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Institut National de la Santé et de la Recherche Médicale, Unité 517, Institut Federatif de Recherche 100, Faculty of Medicine, Dijon, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The experimental progressive/regressive (PRO/REG)3 tumor cell system (4) is a well-suited rat model to determine the precise nature of the cells involved in tumor cell destruction in immunized hosts. PRO and REG tumor cell clones were established from a chemically induced colon carcinoma growing in a strain of inbred rats (4). These clones constitutively differ in immunogenicity and tumorigenicity in syngeneic hosts (4, 5). The PRO clones give rise to progressive, metastatic, and lethal tumors. The REG clones, which yield spontaneously regressive tumors, induce a strong tumor-specific immune response that fully and durably protects the rats against subsequent PRO cell or parental tumor injection (6). The spontaneous regression of the REG tumors is related to a T cell-dependent immune response because REG cells give rise to progressive tumors in the nude mice or cyclosporine-treated syngeneic rats (7). Induction of an efficient anti-tumor immune response by REG cells is now well documented (8, 9). We have demonstrated that tumor Ags liberated by early death of a part of these tumor cells following s.c. injection are endocytosed by phagocytic cells which differentiate into dendritic cells. These cells migrate to the draining lymph nodes where they are able to present these Ags and to stimulate specific anti-tumor T cells. However, REG tumor regression is not associated with the appearance of cytotoxic T cells or the increase in NK cell activity in the spleen and in tumor-associated lymphocytes (10).
A study designed to determine the precise nature of effector cells responsible for ultimate elimination of the REG tumors is limited by the small size of these tumors before complete regression. Furthermore, even though a tumor-specific immune response is the predominant mechanism of REG tumor rejection (7), other factors such as spontaneous death of REG cells (8) may contribute to it. To circumvent this problem, we grafted established PRO tumors in rats that have been previously immunized with REG cells. These tumors continue their progression in naive rats but are invariably rejected in immunized rats. In this rat model system, immunohistochemical analyses with complementary in vitro functional studies led us to identify macrophages as the direct effectors of tumor cell killing. Furthermore, tumor-associated lymphocytes that are unable to destroy tumor cells independently are instrumental for macrophage activation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recombinant rat IFN-
, anti-rat IFN-, anti-rat
TNF-
Abs, and a colorimetric ELISA kit for rat IFN- (Quantikine)
were purchased from R&D Systems (Oxon, U.K.). LPS from
Escherichia coli, serotype 0128:B12, and
NG-methyl-L-arginine
(NMMA) were obtained from Sigma (St. Louis, MO).
Animals, cell lines, and tumorigenicity assays
Animals used in these experiments were BD-IX strain rats bred in
our laboratory by brother-sister mating. Animal use and handling were
approved by the local Veterinary Committee and were performed according
to the French laws for animal experimentation. Two cell lines, the
regressive variant DHD-K12/TSb (REGb) and the progressive variant
DHD-K12/TRb (PROb) were established from the tumor DHD, a colon
adenocarcinoma induced by 1–2 dimethylhydrazine in a BD-IX strain
female rat (4). GV1A1 cell line was established from a
glioma induced by N-ethyl-N-nitrosourea in a
BD-IX rat (11). These tumor cells were cultured in Ham’s
F10 medium (BioWhittaker, Verviers, Belgium) complemented with 10% FBS
(Life Technologies, Paisley, Scotland), as previously described
(5). For the tumorigenicity assays, 1 x
106 PROb tumor cells in 100 µl of serum-free
Ham’s F10 medium were injected s.c. into the anterolateral thoracic
wall of syngeneic BD-IX rats. Four weeks later, the resulting PROb
tumor was resected, and tumor pieces weighing 
50 mg were immediately
grafted s.c. into the thoracic wall, at distance from the site of REGb
cell-immunizing injections. Tumor volume was evaluated weekly, using a
caliper to measure two perpendicular diameters.
Histological study of the tumor cell injection site
Immunized and naive rats were sacrificed 7, 14, or 21 days after the tumor graft. The tumor was resected, embedded in Tissue-Tek (Miles, Elkhart, IN), and snap-frozen in methylbutane cooled in liquid nitrogen. An immunohistochemical study of tumor-infiltrating inflammatory cells was performed on acetone-fixed 5 µM cryostat sections. Murine mAbs to rat leukocyte markers, obtained from Serotec (Oxford, U.K.), were used on serially cut sections. Tumor cells were also labeled with a specific mAb (12C) raised against PROb cells. After incubation with specific mAbs, sections were incubated with biotinylated sheep Ab to mouse IgG (Amersham, Arlington Heights, IL), then with streptavidin-peroxidase and stained with aminoethylcarbazole.
