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Blood, Vol. 94 No. 12 (December 15), 1999:
pp. 4263-4273
By
From the Department of Preclinical Research and Development, Genetics
Institute, Andover, MA; and the Hematopoiesis Department, Holland
Laboratory, American Red Cross, Rockville, MD.
Interleukin-12 (IL-12) is a heterodimeric cytokine mediating a
dynamic interplay between T cells and antigen-presenting cells (APCs).
Preclinical studies have demonstrated that recombinant murine IL-12
(rmIL-12) promotes specific antitumor immunity mediated by T cells in
several types of tumors. However, the in vivo antitumor properties of
IL-12 in acute myeloid leukemia (AML) have not been previously
reported. We show here in a murine AML model that systemic administration of rmIL-12 significantly delays tumor growth but is
incapable of rescuing mice from lethal leukemia. In contrast, AML cells
genetically modified to express IL-12 (IL12-AML) using murine stem cell
virus (MSCV) p40 + p35 elicit very potent antileukemic activity.
Vaccines with lethally irradiated IL12-AML cells protect naive mice
against challenge with wild-type AML cells and, more importantly, can cure mice bearing a considerable leukemic burden. Immunized mice show no signs of systemic IL-12 toxicity and their spleen histology is comparable with naive mice spleen. In vivo depletion of IL-12, interferon-
DESPITE THE BROAD USE of the term
vaccines in tumor immunology, current cell-based cancer vaccines are
always therapeutic, aiming to activate host immune responses against
tumor antigens to which the immune system has already been
exposed.1-3 Under these conditions, for tumor cell vaccines
to confer a favorable clinical outcome, they must circumvent host
immune mechanisms conferring the nonresponsiveness (T-cell tolerance)
to cancer.4,5 Recent molecular immunology studies focused
on pathways of T-cell activation have deciphered several critical steps
that can lead to cancer tolerance. It has been clearly demonstrated
that effective antigen recognition by T cells can only occur when the
surrounding tissues carry danger signals as a result of local
inflammation and tissue damage. The most important carriers of these
signals, the host antigen-presenting cells (APCs), provide T cells with membrane-bound and soluble molecules, which are absolutely necessary for their costimulation and consequent expansion upon encountering new
antigens.6,7 The expression of T-cell costimulatory
molecules on APCs and the type of T-cell activation and differentiation largely depends on the cytokine environment at the time of antigen recognition.8-10
The hypothesis that lack of appropriate costimulatory and
cytokine-mediated signals are major causes enabling most tumor types to
induce T-cell tolerance has been confirmed during recent years in
numerous studies on gene therapy tumor models (reviewed in Chen et
al,11 Dranoff and Mulligan,12 and
Pardoll13). In these studies, tumor cells are genetically
engineered to express genes encoding various cytokine and costimulatory
molecules or the combination of both. In this context, the members of
the B7 family costimulatory molecules CD80 (B7.1) and CD86 (B7.2) have shown efficacy at inducing protective and therapeutic immunity against
a number of murine tumors.14-16 Among the different
cytokine genes that have been used to enhance tumor immunogenicity,
granulocyte-macrophage colony-stimulating factor (GM-CSF) and
interleukin-12 (IL-12) appear to be the most potent
molecules.17-20 It has been recently reported that the
expression of both B7.1 and IL-12 molecules at the tumor site enhances
the antitumor efficacy achieved with either molecule
alone.21,22 A critical issue that has emerged from these
studies is that the sustained local release of low amounts of cytokines
leads to the development of dramatic local tissue changes, mostly
having the character of local inflammation, without causing any adverse
systemic effects.
IL-12, a heterodimeric disulfide-linked glycoprotein consisting of a
35- and a 40-kD subunit, has a relatively short history. It was
originally identified in 1989 as natural killer (NK) cell stimulatory
factor (NKCS),23 and it was soon demonstrated that it
mediates a number of important biological properties, including stimulation of cytotoxicity and proliferation of NK cells and cytotoxic
T lymphocytes (CTL), induction of cytokine and chemokine secretion,
especially interferon- Vitale et al42 and Stine et al43 have recently
demonstrated that IL-12 alone or in combination with low-dose IL-2
promotes the lysis of AML blasts in vitro. However, the in vivo
activity of IL-12 in AML has not been previously reported. The
compelling need to evaluate new therapeutic modalities in AML stems
from the fact that AML remains one of the least responsive tumors to therapy. Despite the fact that effective induction chemotherapy regimens induce remission in 60% to 80% of the patients, long-term survival is only achieved in a minority of patients.44,45
At present, it appears highly unrealistic that even the most promising immunomodulatory approaches will replace remission-induction
chemotherapy protocols in AML. However, it is reasonable to argue that
potent preclinical immunotherapy strategies should be tested as
adjuvant therapy in AML patients in remission, most of whom are
harboring minimal residual disease (MRD).
