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IMMUNOBIOLOGY
Perforin and interferon-␥ activities independently control tumor initiation,
growth, and metastasis
Shayna E. A. Street, Erika Cretney, and Mark J. Smyth
Perforin (pfp) and interferon-␥ (IFN-␥) together in C57BL/6 (B6) and BALB/c mouse
strains provided optimal protection in 3
separate tumor models controlled by innate
immunity. Using experimental (B6, RM-1
prostate carcinoma) and spontaneous
(BALB/c, DA3 mammary carcinoma) models of metastatic cancer, mice deficient in
both pfp and IFN-␥ were significantly less
proficient than pfp- or IFN-␥–deficient mice
in preventing metastasis of tumor cells to
the lung. Pfp and IFN-␥–deficient mice were
as susceptible as mice depleted of natural
killer (NK) cells in both tumor metastasis
models, and IFN-␥ appeared to play an early
role in protection from metastasis. Previous
experiments in a model of fibrosarcoma
induced by the chemical carcinogen methylcholanthrene indicated an important role for
NK1.1ⴙ T cells. Herein, both pfp and IFN-␥
played critical and independent roles in providing the host with protection equivalent to
that mediated by NK1.1ⴙ T cells. Further
analysis demonstrated that IFN-␥, but not
pfp, controlled the growth rate of sarcomas
arising in these mice. Thus, this is the first
study to demonstrate that host IFN-␥ and
direct cytotoxicity mediated by cytotoxic
lymphocytes expressing pfp independently
contribute antitumor effector functions that
together control the initiation, growth, and
spread of tumors in mice. (Blood. 2001;97:
192-197)
© 2001 by The American Society of Hematology
Introduction
Considerable attention has been paid to the direct antitumor
activities of cytotoxic lymphocytes within innate and acquired
components of the cellular immunity.1 However, until recently,
little information was available regarding the relative role of direct
cytotoxicity in immunity against tumor development. The development of gene-targeted mice deficient in perforin (pfp)2 and the
discovery of several members of the tumor necrosis factor (TNF)
superfamily with the capacity to induce tumor cell apoptosis3-5
have paved the way to determining which effector molecules are
relevant in tumor immune surveillance. Thus far, in mouse
experimental tumor systems, pfp has been demonstrated to protect
the host against tumor initiation,6 primary tumor growth,6,7 and
tumor metastasis.8 Perforin enables the entry of granzymes that
play a key role in target cell DNA fragmentation, but these serine
esterases are not critical for tumor cell death mediated by effector
cells.9,10 Concerning innate cytotoxicity mediated by NK cells, a
role for Fas ligand (FasL) and other TNF family members in tumor
immune surveillance is less obvious. Notably, in some models
involving innate antitumor activity, natural killer (NK) celldepleted mice were often significantly more susceptible to tumor
metastasis than pfp-deficient mice, despite the lack of activity of
FasL or TNF against these tumors in vivo.8 These observations
suggested that other pfp-independent effector functions may be
contributing to tumor protection.
In this light, IFN-␥ is a pleiotropic cytokine that plays a central
role in innate and adaptive arms of host immune defense.11,12 IFN-␥
is secreted by NK cells and thymus-derived T cells under certain
conditions of activation.13,14 IFN-␥ acts in various ways on host and
tumor cells to favor tumor regression, but the relative role that
IFN-␥ plays in antitumor responses, compared with direct cytotoxicity by effector molecules such as pfp, has not been previously
evaluated. Two studies have elegantly demonstrated a role for
IFN-␥ in tumor immune surveillance15,16; however, neither of these
determined how important IFN-␥ function was compared with
direct cytotoxicity mediated by innate effector cells. Herein, we
have undertaken the first study to compare directly the relative role
of pfp and IFN-␥ in 3 distinct models of tumor immunity. The 3
tumor models used are distinguished by different relative contributions of NK cells and NK1.1⫹ T (NKT) cells in host protection.
Regardless of the relative role of NK and NKT cells, the creation of
mice doubly deficient for pfp and IFN-␥ has demonstrated that
these 2 effector arms can act independently and equally effectively
yet together account for all the natural antitumor immunity
mediated by NK and NKT cells.
From Cancer Immunology, Peter MacCallum Cancer Institute, Victoria,
Australia.
Institute, Locked Bag 1, A’Beckett Street, Victoria, Australia, 8006; e-mail:
[email protected].