Cytotoxicity studies
Rats were immunized with three s.c. injections of 1 x
106 REGb cells, 1 mo apart. Macrophages were
prepared from spleens of one or two naive or immunized rats, with or
without 14-day-old PROb tumor grafts. Tumor grafts were performed 2 wk
after the last immunizing injection. A spleen cell suspension was
prepared by pressing the spleen through a stainless-steel wire mesh
into RPMI 1640 culture medium (Life Technologies, Cergy-Pontoise,
France) supplemented with 10% FBS. Tumor-infiltrating cells were
purified from tumor grafts resected from groups of five to eight rats,
pooled, cut in small fragments, and dissociated with 0.5 mg/ml
collagenase (type II; Sigma) and 0.05 mg/ml DNase (type II; Sigma) for
1 h at 37°C in 5% CO2 in
poly(2-hydroxymethyl methacrylate)-coated vessels (Sigma) to
prevent macrophage attachment. The subsequent steps for purifying
tumor-infiltrating cells were performed at 4°C. Tumor cells were
depleted by incubating cell suspensions with tumor cell-specific mAb
12C, then harvesting the mAb-labeled cells on anti-mouse IgG-coated
magnetic beads (Dynabeads M450; Dynal Biotech, Oslo, Norway).
Macrophage concentrations in spleen and tumor cell suspensions were
estimated by counting adherent cells after a 1-h attachment period on a
hemocytometer glass surface at 37°C in 5% CO2.
One to 2 x 105 macrophages could be
recovered from each individual tumor. For preparing macrophage culture,
tumor-infiltrating cells containing 1 x 106
macrophages/ml were incubated in flat-bottom 96-well plates (Nunclon,
Roskilde, Denmark). After 2 h of incubation at 37°C in 5%
CO2, nonadherent cells were harvested and the
plates were washed twice with PBS to remove all the nonadherent cells.
Most (>95%) adherent cells were shown to be macrophages according to
their morphology and labeling with mAb ED2. Most harvested nonadherent
cells were lymphocytes. In some experiments, CD4+
and CD8+ T cells were positively selected,
following manufacturer’s instructions, using magnetic cell sorting
after marking the nonadherent cell suspension with MicroBeads (Miltenyi
Biotec, Paris, France) conjugated with mouse mAb anti-rat CD4 and
CD8-chain, respectively. The enrichment in
CD4+ and CD8+ cells was 90
and 98%, respectively, when assayed on rat spleen cells in preliminary
experiments. Macrophages with or without lymphocytes were cocultured in
triplicate with tumor cells (1 x 105
cells/ml) in RPMI 1640 medium supplemented with 10% FBS for 48 h
at 37°C in a 5% CO2 atmosphere. The
lymphocyte/macrophage/tumor cell ratio was 30:10:1 in these
experiments. At the end of this incubation period, culture medium was
harvested, centrifuged, and immediately frozen at -20°C until
cytokine assays were performed. The wells were gently washed twice with
PBS, then the cultures were fixed for 15 min with methanol. The plates
were allowed to dry, and labeled for 30 min at room temperature with
1% crystal violet, a protein-labeling dye that stains tumor cells that
remain attached to the wells after the incubation period. The plates
were carefully washed under tap water and dried, and the dye staining
the residual tumor cells was eluted in 100 µl of 33% acetic acid in
water (v/v). The absorbance of the eluted dye in each well was read at
570 nm on a Labtec scan (Bio-Advance, Emerainville, France). Triplicate
wells were used to determine mean and SD. We checked that absorbance
correlated linearly with the number of remaining viable tumor cells.
Cell survival was determined by comparing cell viability in PROb cells
in mixed culture with that of PROb cells alone. Macrophages did not
contribute to the absorbance as they poorly fixed crystal violet.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PRO tumor pieces weighing 50 mg were invariably accepted when
engrafted in naive syngeneic hosts and continued to grow as progressive
tumors. In contrast, rats immunized by three monthly s.c. injections of
1 x 106 REGb cells systematically rejected
PROb tumors grafted 2 wk after the last REGb cell injection. In these
rats, tumors began to shrink from day 12 until complete disappearance
4–8 wk later (Fig. 1
).
