burden than mice cured with B7.1-AML
vaccines.
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MATERIALS AND METHODS |
Mice.
Female SJL/J mice (H-2s), 6 to 8 weeks old, were purchased
from Jackson Laboratories (Bar Harbor, ME) or Charles River
Laboratories (NCI-Frederick Cancer Research & Development Center,
Frederick, MD). Swiss nu/nu mice were purchased from Taconic
Laboratories (Germantown, NY). The animals were kept at the animal
facility of Dana-Farber Cancer Institute according to the institute's
guidelines.
Murine AML model.
The murine AML model used in this study has been previously
described.27 Briefly, radiation-induced AML
cells34 are maintained by growth in syngeneic SJL/J female
mice. Mice injected intravenously (IV) or intraperitoneally (IP) with
 104 AML develop lethal leukemia in 4 to 5 weeks. In all
experiments, freshly isolated or frozen spleen mononuclear cells from
leukemic mice (killed just before succumbing to leukemic burden) were
used.
Retroviral constructs and producer clones.
A cDNA fragment encoding the entire open reading frame of murine B7.2
was amplified by reverse transcriptase-polymerase chain reaction from
total cellular RNA extracted from the murine B-cell line A20 (activated
for 24 hours with 4 µg/mL lipopolysaccharide). The B7.2-specific
sense and antisense primers had the sequences
5 -ATCGATGAAGCACCCACGATGGAC-3 and
5-ATCGATTCACTCTGCATTTGGTTTTGC-3,
respectively.31 The full-length murine B7.2 cDNA was
subcloned in sense and antisense (mock virus) orientation at the
Cla I unique cloning site of the LNCX retroviral vector (kindly
provided by Dusty Miller, Fred Hutchinson Cancer Research Center,
Seattle, WA). For generating virus producer clones, E-86
packaging cell lines were transfected with LNCX-B7.2 constructs and
E86-B7.2-sense or E86-B7.2-antisense clones secreting high titer of
virus were used to infect AML cells. E-86-B7.1-sense and
E-86-B7.1-antisense producer clones have been previously
described.27 Retroviral constructs
MFG-GM-CSF,19 MFG-IL-4,19 and murine
MFG-TNF- (Dranoff and Mulligan, unpublished data) and
CRIP producer clones, secreting high titers of recombinant retroviruses
encoding GM-CSF, IL-4, and TNF-, have been previously
described.19 Empty MFG vector was used for the preparation
of mock viruses.
Infection of AML cells.
Infection of AML cells with recombinant viruses has been previously
described.27 Briefly, AML cells (5 × 105
/mL) were exposed to viral supernatant for 12 to 24 hours in the
presence of 8 to 10 µg/mL polybrene and 15% WEHI-3B conditioned
media, cultured in fresh media for an additional 24 hours, and then
used for in vivo immunizations. In some experiments, a purified
population of B7.2-expressing AML cells was used. To purify
B7.2+ AML cells, infected cells were stained with a
B7.2-specific (GL1) monoclonal antibody (MoAb; PharMingen, San Diego,
CA), labeled with goat-antirat IgG Microbeads (Milteny Biotec,
Sunnyvale, CA), and selected using magnetic MiniMacs separation columns
(Milteny Biotec). Isolated cells were left in culture for 12 to 14
hours and were then used for in vivo immunizations. Flow cytometry
analysis (fluorescence-activated cell sorting [FACS])
showed that these cells were greater than 95% pure and appeared to be
viable by exclusion of trypan blue and forward/side scatter analysis.
Lymphokine enzyme-linked immunosorbent assays (ELISAs).
Levels of GM-CSF, IL-4, and TNF- secreted by the infected AML cells
cultured for 48 hours at 106 cells/mL were determined using
a sandwich ELISA using specific antimurine MoAbs for capture and
detection (PharMingen). A color reaction was developed using
streptavidin-conjugated horseradish peroxidase (Genzyme, Cambridge,
MA), followed by tetramethylbenzidine (TMB) peroxidase substrate
(Kirkegaard & Perry Laboratories Inc, Gaithersburg, MD). The MoAbs
used for capture and detection were the following: for GM-CSF, purified
MP1-22E9 and biotin-conjugated MP1-31G6; for IL-4, purified
11B11 and biotin-conjugated BVD6-24G2; and for TNF-,
purified MP6-XT22 and biotin-conjugated MP6-XT3. Recombinant mouse
GM-CSF (rGM-CSF) with a specific activity of 104 U/µg and
mouse rIL-4 with a specific activity of 104 U/µg were
obtained from PharMingen. Mouse rTNF- with a specific activity of 2
× 105 U/µg was obtained from Genzyme.
Western blotting.
Total cell lysates from spleen AML cells or control cells were prepared
as previously described.35 Ba/F3 cells, transduced with the
murine GM-CSF receptor (kindly provided by Bernard Mathey-Prevot,
Dana-Farber Cancer Institute), were used as a positive control, and
WEHI-3B cells were used as a negative control. Proteins (40 µg of
protein/lane) were fractionated by electrophoresis on a 12% sodium
dodecyl sulfate (SDS)-polyacrylamide gel and transferred to
nitrocellulose membranes for immunoblotting. The membranes were blocked
for 1 hour in 5% nonfat dry milk at room temperature and probed with
rabbit antiserum against the mouse epitope corresponding to carboxy
terminus of GM-CSFR (Santa Cruz Biotechnology Inc, Santa Cruz, CA)
for 45 minutes at room temperature (0.5 µg/mL). The membranes were
then incubated in Tris buffered saline, 0.05% Tween-20 (TBST) and
horseradish peroxidase-conjugated antirabbit antibody (1:5,000
dilution). Protein bands were detected by use of chemiluminescent
techniques according to the manufacturer's instructions (Amersham Life
Science, Little Chalfont, UK).
Immunostaining and flow cytometry analysis.
Cells were stained as previously described.27 The following
antibodies (PharMingen) were used in this study: CD3 (145-2C11), CD4
(RM4-5), CD8a (53-6.7),  TCR (H57-597),  TCR (GL3), Gr-1
(RB6-8C5), CD2 (RM2-5), CD5 (53-7.3), CD18 (C71/16), CD11b (M1/70),
CD25 (7D4), CD45 (30F11.1), CD44 (IM7), CD45R/B220 (RA3-6B2), CD54
(3E2), CD62L (MEL-14), CD69 (H1.2F3), CD80 (1G10), CD86 (GL1), and CD95
(Jo2).
