Natural killer (NK) cells are thought to provide the first line of defence against tumors, particularly major histocompatibility complex (MHC) class I
variants. We have confirmed in
C57BL/6 (B6) mice lacking perforin that peritoneal growth of MHC class I RMA-S tumor
cells in unprimed mice is controlled by perforin-dependent cytotoxicity mediated by CD3
NK1.1+ cells. Furthermore, we demonstrate that B6 mice lacking tumor necrosis factor (TNF)
are also significantly defective in their rejection of RMA-S, despite the fact that RMA-S is insensitive to TNF in vitro and that spleen NK cells from B6 and TNF-deficient mice are equally
lytic towards RMA-S. NK cell recruitment into the peritoneum was abrogated in TNF-deficient mice challenged with RMA-S or RM-1, a B6 MHC class I prostate carcinoma, compared with B6 or perforin-deficient mice. The reduced NK cell migration to the peritoneum
of TNF-deficient mice correlated with the defective NK cell response to tumor in these mice.
By contrast, a lack of TNF did not affect peptide-specific cytotoxic T lymphocyte-mediated
rejection of tumor from the peritoneum of preimmunized mice. Overall, these data show that
NK cells delivering perforin are the major effectors of class I tumor rejection in the peritoneum, and that TNF is specifically critical for their recruitment to the peritoneum.
Key words:
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Introduction |
atural killer (NK) cells are large granular lymphocytes, distinguishable from T or B lymphocytes by
their surface phenotype, cytokine profile, and the ability
to mediate spontaneous cytotoxicity against a broad range
of targets, including MHC class I tumor cells (1). NK
cells mainly circulate in the blood, where they account for
~5-15% of circulating lymphocytes (2); however, in certain
pathologic conditions, including viral and bacterial infections, NK cells selectively accumulate into the infected tissue sites (3). Several studies have shown that NK cells express many known adhesion molecules that bind to
ligands present on both resting and inflamed endothelium
(6). Furthermore, NK cells express many chemokine receptors and are responsive to several chemokines, including
macrophage inflammatory protein (MIP)-1
, IFN-inducible protein (IP)-10, and monocyte chemoattractant protein (MCP) family members (9). Several cytokines have
been shown to modulate these adhesive as well as chemotactic functions, including IL-2, IL-4, IL-8, IFN-
, IL-12,
and TNF (10). Activated NK cells produce a spectrum
of cytokines and can also respond rapidly to exogenous signals such as those delivered by tumor cells, by upregulation
of cytokine production and by increased migration to tissue
sites (16). This migration has been demonstrated to be dependent on the activation of NK cells; however, the mechanisms underlying selective recruitment of NK cells from
the blood vessels to the tissues have not been elucidated.
The ability to spontaneously lyse tumor cells is the best
known functional attribute of NK cells, and particular tumor escape variants that have lost MHC class I expression
are efficiently controlled in vivo by NK cells (17). This
model of NK cell function has been used in perforin-deficient (P0)1 mice to demonstrate that NK cell-mediated lysis
of class I RMA-S tumor cells in mice is perforin dependent (18). Indeed, perforin gene knockout mice have allowed assessment of the relative contribution of the granule
exocytosis pathway in a variety of immune responses in
vitro and in vivo (19). Activated NK cells also produce
the potentially cytolytic molecules, TNF and Fas ligand
(FasL [1, 23]). Herein, we use TNF-deficient (TNF0) and a
variety of other gene-targeted or mutant mice to define a
critical role for TNF in the specific migration of NK cells mediating MHC class I tumor rejection.
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Materials and Methods |
Mice.
Inbred C57BL/6 mice were purchased from The
Walter and Eliza Hall Institute of Medical Research (Melbourne,
Australia). C57BL/6 perforin-deficient (B6.P0) mice (19; from
Dr. Guna Karupiah, John Curtin School of Medical Research,
Canberra, Australia) and C57BL/6 IL-12p40-deficient (B6.IL-12p400) mice (24; Hoffman-La Roche, Nutley, NJ) were bred at
the Austin Research Institute Biological Research Laboratories
(ARI-BRL). C57BL/6 gld (FasL mutant; breeding colonies obtained from The Jackson Laboratory, Bar Harbor, ME) and
C57BL/6 TNF-deficient (B6.TNF0) mice (25, 26) were obtained
from the Centenary Institute of Cancer Medicine and Cell Biology. Male mice of 5-6 wk of age were used in all experiments,
which were performed according to animal experimental ethics
committee guidelines.
Cell Culture and Reagents.
The mouse WEHI 164 fibrosarcoma (H-2d), RM-1 prostate carcinoma (H-2b; reference 27),
YAC-1 (H-2a) lymphoma, RMA (H-2b) lymphoma, RMA-S
(H-2b) mutant lymphoma (derived from the Raucher virus-
induced murine cell line RBL-5 and defective for peptide loading
of MHC class I molecules [28]), and an E7-transfected clone of
RMA, RMA-E7 (29), were grown in RPMI medium supplemented with 10% (vol/vol) FCS, 2 mM glutamine, 100 U/ml
penicillin, and 100 µg/ml streptomycin (GIBCO BRL, Gaithersburg, MD). Soluble recombinant human FasL (srFasL; produced
in COS cell supernatants and used neat), and mouse Fas-Fc and
human TNF p80 receptor (TNFR)-Fc fusion proteins were a gift
from Dr. David Lynch (Immunex Corp., Seattle, WA). Recombinant human IL-2 was a gift from Chiron Corp. (Emeryville, CA).