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To analyze the regression mechanisms, immunohistological analyses
were performed on regressing tumors in the immunized animals as well as
progressive tumors in the naive rats on days 7, 14, and 21
postgrafting. The most significant differences were observed on day 14,
at the beginning of tumor regression in immunized rats. At this time,
tumor cells were easily identifiable according to their typical
morphology, 12C positivity (Fig. 2A), and the formation of
poorly differentiated dense nodules. Progressive tumors in naive rats
showed a peripheral halo of different inflammatory cells including
ED1+ monocytes and immature dendritic cells (Fig. 2B), and few, if any, ED2+-stained
mature macrophages (Fig. 2C). TCR+ T
cells were also restricted to the tumor periphery (Fig. 2D)
and were either CD4+ (Fig. 2E) or
CD8+ (Fig. 2F). A few cells, also
limited to the tumor periphery, expressed MHC class II molecules (Fig. 2G). In contrast, regressing tumors in immunized rats showed
a spectacular infiltration by ED2+ mature
macrophages within the tumor nodule (Fig. 2C).
ED1+-staining cells (Fig. 2B), as well
as TCR+, CD4+, and
CD8+ T cells (Fig. 2, D–F), which
were more abundant in these regressive tumor nodules, remained at the
periphery of the tumor.
TCR-CD8+ cells
infiltrating the tumor nodules (Fig. 2, D and F)
were observed to be large, irregular cells with a cytoplasmic staining.
These cells are likely to be macrophages as they correspond in
morphology and location to ED2+
tumor-infiltrating macrophages, and rat macrophages were shown to
express CD8 molecules (12). To determine whether the
CD8+ cells found inside the tumor nodules were
CD8+ lymphoid cells (CTL or NK cells) or
CD8+ macrophages, another mAb to rat CD8, G28,
was used. In contrast to OX8 mAb, which labels a rat CD8 hinge
region epitope expressed by both macrophages and lymphoid cells, G28
labels an Ig variable-like region epitope, which is expressed only by
CTL and NK cells (13). Both OX8 and G28 Abs labeled cells
at the periphery of the tumor nodules. Only OX8, but not G28, labeled
CD8+ cells that infiltrated tumor nodules (Fig. 3). These
CD8+G28- cells were also
characterized by their elongated morphology and cytoplasmic staining,
in contrast to the G28+ cells surrounding the
tumor nodules that were characterized by a round shape and a
predominantly cell surface labeling. Like TCR+ T
cells, 3.2.3+ NK cells were more abundant in
regressive than in progressive tumors but remained confined to the
periphery of the tumor nodules (data not shown).
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Cytotoxicity assays were performed in vitro to determine the
effects of tumor-infiltrating and splenic macrophages (adherent cells)
and lymphocytes (nonadherent cells) on PROb tumor cells.
Tumor-infiltrating macrophages isolated 14 days after the graft in
immune rats were strongly cytotoxic as they killed all tumor cells in a
48-h coculture assay at a 10:1 E:T ratio. In comparison,
tumor-infiltrating macrophages isolated 14 days after the graft in
nonimmune rats were also cytotoxic, but to a considerably lower extent,
reducing tumor cell viability by only 32–45%. Splenic macrophages
from naive and immune rats, either tumor-bearing or not, showed no
significant cytotoxicity (Fig. 4A). Nonadherent cells, which
were predominantly lymphocytes, were isolated from the spleen and tumor
of naive or immunized rats. These cells showed no significant
cytotoxicity to tumor cells even when used at a 30:1 E:T ratio (Fig. 4B). Similar results were obtained in five independent
experiments. This macrophage-mediated cytotoxic effect was not
restricted to cells from the colon cancer from which the PRO/REG cell
variants originated. Macrophages from regressing PROb tumors also
demonstrated cytotoxic activity toward a rat glioma cell line (Fig. 5A). We investigated also the
mechanisms used by tumor-infiltrating macrophages for killing PROb
cells. Addition of 1 mM NMMA, an inhibitor of NO generation from
arginine, strongly inhibited PROb cell killing by macrophages from
regressing PROb grafts, whereas macrophage-induced cytotoxicity was not
influenced by an anti-TNF- mAb (5 µg/ml) (Fig. 5B).
