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,
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*
Department of Immunology, Wenner-Gren Institute, University of Stockholm, Stockholm, Sweden;
Microbiology and Tumor Biology Center, Karolinska Institute, Stockholm, Sweden; and
Center for Infectious Medicine, Department of Medicine, Karolinska Institute, Huddinge University Hospital, Stockholm, Sweden
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-converting
enzyme-associated inhibitory protein (FLIPL) and poxvirus
cytokine response modifier A (CrmA) into the MHC class I-deficient T
lymphoma cell line RMA-S. RMA-S cells do not normally express Fas in
vitro, and it was previously postulated that the rejection of these
tumors by NK cells is strictly perforin dependent. We show that
perforin-deficient NK cells directly mediate Fas up-regulation on RMA-S
cells and thereafter kill the cells in a Fas-dependent manner, and that
RMA-S FLIPL and RMA-S CrmA are protected from such killing.
When injected in immunocompetent recipients, RMA-S cells up-regulate
Fas, rendering in vivo-passed mock-transduced cells sensitive to
Fas-mediated apoptosis. Moreover, RMA-S FLIPL and RMA-S
CrmA cells establish aggressive tumors, in contrast to RMA-S mock cells
that are rejected. These results demonstrate that FLIPL and
CrmA function as tumor progression factors by protecting MHC class
I-deficient tumors from rejection mediated by NK cells. Moreover, our
data indicate that death receptor-mediated apoptosis has a more
prominent role in the clearance of NK-sensitive tumors than previously
suggested. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cytotoxicity is mainly mediated through the release of granules containing perforin and granzymes or through the induction of death receptor-mediated apoptosis. In the perforin-dependent pathway, the entry of granzymes into the target cell leads to the activation of both caspase-dependent and caspase-independent apoptotic pathways (8). In death receptor-mediated apoptosis, death ligands on effector cells, such as Fas ligand (FasL3; CD95, Apo1), TNF, or TNF-related apoptosis-inducing ligand (TRAIL), bind to death receptors expressed on the surface of the target cell. Upon death receptor ligation, intracellular adapter molecules are recruited, and these molecules can, in turn, associate with initiator caspases through death effector domain (DED) or caspase recruitment domain interactions (9) leading to their activation, thereby starting the caspase cascade with the final demise of the cell as a result (10).
Several proteins, of both cellular and viral origin, are able to block
apoptosis at different levels. For example, the long form of
Fas-associated death domain-like IL-1-converting enzyme-associated
inhibitory protein (FLIPL), a member of the
Fas-associated death domain-like IL-1-converting enzyme inhibitory
protein family, prevents the association of the initiator caspases 8
and 10 with the adapter molecule Fas-associated death domain through
DED-DED interactions, thereby blocking caspase activation triggered by
death ligands (11, 12). Another example is the cytokine
response modifier A (CrmA), an orthopoxvirus-encoded protein that has
been shown to block death receptor-mediated apoptosis by preventing the
activation of caspase-8 (13).
Previously, we and others have shown that inhibition of death receptor-mediated apoptosis by FLIP in murine tumor models confers resistance to rejection by conventional T cells, leading to uncontrolled tumor progression (14, 15). In this study we use MHC class I-deficient tumors that are normally rejected by NK cells to test the hypothesis that the inhibitors of death receptor-mediated apoptosis FLIPL and CrmA can act as tumor progression factors preventing the cytotoxic activity of NK cells in vitro and in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mouse T lymphoma cell lines RMA and RMA-S and the human retroviral packaging cell line Phoenix-Ampho were grown as previously described (14, 16). RMA-S cells (2, 3) have a mutation in the TAP-2 gene that impairs peptide trafficking into the endoplasmic reticulum (17), resulting in the expression of low levels of unstable MHC class I molecules at the cell surface (18). Sex- and age-matched (4- to 6-wk-old) inbred C57BL/6 mice were obtained from Charles River (Uppsala, Sweden). FasL mutant mice (gld) bred on the C57BL/6 background were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice were maintained in the animal facility at Stockholm University. Perforin-deficient C57BL/6 mice (PKOB) (19) were maintained in the animal facility at Karolinska Institute.