Recently, we and others have shown in preclinical studies that vaccines
with gene-modified AML cells represent a potent immunotherapeutic approach.46-49 We have demonstrated in an SJL primary
leukemia model that vaccines with AML cells expressing costimulatory
molecules (B7.1) or cytokines (GM-CSF) cure leukemic mice and protect
naive mice against subsequent challenge with wild-type
(wt) AML cells.47 The efficacy of the AML
cell-based vaccines provides convincing evidence that leukemia-specific
antigens exist on AML cells and, more importantly, emphasize that the
AML antigens recognized by the immune system do not cross-react with
epitopes on normal hematopoietic progenitors. In this report, we extend
our studies to evaluate the efficacy of the cytokine IL-12 in the SJL
AML model. Our results demonstrate that, within the same model,
systemic and local release of IL-12 confer distinct clinical effects.
Although systemic administration of rmIL-12 leads to significantly
delayed leukemia growth and prolonged survival, ultimately it cannot
prevent the progression from residual to lethal leukemia. In contrast,
vaccines with AML cells expressing IL-12 elicit very potent
antileukemia immunity, leading to the curing of leukemic
mice
Mice.
Female SJL/J mice (H-2s), which were 6 to 8 weeks old, were
purchased from Jackson Laboratories (Bar Harbor, ME). The animals were
kept at the animal facility of Genetics Institute according to the
institute's guidelines.
Murine AML model.
The murine AML model used in this study has been previously
described.46 Briefly, radiation-induced AML cells are
maintained by growth in syngeneic SJL/J female mice. Mice injected
intravenously (IV) or intraperitoneally (IP) with rmIL-12 administration.
Mice were injected IV (tail vein) with live 105 AML cells
and subsequently received subcutaneous (SC) injections of rmIL-12 (Genetics Institute, Andover, MA) at various doses and schedules. Mice
were observed for signs of toxicity and monitored daily for survival.
Retroviral constructs and murine IL-12 transduction of AML cells.
The retroviral vector encoding murine IL-12 and producer clones have
been previously described.50 Briefly, the vector uses the
LTR of the murine stem cell virus (MSCV) and contains the puromycin
N-acetyltransferase (pac) gene under the control of an
internal phosphoglycerate kinase (pgk) gene promoter. An
encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES)
permits the translation of the p35 gene from the same
LTR-directed RNA transcript as the p40 gene. The retroviral
construct was transfected into helper- free ecotropic packaging
GP+E-86 cells and producer clones (GP+E-86/MSCVmIL-12) secreting high titer of virus were used for AML cell infection. Producer cells secreting empty virus (GP+E-86/MSCVpac) were used for infection of control cells. Cells were main- tained at
37°C in Dulbecco's modified Eagle's medium containing 10%
fetal calf serum, 2% glutamine, 1% penicillin-streptomycin, and
1 µg/mL puromycin. Infection of AML cells with recombinant
viruses has been previously described.46 Briefly,
frozen spleen AML cells (5 to 7 × 105/mL) were
exposed twice to viral supernatant for 4 to 6 hours in the presence of
8 µg/mL polybrene and 15% WEHI-3B-conditioned media.
Designated numbers of infected, unselected cells were used for in
vivo injections.
Recombinant murine IL-12 enzyme-linked immunosorbent assay (ELISA).
Levels of rmIL-12 secreted by irradiated and nonirradiated IL12-AML
cells, cultured for 24 hours at 106 cells/mL, were
determined using the InterTest-12X mouse rmIL-12p70 ELISA kit (Genzyme,
Cambridge, MA). The kit is a solid-phase ELISA using the multiple
antibody sandwich principle and is used for the quantitation of mouse
rmIL-12p70 heterodimer levels. The sensitivity of the assay is 5 pg/mL.
In vivo immunization studies.
For the in vivo immunization studies, groups of leukemic or naive SJL/J
mice (8 to 10/group) were injected IV (tail vein) with live or
irradiated (7,335 cGy from a 137Cs source discharging 815 cGy/min) IL12-AML or control pac-AML cells. In most of the experiments,
105 cells were used and were diluted in 200 to 300 µL of
phosphate-buffered saline (PBS).
In vivo depletion studies.
The following antibodies were used for in vivo depletion studies:
C17.15, rat antimouse IL-12 monoclonal antibody (MoAb; Genetics Institute); XGM1.2, rat antimouse IFN- Proliferation assays.