Materials and methods
Mice
Inbred C57BL/6, BALB/c, and BALB/c SCID mice were purchased from
The Walter and Eliza Hall Institute of Medical Research (Melbourne,
Australia). BALB/c.B6Cmv1r (NK1.1⫹) were obtained from the Animal
Resources Centre (Perth, Australia). The following gene-targeted mice were
bred at the Austin Research Institute Biological Research Laboratories
(ARI-BRL): C57BL/6.IFN-␥–deficient (B6.IFN-␥0) (kindly provided by
Genentech, San Francisco, CA)17; C57BL/6.perforin-deficient (B6.pfp0)
mice2 (from Dr Guna Karupiah, John Curtin School of Medical Research,
Submitted May 17, 2000; accepted July 28, 2000.
Supported by a National Health and Medical Research Council (NHMRC) of
Australia project grant. M.J.S. is supported by an NHMRC Principal Research
Fellowship.
Reprints: Mark J. Smyth, Cancer Immunology, Peter MacCallum Cancer
192
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2001 by The American Society of Hematology
BLOOD, 1 JANUARY 2001 䡠 VOLUME 97, NUMBER 1
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BLOOD, 1 JANUARY 2001 䡠 VOLUME 97, NUMBER 1
ANTITUMOR EFFECTOR FUNCTIONS
Canberra, Australia); C57BL/6.IL-12p40–deficient (B6.IL-12p400) mice18
(from Hoffmann-La Roche, Nutley, NJ); C57BL/6.RAG-1–deficient
(B6.RAG-10) mice (from Dr Lynn Corcoran, The Walter and Eliza Hall
Institute of Medical Research); C57BL/6.T-cell receptor (TCR).J␣281deficient (B6.J␣2810) mice (from Dr Masaru Taniguchi, Chiba University
School of Medicine, Chiba, Japan)19; C57BL/6.gld (FasL mutant); BALB/
c.perforin-deficient (BALB/c.pfp0),8 and BALB/c.IFN-␥–deficient (BALB/
c.IFN-␥0).17 C57BL/6 and BALB/c mice doubly deficient for perforin and
IFN-␥ (B6.pfp0.IFN-␥0, BALB/c.pfp0.IFN-␥0) were produced and bred at
the ARI-BRL. Mice of 4 to 8 weeks of age were used in all experiments,
performed according to animal experimental ethics committee guidelines.
Cell culture and chromium
51Cr
release assays
The mouse tumor cell lines and spleen cell cultures used in this study were
maintained as previously described.8,19,20 Cytotoxicity of freshly isolated
mouse spleen cells or IL-2–activated adherent NK cells (greater than 80%
NK1.1⫹ TCR␤⫺) from wild-type or gene-targeted mice were assessed by
4-hour chromium 51Cr release assays against labeled targets as described.7
Each experiment was performed twice with at least 4 different effector–
target ratios using duplicate samples.
Subset depletion
The protocols for depletion of NK1.1⫹ (NK and NKT) cells in B6 mice
using anti-NK1.1 monoclonal antibody (mAb) (PK136) were as previously
described.8,19 Groups of BALB/c and BALB/c.B6Cmv1r (NK1.1⫹) mice
were depleted of asialo GM1⫹ cells using the rabbit anti-asialo GM1
antibody (Wako Chemicals, Richmond, VA) as described.8 The BALB/
c.B6Cmv1r (NK1.1⫹) mice were used to show that this depletion was
effective because DX5 and other markers are not entirely suitable for
detecting NKT cells. Anti-asialo GM1 antibody depleted NK1.1⫹ TCR␤⫺
NK cells, but not NK1.1⫹TCR␤⫹ NK) T cells (Table 1). Assessment of
depletion was performed after the preparation of cell suspensions from
spleen and liver as described.21 Flow cytometry of cells was performed with
allophycocyanin-conjugated anti-␣␤TCR (clone H57-597) and phycoerythrin-conjugated anti-NK1.1, purchased from Pharmingen (San Diego, CA).