In vivo immunization studies.
SJL/J mice or Swiss nu/nu mice were injected IV with live or irradiated
(3,200 cGy from a 137Cs source) transduced AML cells
(B7.1-, B7.2-, GM-, IL-4-, TNF--AML). We have previously shown
that irradiation of AML cells with 3,200 cGy abrogates their
tumorigenicity.27 In most of the experiments,
105 transduced AML cells were used and diluted in 200 to
300 µL of phosphate-buffered saline.
Statistical analysis.
Most individual experiments consisted of 10 mice per treatment
group. The data analyzed represent the results of one or two individual
experiments. Cytokine values secreted by transduced AML cells are the
mean ± standard error of the mean (SEM).
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RESULTS |
Infection of AML cells.
AML cells were exposed to E86-B7.2-sense or E86-B7.2-antisense viral
supernatants as described in the Materials and Methods. Expression of
B7.2 on infected, unselected AML cells was confirmed by surface
staining and flow cytometry (Fig 1A).
Transduction of AML cells with the MFG-cytokine retroviral constructs
resulted in secretion of the gene products and did not alter their in
vitro growth characteristics after 3 to 4 days of culture (data not
shown). The amount of cytokines produced by cytokine-transduced AML
cells (cytokine-AML) was evaluated using specific ELISA assays (Fig
1B). The cytokine levels were comparable to production levels reported
in other tumor vaccine models. A sample of some recent reports is given
in Table 1.36-39 Irradiation of
cytokine-AML cells (3,200 cGy) did not abrogate cytokine secretion in
vitro for at least 4 days (data not shown). AML cells in this model
express the GM-CSF receptor, which is downregulated when the cells are
cultured in the presence of IL-3 (Fig 1C), because GM-CSF and IL-3
cross-compete for cellular binding to AML cells.40,41
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| Fig 1.
(A) CD86 expression on infected AML cells. Spleen AML
cells were infected with either CD86-sense (a) or CD86-antisense (b)
producer clones as described in the Materials and Methods. ( )
Control IgG (rat IgG-PE); ( ) CD86-PE (anti-B7.2) MoAb. The hidden
portion of the control curve in (a) drops monotonically as a function
of fluorescence intensity. (B) Cytokine production by transduced AML
cells. Levels of GM-CSF, IL-4, and TNF- secreted by retrovirally
transduced, unselected AML cells cultured for 48 hours at
106 cells/mL were determined by sandwich ELISA using
specific antimurine MoAbs for capture and detection. Data are shown as
the mean ± SD of six independent experiments. (C) AML cells express
GM-CSF receptor. Total cell lysates (40 µg of protein/lane) from
spleen AML cells from two different mice (lanes 3 and 4) or control
cells (lanes 1 and 5) were fractionated by electrophoresis on a 12%
SDS-polyacrylamide gel, transferred to nitrocellulose membrane, and
probed with polyclonal antibody against mouse GM-CSFR as described
in the Materials and Methods. AML cells cultured for 24 hours in the
presence of IL-3 downregulate GM-CSFR expression (lane 2).
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B7.2-AML cells have reduced tumorigenicity but do not elicit systemic
immunity.
We have previously shown that one IV injection of irradiated B7.1-AML
cells can protect mice from subsequent challenge with wild-type AML
cells and that one exposure to irradiated, B7.1-AML cells can cure
leukemic mice vaccinated up to 1 week after leukemia inoculation (early
vaccination), whereas after 2 weeks of leukemic inoculation (late
vaccination) the same vaccine only delays tumor growth.27
To evaluate the role of B7.2 expression on the leukemic cell growth,
mice were injected IV with increasing numbers of live B7.2-AML cells
and their clinical outcome was monitored. As shown in Fig
2A, mice injected with 105
B7.2-AML cells rejected their tumor, whereas mice injected with
106 B7.2-AML cells developed lethal leukemia. Flow
cytometry showed a consistent population of AML cells (20% to 25%)
not expressing B7.2 after retroviral infection (Fig 1A). Therefore, we
tested if mice injected with 5 × 105 purified
B7.2-AML cells would reject their leukemia. All mice in this experiment
developed lethal leukemia at the expected interval (data not shown). We
next examined if immunization with irradiated B7.2-AML cells could
elicit systemic immunity and protect mice against subsequent challenge
with wild-type AML cells. Mice were immunized with irradiated (3,200
cGy) 105 or 2 × 106 B7.2-AML cells and 2
weeks later were challenged with live 105 wild-type AML
cells. As shown in Fig 2B, challenge was lethal to both groups of
vaccinated mice, and even vaccinations with as high as 2 ×
106 irradiated B7.2-AML cells only prolonged survival for 5
to 7 days.
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| Fig 2.
B7.2-AML cells have reduced tumorigenicity but do not
elicit systemic immunity. (A) SJL/J mice (8 to 10 mice for each type of
experiment) were injected IV with 105 or 106
B7.2-AML cells or 105 control cells. These experiments were
repeated twice. Mice injected with ( ) 105 B7.2-AML cells
rejected their tumor, whereas mice injected with ( ) 106
B7.2-AML or ( ) control cells developed lethal leukemia. (B) SJL/J
mice were immunized IV with () 105 irradiated (3,200
cGy) B7.2-AML or () 2 × 106 B7.2-AML cells or ( )
control cells (solid arrow) and 2 weeks later challenged with
105 live wild-type AML cells (open arrow). Challenge was
lethal to all groups of vaccinated mice. Vaccinations with 2 ×
106 irradiated B7.2-AML cells prolonged survival for 5 to 7
days.
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Finally, we examined whether B7.2-AML cells could rescue leukemic mice
with very small leukemic burden. Mice were injected with
104 AML cells (lowest tumorigenic number) and immunized 2
days later with live or irradiated 105 B7.2-AML cells. All
mice in this experiment developed lethal leukemia after 5 weeks (data
not shown). Taken together, these results show that, in this AML model,
expression of B7.2 on the AML cells can initiate significant
tumor-killing mechanisms, thus reducing their tumorigenicity. However,
B7.2-AML cells (at least the numbers used in this study) do not induce
protective or therapeutic immunity.
Tumorigenicity and immunogenicity of cytokine-AML cells.