Expression vectors and cell transduction
Human FLIPL was amplified by PCR from PBMC, using the oligonucleotides hFLIP5'Mfe (5'-ACT TAG CAA TTG CCA CCA TGT CTG CTG AAG TCA TCC AT-3') and hFLIP3'Mfe (5'-ATT CAC AAT TGT TAT GTG TAG GAG AGG ATA AGT TTC-3'). The fragment was inserted into the EcoRI site of the retroviral expression vector pLXIN (Clontech, Palo Alto, CA). CrmA was amplified by PCR from the CrmA/pRK5 expression vector (a gift from Dr. D. Göddel, Tulank, San Francisco, CA) using the oligonucleotides CrmA5'Eco (5'-TTG GCG AAT TCA CAC AAC AAA ATC GAT TGC CAT G-3') and CrmA3'Eco (5'-GTC AGG AAT TCG AAT GTG ATT TAA TTA GTT GTT GGA GAG-3') and inserted into the EcoRI site of the retroviral expression vector pLXIN. All vectors were then separately used to transiently transfect the Phoenix-Ampho packaging cell line (provided by Dr. G. P. Nolan, Stanford University, Stanford, CA). Supernatants containing recombinant viral particles were used for transduction of RMA-S cells, and stable G418-resistant clones were obtained. mRNA expression was verified by RT-PCR, and the presence of helper virus was excluded by PCR amplification of viral env using the primers 5'-ACCTGGAGAGTCACCAACC-3' and 5'-TACTTTG GAGAGGTCGTAGC-3'.
Flow cytometry
Cells were washed twice in PBS containing 2% FCS and 0.1% NaN3, and incubated with an anti-H-2Kb mAb (AF6-88.5), an isotype control mAb (G235-2356), or an anti-Fas mAb (Jo2), in the presence of anti-CD32/CD16 (Fcblock; all Abs were obtained from PharMingen, San Diego, CA). Propidium iodide was added to discriminate dead cells. Fresh C57BL/6 splenocytes were used as a positive control for MHC class I staining, and A20 cells were used as a positive control for Fas staining. Cells were analyzed in a FACScan (BD Biosciences, San Jose, CA), gating for living cells.
Apoptosis and limiting dilution assays
Sensitivity to Fas-induced apoptosis was assessed by treating
5 x 105 cells with 100 ng/ml of the
anti-mouse Fas mAb Jo2 (PharMingen) for 24–72 h at 37°C.
Alternatively, mouse soluble FasL (sFasL) was added at a 1/2 dilution
(FasL-hCD8
/pSG5 vector, a gift from Dr. H. Yagita, Juntendo
University, Tokyo, Japan). In some assays, 130 or 13 U/ml IL-2
or 10-fold dilutions (from 100 to 1 U/ml) of IFN-
(Genzyme,
Cambridge, MA) were added together with sFasL or the agonistic Jo2 mAb.
The sensitivity to TNF-induced apoptosis was assessed by treating
5 x 105 cells with 10-fold dilutions of
mouse recombinant TNF (Genzyme) starting from 104
U/ml in both the absence and the presence of 10-fold dilutions of
cycloheximide (CHX; Sigma, St. Louis, MO) from 10 µM to 1 nM for
24–72 h at 37°C. CHX-induced apoptosis was assessed by treating
5 x 105 cells with 10 or 1 µM CHX for 3,
6, and 24 h at 37°C. Etoposide-induced apoptosis was assessed by
treating 5 x 105 cells with 10-fold
dilutions of VP16 (Sigma) from 10 µg/ml to 1 ng/ml for 24–42 h at
37°C. TRAIL-induced apoptosis was assessed by treating 5 x
105 cells with 100–600 ng/ml recombinant human
soluble TRAIL (Alexis, San Diego, CA) in the presence of 2 µg/ml
enhancer, as recommended by the manufacturers, for 24–72 h at 37°C.