Spleen cells were harvested from B6, B6.P0, and B6.TNF0 mice,
and IL-2-activated adherent NK cells were cultured and prepared
for adoptive transfer as described previously (30). Recombinant
mouse TNF- was a gift from the Biological Response Modifier's
Program Repository (Frederick Cancer Research and Development Center, Frederick, MD). Polyinosinic-polycytidylic acid
(poly-IC) was purchased from Sigma Chemical Co. (St. Louis,
MO). Peptide of human papilloma virus protein 16 (HPV-16)
protein E749-57 (RAHYNIVTF) was synthesized (>98% pure) on
an automated peptide synthesizer (model 430A; Applied Biosystems, Inc., Foster City, CA). In some experiments, to deplete T
lymphocytes and NK cells in vivo, mice were treated with mAb
(200 µg) anti-CD3 (KT3, rat IgG2a), anti-CD4 (H129.19, rat
IgG2a), anti-CD8 (1803, rat IgG2a), or anti-NK1.1 (PK136,
mouse IgG2a; all from the American Type Culture Collection,
Rockville, MD) on days 
4, 2, 0 (day of tumor inoculation),
and weekly thereafter. Depletions were monitored by the analysis
of spleens of treated mice by immunofluorescence using FITC-labeled mAbs anti-CD3 (29B, rat IgG2b; Sigma Chemical Co.);
anti-CD4 (H129.19; Sigma Chemical Co.); anti-CD8 (53-6.7, rat IgG2a; Sigma Chemical Co.); and anti-NK1.1 (PK136).
51Cr-release Assays.
The cytotoxicity of NK cells, responder
CTL, perforin/granzyme B, soluble FasL, or mouse TNF were
assessed by 51Cr-release assays against labeled targets (RMA-S,
YAC-1, RMA, WEHI 164, or RMA-E7). Spontaneous release
of 51Cr was determined by incubating the target cells with medium alone, and maximum release was determined by adding
SDS to a final concentration of 5%. The percent specific lysis was
calculated as follows: 100 × ([experimental release spontaneous release]/[maximum release spontaneous release]). Each experiment was performed twice using triplicate samples.
Peptide Immunization and Induction of B6, B6.TNF0, and B6.P0
Anti-E749-57 CTL.
100 µg (100 µl of 1 mg/ml peptide solution) peptide was extensively mixed with 100 µl IFA and 0.5%
BSA. The 200 µl mixture was injected subcutaneously in B6,
B6.TNF0, or B6.P0 mice and the procedure repeated after 2 wk.
1 wk after the second immunization, mice were either challenged
with 105 RMA-E7 tumor cells intraperitoneally or killed. The
spleen cells from killed mice were cultured overnight on plastic
flasks to remove adherent cells. Responder cells were then treated
with anti-CD4 (H129.19, rat IgG2a; Sigma Chemical Co.) and
C' (1:30 dilution of normal rabbit serum) as described previously
(30). Stimulator RMA-S cells were cultured at 25°C for 24 h and
incubated with 100 µM E749-57 peptide for 2-4 h at 33°C. Stimulator cells (106) were extensively washed, irradiated (20,000 rad),
and then cultured (25-cm2 culture flasks) with responder cells
(2 × 107) in RPMI supplemented with 10% FCS and 5 µM peptide. After 5 d, the CTL activity of responder cells was determined
in a 4-h 51Cr-release assay using labeled RMA-E7 target cells.
Tumor Control In Vivo.
Groups of five untreated (B6, B6.gld,
B6.TNF0, or B6.P0), mAb-depleted (B6), or peptide-immunized
(B6, B6.TNF0, or B6.P0) mice were injected intraperitoneally
with RMA-S, RMA, or RMA-E7 tumor cells in 0.2 ml PBS
as indicated. Mice were observed daily for tumor growth for
70-100 d by monitoring body weight and development of ascites
in mice. Mice were killed when first distressed by tumor growth.
Flow Cytometry.
For multiparameter analysis of lymphocytes,
cells were stained with anti-CD3-biotin (clone 145-2C11;
PharMingen, San Diego, CA), anti-NK1.1-PE, anti-CD4-FITC
(clone RM4-5; PharMingen), or anti-CD8 APC (clone 53-6.7;
PharMingen). Biotinylated mAbs were detected with streptavidin-conjugated Texas red (SA-TXR; Molecular Probes, Inc.,
Eugene, OR). Analysis was performed on a FACStar PLUS (Becton
Dickinson, San Jose, CA).
NK Cell Migration to the Peritoneum.
The number of NK cells
migrating to the peritoneum was evaluated in groups of five B6,
B6.P0, or B6.TNF0 mice that had received PBS (0.2 ml) or
RMA-S tumor cells (102-104)/0.2 ml i.p. After 24-144 h (72 h
optimal for NK cell migration), mice were killed by CO2 asphyxiation, and their peritoneal cavity flushed with 0.5 ml RPMI
1640 and 10% FCS and aspirated with a syringe. Cytospins were
performed to make sure no blood leakage into the peritoneal cavity had occurred; if so, these mice were discarded. Analysis of
peritoneal NK1.1+ cell content was not practical by flow cytometry due to relatively low cell numbers; thus, single color immunofluorescence was performed upon cells harvested from the peritoneum by fluorescence microscopy. Peritoneal contents were
analyzed for NK1.1+ cell numbers by detecting the percentage of
NK cells with FITC-labeled anti-NK1.1 (PK136; relative to a
negative control) and converting this value into an absolute number of NK1.1+ cells on the basis of a simultaneously obtained cell
count. Results are expressed as the mean ± SE of five mice. The
NK activity and number of cells collected in the peritoneum
were measured against YAC-1 after overnight culture in IL-2
(1,000 U/ml) and the results recorded as LU/107 cells, where one
LU is the number of effector cells required to lyse 25% of the target cells.
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Results |
Class I Tumor Control in the Peritoneum Is Perforin and
TNF Dependent.