To evaluate if cytokine gene transduction of AML cells would have any
effect on their tumorigenicity and immunogenicity, groups of mice were
injected with live 105 to 106 GM-AML,
IL-4-AML, or TNF-- AML cells or with mock-infected control AML
cells. Mice did not develop any signs of toxicity and only mice
injected IV with live 105 to 106 GM-AML cells
developed a transient increase of the white blood cell count (up to 18
× 103/µL, from the normal 10 to 12 ×
103/µL), between weeks 2 and 3 after tumor inoculation.
All mice inoculated with 105 cytokine-AML (GM-, IL-4-, or
TNF--AML) cells rejected their tumors (Fig
3A). From the groups of mice inoculated
with live 106 cells, only the GM-AML group rejected the
leukemic cells, whereas IL-4-AML or TNF--AMLinjected mice only
had prolonged survival for 1 to 2 weeks (Fig 3A).
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| Fig 3.
Tumorigenicity and immunogenicity of cytokine-AML cells.
(A) SJL/J mice (8 to 10 mice for each type of experiment) were injected
IV with 105 or 106 live GM-AML, IL-4-AML, or
TNF--AML cells (cytokine-AML). Control mice were injected with
105 or 106 mock-infected AML cells. All mice
injected with 105 cytokine-AML ( ) and 106
GM-AML cells () rejected their leukemia. Mice injected with
105 ( ) or 106 () control cells developed
leukemia at the expected interval. Mice injected with 106
IL-4-AML () had 1 week and those injected with 106
TNF--AML cells () had 2 weeks of prolonged survival. (B) SJL/J
mice (8 to 10 mice for each type of experiment) were vaccinated IV
(solid arrow) with 105 irradiated (3,200 cGy) GM-AML (),
IL-4-AML (), or TNF--AML cells () or mock-infected control
cells () and were challenged 2 weeks later (open arrow) with
105 live wild-type AML cells. GM-AML-vaccinated mice
survived tumor challenge, whereas challenge was lethal to all other
groups of mice.
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We next examined if vaccinations of mice with irradiated cytokine-AML
cells could elicit systemic immunity. Groups of mice were immunized
with irradiated 105 cytokine-AML (GM-, IL-4-, or
TNF--AML) cells and were challenged 2 weeks later with
105 live wild-type AML cells. As shown in Fig 3B, only
GM-AML-vaccinated mice developed protective immunity and survived the
tumor challenge, whereas the challenge was lethal to all mice immunized
with IL-4-AML or TNF--AML cells. To determine whether higher
numbers of irradiated IL-4- or TNF--AML cells could elicit
protective immunity, the same experiments were repeated with irradiated
2 × 106 IL-4- or TNF--AML cells. All animals in
these experiments succumbed to subsequent challenge with
105 live wild-type AML cells (data not shown). These
results clearly show variability in the efficacy of the 3 different
cytokine vaccines in the same AML model. Whereas significant tumor-cell
killing mechanisms appear to follow each of the cytokine vaccines at
low tumor cell doses (Fig 3A), the ability to elicit protective
immunity is restricted to GM-AML cells (Fig 3B).
Rejection of GM-AML cells is not T-cell-dependent.
In an attempt to confirm that 105 GM-AML cells were as
leukemogenic as wild-type AML cells, we injected Swiss nu/nu mice with
105 GM-AML or mock-infected cells. We have previously shown
that 105 B7.1-AML cells are equally as tumorigenic as
wild-type AML cells in these mice. Surprisingly, all nude mice injected
with GM-AML cells remained healthy and tumor-free, whereas mice
injected with control cells developed leukemia (Fig
4). This clinical outcome indicated that
either 105 GM-AML cells had lost their tumorigenicity or
effector cells other than T cells were responsible for their rejection
in nude mice. To address this question, we irradiated SJL/J mice (600
cGy TBI) and injected them 2 days later with 105 GM-AML or
wild-type AML cells. Both groups of irradiated SJL/J mice developed
lethal leukemia, clearly indicating that 105 GM-AML were
equally leukemogenic as wild-type AML cells (Fig 4).
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| Fig 4.
Rejection of GM-AML cells is not T-cell-dependent. (A)
Swiss nu/nu mice, in groups of 6, were injected IV with 105
GM-AML cells () or 105 mock-infected control cells
(). The former group rejected their tumor, whereas the latter group
developed lethal leukemia. (B) SJL/J mice, in groups of 6, were
irradiated (600 cGy TBI) and injected 2 days later with 105
GM-AML cells () or mock-infected control cells (). Both groups of
SJL/J mice developed lethal leukemia.
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We next examined if we could detect by flow cytometry any cell
population changes in the spleens of nude mice that reject GM-AML
cells. Swiss nu/nu mice were injected with live GM-AML or B7.1-AML
cells (previously shown to be leukemogenic). Control mice were injected
with mock-infected AML cells. The spleens were removed 3 days later,
and spleen mononuclear cells were stained with the MoAbs described in
the Materials and Methods directed against a wide range of
hematopoietic subsets. There were no differences observed for 14 of 20
surface markers studied (including the T-cell markers CD3, CD4, and CD8
that were negative) between naive and experimental mice. However,
GM-AML-injected mice showed an increased expression of CD80, CD86,
IL-2R, CD18, and Mac-1. In addition, a larger population of cells
(25.31% v 18.51% in naive mice) was negative for the
B-cell-specific marker B220 (Fig 5). These
data clearly show that injection of nude mice with GM-AML cells leads
to an influx of non-B cells in the spleen of the animals. Most likely,
the majority of these cells are of myeloid-monocytic origin, expressing
APC/monocytic activation markers such as CD80, CD86, and IL-2R.
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| Fig 5.
FACScan analysis of spleen cells from Swiss nu/nu mice.
Swiss nu/nu mice, in groups of 3, were injected IV with live
105 GM-AML or B7.1-AML or mock-infected AML cells. Three
days later, their spleen cells were stained with a panel of 20 MoAb as
described in the Materials and Methods and compared with spleen cells
from naive Swiss nu/nu mice. A total of 10,000 cells were analyzed by
FACS for each sample. GM-AML-injected mice showed a higher population
of cells negative for B220 (25.31% v 18.51% in naive mice)
and increased expression of CD80, CD86, IL-2R, CD18, and Mac-1.