Tumor surveillance in vivo
Effector function was examined in 3 different tumor models (RM-1, DA3,
and methylcholanthrene [MCA] induction of fibrosarcoma), as described,
using gene-targeted mice or mice depleted of lymphocyte subsets.8,19 In the
RM-1 model, mice were injected subcutaneously with RM-1 tumor cells
(2 ⫻ 106), and tumors were established for 9 days. At this time, subcutaneous tumors were surgically resected, and fresh in vitro cultured RM-1 cells
were injected through the dorsolateral tail vein. Mice were killed 14 days
later, the lungs were removed and fixed in Bouin’s solution, and surface
lung metastases were counted blinded with the aid of a dissecting
microscope. Additionally, in the RM-1 tumor model, groups of B6 mice
were treated with rat antimouse IFN-␥ mAb (R4-6A2) or control rat IgG1
(R3-34; Pharmingen, San Diego, CA) (500 ␮g each) on days ⫺2, 0 (day of
Table 1. Depletion of NK cells using anti-asialo GM1 antibody
in BALB/c.B6Cmv1r mice
n†
No. leukocytes
(⫻10⫺6)‡
% NK1.1⫹
NK cells
% TCR␤⫹
cells
% NK1.1⫹
TCR␤⫹ cells
Spleen
5
105 ⫾ 5§
3.0 ⫾ 0.4
25 ⫾ 2
0.4 ⫾ 0.1
Spleen ⫹ Ab
3
90 ⫾ 7
⬍ 0.2
26 ⫾ 3
0.5 ⫾ 0.2
Liver
5
2.4 ⫾ 0.4
19.7 ⫾ 1.4
43 ⫾ 4
7.7 ⫾ 1.3
Liver ⫹ Ab
3
2.8 ⫾ 0.1
0.3 ⫾ 0.1
44 ⫾ 4
7.5 ⫾ 3.4
Tissue*
Cell suspensions were prepared as described and labeled with mAb specific for
␣␤TCR and NK1.1.
*Mice were untreated or treated with 100 ␮g anti-asialo GM1 one day before
tissue harvest.
†n, number of mice tested.
‡Total number of leukocytes isolated from spleen or liver.21
§Results represent mean ⫾ SE.
193
intravenous tumor inoculation), 2, 7, and 10. Some mice received anti–
mIFN-␥ mAb early (days ⫺2, 0) and others late (days 7, 10). Protocols that
have used similar concentrations and conditions have been shown to
effectively inhibit mIFN-␥ activity in vivo.22 Data were recorded as the
mean number of metastases ⫾ SEM. Significance was determined by a
Mann-Whitney U (rank sum) test.
Results
NK and NKT cell number and NK cytotoxic function
in gene-targeted mice
Multiparameter flow cytometry was used to examine the status of
NK and NKT cells in the various mouse strains in this study. To use
the NK1.1 marker, B6 strains were examined. Cell suspensions
were made from thymus, spleen, liver, bone marrow, lymph nodes,
and peritoneal exudate cells. As expected, NK1.1⫹ cells were
detected in each organ and included both ␣␤TCR⫺ (NK cells) and
␣␤TCR⫹ (NKT cells). Both NK and NKT cells were clearly
present in the appropriate organs of wild-type, B6.IFN-␥0, B6.pfp0,
and B6.pfp0.IFN-␥0 strains (data not shown). Next, the cytotoxic
function of NK cells in both B6 and BALB/c gene-targeted mice
was examined in a 4-hour 51Cr release assay against class
1–deficient, NK-sensitive targets (RMA-S and DA3) and class
1–expressing targets (RMA and P815) (Figure 1A-B). In both
strains (B6 and BALB/c), freshly isolated splenocytes or IL-2–
activated adherent NK cells (greater than 80%) from the spleen of
wild-type or IFN-␥0 mice displayed NK and LAK activity (Figure
1A-B). By contrast, resting or IL-2–activated adherent NK cells
from the spleen of pfp0 or pfp0.IFN-␥0 mice displayed little or no
NK and LAK activity (Figure 1A-B). These data confirmed that
pfp, but not IFN-␥, was critical for direct cytotoxicity by NK cells.