To investigate the effector mechanisms
used to reject class I tumors in vivo, we injected RMA-S
cells intraperitoneally into B6, B6.P0, B6.TNF0, and B6.gld
mice (Fig. 1 A). B6 mice that received 
105 cells died
within 18-33 d, whereas 104 cells were controlled to some
extent (death between 35 and 50 d after inoculation). An
inoculum of 103 injected cells was controlled beyond 100 d
in wild-type B6 mice. By contrast, intraperitoneal injection
of 102 RMA-S tumor cells resulted in uncontrolled tumor
growth in B6.P0 mice (all dead within 22-41 d), and even
as few as 10 RMA-S tumor cells caused the death of 2/5
B6.P0 mice. B6.P0 mice that received greater numbers of
RMA-S cells (>102) all died within 31 d. In accordance
with van den Broek et al. (18), these data confirmed that
MHC class I RMA-S tumor growth is controlled by perforin-dependent cytotoxicity. NK cells have also been
demonstrated to display cytotoxicity via two members of
the TNF superfamily, FasL and TNF (1, 23), and thus the
contribution of these molecules to class I tumor rejection
was examined. FasL mutant B6.gld mice rejected RMA-S
cells in a similar fashion to B6 mice, and there was no apparent role for NK cell FasL in this process (Fig. 1 A). The data were in agreement with the finding that NK cell cytotoxicity against many tumor target cells appears to be predominantly perforin mediated (30), but contrast a possible
role for CTL FasL in tumor control in vivo (32). Clearly
however, B6.TNF0 mice were unable to control the peritoneal growth of RMA-S tumor cells, even at inoculations
as low as 102 cells (death within 38-50 d). The defective
control of RMA-S cells in B6.TNF0 mice was not as dramatic as in B6.P0 mice, but was nonetheless significantly
compromised compared with rejection in B6 and B6.gld
mice. It should be noted that all of these strains of B6 mice
cleared class I+ RMA tumor cells to a similar degree (103
cells; data not shown).
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Fig. 1.
Elimination of intraperitoneally administered MHC
class I syngeneic tumors
(RMA-S) in vivo: dependence
on perforin and TNF. (A) B6,
B6.P0, B6.TNF0, and B6.gld
mice (n = 5/group) were injected intraperitoneally with live
tumor cells (10-106) in 0.2 ml
PBS as indicated. Mice were observed daily for tumor growth
for up to 100 d by monitoring
body weight and development of
ascites in mice. Individual mice
are represented by each symbol.
(B) B6 mice were inoculated
with RMA-S as above, except
that B6 mice were treated on
days 4, 2, the day of tumor inoculation, and weekly thereafter with anti-CD3, anti-CD4, anti-CD8, or anti-NK1.1 mAb. Complete depletion of
lymphocyte subsets was observed throughout the course of the experiment, and anti-NK1.1-treated mice were shown to lack any NK cytotoxicity
against YAC-1 target cells.
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NK1.1+ NK Cells Control Class I Tumor Growth In Vivo.
To exclude the possibility that growth of RMA-S was controlled by T cells expressing perforin and recognizing residual empty MHC class I in an allotype-like reaction, B6
mice were depleted of CD3+, CD4+, CD8+, or NK1.1+
cells before and after tumor inoculation (Fig. 1 B). Depletions were complete (1% remaining) as monitored by fluorescence microscopy and splenic NK activity (data not
shown). Mice depleted of NK1.1+ cells were unable to
control RMA-S tumor growth, with all mice receiving 102
cells surviving only 21-47 d. The remarkably similar profile of B6.P0 mice (Fig. 1 A) and B6 mice treated with anti-NK1.1 (Fig. 1 B) suggested that T cells expressing perforin
were not responsible for RMA-S tumor rejection. This
conclusion was supported by the lack of effect of depleting
CD3+, CD4+, or CD8+ T cells on RMA-S tumor rejection (Fig. 1 B). It should also be noted that B6.P0 mice
were just as ineffective in controlling RMA-S tumor
growth as B6.P0 mice treated with anti-NK1.1 mAb (data
not shown). It is unlikely that IL-12-responsive NK1.1+ T
cells expressing perforin (33, 34) were responsible for the
rejection of RMA-S cells, since RMA-S is CD1, anti-CD3 mAb depletion of T cells was without effect, and
B6.IL-12p400 mice rejected RMA-S in a similar manner to
wild-type B6 mice (data not shown). The use of B6.CD1-deficient mice lacking NK1.1+ T cells (35) will provide
confirmatory evidence of the role of this cytotoxic subset
in RMA-S tumor rejection.
RMA-S Cells Are Not Sensitive to TNF.
A simple explanation for defective class I tumor control in B6.P0 and
B6.TNF0 mice might be that in wild-type mice, RMA-S
tumor cells are directly lysed by perforin and/or TNF derived from NK cells. Indeed, RMA-S cells were sensitive
to a sublytic concentration of perforin combined with increasing concentrations of granzyme B (Fig. 2 A), and NK cells from the spleens of B6.P0 mice were not cytolytic to
RMA-S tumor cells (Fig. 2 D). Although soluble TNF is
not a good effector of TNF cytotoxicity compared with
membrane-bound TNF (36), RMA-S cells were not sensitive to soluble TNF compared with WEHI 164 cells (Fig. 2
B). Furthermore, NK cells from B6.TNF0 mice were as cytolytic towards RMA-S as NK cells from B6 mice, whereas
NK cells from B6.P0 (that presumably still express membrane TNF) were not cytolytic in 4-h (Fig. 2 D) or longer
assays (data not shown). B6 NK cell lysis of RMA-S was
not inhibited by a TNFR-Fc fusion protein that significantly inhibited TNF-mediated lysis of WEHI 164 cells
(data not shown). RMA-S tumor cells were sensitive to
FasL (Fig. 2 C), but NK cells expressing nonfunctional mutant gld-FasL still effectively lysed RMA-S cells (Fig. 2 D).
The data further supported a role for NK cell perforin in
direct lysis of RMA-S in vivo, but suggested that direct lysis by NK cell TNF was not critical. Furthermore, the relative insensitivity of RMA-S to TNF suggested that direct lysis of RMA-S in vivo by other leukocyte subsets expressing TNF, including T cells and activated macrophages, was
unlikely. Together, the in vitro and in vivo data suggested
that the role of TNF in NK cell-mediated tumor rejection
may not be related to direct cytotoxicity.
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Fig. 2.
Sensitivity of RMA-S tumor cells to granule- and FasL-
mediated cytotoxicity. The cytotoxicity of various cytotoxic molecules or
NK cells was assessed by 51Cr-release assays against labeled targets as indicated. (A) Perforin (30 U) and increasing concentrations of granzyme B
(GrzB, 0-2 µg/ml) versus RMA-S, (B) murine TNF (0.002-2 ng/ml)
versus RMA-S or TNF-sensitive WEHI 164, (C) soluble FasL versus
RMA-S or FasL-insensitive EL-4, and (D) NK cells from B6, B6.P0,
B6.TNF0, or B6.gld mice versus MHC class I RMA-S or MHC class I+
RMA. The spontaneous release of 51Cr was always <15%, and each experiment was performed twice using triplicate samples.
