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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 12 (June 15), 1999:
pp. 4328-4335
Activated Dendritic Cells From Bone Marrow Cells of Mice Receiving
Cytokine-Expressing Tumor Cells Are Associated With the Enhanced
Survival of Mice Bearing Syngeneic Tumors
By
Shin-ichiro Fujii,
Hirofumi Hamada,
Koji Fujimoto,
Taizo Shimomura, and
Makoto Kawakita
From the Center for Bone Marrow Transplantation and Immunotherapy,
Institute for Clinical Research, Kumamoto National Hospital, Kumamoto,
Japan; the Department of Molecular Biotherapy Research, Cancer
Chemotherapy Center, Cancer Institute, Tokyo, Japan; and the Second
Department of Internal Medicine, Kumamoto University School of
Medicine, Kumamoto, Japan.
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ABSTRACT |
Dendritic cells (DCs), which phagocytose antigens and subsequently
proliferate and migrate, may be the most powerful antigen-presenting cells that activate naive T cells. To determine their role in the
immune response to tumors, we used WEHI-3B murine leukemia cells
transduced with adenovirus vectors expressing cytokines. We found that
mixtures of irradiated cells expressing granulocyte-macrophage colony-stimulating factor (GM-CSF) plus those expressing interleukin-4 (IL-4) or tumor necrosis factor  (TNF) protected mice against WEHI-3B-induced leukemias. When bone marrow mononuclear cells (BMMNCs)
obtained from mice that had been injected with irradiated, cytokine-expressing tumor cells were injected into tumor-bearing mice,
the survival of the latter was significantly prolonged; the longest
survival was observed in mice receiving BMMNCs containing an increased
number of DCs from animals injected with a mixture of tumor cells
expressing GM-CSF with those expressing IL-4. Assay for antileukemic
effects in spleen of the latter animals showed specific antitumor
cytotoxicity against WEHI-3B, suggesting that DCs from donor mice
activate specific T cells in the tumor-bearing recipients. These
results suggest that the infusion of syngeneic BMMNCs stimulated with
cytokine-expressing tumor cells may be effective in treating certain
types of tumors.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE INTRODUCTION OF genes encoding
cytokines, costimulatory factors, or antigen-presenting molecules into
tumor cells can enhance the host's antitumor immunity and may be
useful in treating patients with cancer.1-5 Vaccination of
using animals with cytokine-expressing tumor cells has shown that, of
the cytokines assayed, the most potent, specific, and long-lasting
inducer of immune responses against non-gene-modified tumor cells is
granulocyte-macrophage colony-stimulating factor
(GM-CSF).2,3 These findings suggested that GM-CSF induces
the production of antigen-presenting cells (APCs), primarily dendritic
cells (DCs), in these hosts. In addition, a high peritumoral level of
GM-CSF, induced by an enhanced localized immune response, can also
attract immune cells (DCs, T lymphocytes, natural killer [NK] cells,
neutrophils, and macrophages) to the tumor site without causing
systemic cytotoxicity.
It has been hypothesized that functional immature DCs in the bone
marrow (BM) may take up tumor-associated antigens6 and, after migrating into the peripheral tissues, such as the lymph nodes or
spleen, may develop into mature DCs.7 These, in turn, may
activate killer T cells, which exhibit antitumor activity in syngeneic
leukemia-bearing mice. DCs can be induced from BM of humans and mice by
either tumor necrosis factor  (TNF) or interleukin-4 (IL-4) in
combination with GM-CSF.8-12 To determine the combination
of cytokines that effectively induces the in vivo activation of T cells
by DCs, we used a murine monocytic leukemic cell line, WEHI-3B,
infected with an adenoviral vector expressing murine GM-CSF, TNF, or
IL-4. We tested the ability of these irradiated, cytokine-expressing
WEHI-3B cells, individually or in combination, to suppress the
induction of tumors by wild-type WEHI-3B cells. We also assayed the
ability of BM cells from mice injected with cytokine-expressing WEHI-3B
cells to suppress the growth of pre-existing leukemic cells in
syngeneic mice.
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MATERIALS AND METHODS |
Cell lines, reagents, and mice.