NK cell–mediated control of spontaneous metastasis
by independent pfp and IFN-␥ activities
The relative importance of pfp and IFN-␥ was next examined in a
model of spontaneous metastasis that is strictly controlled by NK
cells.8 Two different DA3 tumor doses (105 and 5 ⫻ 105) were
inoculated subcutaneously, and after 42 days, lung metastases were
counted. There were significantly increased numbers of metastases
in BALB/c.pfp0 mice or BALB/c.IFN-␥0 mice compared with
wild-type BALB/c mice, but fewer than found in NK cell–depleted
(anti-asialo GM1-treated) mice (Figure 2). Anti-asialo GM1 antibody depletion was complete and specific for NK cells and did not
effectively deplete NKT- or T-cell populations (Table 1). Similar
increases in DA3 lung metastasis were observed in BALB/
c.B6Cmv1r mice depleted with anti-asialo GM1 antibody (Figure 2
legend). There was no increase in DA3 metastases in T-cell– and
NKT-cell–deficient BALB/c.SCID mice (Figure 2). Importantly,
BALB/c.pfp0.IFN-␥0 mice were as susceptible to tumor metastasis
as NK-cell–depleted mice. In addition, qualitatively equivalent
results were obtained in mice simply injected intravenously with
DA3 tumor cells (data not shown), suggesting that pfp and IFN-␥
were both protecting against tumor cell engraftment in the lung
rather than migration from the subcutaneous site of inoculation.
NK- and NKT-cell–mediated control of experimental tumor
metastasis by independent pfp and IFN-␥ activities
Innate protection from metastasis was then examined in the B6
RM-1 tumor model in which NK and NKT cells have previously
been shown to control tumor metastasis.8,19 Confirming previous
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194
STREET et al
BLOOD, 1 JANUARY 2001 䡠 VOLUME 97, NUMBER 1
for FasL or TNF effector molecules was observed in this model of
metastases (Figure 3A).
To evaluate whether the effector functions of pfp, IL-12,
and IFN-␥ were independent or overlapping, B6, B6.J␣2810,
B6.IL-12p400, or B6.pfp0 mice were treated with neutralizing
antimouse IFN-␥ mAb over the course of the challenge with RM-1
tumor cells. Anti–IFN-␥ mAb significantly increased the incidence
of lung metastasis in both B6 and B6.pfp0 mice, but only minor
increases were observed in B6.J␣2810 or B6.IL-12p400 mice
(Figure 3B). The effect was most prominent when mAb was
administered early, rather than late, after the tumor challenge. The
enhanced number of RM-1 lung metastases in B6.pfp0 mice treated
with anti–IFN-␥ was further supported by the number of metastases in B6.pfp0.IFN-␥0 mice. When directly compared (ie, same
experiments), the B6.pfp0.IFN-␥0 mice displayed tumor incidence
equivalent to B6 mice depleted of NK1.1⫹ cells (Figure 3B
compared with Figure 3A). NKT cells have recently been demonstrated to rapidly activate NK cells by their production of IFN-␥.25
The early protective effect of anti–IFN-␥ mAb in the RM-1 tumor
model may also support this concept. Nonetheless, the greater
defect observed in mice depleted of all NK1.1⫹ cells suggested that
NK cells have protective mechanisms independent of NKT cells.
The results in mice deficient for pfp, or pfp and IFN-␥, indicated
this independent antimetastatic function of NK cells may involve
both the cytolytic activity of pfp and antitumor activity of IFN-␥.
Therefore, these data in the RM-1 tumor model supported our
previous results in the DA3 tumor model—that innate effector cells
Figure 1. NK-cell–mediated cytotoxicity of pfp and IFN-␥ gene-targeted mice.
Resting splenocytes and IL-2–activated adherent spleen NK cells were assessed by
4-hour 51Cr release assays using labeled targets as indicated for (A) B6 or (B) BALB/c
strains of mice. An effector–target ratio of 100:1 is shown (4 were examined). The
spontaneous release of 51Cr was always less than 15% (subtracted from test), and
each experiment was performed twice using triplicate samples.