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GM-AML vaccines cure mice with larger tumor burden than B7.1-AML
vaccines and elicit leukemia-specific memory cells. Experiments
were designed to evaluate the effectiveness of different cytokine
vaccines in leukemic mice and to compare them with B7.1-AML vaccines
that can only cure mice with small (1 week) leukemic
burden.27 Mice were inoculated with 105
wild-type AML cells and 1 week later were immunized with irradiated
105 GM-, IL-4-, or TNF--AML cells. All mice vaccinated
with IL-4-AML or TNF--AML cells developed lethal leukemia at the
expected interval (data not shown), whereas 100% of mice vaccinated
with irradiated GM-AML rejected their tumor. We then examined if GM-AML
vaccines administered 2 or 3 weeks after tumor inoculation were able to
rescue mice from lethal leukemia. In this experiment, 90% of mice
vaccinated at week 2 and 20% of mice vaccinated at week 3 rejected
their leukemia and remained tumor-free for an observation period of 4
months (Fig 6
[View Larger Version of this Image (16K GIF file)]
A). To directly
compare the effectiveness of B7.1-AML and GM-AML vaccines in this
model, we performed a head-to-head comparison of the vaccines in the
same experiment. Mice were injected with 105 AML cells and
1 or 2 weeks later they were vaccinated with irradiated 105
B7.1-AML or GM-AML cells; nonvaccinated leukemic mice and leukemic mice
injected with mock-infected AML cells were used as control. All mice
vaccinated at week 1 with either B7.1-AML or GM-AML cells rejected
their leukemia. Vaccinations with B7.1-AML cells at week 2 had no
effect on survival and 100% of the mice developed lethal leukemia. On
the contrary, vaccinations with GM-AML cells at week 2 resulted in 80%
cure and 20% prolonged survival of leukemic mice (Fig 6B). These
results clearly show that, in the SJL/J AML model, GM-AML vaccines
provide more potent antileukemia immunity than do B7.1-AML vaccines. In
an attempt to investigate if leukemia rejection by SJL/J mice involved
immune mechanisms leading to leukemia-specific memory, mice that had
been rescued by week 2 GM-AML vaccines were challenged 4 months later
with 105 wild-type AML cells. This experiment showed that
67% of the mice had developed immunologic memory that enabled them to
survive the AML challenge (Fig 6C).
GM-AML vaccines cure mice with larger tumor
burden than B7.1-AML vaccines and elicit leukemia-specific memory
cells. (A) SJL/J mice were inoculated with live 105
wild-type AML cells (open arrow). One (), 2 (), or 3 weeks ()
later (10 mice per group), they were immunized with 105
irradiated (3,200 cGy) GM-AML cells. Five control mice () were not
vaccinated after leukemia inoculation. (B) Mice were inoculated with
live 105 wild-type AML cells, and 1 or 2 weeks (solid
arrows) later, they were immunized with 105 irradiated
GM-AML or B7.1-AML cells (10 mice per group). Nonvaccinated leukemic
mice and leukemic mice injected with mock-infected AML cells were used
as control (). All mice vaccinated at week 1 with either B7.1-AML
( ) or GM-AML () cells rejected their leukemia. All mice
vaccinated at week 2 with B7.1-AML () cells developed lethal
leukemia. Vaccinations with GM-AML cells at week 2 () resulted in
80% cure and 20% prolonged survival. (C) SJL/L mice (9 mice) that had
been rescued by week 2 GM-AML vaccine were challenged 4 months later
with 105 wild-type AML cells (solid arrow). Six of nine
vaccinated mice (67%; ) survived the challenge with AML cells.
Control mice () developed lethal leukemia.
Fig 6.
| |
DISCUSSION |
In this report we expanded our studies on gene immunotherapy in a
primary murine AML model. Our previous work has shown that leukemia
growth per se does not induce T-cell unresponsiveness or a Th2 cytokine
profile and that B7.1-AML vaccines can cure leukemic mice without a
large leukemic burden. We investigated here the possibility of using
either B7.2- or cytokine-transduced AML cells (GM-CSF, IL-4, and
TNF-) as more effective vaccines. Our results clearly show that, in
this AML experimental model, there is dissociation between
tumor-killing and vaccine-induced tumor immunity and that, from the
molecules studied, only GM-CSF can cure leukemic mice with considerable
leukemic burden and elicit protective memory immune responses.
Furthermore, we show that the antitumor activity of GM-CSF is present
in nude mice, indicating that cell populations other than T cells are
involved in GM-AML cell elimination. Several questions arise from our
data. (1) Why are tumor-cell-induced B7.1 and B7.2 costimulatory
signals different in the same AML model, in that B7.2 is not inducing
T-cell-mediated antileukemia protective and therapeutic immunity? (2)
Why are IL-4- and TNF--AML cells ineffective in providing
antileukemia immunity? (3) Why can GM-CSF vaccines fight a larger tumor
burden than B7.1-AML vaccines? (4) Why do not all GM-CSF-vaccinated
leukemic mice develop antileukemia memory?
There is increasing evidence that the two-signal axiom does not
completely cover the complex process of T-cell activation and that CD28
ligation is not necessary for initial T-cell activation and
proliferation but is required for cell survival.42-44
Indeed, a sequential multiple-step model for T-cell activation has been
proposed.45 According to this model, primary costimulatory
signals can be delivered by several adhesion and costimulatory
molecules, and continued and prolonged B7/CD28 interaction leads to
optimal T activation by inducing cytokine gene transcription and
cytokine mRNA stabilization.44-46 These observations
support our reported data that AML growth induces activation of T cells
in the initial stages of leukemia, despite the fact that AML cells in
the SJL/J model do not express B7 family costimulatory
molecules.30 Another recently reformed concept is that,
although the costimulatory molecules B7.1 and B7.2 both bind to CD28
and CTLA-4, their binding with these receptors mediates distinct
biologic functions.43,44 As opposed to the widely
demonstrated positive effect of CD28 ligation in T-cell activation and
survival, it has been reported that CTLA-4 costimulation delivers
downregulatory signals, either by inhibiting signaling through the
TCR,47 provoking an active CTLA-4-mediated apoptotic
death,48 or by inducing cell cycle arrest in
G1/G0.49 It has also been reported that, although B7.1 and
B7.2 have the same high affinity for CTLA-4, this receptor on T cells
may have a differential response to B7.1 and B7.2
ligation.50,51 However, these differences between the two
costimulatory molecules cannot directly address the question as to why
B7.1 costimulation, when provided by engineered tumor cells, is
superior to B7.2 in several tumor models. It has been hypothesized that
B7.2 costimulation may promote a Th2-type cytokine profile of T
cells.28,52 In studies comparing immune parameters of mice
vaccinated with various types of vaccines, we could not confirm a Th2
cytokine profile in B7.2-AML-vaccinated mice (K. Dunussi-Joannopoulos,
unpublished data). Moreover, it has been recently shown
that B7.1 and B7.2 do not appear to selectively regulate Th1 versus Th2
differentiation.53 In the SJL/J AML model, B7.1-AML
costimulation is capable of eliciting leukemia-specific immunity and
leukemia-specific memory CTLs, whereas B7.2-AML costimulation is not
providing protective or therapeutic antileukemia immunity. We speculate
that the differential clinical outcome mirrors a positive B7.1/CD28
signal, leading to clonal CTL expansion, and a negative B7.2/(CTLA-4?)