Cells were stained with propidium iodide, and apoptosis was monitored
by flow cytometric analysis. For limiting dilution assays, cells were
plated at different cell inputs, starting from 32 to 0.25 cells/well in
100 µl medium. Cells were seeded in 48 wells for each cell input. The
frequency of clonal growth was determined by visual inspection on day
12 and was calculated as previously described (20).
Generation of effector cells
IL-2-activated NK cells were prepared according to the following
protocol: 25 x 106 erythrocyte-depleted
splenocytes were resuspended in MEM containing 10 mM HEPES, 5
x 10-5 M 2-ME, and 10% FCS (all reagents from
Life Technologies, Paisley, U.K.) supplemented with 1000 U/ml human
rIL-2 (PeproTech, Rocky Hill, NJ) and cultured in
25-cm2 tissue culture flasks in 10%
CO2 at 37°C. When NK cells were generated from
perforin-deficient mice, an additional purification step was introduced
at the beginning of culture, using an anti-DX5 Ab linked to MACS
beads (Miltenyi Biotec, Bergisch Gladbach, Germany), according to the
manufacturer’s instruction. After 5 or 6 days, IL-2-activated NK cells
were resuspended by pipetting and scraping and were used for
cytotoxicity assays.
Cytotoxicity assay and Fas up-regulation assay
Effector cells were washed once, resuspended, and used as effectors in standard 4- or 18-h 51Cr release assays. Briefly, target cells were labeled with 10–20 µl 10 mCi/ml Na[51Cr]O4 for 1 h at 37°C and then washed. Cells (5 x 103) were incubated with titrated numbers of effector cells in round-bottom 96-well plates for 4 or 18 h at 37°C in 10% CO2. In some experiments, anti-mouse FasL mAb (MFL3; PharMingen) was added to the culture at 10 µg/ml. After incubation, released radioactivity was measured, and specific lysis was calculated according to the formula: % specific release = [(experimental release - spontaneous release)/(maximum release - spontaneous release)] x 100. In the Fas up-regulation assay, effector cells were incubated with 1 x 105 target cells at a 50:1 ratio for 18 h at 37°C in 10% CO2. Cells were then analyzed in a flow cytometer by staining cells with an NK-specific mAb (2B4; PharMingen), an isotype control (G235-2356), or an anti-Fas mAb (Jo2) in the presence of anti-CD32/CD16 (Fcblock). 2B4-positive cells were then excluded from the analysis for Fas expression.
Injection of mice with tumor cells
Groups of five to eight mice were injected s.c. in the
interscapular region with 1 x 105, 1
x 104, 1 x 103, or
1 x 102 RMA, RMA-S, and RMA-S-transduced
cells. In experiments performed with NK cell-depleted mice, animals
were injected i.p. with 100 µg anti-NK1.1 2 days before tumor
grafting, then 2 days, 1 wk, and 2 wk after tumor challenge. Tumors
were monitored every second day by palpation, for 8 wk. Mice were
killed with CO2 when tumors had reached 
1
cm3, as recommended by the Stockholm ethical
committee for animal experiments or when the experiment was terminated.
Tumor samples were obtained by surgical excision and were used for
immunostaining and apoptosis assays.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The murine tumor model, comprising RMA T lymphoma cells and the MHC class I-deficient variant RMA-S, has previously been used in many studies, contributing to the knowledge of NK cell-mediated rejection of MHC class I-deficient tumors (2, 3, 4, 16, 21, 22).
We, like others (16, 22), have observed that NK cells
generated from wild-type mice readily kill RMA-S cells in a specific
51Cr release assay, whereas NK cells generated
from PKOB fail to kill RMA-S cells after short term incubations (4 h).
