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in Primary and Secondary Immunity Generated by NK Cell-Sensitive Tumor-Expressing CD80 In Vivo1
* Cancer Immunology Program, Sir Donald and Lady Trescowthick Laboratories, Peter MacCallum Cancer Institute, East Melbourne, Victoria, Australia; and
Department of Immunology, Juntendo University School of Medicine, Bunkyo-ku, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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activity were critical
for the rejection of MHC class I-deficient RMA-S-CD80 tumor cells. This
primary rejection process stimulated the subsequent development of
specific CTL and Th1 responses against Ags expressed by the MHC class
I-deficient RMA-S tumor cells. The development of effective secondary T
cell immunity could be elicited by irradiated RMA-S-CD80 tumor cells
and was dependent upon NK cells and IFN- in the priming response.
Our findings demonstrate a key role for IFN- in innate and adaptive
immunity triggered by CD80 expression on tumor
cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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secretion plays in immune regulation has been given far less
attention (10, 11, 12). As NK cells produce a number of
inflammatory cytokines, it has been suggested that these cells may
modulate the development of adaptive immune responses
(13). Nevertheless, acceptance of this hypothesis has been
limited, and a molecular demonstration that NK cell cytokine secretion
shapes the adaptive immune response has not been provided.
Cells with impaired TAP function express low levels of cell surface MHC
class I molecules and are generally sensitive to lysis by NK cells. We
have investigated the primary and secondary immunity elicited by
TAP-deficient RMA-S tumors expressing CD80 that interact with CD28,
CD152, or a related molecule expressed on NK cells (12).
We demonstrate that the primary rejection of MHC class I-deficient
RMA-S-CD80 tumor cells was mediated by NK cells and perforin
(pfp)3- and
IFN--dependent mechanisms. This NK cell-mediated process also very
efficiently evoked the subsequent development of tumor-specific CTL and
Th1 responses against Ags expressed by the parental tumor, and priming
of the secondary response was also IFN- dependent.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Inbred wild-type C57BL/6 (WT) mice were purchased from The
Walter and Eliza Hall Institute of Medical Research (Melbourne,
Australia). The following gene-targeted mice were bred at The Peter
MacCallum Cancer Institute: C57BL/6 IFN--deficient
(IFN--/-) mice (provided by Genentech, South
San Francisco, CA), C57BL/6 pfp-deficient
(pfp-/-) mice, and C57BL/6 RAG-1-deficient
(RAG-1-/-) mice (from Dr. L. Corcoran, The
Walter and Eliza Hall Institute of Medical Research). C57BL/6 mice
doubly deficient for pfp and RAG-1
(pfp-/-RAG-1-/-) were
produced and bred at The Peter MacCallum Cancer Institute. Mice 6–12
wk of age were used in all experiments, which were performed according
to animal experimental ethics committee guidelines.
Abs and reagents
Purified mAbs reactive with mouse CD4 (GK1.5), mouse CD8
(53-6.7), mouse CD16/32 (2.4G2), mouse NK1.1 (PK136), and mouse IFN-
(R4-6A2) were all purified from hybridomas. Isotype control for rat
IgG1 (R3-34), rat IgG2a (R35-95; BD PharMingen, San Diego, CA), and
PE-labeled goat anti-rat IgG (Caltag Laboratories, Burlingame, CA)
were purchased. Rabbit asialoGM1 Ab was purchased from WAKO (Richmond,
VA). Human IL-2 was provided by Chiron (Emeryville, CA).
Cell lines
All cell lines used in this study were derived from C57BL/6 (B6, H-2b) mice. RMA and RMA-S cell lines were T cell lymphomas derived from the Rauscher murine leukemia virus (MuLV)-induced RBL-5 cell line (14). MBL-2 was isolated from a Moloney MuLV-inoculated B6 mouse (15). EL-4 is a dimethylbenzanthracene-induced thymoma cell line (16). B16F10 mouse melanoma cells have been described previously (17). All cell lines were grown in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, Gaithersburg, MD) as previously described (18, 19). RMA-S-m and RMA-S-CD80 infectants were prepared using a previously described method (20). Briefly, mouse CD80 cDNA was cloned into the retroviral vector plasmid MSCV. The plasmid was cotransfected with an amphotropic packaging plasmid into 293T cells by calcium phosphate precipitation. After 48 h, the supernatant was harvested and added to tumor cells every 8 h for 3 days. The cells were allowed to recover and then analyzed for green fluorescence protein (GFP) expression by flow cytometry. The highest 10% of cells were sterilely sorted, expanded, resorted, and subsequently expanded in oligoclonal pools. Cells were subsequently screened for expression of CD80 and sorted by flow cytometry using the 1G10 anti-mCD80 mAb. Parental tumor cells were also infected with the empty MSCV vector alone, and GFP-expressing cells were similarly selected (RMA-S-m). GFP expression had no effect on the Kb or Db expression on tumor cells.