AML cells were cultured at 4 × 105 cells/well in
U-bottomed 96-well plates in the presence of rmIL-12 (10 ng/mL) or
rmIFN- Immunostaining and flow cytometry analysis.
AML cells were cultured at 2 × 106 cells/mL in the
presence of rmIL-12 (10 ng/mL) or rmIFN- Spleen histology-immunohistochemistry.
Spleens were collected and bisected; one half was cryopreserved in
optimum cutting temperature (OCT; Tissue-Tek; Sakura Finetek, Inc,
Torrance, CA) by liquid nitrogen-cooled isopentane method, and the other half was fixed in 10% neutral-buffered formalin. For
histological evaluation, 5-µm sections from paraffin-embedded tissues
were stained with hematoxylin and eosin (H+E). For
immunohistochemistry, cryopreserved samples were cryosectioned onto
capillary gap microslides and fixed in acetone before storing at
51Cr release CTL assays.
Spleens were collected and single-cell suspensions were prepared.
Splenocytes (5 × 106) were cocultured with irradiated
(7,335 cGy) AML cells (1 × 105) in 2 mL complete
RPMI/well of a 24-well tissue culture plate (Costar, Cambridge, MA).
Six days later, splenocytes were harvested and used as effector cells
in CTL assays. Autologous AML cells or control EL-4 tumor cells (2 × 106) were labeled with 200 µCi of
51Cr (New England Nuclear, Boston, MA) for 90 minutes,
washed twice, and used as targets (5,000/well) in the CTL assays. The
standard 4-hour CTL assays were set up with various effector to target (E:T) ratios in a total volume of 0.2 mL/well in a 96-well microtiter plate. All conditions were set up in quadruplicate. After 4 hours of
incubation, 75 µL of supernatant was harvested from each well and
mixed with 125 µL of liquid scintillation cocktail (Optiphase Suprmix; Wallac, Turku, Finland), and the quantity of 51Cr
was determined using a microbeta counter. Results are expressed as the
percentage of specific lysis, which was calculated as follows: 100 × (sample cpm Statistical analysis.
Most individual experiments consisted of 10 mice per treatment group.
The data analyzed represent the results of 1 or 2 individual experiments. The statistical survival analysis was performed using the
standard Mantel-Cox logrank test. Cytokine values secreted by
transduced AML cells and AML cells proliferation results are mean ± SD. The statistical significance between various groups was analyzed
using the Student's t-test.
Systemic rmIL-12 administration significantly delays leukemia growth.
We first investigated the effect of rmIL-12 administration on the tumor
growth. In this AML model, untreated mice develop lethal leukemia 4 to
5 weeks after AML inoculation. Various doses and schedules of rmIL-12
administration were tested (including continuous low dose of 0.5 µg/d
during the entire period of the experiment), and the best results were
achieved when leukemic mice received 2.5 µg/injection of rmIL-12 SC
on days 0 through 4, 14 through 18, and 28 through 32. During the
course of rmIL-12 administration, mice did not develop any clinical
signs of toxicity. Spleen histology and fluorescence-activated cell
sorting (FACS) analysis 4 weeks after leukemia
inoculation showed that rmIL-12-treated mice had comparable spleen
histology and cell subsets with naive mice (data not shown). However,
although IL-12-treated mice had a statistically significant prolonged
survival compared with control untreated animals (P < .001),
this type of treatment could not prevent the development of lethal
leukemia (Fig 1).
IL-12 gene transduction of AML cells.
The levels of rmIL-12 secreted by IL-12-transduced AML cells
(IL12-AML) were 2.2 ± 3.4 ng/106 cells/mL/24 h and were
comparable to production levels reported in other IL-12 gene transfer
studies. wt AML cells cultured under the same conditions did not
secrete detectable amounts of rmIL-12. Irradiation of IL12-AML cells
did not abrogate rmIL-12 secretion in vitro for at least 4 days (data
not shown). After 3 to 5 days of culture, the in vitro growth
characteristics of IL12-AML cells, including cell morphology and growth
rate, as well as MHC, costimulatory, and adhesion molecule expression,
were similar to those of mock-infected AML cells (data not shown).
IL12-AML cells have reduced tumorigenicity.
Studies on gene therapy tumor models have shown that a dissociation may
exist between the tumorigenicity of live cells and the immunogenicity
of irradiated transduced tumor cells, suggesting that mechanisms
responsible for tumor growth inhibition and immune memory may not be
the same.51,52 In this context, Dranoff et al17
have demonstrated that irradiated, GM-CSF-transduced murine melanoma
cells are potent prophylactic vaccines, whereas live transduced cells
are not rejected. In contrast, we have shown in previous studies that
leukemogenic numbers of live B7.2-expressing, IL-4-expressing, and
tumor necrosis factor-
Irradiated IL12-AML cells induce prophylactic and therapeutic
immunity.