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Defective Migration of NK Cells in Response to Tumor in the
Peritoneum of B6.TNF0 Mice.
Given that TNF has previously been reported to be chemokinetic for NK cells in
vitro (37), a possible mode of action of TNF could be to
recruit NK cells to the peritoneum upon tumor inoculation. Since members of the TNF family of cytokines influence lymphoid architecture (26, 38, 39), it was important
to initially evaluate B6 and B6.TNF0 mice for NK1.1+ cell
number and distribution. Flow cytometric analysis of
NK1.1+ NK cells and NK1.1+ T cells in the spleen, thymus, lymph nodes, blood, and bone marrow of unchallenged B6 versus B6.TNF0 mice revealed no statistical differences (data not shown). Migration of NK1.1+ cells to
the peritoneum was observed in B6 mice that had received RMA-S tumor cells (Table 1). Inoculation with RMA-S
cells increased NK1.1+ cell numbers by almost 10-fold after 72 h. By contrast, NK cell recruitment to the peritoneum was significantly reduced and delayed in B6.TNF0
mice receiving RMA-S tumor cells. Despite the reduction
in NK1.1+ cell migration, RMA-S tumor inoculation
stimulated similar total leukocyte migration in B6.TNF0
and B6 mice (Table 1). The NK activity recovered, as
measured by LU25, correlated with the number of NK1.1+
cells in the peritoneum, thus indicating that the NK cells
migrating to the peritoneum by 72 h displayed NK cell cytotoxicity (Table 1). Irrespective of the RMA-S cell number inoculated (102-104), migration of NK1.1+ cells to the
peritoneum was observed in both B6 and B6.P0 mice, but
was abrogated in B6.TNF0 mice (Table 2). NK cell cytotoxicity recovered from B6.TNF0 mice was minimal but
measurable compared with undetectable levels of NK cytotoxicity in the peritoneum of B6.P0 mice challenged with
RMA-S cells (Table 3). NK cell migration in response to
RMA-S tumor inoculation appeared normal in B6.P0,
B6.IL-12p400, and B6.gld mice (Table 3).
It was important to establish that the role of TNF in tumor rejection and NK cell recruitment was not specific for
RMA-S tumor target cells. We have previously demonstrated that the B6 MHC class I prostate carcinoma RM-1
was NK cell sensitive and when injected into the peritoneum was cleared by perforin expressing CD3NK1.1+
cells in a TNF-dependent manner (data not shown). Migration of NK1.1+ cells to the peritoneum was also observed in B6 mice in response to class I RM-1 prostate
carcinoma cells (Table 4). Inoculation with RM-1 cells increased NK1.1+ cell numbers by approximately sixfold after 72 h. RM-1 tumor inoculation again stimulated similar
total leukocyte migration in B6.TNF 0 and B6 mice (Table
4), however NK cell recruitment to the peritoneum was
significantly reduced in B6.TNF0 mice receiving RM-1
tumor cells. The data indicated that endogenous TNF was
a major contributor to NK cell accumulation in the peritoneum specifically in response to syngeneic class I tumor
cells.
Consistent with earlier reports (37), cell recruitment, in
particular NK1.1+ cell migration, was greatly enhanced in B6
mice inoculated with poly-IC, a powerful stimulator of NK
cell migration and cytotoxicity (Table 4). Peritoneal inoculation with poly-IC increased total leukocyte migration in
B6.TNF0 and B6 mice by 3-5-fold and NK1.1+ cell numbers in B6 mice by >15-fold (Table 4). In contrast to tumor
inoculation, poly-IC did stimulate significant NK1.1+ cell
numbers in the peritoneum of B6.TNF0 mice (~11-fold
above controls); however, numbers of NK1.1+ cells were still
reduced compared with B6 mice inoculated with poly-IC.
These data further served to illustrate that TNF is not critical
for all NK cell recruitment, even into the peritoneum.
CTL-mediated Tumor Rejection in the Peritoneum Is Normal
in B6.TNF0 Mice.
The striking need for TNF in effective
NK cell-mediated rejection of class I RMA-S cells
prompted the question whether TNF was also necessary for CTL rejection of tumor localized in the peritoneum.
To minimize tumor variation, RMA cells expressing the
HPV-16 E7 protein were used as the tumor challenge to
mice preimmunized with the E749-57 H-2Db-presented
9-mer peptide. Importantly, mouse CD8+ CTL reactive
with E749-57 were generated from the spleen cells of preimmunized B6, B6.P0, and B6.TNF0 mice (Fig. 3 A). Thus,
TNF was not absolutely necessary for the generation of
peptide-specific CTL. CD8+ CTL generated from B6 and
B6.TNF0 mice displayed peptide-specific perforin- and
FasL-mediated lysis of RMA-E7 (as determined by inhibition with Mg-EGTA or Fas-Fc, respectively), whereas
CD8+ CTL from B6.P0 mice lysed RMA-E7 via FasL exclusively (data not shown). Preimmunized B6 and B6.TNF0
mice effectively cleared intraperitoneal challenge with 105
RMA-E7 cells, whereas B6.P0 mice did not and died
within 20-30 d (Fig. 3 B). Unprimed B6, B6.TNF0, and
B6.P0 mice challenged with RMA-E7 all died within 17-
28 d. Thus, the data indicated that RMA-E7 tumor rejection in the peritoneum was mediated by CTL expressing
perforin and showed that a lack of host TNF did not reduce CTL effector function at this site in vivo.
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Fig. 3.