The murine monocytic leukemic cell line, WEHI-3B, was obtained from the
Institute for Fermentation (Osaka, Japan) and A20 was obtained from
American Type Culture Collection (ATCC TIB-208; ATCC, Rockville, MD).
Hamster antimouse antibody CD11c (N418) was purchased from PharMingen
(San Diego, CA) and goat antihamster IgG FITC were purchased from
Southern Biotechnology Associates (Birmingham, AL). Antimouse CD80- and
CD86-fluorescein isothiocyanate (FITC) were purchased from Immunotech
(Marseille, France). Rat antimouse monoclonal antibody (MoAb) against
CD4, CD8, Mac-1, B220, Gr-1, goat antirat MoAb-FITC all were purchased
from PharMingen. DEC205 (NDLC-145) was kindly provided by Dr Ralph
Steinman (The Rockefeller University, New York, NY).
Female BALB/c mice, 7 to 8 weeks old, were obtained from SLC (Shizuoka,
Japan) and housed under specific pathogen-free conditions in the
Kumamoto University Animal Center (Kumamoto, Japan).
Generation of recombinant adenoviruses.
Murine GM-CSF, TNF, and IL-4 cDNA were each subcloned into a
plasmid, pCAGGS, that consists of a cytomegalovirus enhancer, a chicken
 -actin promoter, cDNA cloning sites, and an anti-(rabbit -globin) poly(A) signal sequence. Each expression cassette was subcloned into the Swa I site of pAdex1cw, a 42-kb cosmid
containing the 31-kb adenovirus type 5 genome that lacks the E1A, E1B,
and E3 genes. Each cosmid was transfected into 293 cells (ATCC;
CRL1573), and positive clones were picked using standard
techniques.13
Transduction of adenovirus vector into WEHI-3B.
Exponentially growing WEHI-3B cells in complete medium (CM), consisting
of Dulbecco's modified Eagle's medium (DMEM) containing 100 U/mL penicillin, 100 µg/mL kanamycin, 2 mmol/L L-glutamine, and
10% fetal calf serum (FCS), were transduced for 1 hour
with a recombinant adenovirus expressing murine GM-CSF,
TNF, or IL-4, at a multiplicity of infection (moi) of 0 to 4,000 with frequent gentle shaking; these cells, designated WEHI-3B-GM-CSF,
WEHI-3B-TNF, and WEHI-3B-IL-4 cells, respectively, were
subsequently incubated for 48 hours at 37°C in CM in a 5%
CO2 incubator. Cells transduced with recombinant
adenovirus-lacZ (Ad-lacZ) and donated WEHI-3B-lacZ cells were used as
a control in all experiments. Efficiency of transduction was verified
by fluorescent-activated cell sorting (FACS) of
-galactosidase-stained cells. Briefly, cells were fixed with 1%
glutaraldehyde for 5 minutes, rinsed once with phosphate-buffered saline, and stained for 8 hours in X-Gal buffer containing 5 mmol/L K4Fe(CN)6, 5 mmol/L
K3Fe(CN)6, 2 mmol/L MgCl2, and 1 mg/mL 5-bromo-4-chloro-3-indolyl -galactoside (X-Gal). The number of
blue cells was scored visually and analyzed with a FluoReporter
lacZ Flow Cytometry Kit (Molecular Probes, Eugene, OR). The
fluorescence intensity of individual cells was measured as relative
fluorescence unit(s) (FU).14
Cytokine production by modified WEHI-3B cells.
One million WEHI-3B cells in 10 mL CM were infected with adenovirus
expressing GM-CSF, TNF, or IL-4 at an moi of 40, 400, or 4,000. The
medium was removed after 48 hours and cytokine expression was measured
by enzyme-linked immunosorbent assay (ELISA) in a 96-well microtiter
plate (Nunc, Roskilde, Denmark), using murine GM-CSF, TNF, and IL-4
ELISA testing kits (Amersham, Little Chalfont, Buckinghamshire, UK).
Vaccination with irradiated, modified WEHI-3B cells.
To assess whether tumor cells expressing cytokines can induce systemic
immunity, BALB/c mice were injected with WEHI-3B-lacZ cells or with
WEHI-3B-GM-CSF cells, alone or in combination with WEHI-3B-TNF or
with WEHI-3B-IL-4 cells, before injection of wild-type WEHI-3B cells.