reports, B6 mice depleted of NK1.1⫹ cells displayed a significantly
higher number of RM-1 lung metastases than B6 mice deficient for
T cells and NKT cells (RAG-10) or NKT cells alone (J␣2810)
(Figure 3A). These data suggested that NK cells were the primary
effector population, but clearly NKT cells play some role in RM-1
tumor protection because J␣2810 mice displayed significantly more
metastases than untreated B6 mice. The major effector molecules
involved in host protection were pfp, IL-12, and IFN-␥, as
evidenced by the significant increase in RM-1 lung metastases in
mice deficient for each of these molecules (Figure 3A). Nevertheless, B6.pfp0, B6.IL-12p400, and B6.IFN-␥0 mice had significantly
fewer RM-1 lung colonies than B6 mice depleted of NK1.1⫹ cells
(Figure 3A). In this model, we have already demonstrated that
B6.pfp0 mice or B6.IFN-␥0 mice treated with anti-NK1.1 mAb do
not develop more metastases than B6 mice treated with anti-NK1.1
mAb; thus, pfp and IFN-␥ activities are not independent of NK cell
function.23 Although IFN-␥ can clearly potentiate the apoptotic
activity of TNF superfamily molecules,12 including TNF-related,
apoptosis-inducing ligand24 (not assessed herein), no apparent role
Figure 2. NK-cell–mediated control of spontaneous metastasis by independent
pfp and IFN-␥ activities. BALB/c, BALB/c.SCID, BALB/c.IFN-␥0, BALB/c.pfp0, and
BALB/c pfp0.IFN-␥0 mice or BALB/c mice treated with anti-asialo GM1 antibody (100
␮g/injection) were inoculated subcutaneously with DA3 tumor cells (105 or 5 ⫻ 105)
as indicated. Mice depleted of subsets in vivo were treated on days ⫺4, 0 (day of
subcutaneous tumor inoculation), and weekly thereafter. Forty-two days after tumor
inoculation, the lungs of these mice were harvested and fixed, and colonies were
counted and recorded as the mean number of colonies ⫾ SE. Asterisks indicate the
groups that are significantly different from BALB/c-untreated mice (*P ⬍ .0001;
**P ⬍ .005; Mann-Whitney U test). A significant difference was also detected
between the BALB/c.P0 mice or BALB/c.IFN-␥0 mice and BALB/cP0.IFN-␥0 mice
(P ⬍ .0001). The number of mice per group is shown in parentheses. Note that for
groups of 5 mice receiving 105 DA3 tumor cells, the numbers of DA3 lung colonies
were as follows: BALB/c.B6Cmv1r ⫽ 8.0 ⫾ 2.6; BALB/c.B6Cmv1r ⫹ anti-asialo GM1
antibody ⫽ 62.4 ⫾ 6.6.
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BLOOD, 1 JANUARY 2001 䡠 VOLUME 97, NUMBER 1
protected the host from lung metastases and that pfp and IFN-␥
were independent effector mechanisms that contributed to
protection.
NKT-cell–mediated control of MCA-induced fibrosarcoma
by independent pfp and IFN-␥ activities
Induction of fibrosarcoma by MCA has previously been demonstrated to be controlled by effector cells in a pfp-dependent and an
IFN-␥ receptor–dependent manner.2,16,19 By examining the relative
deficiencies of B6 mice depleted of NK1.1⫹ cells and B6 mice
specifically deficient in V␣14 TCR NK1.1⫹ T cells (J␣2810), we
ANTITUMOR EFFECTOR FUNCTIONS
195
have been able to establish that these NKT cells play a major role in
protection from MCA-induced sarcoma.19 Herein we created B6
mice doubly deficient for pfp and IFN-␥. Groups of B6 and B6
gene–targeted mice were injected subcutaneously with 2 different
doses of MCA, and fibrosarcoma development was monitored for
180 days (Figure 4A). At the doses examined, B6.pfp0 mice and
B6.IFN-␥0 mice developed tumors significantly more frequently
than B6 control mice. These data in B6 mice confirmed a role for
pfp6,8 and IFN-␥,16 previously suggested by studies in this strain. In
addition, at the 2 doses examined, a greater proportion (90% and
80%) of B6.pfp0.IFN-␥0 mice succumbed to MCA-induced sarcoma than either B6.pfp0 mice or B6.IFN-␥0 mice (Figure 4A). The
B6.pfp0.IFN-␥0 mice displayed a similar sarcoma incidence to
NKT-cell–deficient B6.J␣2810 mice (Figure 4A). Thus, in a third
model involving innate protection from tumor initiation, the
combined deficiency of pfp and IFN-␥ compromised the host to a
greater extent than loss of pfp or IFN-␥ alone. A random group of
10 mice was monitored for individual tumor growth in groups
receiving 100 ␮g MCA (Figure 4B) or 25 ␮g MCA (data not
shown). Strikingly, the growth of sarcomas in B6.IFN-␥0,
B6.pfp0.IFN-␥0, or B6.J␣2810 mice was significantly greater than
that compared with B6 or B6.pfp0 mice. This increase in the growth
rate of sarcomas in IFN-␥–deficient mice occurred despite similar
rates of incidence between B6.pfp0 and B6.IFN-␥0 mice (Figure
4A). Similar data have been obtained in the same BALB/c strains
of mice treated with MCA (data not shown). Therefore, though
IFN-␥ was important in the control of sarcoma initiation, it appears
that IFN-␥ and NKT cells were also very important in the control of
sarcoma growth.