CTL-mediated
peritoneal tumor rejection is
normal in B6.TNF0 mice. B6,
B6.P0, and B6.TNF0 mice were
immunized twice (2 wk apart)
with E7 peptide and their spleen
cells harvested 1 wk later and
stimulated in vitro (as described
in Materials and Methods). 5 d
after stimulation, the CTL activity of responder cells was determined in a 4-h 51Cr-release assay
(at E/T ratios indicated) using labeled RMA-E7 target cells,
RMA-S pulsed with E749-57 peptide, or RMA-S pulsed with OVA257-264. The spontaneous release of 51Cr was <15%, and the experiment was performed
using triplicate samples. (B) Untreated B6, B6.P0, and B6.TNF0 mice (n = 5/group) or those immunized as above were challenged intraperitoneally 1 wk
after the second immunization with 105 RMA-E7 tumor cells in 0.2 ml PBS. Mice were observed daily for tumor growth for up to 70 d by monitoring
body weight and development of ascites in mice. Individual mice are represented by each symbol.
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Discussion |
NK cells have been shown to accumulate locally and in
various organs after animals are exposed to viruses (4, 40)
or biological response modifiers (37, 41, 42). Where evaluated, it has become apparent that TNF may (37) or may
not (40, 42) be critical for NK cell migration in response to
these stimuli; therefore, NK cell emigration does not appear to be generally affected by TNF. However, the role of
TNF in NK cell recruitment to and rejection of tumor has
been comparatively poorly understood. We have used two
different MHC class I tumors and TNF0 mice to illustrate
an essential role for TNF in NK cell antitumor activity in
the peritoneum. Given a previously demonstrated role for
TNF in leukocyte trafficking (25, 26, 38, 39, 43) and our
observation that B6.TNF0 mice displayed reduced numbers
of NK1.1+ cells in the peritoneum in response to tumor
inoculation, we believe TNF is specifically vital for the migration of NK cells into the peritoneum.
Several other possibilities may explain a critical role for
TNF in NK cell-mediated tumor rejection, including (a) a
direct effect of NK cell TNF secretion on tumor growth;
(b) a direct enhancement of the NK cell activity of resident
NK cells by TNF; (c) local proliferation or enhanced survival of peritoneal NK cells in response to TNF; or (d) decreased adherence of NK cells to endothelial cells in the
presence of TNF. However, most experimental evidence
that we and others have generated would undermine these possibilities. A direct cytotoxic effect of NK cell, or host, TNF on RMA-S tumor growth is unlikely, given that this
tumor appears insensitive to TNF. NK cells from TNF0
mice have normal levels of NK activity in the spleen and
thus a general defect in the activation of NK cell cytotoxic
potential in mice lacking TNF was not apparent. In addition, when spleen NK cells from B6.TNF0 mice were expanded by culture in IL-2, the proliferation of NK1.1+
cells and their lytic activity were comparable with the same cells from wild-type B6 mice (data not shown). Similarly,
exogenous TNF did not support the survival of B6 or
B6.TNF0 peritoneal cells taken 24-72 h after tumor inoculation (data not shown). Furthermore, when adoptively
transferred intraperitoneally into B6.TNF0 mice, cultured
B6.TNF0 and B6 NK cells, but not B6.P0 NK cells, were
capable of mediating RMA-S tumor rejection (data not
shown). Therefore, our data would support a previous
study that demonstrated TNF did not enhance NK cell survival (44). Whether exogenous soluble TNF could mimic
local signals that control cytokine networks, resulting in
NK cell emigration, is debatable; however, B6 or B6.TNF0
mice that received TNF intraperitoneally did not display
increased numbers of NK cells or enhanced lytic activity in
response to RMA-S (data not shown). It appears doubtful
that TNF reduced adhesion and increased the peritoneal lavage recovery of NK1.1+ cells considering that TNF has
previously been observed to increase NK cell adherence
(37).
There may be several processes whereby TNF regulates
NK cell trafficking to tumor in the peritoneum. We have
not attempted to determine the original source of NK1.1+
NK cells which accumulate in the peritoneal cavity after tumor inoculation; however, previous studies of NK cell migration in response to viral infection would favor the bone
marrow as a source over peripheral lymphoid organs (45,
46). TNF induces expression of the vascular adhesion molecule (VCAM-1) on vascular endothelial cells, and VCAM-1/
very late antigen (VLA)-4 interactions are required for migration of NK cells from the blood into lung and liver after
poly-ICLC treatment (41, 47). Nonetheless, binding to vascular endothelium is only the first step in NK cell infiltration, and subsequent events must result in transit across the basement membrane and chemotaxis to the tumor. Recent studies in autoimmune inflammation in the B6.TNF0 central
nervous system (CNS) indicated not only that VCAM-1 is upregulated on CNS endothelium normally (25), but that
leukocytes extravasate and accumulate in the CNS perivascular region yet fail to move out into the tissue in a normal
fashion (25, 43). Specific leukocyte populations infiltrating
lesions were not defined in these studies, but the contrasting
roles of NK1.1+ cells reportedly controlling the severity of
experimental allergic encephalomyelitis (EAE [48]) and TNF
increasing the onset of EAE (43) suggest that TNF is not
regulating EAE by affecting NK1.1+ cell emigration.
NK cell availability, activity, and chemotaxis have been
demonstrated to be regulated by cytokines of the C-C family made in NK cells (49), and macrophages or dendritic
cells (15, 51, 52). Thus, it is possible that TNF may serve to
regulate macrophages or dendritic cell production of
chemokines that in turn recruit NK cells. Although IL-12
does not appear necessary, tumor rejection experiments in
various C-C chemokine-deficient mice may be very informative. Pathways for NK cell trafficking are likely to be
complex, and the chemokine network elicited will very
much depend on the form and site of the challenge. Our
data indicated that NK1.1+ cell recruitment in response to
tumor was virtually abolished in the absence of TNF,
whereas NK cell migration to the peritoneum was still significant in response to poly-IC. Poly-IC elicits many cytokines that directly activate NK cells; thus, TNF may play
a relatively minor role in the migration of NK cells into the
peritoneum in response to poly-IC. Subcutaneous inoculation of various gene-targeted mice or B6 mice treated with
anti-NK1.1 mAb with RMA-S tumor cells revealed that
TNF was not necessary for NK1.1+ cells to reject RMA-S
in a perforin-dependent manner (data not shown). Thus, in
the absence of further data at other tumor sites, it appears
that the absolute requirement for TNF in NK cell antitumor activity may be restricted to the peritoneum.