Briefly, adenovirus-transduced WEHI-3B cells were irradiated to 70 Gy
by a 137Cs source, and 5 × 105 of these
cells were injected into the tail vein of each mouse. Seven days later,
5 × 105 wild-type WEHI-3B cells were injected into
each tail vein for measurement of survival in the first group
(Fig 1A and B). On the same day, a second
group of 5 animals in each group were killed, and the B cells, T cells,
and DC content in the BM of each mouse were analyzed by flow cytometry.
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| Fig 1.
Schematic diagrams of in vivo experiments in leukemic
mice. (A) Mice were injected with wild-type WEHI-3B cells and survival
was measured. (B) Mice were injected with irradiated
WEHI-3B-lacZ, WEHI-3B-GM-CSF, WEHI-3B-GM-CSF plus WEHI-3B-IL-4, or
WEHI-3B-GM-CSF plus WEHI-3B-TNF cells. One week later, mice
were injected with wild-type WEHI-3B cells and survival was
measured. (C) One group of mice was injected with wild-type WEHI-3B
cells. A second group of mice was injected with irradiated, modified
cells as described in (B), above. After 7 days, the latter mice were
killed and BMMNCs from these animals were injected into the first group
of mice.
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Effect of BM mononuclear cells (BMMNCs) from mice injected with
irradiated, modified WEHI-3B cells on syngeneic mice previously
inoculated with wild-type WEHI-3B cells.
Wild-type WEHI-3B cells (5 × 105/mouse) were
inoculated into one group of 10 mice. On the same day, other groups of
10 mice each were injected with 5 × 105 WEHI-3B-lacZ
or WEHI-3B-GM-CSF cells or with WEHI-3B-GM-CSF cells plus
WEHI-3B-TNF or WEHI-3B-IL-4 cells, irradiated as described above.
After 7 days, these mice in the latter four groups were killed, and 5 × 105 or 106 BMMNCs were separated from
each femur sample by centrifugation for 30 minutes at 1,500 rpm on a
Lympholyte-M (Cedarlane Laboratories, Hornby, Ontario,
Canada) and were injected into the tail vein of the first
group of mice (Fig 1C). Survival was measured in one group of recipient mice.
Cell-mediated cytotoxicity assay.
WEHI-3B-lacZ or WEHI-3B-GM-CSF cells, or WEHI-3B-GM-CSF cells plus
WEHI-3B-TNF or WEHI-3B-IL-4 cells, irradiated as described above,
were inoculated into one group of 3 mice (5 × 105/mouse). After 7 days, these mice were killed, and
106 BMMNCs were injected into the tail vein of the other
group of mice. Moreover, after 7 days, these mice were killed for the
preparation of flow cytometric analysis and cytotoxicity assay (see Fig
6A). Cytotoxicity of tumor cells by spleen cells was measured in vitro using the lactate dehydrogenase (LDH) release assay and CytoTox 96 nonradioactive cytotoxicity assay (Promega, Madison, WI). The spleen
cells were tested for their cytotoxicity against WEHI-3B, A20, and
BMMNC from the identical mice in an LDH assay at an effector:target (E:T) ratio of 1:1, 3:1, 5:1, 25:1, and 50:1. Target cells were cocultured with effector cells at various ratios for 6 hours in 96-well
round-bottomed plates (Nunc) in phenol red-free RPMI (GIBCO BRL, Grand
Island, NY) containing 0.5% FCS. Spontaneous release of
effector and target cells was controlled by separate incubation of the
respective populations. Maximal LDH enzyme release was measured after
lysis of the target cells with lysis solution. Cell-free supernatants
were incubated in a separate 96-well plate (Nunc) with LDH substrate
for 30 minutes before measuring absorbance using a microplate reader
(SOFTmax; Molecular Devices Corp, Sunnyvale, CA) at 490 nm
with 650 nm reference. The percentage of cytotoxicity was calculated
according to the following formula: % Cytotoxicity = ([E  St Se]/[M St]) × 100 (with E being the LDH
release by effector-target coculture, St the spontaneous release by
target cells, Se the spontaneous release by effector cells, and M the maximal release by target cells). Target cells are WEHI-3B, A20, and
BMMNCs derived from the syngeneic mouse.