Interestingly, NK cells generated from PKOB induced specific lysis in
RMA-S cells after lengthy incubation (18 h), as shown in Fig. 1
A. Moreover, when an
anti-FasL Ab was present during the incubation with NK cells under
similar conditions, cytotoxicity against RMA-S cells was efficiently
blocked (Fig. 1A). These results indicate that Fas-mediated
apoptosis is used as a mechanism for cytotoxicity, as well as granzyme
B-mediated apoptosis.
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Mock-, FLIPL-, and CrmA-transduced RMA-S
cells were mixed with NK cells, and specific lysis was measured by
means of 51Cr release. As shown in Fig. 1
B, the mock-transduced RMA-S cells along with the
FLIPL- and CrmA-transduced cells were readily
killed by NK cells after 4-h incubation. Similar results were obtained
when NK cells from gld mice were used (data not shown). NK
cells generated from PKOB also killed RMA-S mock cells after long-term
incubation (18 h), whereas RMA-S FLIPL and RMA-S
CrmA cells were protected against cytotoxicity, as shown in Fig. 1C. The cytotoxicity mediated through the perforin-dependent
pathway is fast and is most likely masking the effect of Fas-mediated
apoptosis. However, the contribution of the Fas-mediated pathway is
considerable and enough to kill the cells, as shown by the percentage
of specific 51Cr release in Fig. 1, A
and C. These findings suggest that the perforin-independent
cytotoxic activity of NK cells is mediated through the Fas/FasL
pathway, and that it can be blocked by inhibitors of death
receptor-mediated apoptosis.
NK cells mediate the up-regulation of Fas on RMA-S cells
RMA-S cells do not normally express detectable levels of Fas when
grown in vitro, as shown in Fig. 2A. As a consequence, they are
insensitive to Fas-induced apoptosis when sFasL or an agonistic
anti-Fas mAb is added (Table I).
Neither IL-2 nor IFN-, alone or in combination, could induce Fas
up-regulation. IL-3 or supernatant from Con A-stimulated splenocytes
also failed to induce Fas up-regulation (data not shown). RMA-S cells
were also insensitive to other death ligands, such as TNF- and
TRAIL, although apoptosis could be induced by other agents, such as CHX
and the DNA-damaging agent VP16 (Table I). Interestingly, NK cells were
able to directly mediate the up-regulation of Fas on RMA-S cells, as
shown in Fig. 2B. This finding adds further explanation to
the observation that perforin-deficient NK cells need longer incubation
periods with target cells to induce Fas-mediated apoptosis. These
results suggest that the cytotoxic activity of NK cells against
Fas-negative targets is a two-step process in which NK cells actively
induce the expression of this death receptor and subsequently kill the
targets by apoptosis.
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Mock-, FLIPL-, and CrmA-transduced RMA-S
cells were injected s.c. in immunocompetent syngeneic C57BL/6
recipients, and tumor samples were analyzed for Fas expression. As
shown in Fig. 2C, all tumors had up-regulated Fas after in
vivo passage. Tumor cells were then tested for sensitivity to
Fas-induced apoptosis. As shown in Fig. 3, mock-transduced tumor cells were
readily killed by sFasL, whereas FLIPL- and
CrmA-transduced tumor cells were protected. These findings show that
Fas is up-regulated in vivo, suggesting that death receptor-mediated
apoptosis may play an important role in the rejection of tumors by NK
cells.