However, NK1.1+ cells with antitumor activity are heterogeneous (18, 33, 34), as are dendritic cells and macrophages in various tissues (52); therefore, future studies will be required to resolve the apparent anomaly that TNF is not universally critical for all sites of tumor rejection. Although all
the mediators involved in NK cell responses to class I
tumor are unclear, our work has highlighted a critical role
for TNF in NK cell rejection of class I-deficient tumors by
virtue of its capacity to regulate cell recruitment. Importantly, the requirement for TNF appears specific for NK cells
responding to tumor. This finding now will stimulate further
examination of a possible role for TNF in controlling NK
cell responses to tumor initiation, growth, and metastasis.
Address correspondence to Mark Smyth, Cellular Cytotoxicity Laboratory, The Austin Research Institute,
Studley Road, Heidelberg, Victoria 3084, Australia. Phone: 61-3-92870655; Fax: 61-3-92870600; E-mail:
m.smyth{at}ari.unimelb.edu.au
We thank Dr. Joe Trapani for purified perforin and granzyme B, Dr. David Lynch for recombinant FasL,
Dr. Timothy Thompson for the RM-1 prostate carcinoma cells, and Drs. Ricky Johnstone and Thomas
Sayers for helpful discussion. We also thank Dr. Jeanne Magram (Hoffman-La Roche, Nutley, NJ) for providing the IL-12 gene knockout mice.
| 1.
|
Trinchieri, G..
1989.
Biology of natural killer cells.
Adv. Immunol.
47:
187-376
[Medline].
|
| 2.
|
Whiteside, T.L., and
R.B. Herberman.
1994.
Human natural
killer cells in health and disease. Biology and therapeutic potential.
Clin. Immunother.
1:
56-66
.
|
| 3.
|
Holmberg, L.A.,
K.A. Springer, and
K.A. Ault.
1981.
Natural killer cell activity in the peritoneal exudate of mice infected with Listeria monocytogenes.
J. Immunol.
127:
1792-1799
[Abstract].
|
| 4.
|
McIntyre, K.W., and
R.W. Welsh.
1986.
Accumulation of
natural killer and cytotoxic T large granular lymphocytes in
the liver during virus infection.
J. Exp. Med.
164:
1667-1681
[Abstract/Free Full Text].
|
| 5.
|
Nemlander, A.,
E. Saksela, and
P. Hayrj.
1983.
Are natural
killer cells involved in allograft rejection?
Eur. J. Immunol.
13:
348-350
[Medline].
|
| 6.
|
Bender, J.R.,
R. Pardi,
M.A. Karasek, and
E.G. Engleman.
1987.
Phenotypic and functional characterization of lymphocytes that bind human microvascular endothelial cells in
vitro.
J. Clin. Invest.
79:
1679-1688
.
|
| 7.
|
Allavena, P.,
C. Paganin,
I. Martin-Padura,
G. Peri,
M. Gaboli,
E. Dejana,
P.C. Marchisio, and
A. Mantovani.
1991.
Molecules and structures involved in the adhesion of natural killer
cells to vascular endothelium.
J. Exp. Med.
173:
439-448
[Abstract/Free Full Text].
|
| 8.
|
Gismondi, A.,
S. Morrone,
M.J. Humphries,
M. Piccoli,
L. Frati, and
A. Santoni.
1991.
Human natural killer cells express VLA-4 and VLA-5, which mediate their adhesion to fibronectin.
J. Immunol.
146:
384-392
[Abstract].
|
| 9.
|
Taub, D.D.,
T.J. Sayers,
C.R.D. Carter, and
J.R. Ortaldo.
1995.
and  chemokines induce NK cell migration and enhance NK-mediated cytolysis.
J. Immunol.
155:
3877-3888
[Abstract].
|
| 10.
|
Maghazachi, A.A..
1991.
Tumor necrosis factor- is chemokinetic for lymphokine-activated killer cells: regulation by cyclic adenosine monophosphate.
J. Leukocyte Biol.
49:
302-308
[Abstract].
|
| 11.
|
Bianchi, G.,
M. Sironi,
E. Ghibaudi,
C. Selvaggini,
M. Elices,
P. Allavena, and
A. Mantovani.
1993.
Migration of NK cells
across endothelial cell monolayers.
J. Immunol.
151:
5135-5144
[Abstract].
|
| 12.
|
Bottazzi, B.,
M. Introna,
P. Allavena,
A. Villa, and
A. Mantovani.
1985.
In vitro migration of human large granular
lymphocytes.
J. Immunol.
134:
2316-2321
[Abstract].
|
| 13.
|
Pohajdak, B.,
J. Gomez,
F.W. Orr,
N. Khalil,
M. Talgoy, and
A.H. Greenberg.
1986.
Chemotaxis of large granular
lymphocytes.
J. Immunol.
136:
278-284
[Abstract].
|
| 14.
|
Sebok, K.,
D. Woodside,
A. al-Aoukaty,
A.D. Ho,
S. Gluck, and
A.A. Maghazachi.
1993.
IL-8 induces the locomotion of human IL-2-activated natural killer cells. Involvement of a guanine
nucleotide binding (Go) protein.
J. Immunol.
150:
1524-1534
[Abstract].
|
| 15.
|
Allavena, P.,
C. Paganin,
D. Zhou,
G. Bianchi,
S. Sozzani, and
A. Mantovani.
1994.
Interleukin-12 is chemotactic for
natural killer cells and stimulates interaction with vascular endothelium.
Blood.
84:
2261-2268
[Abstract/Free Full Text].
|
| 16.
|
Whiteside, T.L., and
R.B. Herberman.
1992.
Extravasation
of antitumor effector cells.
Invasion Metastasis.
12:
128-146
[Medline].
|
| 17.
|
Karre, K.,
H.G. Ljunggren,
G. Piontek, and
R. Kiessling.
1986.
Selective rejection of H-2-deficient lymphoma variants
suggests alternative immunedefence strategy.
Nature.
319:
675-678
[Medline].
|
| 18.
|
van den Broek, M.F.,
D. Kagi,
R.M. Zinkernagel, and
H. Hengartner.
1995.