signal resulting in absence (elimination?) of leukemia-specific T-cell
responses. A recent report that treatment of mice with CTLA-4 MoAb
prevented tumor outgrowth and induced the regression of established
tumors54 strongly suggests that, at least in tumor models,
a negative signal mediated by CTLA-4 plays a more decisive role than a
positive signal mediated by CD28. However, this phenomenon still
suffers from lack of cellular and structural detail.
Another issue for discussion is the efficacy of cytokine gene
immunotherapy in murine AML. Studies in murine, mostly
nonhematopoietic, tumor models have shown that certain cytokines
produced by genetically engineered tumor cells lead to decreased
tumorigenicity and increased immunogenicity of the transduced tumor
cells.15-19 A variety of cytokines have been described that
augment host antitumor immunity. We show that secretion of the
cytokines GM-CSF, IL-4, or TNF- in this AML model is each able to
initiate immune responses that inhibit the in vivo growth of transduced
AML cells. However, the desirable clinical outcome of tumor vaccines,
ie, recruitment of tumor-specific protective and therapeutic immunity,
was only achieved with GM-CSF gene immunotherapy, which suggests that
tumor growth inhibition and antitumor immunity may be mediated by
different cell populations in cytokine tumor models.55
Activated natural killer cells, macrophages, neutrophils, and
eosinophils may be involved in direct killing of cytokine-AML cells, as
shown by GM-AML cell experiments. Rejection of GM-AML cells in Swiss
nude mice correlates with detection of a non-B-cell splenic population
expressing myeloid and activated monocytic/APC surface markers,
confirming that T cells are not necessary for tumor rejection.
Participation of both innate and T-cell-mediated immunity in GM-CSF
immunotherapy may partially explain its superiority as compared with
B7.1-AML vaccines in this experimental AML model. On the contrary, the
lack of immunogenicity of the IL-4- or TNF--AML cells (at least
the numbers used in this study) suggests that the cytokines released by
transduced cells may initiate rapid tumor clearance mechanisms,
possibly resulting in limited loading of APCs with tumor antigen(s) and
ineffective priming of leukemia-specific CTLs.21
Alternatively, mechanisms of T-cell immunosupression may govern this
outcome. We are currently investigating immune parameters in
cytokine-AML-vaccinated mice and the role of combined cytokine gene
immunotherapy in the SJL/J AML model.
The introduction of the hematopoietic cytokine GM-CSF into treatment
regimens for AML raised concerns several years ago, because it was well
established that AML progenitor cells require hematopoietic growth
factors (HGF) for survival and proliferation,56,57 although
they usually show little maturation under the influence of these
regulators.58 The demonstration in the early 1990s that HGF
could shorten the duration of neutropenia after intensive chemotherapy
for solid tumors provoked the introduction of GM-CSF and G-CSF into AML
clinical trials.8 GM-CSF has been used in three general
clinical situations in AML: for attenuation of neutropenia, for
sensitization of leukemic cells to cytotoxic therapy, and for the
induction of terminal differentiation of leukemic cells.4,8
The conclusions from numerous clinical trials that have been conducted
during the last years are still debatable.4 However, most
importantly, early concerns about a possible proliferation of the
blasts have not been confirmed.4,8
Several recent studies on tumor models have shown that GM-CSF gene
immunotherapy is highly therapeutic.37,59-61 Based on our
data on the AML model, a two-step use for GM gene immunotherapy in AML
is feasible. First, administration of GM-CSF transduced AML cells
during induction as a substitute for the systemic cytokine
administration. GM-CSF secretion by AML cells in the bone marrow
microenvironment will address the issue of dose limitations raised by
the toxic effects of systemic cytokine administration.8
When patients are in remission having minimal residual disease, an
additional dose(s) of GM-AML vaccines will need to be administered to
achieve therapeutic benefits mediated by the immune system. However,
certain concerns arise from our observation that long-lasting
antileukemia memory was not maintained by all vaccinated mice. Model
systems in which the fate of tumor-specific T cells can be monitored in
vivo, with or without the presence of tumor antigen(s), would greatly
enhance our understanding of tumor-related memory immunity. It may turn
out to be necessary that gene immunotherapy-treated patients will need
boosts with irradiated, wild-type tumor cells to secure the longevity
of tumor-specific immunity.
| |
FOOTNOTES |
Submitted May 5, 1997;
accepted September 8, 1997.
Supported in part by the Andrew F. Gaffney Foundation.
Address reprint requests to Kyriaki Dunussi-Joannopoulos, MD, PhD,
Genetics Institute, 1 Burrt Rd, Andover, MA 01810.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely
to indicate this fact.
| |
REFERENCES |
1.
Russel NH:
Biology of acute leukemia.
Lancet
349:118,
1997[Medline]
[Order article via Infotrieve]
2.
Zeleznik-Le NJ,
Nicifora G,
Rowley JD:
The molecular biology of myeloproliferative disorders as revealed by chromosomal abnormalities.
Semin Hematol
32:201,
1995[Medline]
[Order article via Infotrieve]
3.
Sawyers CL:
Molecular genetics of acute leukemia.
Lancet
349:196,
1997[Medline]
[Order article via Infotrieve]
4.
Burnett AK,
Eden OB:
The treatment of acute leukemia.
Lancet
349:270,
1997[Medline]
[Order article via Infotrieve]
5.
Del Poeta G,
Stasi R,
Aronica G,
Venditti A,
Cox MC,
Bruno A,
Buccisano F,
Masi M,
Tribalto M,
Amadori S,
Papa G:
Clinical relevance of P-glycoprotein expression in de novo acute myeloid leukemia.
Blood
87:1997,
1996[Abstract/Free Full Text]
6.
Bosch I,
Croop JM:
P-glycoprotein multidrug resistance and cancer.