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Mock-, FLIPL-, and CrmA-transduced RMA-S
cells were injected s.c. in immunocompetent syngeneic C57BL/6
recipients to investigate whether FLIPL or CrmA
would enhance tumor progression in vivo. RMA cells were used as a
positive control for tumor take and RMA-S cells were used as a control
to exclude that retroviral transduction could interfere with tumor
rejection. The inoculum of injected cells ranged from
102 to 105 cells. When
105 cells were injected, all transduced cells
formed tumors that quickly reached the maximally allowed size of 1
cm3 (Table II). At
lower cell doses (
104 cells), differences in
tumor take were seen between RMA-S mock cells and the RMA-S
FLIPL or RMA-S CrmA cells (Table II). At
103 cells, the rejection frequency was higher for
the mock-transduced cells, and at 102 cells, 96%
of mock tumors were rejected, while a large proportion of the mice that
had been injected with FLIPL- or CrmA-transduced
cells developed aggressive tumors that grew uncontrolled (Table II). In
Fig. 4 the percent tumor take for the
mock-transduced cells at a 102 cell inoculum is
compared with that for FLIPL (Fig. 4A)
and CrmA-transduced tumor cells (Fig. 4B). RMA-S
FLIPL and RMA-S CrmA cells generated tumors
quickly after injection (days 12–14), and by day 30 all mice having
tumors had been euthanized. To the contrary, the only mock tumor was
detectable on day 24 and had not attained the size of 1
cm3 on day 34, when it was taken for ex vivo
experiments. These findings indicate that the inhibitors of death
receptor signaling FLIPL and CrmA can act as
tumor progression factors and promote tumor survival.
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RMA-S cells were injected in mice depleted of NK cells to directly
test the hypothesis that FLIPL and CrmA act by
protecting the tumor cells from NK cell-mediated rejection. When
injected into NK cell-depleted recipients, mock-,
FLIPL-, and CrmA-transduced RMA-S cells grew as
fast and to the same extent, as shown in Fig. 4, C and
D, demonstrating the strict dependence on NK cells for the
rejection process. These results also demonstrate that
FLIPL and CrmA do not confer any proliferative or
survival advantage to the cells in vivo in the absence of NK cells, in
line with limiting dilution analysis in vitro, in which all RMA-S cells
showed similar frequencies of clonable cells (mock, 1/1.9; 95%
confidence interval, 1/1.6 to 1/2.8; FLIPL,
1/1.8; 95% confidence interval, 1/1.5 to 1/2.3; CrmA, 1/1.5; 95%
confidence interval, 1/1.3 to 1/1.9). As an additional control, tumor
samples were extracted by surgical excision and analyzed for MHC class
I expression to exclude the unlikely possibility that
FLIPL or CrmA could lead to the up-regulation of
MHC class I, thereby rendering the tumors insensitive to NK
cell-mediated cytotoxicity in vivo. As shown in Fig. 5, the RMA cells expressed MHC class I on
their surface, whereas all transduced RMA-S cells stained negatively
for this Ag.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous results by Smyth et al. (22) suggest that Fas- and TNF-induced apoptosis seem to be dispensable in this tumor system, as mice defective in either FasL (gld) or TNF can reject RMA-S tumors to the same extent as wild-type mice. Our results show that Fas is up-regulated in vivo, and that RMA-S cells are killed in a Fas-dependent manner by perforin-deficient NK cells in vitro. These data argue for a more prominent role of death receptor-mediated apoptosis in the clearance of MHC class I-deficient tumors than previously thought. The slower kinetics seen in the in vitro experiments with perforin-deficient NK cells are due to the fact that Fas needs to be up-regulated on RMA-S cells before they can be killed. Indeed, RMA-S cells do not show detectable expression levels of Fas in vitro, whereas they up-regulate Fas after being exposed to NK cells. The induction of Fas expression in target cells seem to be a general mechanism for cytotoxic lymphocytes, as a recent report shows that CTLs mediate Fas up-regulation on target cells. Moreover, the induction of Fas expression occurs regardless of the origin of the target cell, as both lymphoid and nonlymphoid targets up-regulate Fas when they encounter CTLs (23, 24). It is likely that Fas is up-regulated in vivo at an early time point, as a recent study indicates that Fas expression is detectable as soon as 4 days after in vivo grafting (23). More studies are needed to examine the mechanism through which NK cells induce Fas expression in vitro, although preliminary results obtained from double-chamber experiments indicate that NK cells need direct contact with target cells to induce Fas up-regulation. Recently, it was shown that NK cells express TRAIL, and that liver NK cells induce apoptosis through this death ligand during the rejection of tumor metastasis (25, 26). It would be interesting to investigate whether NK cells can also mediate the up-regulation of TRAIL receptors on target cells.