Statistical analysis.
Survival curves of mice were prepared by the Kaplan-Meier method, and
the differences between the survival curves were evaluated using
log-rank tests.
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RESULTS |
Transduction efficiencies and expression of cytokine genes in WEHI-3B
cells.
Adenovirus constructs expressing lacZ and the three cytokines (GM-CSF,
IL-4, and TNF) were transduced into WEHI-3B cells. Each construct
showed a similar transduction efficiency, as determined by
-galactosidase staining and flow cytometric analysis
(Fig 2). When
we assayed cytokine production in the culture supernatants, we found
that transduction of cells with each construct led to the production of
that cytokine, but not of the others, and that cytokine production was
dependent on the moi of infection (Table 1). We also
observed that irradiation of these cells did not markedly affect
cytokine production, and the stability of cytokine's secretion at
these concentrations continued for approximately 5 days.
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| Fig 2.
Efficiency of transduction of recombinant adenoviruses
into WEHI-3B cells. (Panel I) -gal staining of cells transduced with
virus at moi of (a) 4, (b) 40, (c) 400, and (d) 4,000. (Panel II) Flow
cytometric analysis of fluorescein-stained cells transduced with virus
at moi of (A) 0, (B) 4, (C) 40, (D) 400, and (E) 4,000 and exposed to
20 mmol/L fluorescein di--D-galactopyranoside (FDG) at
37°C for 1 hour. The rate of transduction of cells at each moi was
(A) 0.38%, (B) 0.55%, (C) 1.20%, (D) 3.74%, and (E) 29.69%.
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Immune system cells in BMMNCs involved in the response to
cytokine-expressing WEHI-3B cells.
To determine the types of immune cells involved in the response to the
injection of cytokine-gene modified WEHI-3B cells, we injected
irradiated adenovirus-transduced WEHI-3B cells into BALB/c mice. Seven
days later, we assayed the number of DCs and the number of
CD4+ and CD8+ T lymphocytes in the BMMNCs by
flow cytometry (Fig 3). The
number of DCs in the BM, determined by measuring cells expressing DC marker, CD11c, and DEC205, was higher in mice injected with
WEHI-3B-GM-CSF and WEHI-3B-IL-4 cells (4.12% of these cells were
CD11c+, and 10.60% of these cells were
DEC205+) than in mice injected with WEHI-3B-GM-CSF cells
alone (3.15% of these cells were CD11c+, and 0.32% of
these cells were DEC205+, respectively) or in combination
with WEHI-3B-TNF cells (1.74% of these cells were
CD11c+, and 0.39% of these cells were
DEC205+). However, they expressed little Mac-1, CD4, CD8,
and mature DC marker, CD80 (B7-1) and CD86 (B7-2) (Fig 3). We showed
May-Giemsa stain of the BMMNCs, indicating the proliferation in
granulocytes owing to the effects of GM-CSF (Fig 4).
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| Fig 3.
Analysis of immune cell phenotypes of BMMNCs in mice
injected with WEHI-3B cells expressing exogenous cytokines. BALB/c mice
were injected with irradiated WEHI-3B-lacZ, WEHI-3B-GM-CSF,
WEHI-3B-GM-CSF plus WEHI-3B-TNF, or WEHI-3B-GM-CSF plus
WEHI-3B-IL-4 cells. Seven days later, immune cell phenotypes were
assayed by flow cytometry.
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| Fig 4.
May-Giemsa stain of BMMNCs from mice injected with
WEHI-3B cells expressing exogenous cytokines. BALB/c mice were injected
with irradiated WEHI-3B-lacZ, WEHI-3B-GM-CSF plus WEHI-3B-TNF, or
WEHI-3B-GM-CSF plus WEHI-3B-IL-4 cells. Seven days later, each BMMNC
was analyzed by May-Giemsa stain.
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Effect of preinjection of cytokine-expressing WEHI-3B cells on
tumorigenesis.