In clinical trials with tumor cell vaccines, cancer patients are
vaccinated with irradiated, genetically modified tumor cells. Therefore, we evaluated the capacity of irradiated IL12-AML cells to
protect naive mice from live wt AML challenge or to cure mice with
already established leukemia. In this model, vaccinations with
irradiated wt AML cells confer no detectable antileukemia immunity.46 Mice were injected with 105
irradiated (7,335 cGy) IL12-AML or pac-AML cells and 1 week later were
challenged with 105 live wt AML cells. All mice immunized
with irradiated IL12-AML cells developed antileukemia immunity and
survived the AML challenge, whereas the challenge was lethal to all
mice immunized with irradiated pac-AML cells
(Fig 3A).
Vaccinated leukemic mice have spleen histopathology comparable with
naive mice.
In our studies, mice injected with either live or irradiated IL12-AML
cells never developed any clinical signs of toxicity. To obtain
information on organ histology of vaccinated mice, we compared spleens
from IL12-AML or pac-AML vaccinated mice and naive SJL mice. In this
experiment, mice were injected with live wt AML cells and 1 week later
were immunized with irradiated IL12-AML or control pac-AML cells. The
spleens were removed 31 days after leukemia inoculation, at the time
when the control (pac-AML) vaccinated mice had become moribund. Spleen
histology showed that the naive spleen and the spleen from IL12-AML
vaccinated mouse were comparable, both having appreciable hematopoiesis
present and normal complements of what appeared to be relatively active
lymphoid tissue or white pulp. In contrast, the spleen from the control
vaccinated mouse had virtually complete obliteration of splenic
architecture with a diffuse infiltrate of myeloid leukemic cells (data
not shown). Spleen immunohistochemistry using the lymphoid antigens
CD3, CD4, CD8, and B220 and the myeloid antigen Gr-1 (it is not
expressed on lymphoid and erythroid cells) confirmed comparable normal
architecture and distribution of lymphoid and myeloid cells in the
naive and IL12-AML spleen. In the control vaccinated spleen, very few
cells stained positive for lymphoid markers and the spleen was
diffusely infiltrated by Gr-1-positive cells
(Fig 4). Taken together, spleen histology
demonstrates that the local secretion of rmIL-12 by irradiated tumor
cells, which presumably leads to transient local recruitment of immune
cells and consequent tumor rejection, does not result in any long-term
microscopic alterations from the normal splenic architecture.
In vivo depletion of IL-12 or IFN-
In vitro effects of rmIL-12 and rmIFN-
IL12-AML cell rejection is CD8+ T-cell dependent and
supports the development of long-lasting, leukemia-specific
CTL activity.
The mechanisms responsible for tumor rejection in cytokine gene
immunotherapy may not be the same as those required for immune memory.
Several types of immune cells, such as macrophages, eosinophils, and
neutrophils, that are recruited locally by the secreted cytokine may
participate in tumor killing, but the induction of memory cells is
exclusively CD8+ T-cell dependent.12,51 To
delineate the role of T-cell subsets in the rejection of live IL12-AML
cells, we performed in vivo depletion of CD4+ or
CD8+ T cells. As shown in Fig
7A, all CD8+-depleted vaccinated mice developed leukemia at
the same time as control animals, whereas from the
CD4+-depleted group only 20% developed leukemia, with 80%
of the mice rejecting live IL12-AML cells. These results demonstrate
the absolute requirement for CD8+ T cells in tumor
rejection, whereas the participation of CD4+ T cells was
not necessary in most of the vaccinated mice. In an attempt to address
the issue of CTL memory persistence in the AML model, we challenged
mice with wt AML cells 4 months after the rejection of live IL12-AML
cells. All the mice in this experiment were resistant to the challenge
(Fig 7B). Three months later, spleens from these mice were removed and
assayed for in vitro leukemia-specific CTL activity. As shown in Fig
7C, splenocytes from immunized mice generated a cytolytic response upon
stimulation with wt AML cells. The response was AML-specific, because
the same cells did not lyse alloantigen-presenting EL-4
(H-2b) cells. These results indicate that, in this model,
the rejection of live IL12-AML cells is mediated almost exclusively by
CD8+ T cells, which then develop into long-lived
leukemia-specific memory cells.