Flow cytometry
Mononuclear cells were prepared from the spleen as previously
described (21). To avoid the nonspecific binding of Abs to
FcR, the cells were preincubated with anti-mouse CD16/32 (2.4G2)
before staining. Flow cytometric sorting of cells was performed
following staining with FITC -conjugated anti-
TCR (clone
H57-597) and PE-conjugated anti-NK1.1 (clone PK-136). All flow
cytometry reagents were purchased from BD PharMingen, unless otherwise
indicated. Expression of CD80 was determined on infectants using the
anti-mCD80 mAb (IG10) and a secondary PE-labeled goat anti-rat
IgG Ab.
Peptides
The following purified peptides purchased from Auspep (Parkville, Australia) were used to determine CTL and Th1 functions: the MuLV gag leader (gagL) 75–83 (CCLCLTVFL) epitope presented by H-2Db, the MuLV envelop (env) 189–196 (SSWDFITV) epitope presented by H-2Kb (22), the RMA/EL-4 common tumor (NKGENAQL) epitope presented by H-2Db (23), the control OVA (SIINFEKL) epitope presented by H-2Kb, the env-H19 (EPLTSLTPRCNTAWNRLKL) epitope presented by I-Ab, and the control OVA-H (ISQAVHAAHAEINEAGR) epitope presented by I-Ab.
Cell culture
Spleen cells from RAG-1-/- and
pfp-/-RAG-1-/- mice
were incubated in tissue culture dishes at 37oC
for 1 h to deplete adherent cells. Harvested cells were routinely
>95% NK1.1+TCR- as
estimated by flow cytometry. Purified NK cells were cultured in RPMI
1640 medium supplemented with 10% heat-inactivated FCS, 2 mM
L-glutamine, and 25 mM sodium bicarbonate
(NaHCO3) in humidified 5%
CO2 at 37oC as previously
reported (24). In coculture experiments, purified NK cells
(3 x 105/well) were cocultured with
irradiated (15,000 rad) RMA-S-m or RMA-S-CD80 cells (3 x
104/well) in 96-well, round-bottom culture plates
in the presence of 25 U/ml IL-2. Before incubation, some NK cell
cultures were depleted of NK1.1+ cells by
anti-NK1.1 (10 µg/ml) and complement (C'). CTL, and Th1 responses
were analyzed in splenocytes from mice that had been primed 5 wk
earlier with 5 x 105 live RMA-S-CD80 tumor
cells, 107 irradiated (15,000 rad) RMA-S-CD80
tumor cells, or 107 irradiated (15,000 rad)
RMA-S-m tumor cells. For CTL assays, splenocytes from each group of
mice were pooled, and cell cultures were incubated overnight to delete
adherent cells. Responder cells were treated with anti-CD4 (GK1.5)
and complement as previously described (25), and
quadruplicate cultures for each group were then incubated for 7 days
with irradiated RMA tumor cells in RPMI containing 5% FCS before assay
for cytotoxic activity (see below). For Th1 assays, splenocytes from
each group of immunized mice were pooled, and cell cultures (3 x
106/well of 24-well plates) were stimulated with
an equal number of irradiated B6 spleen cells (3,000 rad) and
irradiated RMA (15,000) tumor cells. Seven days later, bulk cultures
(by flow cytometry, 70–80% CD4+) were assessed
for proliferative response and IFN- production as described
below.
Proliferation assays
Proliferation assays were conducted in 96-well U-bottom plates.
The NK cell cultures were incubated for 2 days and pulsed with 0.5
µCi/well [3H]thymidine (Amersham, Little
Chalfont, U.K.) for the last 16 h. The bulk
CD4+ cultures at 1 x
105, 5 x 104, and
104 cells/well were stimulated with irradiated
spleen cells (105/well) with or without a Th
peptide (5 µg/ml) or irradiated RMA tumor cells (5 x
104/well). No exogenous IL-2 was added, and after
3 days, the cultures were pulsed with 0.5 µCi/well
[3H]thymidine (Amersham) for the last 16
h. Incorporation of radioactivity was measured in a Tri-Carb 2100TR
liquid scintillation counter (Packard Instrument, Downers Grove, IL).
Cell-free supernatants from both NK and CD4+
cultures were harvested after incubation for 3 days and subjected to
IFN- ELISA.
ELISA
IFN- levels in the culture supernatants were evaluated using
a mouse IFN--specific ELISA kit (BD PharMingen) according to the
manufacturer’s instructions.
Cytotoxicity assay
Cytotoxic activity of NK cells and CTL cultures was assessed against 51Cr-labeled tumor target cells in a standard 4-h 51Cr release assay (18). RMA-S-m target cells were cultured for 24 h at 25°C and then pulsed with 100 µM peptide for 2–4 h at 33°C. All cells were then labeled with 50 µCi 51Cr for 1 h at 37°C and washed three times. B16F10 target cells were pulsed with 100 µM peptide for 1 h at 37oC before the assay, and peptides remained present during the assay. Effectors and targets were coincubated at various E:T cell ratios for 4 h as previously described (18). Spontaneous release of 51Cr was determined by incubating the target cells with medium alone and was always <15%. Maximum release was determined by adding SDS to a final concentration of 5%. The percent 51Cr release was calculated as follows: 100 x ((experimental release - spontaneous release)/(maximum release - spontaneous release)). Each experiment was performed twice using triplicate samples.