When we assayed the ability of transduced, irradiated cells to inhibit
tumorigenesis in BALB/c mice, we found that mice injected with 5 × 105 untransduced or WEHI-3B-lacZ cells died 45 to
57 days after inoculation; these mice had a mean survival of 27.2 and
39.0 days, respectively (P > .05;
Fig 5A). Mice inoculated with
WEHI-3B-GM-CSF cells alone showed a significantly prolonged survival
(mean, 56 days; P < .05; Fig 5A). To determine the optimal
amounts of GM-CSF plus TNF and of GM-CSF plus IL-4 needed to enhance
survival, we injected mice with cells that secreted various
concentrations of these cytokines. In mice injected with
WEHI-3B-GM-CSF and WEHI-3B-TNF cells, optimal survival was
observed with cells secreting 10 ng/mL GM-CSF and 1 ng/mL TNF (Fig
5B). In mice injected with WEHI-3B-GM-CSF and WEHI-3B-IL-4 cells,
optimal survival was conferred by cells secreting 500 ng/mL GM-CSF and
100 ng/mL IL-4 (Fig 5C). Both of these were significantly superior to
control (mice injected with WEHI-3B-lacZ cells; P < .007).
In these vaccine models, other statistical analysis showed that, in Fig
5B, the group of mice injected with WEHI-3B-GM-CSF secreting 10 ng/mL
and WEHI-3B-TNF secreting 1 ng/mL (10 ng/mL GM-CSF-WEHI-3B plus 1 ng/mL TNF-WEHI-3B) survived other groups with statistical
significance (P < .05). In Fig 5C, statistically significant
differences were shown between mice of wild-type and mice of 500 ng/mL
GM-CSF-WEHI-3B plus 10 ng/mL IL-4-WEHI-3B, 500 ng/mL GM-CSF-WEHI-3B
plus 100 ng/mL IL-4-WEHI-3B, and 10 ng/mL GM-CSF-WEHI-3B plus 100 ng/mL
IL-4-WEHI-3B (P < .05); however, the mouse group of 500 ng/mL
GM-CSF-WEHI-3B plus 10 ng/mL IL-4-WEHI-3B survived the groups of 100 ng/mL GM-CSF-WEHI-3B plus 10 ng/mL IL-4-WEHI-3B, 100 ng/mL
GM-CSF-WEHI-3B plus 100 ng/mL IL-4-WEHI-3B, and 500 ng/mL
GM-CSF-WEHI-3B plus 100 ng/mL IL-4-WEHI-3B, but without statistical
significance (P > .05). However, 7 days after inoculation, we
did not detect any increase in serum concentrations of the three
cytokines in mice inoculated with cytokine-expressing cells (data not
shown).
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| Fig 5.
Survival of mice inoculated with irradiated WEHI-3B cells
expressing exogenous cytokines. (A) Mice were injected with 5 × 105 wild-type WEHI-3B cells (X). Mice were injected with 5 × 105 irradiated WEHI-3B-lacZ (x) or WEHI-3B-GM-CSF
( ) cells and then with 5 × 105 wild-type WEHI-3B
cells. (B) Mice were injected with 5 × 105 irradiated
WEHI-3B-lacZ cells (X) or with WEHI-3B-GM-CSF and WEHI-3B-TNF
cells expressing 500 ng/mL GM-CSF and 10 ng/mL TNF (x); 10 ng/mL
GM-CSF and 1 ng/mL TNF ( ); 100 ng/mL GM-CSF and 1 ng/mL TNF
( ); 500 ng/mL GM-CSF and 1 ng/mL TNF (); 10 ng/mL GM-CSF and
10 ng/mL TNF ( ); or 100 ng/mL GM-CSF and 10 ng/mL TNF ( ).