Biochem Biophys Acta
1288:37,
1996
7.
Caron PC,
Scheinberg DA:
Immunotherapy for acute leukemias.
Curr Opin Oncol
6:14,
1994[Medline]
[Order article via Infotrieve]
8.
Schiffer CA:
Hematopoietic growth factors as adjuncts to the treatment of acute myeloid leukemia.
Blood
88:3675,
1996[Abstract/Free Full Text]
9.
Boon T,
Cerottini JC,
Van den Eynde B,
Van der Bruggen P,
Van Pel A:
Tumor-antigens recognized by T lymphocytes.
Annu Rev Immunol
12:337,
1994[Medline]
[Order article via Infotrieve]
10.
Dranoff G,
Mulligan RC:
Gene transfer as cancer therapy.
Adv Immunol
58:417,
1995[Medline]
[Order article via Infotrieve]
11.
Chen L,
Linsley PS,
Hellström KE:
Costimulation of T cells for tumor immunity.
Immunol Today
14:483,
1993[Medline]
[Order article via Infotrieve]
12.
Pardoll DM:
Paracrine cytokine adjuvants in cancer immunotherapy.
Annu Rev Immunol
13:399,
1995[Medline]
[Order article via Infotrieve]
13.
Chen L,
Ashe S,
Brady WA,
Hellström I,
Hellström KE,
Ledbetter JA,
McGowan P,
Linsley P:
Costimulation of antitumor immunity by the B7 counterreceptor for the T lymphocyte molecules CD28 and CTLA-4.
Cell
71:1093,
1992[Medline]
[Order article via Infotrieve]
14.
Townsend SE,
Allison JP:
Tumor rejection after direct costimulation of CD8+ T cells by B7-transfected melanoma cells.
Science
259:368,
1993[Abstract/Free Full Text]
15.
Gansbacher B,
Zier K,
Daniels B,
Cronin K,
Bannerji R,
Gilboa E:
Interleukin-2 gene transfer into tumor cells abrogates tumorigenicity and induces protective immunity.
J Exp Med
172:1217,
1990[Abstract/Free Full Text]
16.
Tepper RI,
Pattengale PK,
Leder P:
Murine interleukin-4 displays potent anti-tumor activity in vivo.
Cell
57:503,
1989[Medline]
[Order article via Infotrieve]
17.
Sun WH,
Kreisle RA,
Philips AW,
Ershler WB:
In vivo and in vitro characteristics of interleukin 6-transfected B16 melanoma cells.
Cancer Res
52:5412,
1992[Abstract/Free Full Text]
18.
Watanabe Y,
Kuribayashi K,
Miyatake S,
Nishikhara K,
Nakayama E,
Taniyama T,
Sakata T:
Exogenous expression of mouse interferon gamma cDNA in mouse neuroblastoma C1300 cells results in reduced tumorigenicity by augmented anti-tumor immunity.
Proc Natl Acad Sci USA
86:9456,
1989[Abstract/Free Full Text]
19.
Dranoff G,
Jaffee E,
Lazenby A,
Golumbek P,
Levitsky H,
Brose K,
Jackson V,
Hamada H,
Pardoll D,
Mulligan R:
Vaccination with irradiated tumor cells engineered to secrete murine granulocyte-macrophage colony-stimulating factor stimulates potent, specific, and long-lasting anti-tumor immunity.
Proc Natl Acad Sci USA
90:3539,
1993[Abstract/Free Full Text]
20.
Colombo MP,
Forni G:
Cytokine gene transfer in tumor inhibition and tumor therapy: Where are we now?
Immunol Today
15:48,
1994[Medline]
[Order article via Infotrieve]
21.
Musiani P,
Modesti A,
Giovarelli M,
Cavallo F,
Colombo MP,
Lollini PL,
Forni G:
Cytokines, tumour-cell death and immunogenicity: A question of choice.
Immunol Today
18:32,
1997[Medline]
[Order article via Infotrieve]
22.
Abe J,
Wakimoto H,
Aoyagi M,
Hirakawa K,
Hamada H:
Cytokine-gene-modified tumor vaccination intensified by a streptococcal preparation OK-432.
Cancer Immunol Immunother
41:82,
1995[Medline]
[Order article via Infotrieve]
23.
Steinman RM:
The dendritic cell system and its role in immunogenicity.
Annu Rev Immunol
9:271,
1991[Medline]
[Order article via Infotrieve]
24.
Inaba K,
Inaba M,
Romani N,
Aya H,
Deguchi M,
Ikehara S,
Muramatsu S,
Steinman RM:
Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor.
J Exp Med
176:1693,
1992[Abstract/Free Full Text]
25.
Caux C,
Dezutter-Dambuyant C,
Schmitt D,
Banchereau J:
GM-CSF and TNF-alpha cooperate in the generation of dendritic Langerhans cells.
Nature
360:258,
1992[Medline]
[Order article via Infotrieve]
26.
Matulonis UA,
Dosiou C,
Lamont C,
Freeman GJ,
Mauch P,
Nadler LM,
Griffin JD:
Role of B7-1 in mediating an immune response to myeloid leukemia cells.
Blood
85:2507,
1995[Abstract/Free Full Text]
27.
Dunussi-Joannopoulos K,
Weinstein HJ,
Nickerson PW,
Strom TB,
Burakoff SJ,
Croop JM,
Arceci RJ:
Irradiated B7-1 transduced primary acute myelogenous leukemia (AML) cells can be used as therapeutic vaccines in murine AML.
Blood
87:2938,
1996[Abstract/Free Full Text]
28.
Matulonis U,
Dosiou C,
Freeman G,
Lamont C,
Mauch P,
Nadler LM,
Griffin JD:
B7-1 is superior to B7-2 costimulation in the induction and maintenance of T cell-mediated anti-leukemia immunity.
J Immunol
156:1126,
1996[Abstract]
29.
Boyer MW,
Vallera DA,
Taylor PA,
Gray GS,
Katsanis E,
Gorden K,
Orchard PJ,
Blazar BR:
The role of B7 costimulation by murine acute myeloid leukemia in the generation and function of a CD8+ T-cell line with potent in vivo graft-versus-leukemia properties.
Blood
89:3477,
1997[Abstract/Free Full Text]
30.
Dunussi-Joannopoulos K,
Krenger W,
Weinstein HJ,
Ferrara JLM,
Croop JM:
CD8+ T cells activated during the course of murine AML elicit therapeutic responses to late B7 vaccines after cytoreductive treatment.