In this report, we have used a murine primary AML model to assess the
in vivo activity of the cytokine IL-12. The results we present show
that local release of rmIL-12 by retrovirally transduced AML cells
supports the generation of both therapeutic and long-lasting protective
immunity. Furthermore, we demonstrate that, in this AML model, the
antileukemia immune responses mediated by IL12-AML vaccines are both
IL-12 and IFN- The authors thank Sharon Hunter and Joyce Johnson for technical help,
Dr Brian Clancy for the gene chip experiments, Dr Page Bouchard for
reviewing the spleen histology slides, and Dr Stan Wolf for critical
review of the manuscript.
Submitted April 19, 1999; accepted August 23, 1999.
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.
Address reprint requests to Kyriaki Dunussi-Joannopoulos, MD, PhD,
Genetics Institute, 1 Burtt Rd, Andover, MA 01810.
1.
Pardoll DM:
Cancer vaccines.
Nat Med
4:525, 1998
[Medline]
[Order article via Infotrieve]
2.
Hodi FS, Dranoff G:
Genetically modified tumor cell vaccines.
Cancer Gene Ther
7:471, 1998
3.
Simons JW, Mikhak B:
Ex-vivo gene therapy using cytokine-transduced tumor vaccines: Molecular and clinical pharmacology.
Semin Oncol
25:661, 1998
[Medline]
[Order article via Infotrieve]
4.
Nanda NK, Sercarz EE:
Induction of anti-self-immunity to cure cancer.
Cell
82:13, 1995
[Medline]
[Order article via Infotrieve]
5.
Sotomayor EM, Borrello I, Levitsky HI:
Tolerance and cancer: A critical issue in tumor immunology.
Crit Rev Oncog
7:433, 1996
[Medline]
[Order article via Infotrieve]
6.
Schwartz RH:
Costimulation of T lymphocytes: The Role of CD28, CTLA-4, and B7//BB1 in interleukin-2 production and immunotherapy.
Cell
71:1065, 1992
[Medline]
[Order article via Infotrieve]
7.
June CH, Bluestone JA, Nadler LM, Thompson CB:
The B7 and CD28 receptor families.
Immunol Today
15:321, 1994
[Medline]
[Order article via Infotrieve]
8.
Palm N, Germann T, Goedert S, Hoehn P, Koelsch S, Rude E, Schmitt E:
Co-development of naive CD4+ cells towards T helper type 1 or T helper type 2 cells induced by a combination of IL-12 and IL-4.
Immunobiology
196:475, 1996
[Medline]
[Order article via Infotrieve]
9.
Wenner CA, Guler ML, Macatonia SE, O'Garra A, Murphy KM:
Roles of IFN-gamma and IFN-alpha in IL-12-induced T helper cell-1 development.
J Immunol
156:1442, 1996
[Abstract]
10.
Bradley LM, Dalton DK, Croft M:
A direct role for IFN-gamma in regulation of Th1 cell development.
J Immunol
157:1350, 1996
[Abstract]
11.
Chen L, Linsley PS, Hellström KE:
Costimulation of T cells for tumor immunity.
Immunol Today
14:483, 1993
[Medline]
[Order article via Infotrieve]
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TOP
ABSTRACT
INTRODUCTION
(IFN-
104 AML
cells develop systemic 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 leukemia) were used.
Flow cytometry shows that essentially 100% of these cells express the
myeloid-specific antigen Gr-1.
20°C. Immediately before staining, stored cryosections were
fixed in cold acetone for 5 minutes, air-dried, blocked with
avidin/biotin block (Zymed Laboratories, Inc, South Francisco, CA), and
finally washed in PBS. Next, the slides were assembled on an automated
immunostainer (Ventana Tech Mate 500; Ventana Medical
Systems, Inc, Tucson, AZ) and stained with various MoAbs diluted to
final concentrations ranging from 1 to 5 µg/mL. The following MoAbs
were used: Gr-1 (RB6-8C5), CD4 (L3T4), CD8a (53-6.7), and CD45R/B220
(RA3-6B2) (PharMingen). Serial sections stained with the appropriate
isotype Igs were used as negative controls. An indirect
streptavidin-peroxidase method was used with DAB (3, 3'
diaminobenzidine tetrahydrochloride) as color chromogen. For the B220
staining, paraffin-embedded samples were stained using AEC
(3-amino-9-ethylcarbazole) as the color chromogen.
). Control mice (
) received injections with PBS. A solid
arrow indicates the time of spleen histology and FACScan analysis. This
graph is representative of 4 independent experiments.
-expressing AML cells are
rejected, whereas irradiated cells do not protect mice against wt AML
challenge.
) or 2 × 106 (
), or 15 (