Seven days later, each mouse was injected with 5 × 105
wild-type WEHI-3B cells. (C) Mice were injected with 5 × 105 irradiated WEHI-3B-lacZ cells (X) or with
WEHI-3B-GM-CSF and WEHI-3B-IL-4 expressing 10 ng/mL GM-CSF and 100 ng/mL IL-4 (x); 100 ng/mL GM-CSF and 10 ng/mL IL-4 (); 10 ng/mL
GM-CSF and 10 ng/mL IL-4 (); 500 ng/mL GM-CSF and 100 ng/mL IL-4
(); 100 ng/mL GM-CSF and 100 ng/mL IL-4 (); or 500 ng/mL GM-CSF
and 10 ng/mL IL-4 (). Seven days later, each mouse was injected with
5 × 105 wild-type WEHI-3B cells. (D) Mice were injected
with 5 × 105 wild-type WEHI-3B cells (X). Seven days
later, they were injected with 5 × 105 BMMNCs from
syngeneic mice inoculated with 5 × 105 irradiated
WEHI-3B-LacZ (x) or cytokine transfectants expressing 10 ng/mL GM-CSF
and 1 ng/mL TNF () or 500 ng/mL GM-CSF and 10 ng/mL IL-4 () or
with 106 BMMNCs from mice inoculated with 5 × 105 WEHI-3B-GM-CSF and WEHI-3B-IL-4 cells expressing 500 ng/mL GM-CSF and 10 ng/mL IL-4 ().
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Effect of BMMNCs from mice inoculated with cytokine-expressing cells
on tumor-bearing mice.
Because cytokine-expressing WEHI-3B cells inhibited tumor growth when
injected before wild-type WEHI-3B cells, we sought to determine whether
the BMMNCs from mice injected with cytokine-expressing cells could
inhibit pre-existing tumors. We therefore injected BMMNCs from mice
inoculated with irradiated cytokine-secreting WEHI-3B cells into
syngeneic mice that had received wild-type WEHI-3B cells 7 days before.
Compared with the injection of BMMNCs from mice receiving irradiated
WEHI-3B or WEHI-3B-lacZ cells (mean survival, 52.8 days), the
injection of cells from mice receiving cytokine-expressing WEHI-3B led
to a highly significant (P < .0001) enhancement of survival
(Fig 5D). This was especially notable with BMMNCs from mice injected
with WEHI-3B-GM-CSF and WEHI-3B-IL-4 cells: 80% of the animals
inoculated with 5 × 105 BMMNCs and 100% of the
animals receiving 106 BMMNCs survived more than 100 days
(Fig 5D). There are some statistically significant differences between
survival curves: infused mice group with modified BMMNCs (by
WEHI-3B-GM-CSF and WEHI-3B-TNF/IL-4) survived the infused group
with BMMNCs (by WEHI-3B-lacZ) (P < .0009) or control group
(P < .0001), whereas 1 × 106 cells infused
mice group with BMMNCs (by WEHI-3B-GM-CSF and WEHI-3B-IL-4) survived
the 5 × 105 cell infused mice group without
statistical significance (P > .05), but apparently both
groups significantly survived the infused mice group with modified
BMMNCs (by GM-CSF-WEHI-3B and TNF-WEHI-3B) (P < .0004). Simple BMMNCs (by WEHI-3B-lacZ) infused mice group survived
the control group, but without statistical significance (P > .05).
Cell-mediated cytotoxicity assay.
BMMNCs and spleen cells of mice injected with irradiated cytokine
gene-expressing leukemic cells showed weak cytotoxicity against WEHI-3B
in vitro using CytoTox 96 (15.2% ± 3.3% and 25.5% ± 2.3% in
BMMNCs and spleen cells at an E/T ratio of 50:1, respectively), and
T-cell-depleted BMMNCs by negative selection using antimouse CD4 MoAb,
antimouse CD8 MoAb, and antirat IgG MoAb-coated immunomagnetic beads
(Dynal, Oslo, Norway) showed weak cytotoxicity (11.7% ± 0.10% at
an E/T ratio of 50:1). BMMNCs from mice inoculated with BM cells from
mice injected with cytokine gene transfectants also showed weak
cytotoxicity (12.3% ± 3.1% at an E/T ratio of 50:1). However,
spleen cells from mice administered BMMNCs from mice injected with a
mixture of tumor cells expressing GM-CSF with those expressing IL-4 or
those expressing TNF cells expressed mature DC phenotypic marker,
B7-1, and B7-2 (Fig 6B) as well as DC-specific marker DEC205, and CD11c (data not shown), and they could
exhibit the specific cytotoxicity against WEHI-3B (62.9% ± 5.8%
and 53.4% ± 4.9% at an E/T ratio of 50:1, respectively), whereas
less than 12% of nonspecific lysis were observed at the same E/T ratio
when A20 and syngeneic BMMNCs were used as targets. This suggests that
40% to 50% of killing activity was due to the activity of specific
cytotoxic T lymphocytes (CTLs) against WEHI-3B (Fig 6C and
D).