Blood
89:2915,
1997[Abstract/Free Full Text]
31.
Hodge JW,
Abrams S,
Schlom J,
Kantor JA:
Induction of antitumor immunity by recombinant vaccinia viruses expressing B7-1 or B7-2 costimulatory molecules.
Cancer Res
54:5552,
1994[Abstract/Free Full Text]
32.
Martin-Fontecha A,
Cavallo F,
Bellone M,
Heltai S,
Iezzi G,
Tornaghi P,
Nabavi N,
Forni G,
Dellabona P,
Casorati G:
Heterogeneous effects of B7-1 and B7-2 in the induction of both protective and therapeutic anti-tumor immunity against different mouse tumors.
Eur J Immunol
26:1851,
1996[Medline]
[Order article via Infotrieve]
33.
Gajewski TF:
B7-1 but not B7-2 efficiently costimulates CD8+ T lymphocytes in the P815 tumor system in vitro.
J Immunol
156:465,
1996[Abstract]
34.
Resnitzky P,
Estrov Z,
Haran-Ghera N:
High incidence of acute myeloid leukemia in SJL/J mice after x-irradiation and corticosteroids.
Leuk Res
9:1519,
1985[Medline]
[Order article via Infotrieve]
35.
Arceci RJ,
Stieglitz K,
Bras J,
Schinkel A,
Baas F,
Croop J:
A monoclonal antibody to an external epitope of the human mdr1-p-glycoprotein.
Cancer Res
53:310,
1993[Abstract/Free Full Text]
36.
Wakimoto H,
Abe J,
Tsunoda R,
Aoyagi M,
Hirakawa K,
Hamada H:
Intensified antitumor immunity by a cancer vaccine that produces granulocyte-macrophage colony-stimulating factor plus interleukin 4.
Cancer Res
56:1828,
1996[Abstract/Free Full Text]
37.
Levitsky HI,
Montgomery J,
Ahmadzadeh M,
Staveley-O'Carroll K,
Guarnieri F,
Longo DL,
Kwak LW:
Immunization with GM-CSF-transduced, but not B7-1-transduced lymphoma cells primes idiotype-specific T cells and generates potent anti-tumor immunity.
J Immunol
156:3858,
1996[Abstract]
38.
Vieweg J,
Rosenthal FM,
Bannerji R,
Heston WDW,
Fair WR,
Gansbacher B,
Gilboa E:
Immunotherapy of prostate cancer in the Dunning rat model: Use of cytokine gene modified tumor vaccines.
Cancer Res
54:1760,
1994[Abstract/Free Full Text]
39.
Pericle F,
Giovarelli M,
Colombo MP,
Ferrari G,
Musiani P,
Modesti A,
Cavallo F,
Di Pierro F,
Novelli F,
Forni G:
An efficient Th2-type memory follows CD8+ lymphocyte-driven and eosinophil-mediated rejection of a spontaneous mouse mammary adenosarcoma engineered to release IL-4.
J Immunol
153:5659,
1994[Abstract]
40.
Budel LM,
Elbaz O,
Hoogerbrugge H,
Delwel R,
Mahmoud RA,
Löwenberg B,
Touw IP:
Common binding structure for granulocyte macrophage colony-stimulating factor and interleukin-3 on human acute myeloidleukemia cells and monocytes.
Blood
75:1439,
1990[Abstract/Free Full Text]
41.
Nicola NA:
Receptors for colony-stimulating factors.
Br J Haematol
77:133,
1991[Medline]
[Order article via Infotrieve]
42.
Sperling AI,
Auger JA,
Ehst BD,
Rulifson IC,
Thompson CB,
Bluestone JA:
CD28/B7 interactions deliver a unique signal to naive T cells that regulates cell survival but not early proliferation.
J Immunol
157:3909,
1996[Abstract]
43.
Boussiotis VA,
Freeman GJ,
Gribben JG,
Nadler LM:
The role of B7-1/B7-2:CD28/CTLA-4 pathways in the prevention of anergy, induction of productive immunity and down-regulation of the immune response.
Immunol Rev
153:5,
1996[Medline]
[Order article via Infotrieve]
44.
Sperling A,
Bluestone JA:
The complexities of T-cell co-stimulation: CD28 and beyond.
Immunol Rev
153:155,
1996[Medline]
[Order article via Infotrieve]
45.
Van Gool SW,
Vandenberghe P,
De Boer M,
Ceuppens JL:
CD80, CD86 and CD40 provide accessory signals in a multiple-step T-cell activation model.
Immunol Rev
153:47,
1996[Medline]
[Order article via Infotrieve]
46.
Reinhold MI,
Lindberg FP,
Kersh GJ,
Allen PM,
Brown EJ:
Costimulation of T cell activation by integrin-associated protein (CD47) is an adhesion-dependent, CD28-independent signaling pathway.
J Exp Med
185:1,
1997[Abstract/Free Full Text]
47.
Marengere LEM,
Waterhouse HW,
Duncan GS,
Mittrucker HW,
Feng GS,
Mak TW:
Regulation of T cell receptor signalling by tyrosine phosphatase SYP association with CTLA-4.
Science
272:1170,
1996[Abstract]
48.
Gribben JG,
Freeman GJ,
Boussiotis VA,
Rennert P,
Jellis CL,
Greenfield E,
Barber M,
Restivo VAJ,
Ke X-Y,
Gray GS,
Nadler LM:
CTLA-4 mediates antigen-specific apoptosis of human T cells.
Proc Natl Acad Sci USA
92:811,
1995[Abstract/Free Full Text]
49.
Chambers CA,
Krummel MF,
Boitel B,
Hurwitz A,
Sullivan TJ,
Fournier S,
Cassel D,
Brunner M,
Allison JP:
The role of CTLA-4 in the regulation and initiation of T-cell responses.
Immunol Rev
153:27,
1996[Medline]
[Order article via Infotrieve]
50.
Linsley PS,
Greene IL,
Brady W,
Bajorath J,
Ledbetter JA,
Peach R:
Human B7-1 (CD80) and B7-2 (CD86) bind with similar avidities but distinct kinetics to CD28 and CTLA-4 receptors.
Immunity
1:793,
1994[Medline]
[Order article via Infotrieve]
51.
Morton PA,
Fu XT,
Stewart JA,
Giacoletto KS,
White SL,
Leysath CE,
Evans RJ,
Shieh JJ,
Karr RW:
Differential effects of CTLA-4 substitutions on the binding of human CD80(B7-1) and CD86(B7-2).