Perforin dependence of natural killer cell-mediated tumor control in vivo.
Eur. J. Immunol.
25:
3514-3516
[Medline].
|
| 19.
|
Kagi, D.,
B. Ledermann,
K. Burki,
P. Seiler,
B. Odermatt,
K.J. Olsen,
E.R. Podack,
R.M. Zinkernagel, and
H. Hengartner.
1994.
Cytotoxicity mediated by T cells and natural
killer cells is greatly impaired in perforin-deficient mice.
Nature.
369:
31-37
[Medline].
|
| 20.
|
Kagi, D.,
B. Odermatt,
P.S. Ohashi,
R.M. Zinkernagel, and
H. Hengartner.
1996.
Development of insulitis without diabetes in transgenic mice lacking perforin-dependent cytotoxicity.
J. Exp. Med.
183:
2143-2152
[Abstract/Free Full Text].
|
| 21.
|
Kagi, D.,
B. Ledermann,
K. Burki,
H. Hengartner, and
R.M. Zinkernagel.
1994.
CD8+ T cell-mediated protection
against an intracellular bacterium by perforin.
Eur. J. Immunol.
24:
3068-3072
[Medline].
|
| 22.
|
Schulz, M.,
H.J. Schuurman,
J. Joergensen,
C. Steiner,
T. Meerloo,
D. Kagi,
H. Hengartner,
R.M. Zinkernagel,
M. Schreier,
K. Burki, and
B. Ledermann.
1995.
Acute rejection
of vascular heart allografts by perforin-deficient mice.
Eur. J. Immunol.
25:
474-480
[Medline].
|
| 23.
|
Oshimi, Y.,
S. Oda,
Y. Honda,
S. Nagata, and
S. Miyazaki.
1996.
Involvement of Fas ligand and Fas-mediated pathway
in the cytotoxicity of human natural killer cells.
J. Immunol.
157:
2909-2915
[Abstract].
|
| 24.
|
Magram, J.,
S.E. Connaughton,
W.W. Warrier,
D.M. Carvajal,
C.Y. Wu,
J. Ferrante,
C. Stewart,
U. Sarmiento,
D.A. Faherty, and
M.K. Gately.
1996.
IL-12-deficient mice are
defective in IFN- production and type 1 cytokine responses.
Immunity.
4:
471-481
[Medline].
|
| 25.
|
Korner, H.,
D.S. Riminton,
D.H. Strickland,
F.A. Lemckert,
J.D. Pollard, and
J.D. Sedgwick.
1997.
Critical points of tumor necrosis factor action in central nervous system autoimmune inflammation defined by gene targeting.
J. Exp. Med.
186:
1585-1590
[Abstract/Free Full Text].
|
| 26.
|
Korner, H.,
M. Cook,
D.S. Riminton,
F.A. Lemckert,
B. Hoek,
B. Ledermann,
F. Kontgen,
B. Fazekas de St,
Groth, and
J.D. Sedgwick.
1997.
Distinct roles for lymphotoxin-
and tumor necrosis factor in organogenesis and spatial organization of lymphoid tissue.
Eur. J. Immunol.
27:
2600-2609
[Medline].
|
| 27.
|
Baley, P.A.,
K. Yoshida,
W. Qian,
I. Sehgal, and
T.C. Thompson.
1995.
Progression to androgen insensitivity in a novel in
vitro mouse model for prostate cancer.
J. Steroid Biochem.
Mol. Biol.
52:
403-413
[Medline].
|
| 28.
|
Ljunggren, H.G., and
K. Karre.
1985.
Host resistance directed selectively against H-2-deficient lymphoma variants.
Analysis of the mechanism.
J. Exp. Med.
162:
1745-1759
[Abstract/Free Full Text].
|
| 29.
|
Zhu, X.,
M. Tommasino,
K. Vousden,
E. Sadovnikava,
R. Rappuoli,
L. Crawford,
M. Kast,
C.J. Melief,
P.C. Beverley, and
H.J. Stauss.
1995.
Both immunization with protein and
recombinant vaccinia virus can stimulate CTL specific for the
E7 protein of human papilloma virus 16 in H-2d mice.
Scand.
J. Immunol.
42:
557-563
[Medline].
|
| 30.
|
Smyth, M.J.,
K.Y.T. Thia, and
M.H. Kershaw.
1997.
Xenogeneic mouse anti-human NK cytotoxicity is mediated via
perforin.
Xenotransplantation.
4:
78-84
.
|
| 31.
|
Smyth, M.J.,
V.R. Sutton,
M.H. Kershaw, and
J.A. Trapani.
1996.
Xenospecific cytotoxic T lymphocytes use perforin-
and Fas-mediated lytic pathways.
Transplantation (Baltimore).
62:
1529-1532
[Medline].
|
| 32.
|
van den Broek, M.F.,
D. Kagi,
F. Ossendorp,
R. Toes,
S. Vamvakas,
W.K. Lutz,
C.J.M. Melief,
R.M. Zinkernagel, and
H. Hengartner.
1996.
Decreased tumor surveillance in
perforin-deficient mice.
J. Exp. Med.
184:
1781-1790
[Abstract/Free Full Text].
|
| 33.
|
Cui, J.,
T. Shin,
T. Kawano,
H. Sato,
E. Kondo,
I. Toura,
Y. Kaneko,
H. Koseki,
M. Kanno, and
M. Taniguchi.
1997.
Requirement for V14 NKT cells in IL-12-mediated rejection of tumors.
Science.
278:
1623-1626
[Abstract/Free Full Text].
|
| 34.
|
Kawamura, T.,
K. Takeda,
S.K. Mendiratta,
H. Kawamura,
L. Van Kaer,
H. Yagita,
T. Abo, and
K. Okumura.
1998.
Critical role of NK1+ T cells in IL-12-induced immune responses in vivo.
J. Immunol.
160:
16-19
[Abstract/Free Full Text].
|
| 35.
|
Chen, Y.-H.,
N.M. Chiu,
M. Mandal,
N. Wang, and
C.-R. Wang.
1997.