Discussion
In 3 distinct models of tumor initiation and metastasis, the
sensitivity of mice deficient in pfp and IFN-␥ indicated that both
effector mechanisms independently contribute to innate tumor
immunity mediated by NK1.1⫹ NK or NK1.1⫹ T cells. For some
time it has been recognized that IFN-␥ production and pfpmediated cytotoxicity may be distinct events that contribute to
innate immunity. In particular, in viral infection, IL-12 is required
for NK cell IFN-␥ production but not cytotoxicity, whereas
IFN-␣/␤ production is dominant for enhanced NK lytic activity.26
In T-cell–mediated responses, a critical balance exists between
direct cytotoxicity mediated by pfp and IFN-␥ secretion that
Figure 3. NK- and NKT-cell–mediated control of experimental tumor metastasis
by independent pfp and IFN-␥ activities. (A) B6, B6.pfp0, B6.RAG-10, B6.TNF0,
B6.IFN-␥0, and B6.gld mice or B6 mice treated with anti-NK1.1 were inoculated
subcutaneously between the shoulder blades with RM-1 tumor cells (2 ⫻ 106), and
tumors were allowed to establish for 9 days. Subcutaneous tumors were then
resected and a dose range of RM-1 cells (as indicated) injected through the tail vein.
Mice were killed 14 days later, the lungs were removed and fixed, and colonies were
counted and recorded as the mean number of colonies ⫾ SE. A group of B6 mice was
depleted of NK1.1⫹ cells in vivo by mAb treatment (100 ␮g/injection) on days ⫺2, 0
(day of intravenous tumor inoculation), 2, and 9. (B) B6, B6.pfp0, B6.IFN-␥0, and
B6.pfp0.IFN-␥0 mice or B6 and B6.pfp0 mice treated with anti–mIFN-␥ mAb (500
␮g/injection) or control mAb (500 ␮g/injection) were treated as above. Mice received
anti–IFN-␥ mAb on days ⫺2, 0 (day of intravenous tumor inoculation), 2, 7, and 10,
with some groups receiving anti–mIFN-␥ mAb early (days ⫺2, 0) or late (days 7, 10).
(A) Significant differences from the B6 group were determined by a Mann-Whitney U
test (*P ⬍ .0001). Additionally, mice treated with anti-NK1.1 had significantly more
metastases than any other group, including B6.pfp0 and B6.IFN-␥0 mice (P ⬍ .0001).
(B) Significant differences from the B6.pfp0 group were determined by a MannWhitney U test and denoted (*P ⬍ .0001). The number of mice per group is shown in
parentheses.
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196
STREET et al
Figure 4. NKT-cell–mediated control of MCA-induced fibrosarcoma by independent pfp and IFN-␥ activities. (A) Groups of 10 to 43 B6, B6.J␣2810, B6.pfp0,
B6.IFN-␥0, or B6 pfp0.IFN-␥0 mice were injected subcutaneously in the hind flank with
100 ␮g or 25 ␮g MCA diluted in 0.1 mL corn oil. Mice were observed weekly for tumor
development over the course of 50 to 180 days. Tumors larger than 4 mm in diameter
and demonstrating progressive growth over 3 weeks were counted as positive.
Groups with statistically higher incidence than B6 mice were noted (*P ⬍ .01;
**P ⬍ .05; Fisher exact test). (B) Representative individual fibrosarcomas from
groups of 10 mice (above) were compared with a group of B6 mice receiving 400 ␮g
MCA. Tumor size was measured daily with a caliper square as the product of 2
diameters, and results were recorded as the tumor size (cm2). The mean growth rate
of tumors was determined from the gradients of individual plots and was normalized
against the mean of the B6 (400 ␮g) group ⫽ 1.0. Data are plotted as the mean ⫾ SE,
and significant differences to the B6 (400 ␮g) control were determined by an unpaired
t test with Welch correction (*P ⬍ .01)
BLOOD, 1 JANUARY 2001 䡠 VOLUME 97, NUMBER 1
determines the outcome of viral infection and dictates immune
homeostasis.27,28 In tumor models, each of pfp and IFN-␥ have
been demonstrated to contribute to the control of tumor incidence
and metastasis,6-8,15,16,19,23,29,30 but no previous studies have directly
compared their relative activities in the same model or examined
the effect of removing both activities. Herein, in innate antitumor
responses that involve relatively different contributions by NK and
NKT cells, we have demonstrated for the first time that the killer
cell pfp and cytokine IFN-␥ constitute independent mechanisms
that together control tumor initiation and metastasis.