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| Fig 6.
Flow cytometric analysis and cytotoxicity assay of spleen
cells from mice receiving BMMNCs from animals injected with
cytokine-expressing WEHI-3B cells. (A) Schematic diagrams of biological
analysis of mice transfused with modified BMMNCs. (B) Expression of
CD80 (B7-1) as well as CD86 (B7-2) in the spleen cells. Control
(isotype IgG), mouse injected with modified BMMNCs by WEHI-3B-lacZ,
mouse injected with modified-BMMNCs by WEHI-3B-GM-CSF plus
WEHI-3B-IL-4, and mouse injected with modified BMMNCs by
WEHI-3B-GM-CSF plus WEHI-3B-TNF. (C) Cytotoxic activity by spleen
cells of mouse injected with modified-BMMNCs by WEHI-3B-GM-CSF plus
WEHI-3B-IL-4. (D) Cytotoxic activity by spleen cells of mouse injected
with modified-BMMNCs by WEHI-3B-GM-CSF plus WEHI-3B-TNF.
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DISCUSSION |
We evaluated the effects of BM cells obtained from mice injected with
irradiated cytokine gene-expressing leukemic cells on syngeneic
tumor-bearing animals. The expansion of T-cell clones in recipient mice
as well as their prolonged survival suggests that therapy with
activated BMMNCs containing modified tumor cells (eg, cytokine
gene-expressing cells) may be a promising approach to eliminate minimum
residual disease (MRD) in leukemic patients.
Studies with vaccine models have indicated the efficacy of antitumor
immunotherapy.15-19 The induction of DCs by tumor cells expressing GM-CSF has been found to lead to the activation of T
cells,2,3,17 whereas a deficiency of DCs in tumor-bearing mice has been observed to underlie both the observed nonresponsiveness of T cells to tumor antigens and the resulting rapid progression of
tumor.20 However, the stimulation of T cells by DCs
generated by GM-CSF from hematopoietic precursors in tumor-bearing mice almost completely reversed this nonresponsiveness of the T cells, resulting in an arrest of tumor growth and a dramatic prolongation of
survival.21
Tumor-specific immunity by tumor cells expressing GM-CSF has been shown
to involve both CD4+ and CD8+ T
cells,2 whereas CD4+ T cells and NK cells have
an effect on MHC class I-negative tumors.22 In this study,
CD4+ T cells and CD8+ T cells in spleen also
performed a specific cytotoxicity against leukemic cells (Fig 6).
However, the direct systemic administration of cytokines would probably
meet with limited success for severe toxicities. The local production
of GM-CSF may therefore be associated with the ability of this cytokine
to induce the differentiation of DC precursors into dedicated APCs in
the peritumor area.17,23 Because the combination of GM-CSF
with IL-4 or of GM-CSF with TNF is more efficient in inducing DCs
from the hematopoietic stem cells than is GM-CSF alone in
vitro,8-12 the localized production of the same combination
of cytokines as described above may be more effective than GM-CSF alone
in treating tumors in vivo (Fig 5).23
We performed a set of experiments in which WEHI-3B cells were
transduced with GM-CSF gene or GM-CSF plus IL-4 genes or GM-CSF plus
TNF genes. In assaying the antitumor activity of cytokine-expressing cells, we found that the combination of cells that secreted GM-CSF and
IL-4 was more effective than that of cells that expressed GM-CSF and
TNF (Figs 5 and 6). These findings suggest that GM-CSF and IL-4 may
be superior to GM-CSF and TNF in the in vivo generation of
functional DCs from BMMNCs. Although 10 mice that were injected with
irradiated cells secreting GM-CSF and IL-4 demonstrated an enhanced
survival, there was a significant increase in the number of immature
DCs (positive cells for DEC-205 are 10.6%; however, they express
little CD80 and CD86) in BMMNCs (Fig 3). Even simply the BMMNC fraction
as well as T-cell-depleted BMMNCs perform little cytotoxic effects,
which suggests that the relevant cells in the BMMNC fraction, including
slightly immature DCs, do not exert antileukemic effects.