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When nonadherent cells infiltrating PROb tumors grafted in immune
animals were added to splenic macrophages from normal, immune, or
tumor-bearing rats, toxicity against tumor cells was dramatically
increased and all the tumor cells were eliminated (Fig. 6A). Interestingly, when
nonadherent cells infiltrating PROb tumors grafted in immune animals
were added to macrophages isolated from PROb tumors grafted in naive
rats, cytotoxicity to PROb tumor cells was still observed, although
this cytotoxic effect was more limited (Fig. 6A). In
contrast, nonadherent cells isolated from PROb tumors grafted in naive
animals or from spleen of naive rats did not increase the low cytotoxic
effect of macrophages isolated from progressive tumor grafts in naive
rats (Fig. 6A column 5, compared with Fig. 4
A
column 4). These results were reproduced in three independent
experiments. T cells were responsible for the macrophage-activating
effect of nonadherent cells isolated from regressing tumor grafts
because CD4+ or CD8+ T
lymphocytes isolated from these nonadherent cells activated macrophages
as efficiently as the whole nonadherent cell fraction, without
any significant difference between CD4+ and
CD8+ cells (Fig. 6B). Surprisingly,
addition of both CD4+ and
CD8+ cells had no more macrophage-activating
activity than addition of CD4+ or
CD8+ cells alone. This could indicate that each T
cell subpopulation maximally activated splenic macrophages in this
experiment. We did not determine whether immune T cells activated
macrophages through a direct cell contact or through production of
cytokines in the tumor microenvironment.
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is a lymphokine known for its capacity for inducing rat
macrophages to become cytotoxic against cancer cells. We found that
lymphocytes isolated from PROb tumors grafted in immune rats secreted
significant amounts of IFN- (455 ± 78 pg/ml) in the culture
medium when cultured for 48 h with PROb cells and splenic
macrophages.
Tumoricidal activity of tumor-infiltrating and splenic macrophages
from rats bearing progressive tumors can be induced by exposure to
IFN- and bacterial LPS
As neither splenic macrophages nor macrophages that infiltrated
progressive tumors in nonimmunized rats were cytotoxic to tumor cells,
we investigated whether tumoricidal activity could be induced by
exogenous activators. Addition of IFN- and/or LPS strongly increased
macrophage cytotoxicity to PROb cells (Fig. 7). IFN- used alone did not induce a
cytotoxicity sufficient to destroy all the tumor cells established in a
monolayer, whereas LPS alone was almost as effective as LPS + IFN-
for inducing macrophage-mediated cytotoxicity.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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epitope expressed only by lymphoid cells
(13). These macrophages were in direct contact with tumor
cells and strongly cytotoxic to them in vitro.
The capacity of activated macrophages to destroy tumor cells in vitro
has been known for a long time (21, 22, 23). However, to the
best of our knowledge, the role of macrophages in tumor rejection
observed in specifically tumor-immunized hosts has never been analyzed.
The mechanisms by which activated macrophages kill neoplastic cells
include NO production through activation of inducible NO synthase, and
TNF- secretion (21, 22, 23). We demonstrate here that the
cytotoxic effect of activated macrophages isolated from regressing
tumors is suppressed by NMMA, an inhibitor of NO production, whereas
this cytotoxic effect is not influenced by a blocking anti-TNF-
mAb, probably because PRO cells have no receptor to TNF-
(24). On paraffin-embedded sections, the damaged tumor
cells were not stained after terminal deoxynucleotidyl
transferase-mediated nick end labeling (TUNEL), demonstrating that
macrophage-induced tumor cell death does not imply DNA internucleosomal
cleavage (result not shown).
Tumoricidal activity was restricted to macrophages infiltrating tumors engrafted in immunized hosts. Splenic macrophages from immunized rats were not cytotoxic. Macrophages infiltrating tumors in naive rats were few and sparse. Most of them were not in contact with the cancer cells and demonstrated only a limited cytotoxic effect toward tumor cells in vitro. These observations suggest that macrophages have to undergo in situ activation to exert tumoricidal function. Interestingly, tumor-associated lymphocytes from immunized hosts induce the tumoricidal activity of inactive splenic macrophages from normal or tumor-bearing rats, as well as in macrophages isolated from PROb tumors grown in naive rats. This observation strongly suggests that lymphocytes that surround tumor nodules in immunized rats are stimulated in this local microenvironment, then deliver activation signals to macrophages that infiltrate these tumors.