The critical role of pfp in the direct cytolysis of tumor cells
by NK cells is undisputed2; however, the mechanism(s) of
IFN-␥ action may be more diverse,12,31-36 and exactly which
function is most important remains controversial. Although
originally defined as an agent with direct antiviral activity,37
IFN-␥ regulates several aspects of the immune response,
including the stimulation of antigen presentation by upregulation of major histocompatability complex (MHC) class I
and II molecules,12 the orchestration of leukocyte–endothelium
interactions,12,31 the effects on cell proliferation32 and sensitivity
to apoptosis,33,34 the stimulation of the bactericidal activity of
phagocytes,12 and anti-angiogenesis.35,36 A recent study has also
indicated that IFN-␥ may contribute directly to pfp-mediated
apoptosis by altering target cell sensitivity.38 From this and
many other studies, it is clear the roles of IFN-␥ in host
responses to tumor are complex, and it will not be easy to dissect
the relative role of these molecules in destroying tumors,
activating innate cells, or inhibiting tumor angiogenesis. Much
of the reason for the controversies about which IFN-␥ function
is most important is the great variety of settings in which IFN-␥
has been studied. We chose to focus on tumor models involving
several different host innate responses, with the knowledge that
each tumor may be controlled by a unique combination of
NK/NKT cell mechanisms. Although our study has not contributed greatly to our knowledge of how IFN-␥ may control
tumors, in at least one model (DA3), the enhancement of tumor
immunogenicity by IFN-␥ up-regulation of MHC class I and II
molecules seems unlikely given that T cells played no part in
host protection from tumor metastasis.
One outstanding feature of tumor behavior in mice deficient for
IFN-␥ was the increased growth rate of MCA-induced sarcomas.
The fact that NKT-cell–deficient mice, but not pfp-deficient mice,
also demonstrated increased sarcoma growth further suggested that
IFN-␥ may be an important feature of the NKT-cell response. The
effect of IFN-␥ was distinct from that observed in a study
comparing IFN-␥–responsive and –unresponsive mammary carcinomas that enlarged at similar rates.35 However, we (Smyth,
unpublished data) and others15,29 have observed that IFN-␥ is an
important molecule in the control of tumor growth in other models.
The mechanism by which IFN-␥ controls sarcoma tumor growth
remains unknown, but we did note that in vitro stimulation of cell
lines derived from MCA-induced tumors with IFN-␥ did not reveal
any decrease in proliferation (data not shown). Additional combinations of cytokines may be required to determine whether any
combination can suppress tumor cell proliferation. The possibility
that IFN-␥ has anti-angiogenic activity is also being explored;
however, previous data using the MCA tumor model suggested that
the key targets of the action of IFN-␥ were the tumor cells
themselves.16 IFN-␥ may also have been controlling tumor growth
in the RM-1 and DA3 metastasis models. Thus, in the absence of an
assay to measure micrometastases for these experimental tumors, it
is possible that numbers of surface lung tumor colonies measured at
From www.bloodjournal.org by guest on June 12, 2017. For personal use only.
BLOOD, 1 JANUARY 2001 䡠 VOLUME 97, NUMBER 1
ANTITUMOR EFFECTOR FUNCTIONS
defined points in time may have underestimated tumor burden in
wild-type and B6.pfp0 mice.
In summary, we have used the most rigorous and specific means
available to distinguish the relative roles of NK and NKT cells in
their control of tumor initiation, metastasis, and growth. Regardless
of the varying relative roles of NK and NKT cells in each of the
models used, host pfp and IFN-␥ were consistently demonstrated to
contribute independently to tumor control. Future efforts will focus
197
on defining how pfp-independent NK/NKT-cell antitumor functions are orchestrated by IFN-␥.
Acknowledgment
We thank the staff of the ARI-BRL for their maintenance and care
of the mice in this project.
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From www.bloodjournal.org by guest on June 12, 2017. For personal use only.
2001 97: 192-197
doi:10.1182/blood.V97.1.192
Perforin and interferon-γ activities independently control tumor
initiation, growth, and metastasis
Shayna E. A. Street, Erika Cretney and Mark J. Smyth
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