Several groups have argued that the perforin-dependent pathway is predominantly used for the rejection of RMA-S tumor cells in vivo, as shown by experiments in perforin knockout mice (16, 22). Our experiments in normal mice show that both pathways are responsible for the clearance of RMA-S tumor, as FLIPL- and CrmA-expressing cells are protected from NK cell-mediated rejection and can establish aggressive tumors. It is not known whether FLIPL can block the perforin-dependent pathway in vivo, although in vitro studies exclude this possibility (15, 27). It has also been debated whether CrmA can block the perforin-dependent pathway in vivo. In fact, all reports to date indicate that CrmA is unable to protect from the perforin-dependent pathway induced by CTLs despite the fact that CrmA is a substrate and inhibitor of granzyme B in a cell-free system (28). Nevertheless, it is noteworthy to mention that PI-9, a cellular homologue to CrmA that inhibits granzyme B, is up-regulated in CTLs. It is hypothesized that PI-9 protects these lymphocytes against autocrine and paracrine-like killing when cytotoxic granules are released (29). Our in vitro data indicate that neither FLIPL nor CrmA can protect target cells from the perforin-dependent pathway, whereas they efficiently inhibit Fas-mediated apoptosis. In fact, it has previously been shown that FLIPL and CrmA protect cells from death receptor-mediated apoptosis by blocking caspase-8 activation (11, 12, 13, 30). As a reasonable deduction it can be argued that these two inhibitors of apoptosis exert their tumor-progressive activity by blocking the immune response induced through death receptor-mediated apoptosis. FLIPL and CrmA can only block one pathway of cytotoxicity mediated by NK cells. Moreover, NOs (inducible NO synthase) produced by NK cells can also be involved in cell lysis (31), and it is unclear whether inhibitors of apoptosis can interfere with this type of cell death. Nevertheless, the presence of an inhibitor of death receptor-mediated apoptosis is enough to confer an advantage to tumor cells and to protect them from the immune response.
Taken together, the data presented here complement our previous results (14, 15) and emphasize the pivotal role of inhibitors of death receptor signaling in tumor progression by a mechanism of immune evasion regardless of the lymphocyte population that is responsible for the clearance of tumors. Moreover, these results underscore the role of death receptor-induced apoptosis in tumor clearance mediated by NK cells by a multistep mechanism that includes the induction of Fas expression on Fas-negative targets and the subsequent induction of apoptosis through this death receptor.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Valentina Screpanti, Department of Immunology, Wenner-Gren Institute, University of Stockholm, S-10691 Stockholm, Sweden. E-mail address: valli.screpanti{at}imun.su.se
3 Abbreviations used in this paper: FasL, Fas ligand; sFasL, soluble FasL; TRAIL, TNF-related apoptosis-inducing ligand; FLIPL, long form of Fas-associated death domain-like IL-1-converting enzyme-associated inhibitory protein; CrmA, cytokine response modifier A; PKOB, perforin-deficient C57BL/6 mice; CHX, cycloheximide; DED, death effector domain.
Received for publication February 20, 2001. Accepted for publication June 7, 2001.
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) or the presence (•) of 10 µg/ml anti-FasL
mAb, and lysis was measured after 18 h. B, NK cells
generated from C57BL/6 splenocytes were mixed with
51Cr-labeled RMA (
), RMA-S mock (
) cells, and lysis was
measured after 4 h. RMA cells were used as control targets to
exclude any contribution by CD8+ CTLs to the measured cell
lysis, as splenocytes from C57BL/6 mice were not sorted to obtain an
enriched NK cell population. C, NK cells generated from
PKOB splenocytes were mixed with 51Cr-labeled RMA-S mock
(