In contrast with macrophages, T lymphocytes were located at the
periphery of the regressing tumor nodules, were only infrequently
observed to be in contact with the tumor cells, and had no direct
cytotoxic effect against tumor cells, even at E:T ratios as high as
30:1. Passively transferred CD8+ T cells from
tumor-immunized donors have been shown to induce complete, Th1
cytokine-dependent tumor regression in the recipient in the absence of
any direct cytotoxic effect (1). Furthermore, the
therapeutic effectiveness of tumor-infiltrating
CD8+ T lymphocyte cultures correlates with their
ability to secrete lymphokines rather than their cytotoxic capacity in
vitro (25, 26, 27). CD8+ T cells, as
well as CD4+ Th1 cells (28), are a
source of cytokines such as IFN-, GM-CSF, and TNF-,
which can stimulate the tumoricidal activity of host cells, including
tumor-associated macrophages. In agreement with these findings, IFN-
levels measured in 48-h supernatants of macrophage, tumor cell, and TIL
cocultures were higher for TIL isolated from immunized than from naive
animals. Moreover, addition of IFN-, as well as endotoxin, to
splenic or tumor-associated macrophages from naive animals generated
significant cytotoxic activity in these otherwise noncytotoxic
macrophages. In contrast to mouse macrophages that require two signals
(a priming signal from IFN- and a progression signal from LPS) to
become cytotoxic against cancer cells (29), a single
exogenous stimulus, IFN- or LPS, activates rat resident macrophages
for NO production and tumor cytotoxicity (30).
Progressive human tumors are frequently infiltrated by macrophages
(31). However, these macrophages are not potent effectors
of tumoricidal activity in the absence of stimulation and could even
enhance tumor growth (reviewed in Ref. 32). Tumor cells
could adversely alter macrophage tumoricidal activity
(33). Interestingly, tumoricidal activity of macrophages
isolated from progressive PROb tumors in naive animals
could be restored in vitro by either exposure to lymphocytes isolated
from the regressive tumors of immune rats, or exogenous stimuli such as
IFN- and bacterial endotoxins. The effect of endotoxins is
particularly interesting because peritoneal carcinomatosis that results
from PROb cell i.p. injections into naive rats can be definitely cured
upon multiple administrations of lipid A, an endotoxin component
(34, 35).
We have previously demonstrated that tumor proteins released as a
consequence of initial events following injection of REG cells in naive
syngeneic rats are engulfed by inflammatory cells and transported to
lymph node T cell areas (9). However, the protective
immune response following REG tumor regression was not accompanied by a
detectable increase in the specific or nonspecific cytotoxic activity
of splenic and tumor-associated T lymphocytes and NK cells
(10). In the present study, which was performed on the
same rat tumor model, we clarified the downstream events and defined
the effector cells involved in the final elimination of the tumors in
immunized hosts. These events involve migration of activated
tumor-specific T cells to the tumor site, local production of IFN-
and possibly other cytokines that activate tumor-associated
macrophages, and subsequent elimination of tumor cells by these
macrophages. The present work also demonstrates that macrophages that
infiltrate progressive tumors have the potential to destroy tumor
cells, provided they receive appropriate T cell-mediated activation
signals, such as cytokines, or nonimmune stimuli, such as bacterial
endotoxins.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. François Martin, Institut National de la Santé et de la Recherche Médicale, Unité 517, Faculty of Medicine, 7 Boulevard Jeanne d’Arc, BP87900, 21079 Dijon, France. E-mail address: fmartin{at}u-bourgogne.fr
3 Abbreviations used in this paper : PRO, progressive; REG, regressive; NMMA, NG-methyl-L-arginine; REGb, regressive variant DHD-K12/TSb; PROb, progressive variant DHD-K12/TRb; TIL, tumor-infiltrating lymphocyte.
Received for publication February 16, 2001. Accepted for publication August 27, 2001.
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and tumor necrosis factor have a role in tumor regression mediated by murine CD8+ tumor-infiltrating lymphocytes. J. Exp. Med. 173:647.
production. J. Immunol. 164:1783. and tumor necrosis factor have a role in tumor regressions mediated by murine CD8+ tumor-infiltrating lymphocytes. J. Exp. Med. 173:647.
-interferon plays a key role in T-cell-induced tumor regression. Cancer Res. 53:833. and lipopolysaccharide. J. Biol. Chem. 268:1908.This article has been cited by other articles:
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) and animals
immunized by three injections of REGb cells, 1 mo apart, 2 wk before
the PROb tumor graft (•). The curves represent the mean ± SD of
two independent experiments on a total of 10 rats in each group. All
the tumors that were grafted in naive rats progressed, and rats were
sacrificed at the 4th wk. All the tumors that were grafted in immunized
rats completely disappeared by the 8th wk.