J Immunol
156:1047,
1996[Abstract]
52.
Gajewski TF,
Fallarino F,
Uyttenhove C,
Boon T:
Tumor rejection requires a CTLA-4 ligand provided by the host or expressed on the tumor: Superiority of B7-1 over B7-2 for active tumor immunization.
J Immunol
156:2909,
1996[Abstract]
53.
Schweitzer AN,
Borriello F,
Wong RCK,
Abbas AK,
Sharpe AH:
Role of costimulators in T cell differentiation.
J Immunol
158:2713,
1997[Abstract]
54.
Leach DR,
Krummel MF,
Allison JP:
Enhancement of antitumor immunity by CTLA-4 blockade.
Science
271:1734,
1996[Abstract]
55.
Cavallo F,
Giovarelli M,
Gulino A,
Vacca A,
Stoppacciaro A,
Modesti A,
Forni G:
Role of neutrophils and CD4+ T lymphocytes in primary and memory response to nonimmunogenic murine mammary adenocarcinoma made immunogenic by IL-2 gene.
J Immunol
149:3627,
1992[Abstract]
56.
Vellenga E,
Ostapovicz D,
O'Rourke B,
Griffin JD:
Effects of recombinant IL-3, GM-CSF, and G-CSF on the proliferation of leukemic clonogenic cells in short-term and long-term cultures.
Leukemia
1:584,
1987[Medline]
[Order article via Infotrieve]
57.
Delwel R,
Salem M,
Dorssers L,
Wagemaker G,
Clark S,
Löwenberg B:
Growth regulation of human acute myeloid leukemia: Effects of five recombinant hematopoietic growth factors in a serum-free culture system.
Blood
72:1944,
1988[Abstract/Free Full Text]
58.
Lowenberg B,
Touw IP:
Hematopoietic growth factors and their receptors in acute leukemia.
Blood
81:281,
1993[Free Full Text]
59.
Qin H,
Chatterjee SK:
Cancer gene therapy using tumor cells infected with recombinant vaccinia virus expressing GM-CSF.
Hum Gene Ther
7:1853,
1996[Medline]
[Order article via Infotrieve]
60.
Armstrong CA,
Botella R,
Galloway TH,
Murray N,
Kramp JM,
Song IS,
Ansel JC:
Antitumor effects of granulocyte-macrophage colony-stimulating factor production by melanoma cells.
Cancer Res
56:2191,
1996[Abstract/Free Full Text]
61.
Gunji Y,
Tagawa M,
Matsubara H,
Takenaga K,
Shimada H,
Kondo F,
Suzuki T,
Nakajima K,
Sugaya M,
Asano T,
Ochiai T,
Isono K,
Kageyama H,
Nakamura Y,
Sakiyama S:
Antitumor effect of murine colon carcinoma cells retrovirally transduced with interleukin-4 and granulocyte macrophage-colony stimulating factor genes.
Oncology
54:69,
1997[Medline]
[Order article via Infotrieve]
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|
|
C. Bello-Fernandez, J. Stasakova, A. Renner, N. Carballido-Perrig, M. Koening, M. Waclavicek, O. Madjic, L. Oehler, O. Haas, J. M. Carballido, et al.
Retrovirus-mediated IL-7 expression in leukemic dendritic cells generated from primary acute myelogenous leukemias enhances their functional properties
Blood,
March 15, 2003;
101(6):
2184 - 2190.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Curti, M. Parenza, and M. P. Colombo
Autologous and MHC class I-negative allogeneic tumor cells secreting IL-12 together cure disseminated A20 lymphoma
Blood,
January 15, 2003;
101(2):
568 - 575.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Dunussi-Joannopoulos, K. Zuberek, K. Runyon, R. G. Hawley, A. Wong, J. Erickson, S. Herrmann, and J. P. Leonard
Efficacious immunomodulatory activity of the chemokine stromal cell-derived factor 1 (SDF-1): local secretion of SDF-1 at the tumor site serves as T-cell chemoattractant and mediates T-cell-dependent antitumor responses
Blood,
August 13, 2002;
100(5):
1551 - 1558.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Teshima, N. Mach, G. R. Hill, L. Pan, S. Gillessen, G. Dranoff, and J. L. M. Ferrara
Tumor Cell Vaccine Elicits Potent Antitumor Immunity after Allogeneic T-Cell-depleted Bone Marrow Transplantation
Cancer Res.,
January 1, 2001;
61(1):
162 - 171.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
F. R. Appelbaum, J. M. Rowe, J. Radich, and J. E. Dick
Acute Myeloid Leukemia
Hematology,
January 1, 2001;
2001(1):
62 - 86.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Urashima, H. Suzuki, Y. Yuza, M. Akiyama, N. Ohno, and Y. Eto
An oral CD40 ligand gene therapy against lymphoma using attenuated Salmonella typhimurium
Blood,
February 15, 2000;
95(4):
1258 - 1263.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Dunussi-Joannopoulos, K. Runyon, J. Erickson, R. G. Schaub, R. G. Hawley, and J. P. Leonard
Vaccines With Interleukin-12-Transduced Acute Myeloid Leukemia Cells Elicit Very Potent Therapeutic and Long-Lasting Protective Immunity
Blood,
December 15, 1999;
94(12):
4263 - 4273.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. W. Simons, B. Mikhak, J.-F. Chang, A. M. DeMarzo, M. A. Carducci, M. Lim, C. E. Weber, A. A. Baccala, M. A. Goemann, S. M. Clift, et al.
Induction of Immunity to Prostate Cancer Antigens: Results of a Clinical Trial of Vaccination with Irradiated Autologous Prostate Tumor Cells Engineered to Secrete Granulocyte-Macrophage Colony-stimulating Factor Using ex Vivo Gene Transfer
Cancer Res.,
October 1, 1999;
59(20):
5160 - 5168.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. M. Anderson, S. N. Markovic, J. A. Sloan, M. L. Clawson, M. Wylam, C. A. S. Arndt, W. A. Smithson, P. Burch, M. Gornet, and E. Rahman
Aerosol Granulocyte Macrophage-Colony Stimulating Factor: A Low Toxicity, Lung-specific Biological Therapy in Patients with Lung Metastases
Clin. Cancer Res.,
September 1, 1999;
5(9):
2316 - 2323.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Abstract
Introduction
Abstract