They may have the potential to induce cytotoxic activity against tumor
cells and show effective proliferation in syngeneic mice (Figs 5 and
6).
The development of a local immunological microenvironment during
therapy may increase the immunogenicity of tumor cells and may enhance
the effectiveness of the systemic immune response. The excellent immune
response observed in BMMNCs from syngeneic mice that were inoculated
with cytokine-expressing tumor cells suggests that the homing sites of
modified tumor cells are important (ie, BM for leukemic cells and
regional lymph nodes for melanoma cells). Alternatively, it may suggest
that the importance of the migration of modified tumor cells into the
BM may be associated with the production of immature DCs from BM
hematopoietic stem cells. We demonstrated that the transduced tumor
cells home to the BM at significant enough numbers to create a
significant effect by GM-CSF with or without IL-4 or TNF on local BM
differentiation (Figs 3 and 4). These findings suggest that the
presence of GM-CSF and IL-4 at the site of leukemic cells in the BM can
lead to the production of DCs and to the attraction of immature DCs
from the surrounding hematopoietic progenitor cells in the BM. The
combination of leukemic cells and immature DCs in the BMMNC
preparations we have used may induce the maturation of these functional
DCs. This may occur partly in the BM, due to the uptake of tumor
extracts in specific stages from precursors, but final maturation after uptake of exogenous antigens is thought to take place mainly in the
peripheral tissues,24 including the spleen and lymph nodes, after the migration of the immature DCs to these organs. Mature DCs
expressing B7-1 as well as B7-2 have been shown to enhance the
induction of specific antitumor immune responses in both the spleen and
lymph nodes (Figs 5 and 6).25
We then injected these cells into mice intravenously and showed that
DCs from the BM can be transferred to third-party recipient mice and
transfer antitumor immunity. These transfers putatively result in
expansion of various V TCR segments in the third-party recipient
mice, suggesting that some of the proliferating T-lymphocyte clonotypes
in the recipient mice may be involved in the recognition of
leukemia-associated antigens (data not shown). In fact, in the spleens
of mice receiving BMMNCs, the mature DCs (DEC205+
B7-1+ cells of these cells are 15% in the control group,
are 35% in the GM-CSF + IL-4 group, and are 30% in the GM-CSF + TNF group) were proliferated and the T lymphocytes were activated,
probably due to the ability of the immature DCs in these BMMNC
preparations to migrate to the lymph nodes or spleen, expand, and
interact with the T lymphocytes in the peripheral tissues. The
generation of a specific antitumor response requires that these
functional DCs encounter the relevant tumor-associated antigens and
reflux into the peripheral tissues, thus providing these cells with the greatest potential for presenting the relevant antigens to the T
lymphocytes that possess the appropriate T-cell receptors (data not
shown).26,27 Host T lymphocytes should thus enhance the specific antitumor immune response, ie, they demonstrated the strong
cytotoxicity assay against parental tumors with increasing the number
of mature DCs expressing B7-1 and B7-2 and with presenting tumor
antigens (Fig 6).25
Our results thus suggest that effective immunity may be induced by
increasing the in vivo activity of functional DCs. For application of
this approach to clinical study, we have further studied the
vaccination using DCs following autologous transplantation models in
which leukemic cells at de novo were cryopreserved and, after they were
genetically engineered with cytokine genes and irradiated, they were
infused to tumor-bearing animals as a vaccine therapy after autologous
BM transplantation. Moreover, because this approach should
be relatively general, efficacy may be observable with a variety of
syngeneic tumors.
These findings also suggest that the infusion of syngeneic or
autologous BM cells stimulated with cytokine-expressing tumor cells
without chemotherapy may be a novel and promising immunotherapeutic strategy for managing patients with certain types of cancer.
| |
FOOTNOTES |
Submitted November 3, 1998; accepted February 12, 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 Shin-ichiro Fujii, MD, PhD, The Center for
Bone Marrow Transplantation and Immunotherapy, Institute for Clinical
Research, Kumamoto National Hospital, 1-5 Ninomaru, Kumamoto 860-0008, Japan.
| |
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