Impaired NK1+ T cell development and early
IL-4 production in CD1-deficient mice.
Immunity.
6:
459-467
[Medline].
|
| 36.
|
Grell, M.,
E. Douni,
H. Wajant,
M. Lohden,
M. Clauss,
B. Maxeiner,
S. Georgopoulos,
W. Lesslauer,
G. Kollias,
K. Pfizenmaier, and
P. Scheurich.
1995.
The transmembrane
form of tumor necrosis factor is the prime activating ligand of
the 80 kDa tumor necrosis factor receptor.
Cell.
83:
793-802
[Medline].
|
| 37.
|
Pilaro, A.M.,
D.D. Taub,
K.L. McCormick,
H.M. Williams,
T.J. Sayers,
W.E. Fogler, and
R.H. Wiltrout.
1994.
TNF-
is a principal cytokine involved in recruitment of NK cells to
liver parenchyma.
J. Immunol.
153:
333-342
[Abstract].
|
| 38.
|
Pasparikis, M.,
L. Alexopoulou,
V. Episkopou, and
G. Kollias.
1996.
Immune and inflammatory responses in TNF--
deficient mice: a critical requirement for TNF- in the formation of primary B cell follicles, follicular dendritic cell
networks and germinal centers, and in the maturation of the
humoral immune response.
J. Exp. Med.
184:
1397-1411
[Abstract/Free Full Text].
|
| 39.
|
Matsumoto, M.,
Y.X. Fu,
H. Molina, and
D.D. Chaplin.
1997.
Lymphotoxin--deficient and TNF receptor-I-deficient mice define developmental and functional characteristics of germinal centers.
Immunol. Rev.
156:
137-144
[Medline].
|
| 40.
|
Salazar-Mather, T.P.,
J.S. Orange, and
C.A. Biron.
1998.
Early murine cytomegalovirus (MCMV) infection induces
liver natural killer (NK) cell inflammation and protection
through macrophage inflammatory protein 1 (MIP-1)-
dependent pathways.
J. Exp. Med.
187:
1-14
[Abstract/Free Full Text].
|
| 41.
|
Wiltrout, R.H.,
B.J. Mathieson,
J.E. Talmadge,
C.W. Reynolds,
S.R. Zhang,
R.B. Herberman, and
J.R. Ortaldo.
1984.
Augmentation of organ-associated natural killer activity
by biological response modifiers. Isolation and characterization of large granular lymphocytes from the liver.
J. Exp.
Med.
160:
1431-1449
[Abstract/Free Full Text].
|
| 42.
|
Sayers, T.J.,
L.L. Mason, and
T.A. Wiltrout.
1990.
Trafficking and activation of murine natural killer cells: differing roles
for IFN- and IL-2.
Cell. Immunol.
127:
311-326
[Medline].
|
| 43.
|
Riminton, D.S.,
H. Korner,
D.H. Strickland,
F.A. Lemckert,
J.D. Pollard, and
J.D. Sedgwick.
1998.
Challenging cytokine
redundancy: inflammatory cell movement and clinical course
of experimental autoimmune encephalomyelitis are normal in
lymphotoxin-deficient but not TNF-deficient mice.
J. Exp.
Med.
187:
1517-1528
[Abstract/Free Full Text].
|
| 44.
|
Carson, W.E.,
T.A. Fehniger,
S. Haldar,
K. Eckhert,
M.J. Lindemann,
C.F. Lai,
C.M. Croce,
H. Baumann, and
M.A. Caligiuri.
1997.
A potential role for interleukin-15 in the
regulation of human natural killer cell survival.
J. Clin. Invest.
99:
937-943
[Medline].
|
| 45.
|
Biron, C.A.,
G. Sonnenfeld, and
R.M. Welsh.
1984.
Interferon induces natural killer cell blastogenesis in vivo.
J. Leukoc. Biol.
35:
31-37
[Abstract].
|
| 46.
| Hackett, J., Jr., M. Tutt, M. Lipscomb, M. Bennett, G. Koo,
and V. Kumar. Origin and differentiation of natural killer
cells. II. Functional and morphologic studies of purified NK-1.1+ cells. J. Immunol. 136:3124-3131.
|
| 47.
|
Fogler, W.E.,
K. Volker,
K.L. McCormick,
M. Watanabe,
J.R. Ortaldo, and
R.H. Wiltrout.
1996.
NK cell infiltration
into lung, liver, and subcutaneous B16 melanoma is mediated
by VCAM-1/VLA-4 interaction.
J. Immunol.
156:
4707-4714
[Abstract].
|
| 48.
|
Zhang, B.,
T. Yamamura,
T. Kondo,
M. Fujiwara, and
T. Tabira.
1997.
Regulation of experimental autoimmune encephalomyelitis by natural killer (NK) cells.
J. Exp. Med.
186:
1677-1687
[Abstract/Free Full Text].
|
| 49.
|
Hedrick, J.A.,
V. Saylor,
D. Figueroa,
L. Mizoue,
Y. Xu,
S. Menon,
J. Abrams,
T. Handel, and
A. Zlotnik.
1997.
Lymphotactin is produced by NK cells and attracts both NK cells
and T cells in vivo.
J. Immunol.
158:
1533-1540
[Abstract].
|
| 50.
|
Allavena, P.,
G. Bianchi,
D. Zhou,
J. van Damme,
P. Jilek,
S. Sozzani, and
A. Mantovani.
1994.
Induction of natural killer
cell migration by monocyte chemotactic protein-1, -2 and
-3.
Eur. J. Immunol.
24:
3233-3236
[Medline].
|
| 51.
|
Loetscher, P.,
M. Seitz,
I. Clark-Lewis,
M. Baggiolini, and
B. Moser.
1996.
Activation of NK cells by CC chemokines.
Chemotaxis, Ca2+ mobilization, and enzyme release.
J. Immunol.
156:
322-327
[Abstract].
|
| 52.
|
Cella, M.,
F. Sallusto, and
A. Lanzavecchia.
1997.
Origin,
maturation and antigen presenting function of dendritic cells.
Curr. Opin. Immunol.
9:
10-16
[Medline].
|
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