Tumor growth assays
Groups of five untreated (WT, RAG-1-/-,
IFN--/-, pfp-/-, or
Ab-treated WT (see Lymphocyte subset depletion) mice were
injected s.c. with vector alone-infected or CD80-infected tumor cells
(range, 105–107 cells) in
0.2 ml PBS as indicated. On occasion some mice initially received a
nonlethal dose of live parental tumor or irradiated (20,000 rad)
parental tumor cells. Mice were observed every 2 days for tumor growth
using a caliper square measuring along the perpendicular axes of the
tumors (the product of two diameters ± SE). Mice were sacrificed
when tumors reached >12 mm in diameter, and no signs of rejection were
observed. Following the primary rejection of CD80-expressing tumor
cells, the same mice were secondarily challenged (4 or 12 wk from the
first challenge) in the opposite flank with either parental tumor cells
or irrelevant tumor cells as indicated. Tumors were measured as
described in this section. Mice without any signs of tumor
growth were kept under observation for at least 40 days after secondary
tumor inoculation.
Lymphocyte subset depletion
In some experiments in vivo, to deplete T cells and NK cells,
mice were treated with Ab on days -1, 0, and 7 (where day 0 is the day
of primary or secondary tumor inoculation). The following doses of Abs
were used: anti-CD4 (GK1.5), 100 µg i.p.; anti-CD8 (53-6.7),
100 µg i.p.; anti-NK1.1 (PK136), 100 µg i.p.; and rabbit
anti-asGM1, 200 µg i.p. These protocols have previously been
shown to effectively deplete T and NK cell subsets in C57BL/6 mice
(18, 26). Alternatively, some groups of mice were treated
as described above with 200 µg control rabbit Ig or twice weekly with
0.5 mg control IgG or anti-mIFN- mAb.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RMA-S, a MHC class I-deficient variant of RMA, is the prototypic
NK cell-sensitive target for in vitro and in vivo studies (18, 27, 28). To examine the effect of CD80 on NK cell-mediated tumor
rejection, we prepared by retroviral infection, a series of RMA-S cell
lines that expressed increasing levels of CD80. Infectant populations
with high CD80 expression and vector alone-infected controls (-m) were
chosen for further studies (Fig. 1
). The
control and CD80-expressing cell lines had similar growth kinetics in
vitro, as determined by several different assays of
proliferation (data not shown).
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a). Similar
results were obtained at higher (106 cells, Fig. 2b; 107 cells, Fig. 2c)
doses and using anti-NK1.1 mAb to deplete NK cells (data not
shown). In contrast, RMA-S-CD80 tumor cells were rejected, and this
process was mediated mostly by NK cells at the lowest tumor challenge
doses (Fig. 2a). Despite the limited ability of NK cells to
control larger tumor cell numbers, RMA-S-CD80 tumor cell growth was
even inhibited at higher doses of RMA-S-CD80 tumor cells
(107, Fig. 2c), but rejection in this
case required both T cells and NK cells (Fig. 2c). Overall,
there was a >98% reduction in the growth of RMA-S-CD80 tumor in WT
mice compared with RMA-S-m tumor. These data indicated that CD80
expression could enhance both primary NK cell- and/or T cell-mediated
tumor rejection depending upon the cell number inoculated.
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We then demonstrated that CD80 expression on RMA-S cells could
induce the proliferation and IFN- production of NK cells (Fig. 3, a and b).
Increased proliferation (Fig. 3a) and IFN- production
(Fig. 3b) were abrogated by anti-CD80 or the depletion
of NK cells using anti-NK1.1 (data not shown). CD80-stimulated NK
cell proliferation was not inhibited by an anti-IFN- or control
mAb (Fig. 3b). While NK cells from
pfp-/-RAG-1-/- mice
could produce IFN- following interaction with RMA-S-CD80 cells (Fig. 3b), these NK cells were not cytotoxic toward either RMA-S-m
or RMA-S-CD80 target cells (Fig. 3c). Notably, RMA-S-CD80
were considerably more sensitive than RMA-S-m target cells to NK
cell-mediated cytolysis in 4-h cytotoxicity assays in vitro (Fig. 3c). IFN- production, proliferation, and cytotoxicity of
NK cells derived from CD28-deficient mice were not augmented when
cocultured with RMA-S-CD80 tumor cells (data not shown). Collectively,
these data indicated that ligation of CD28 on NK cells by CD80 on tumor
cells induced IFN- production, proliferation, and cytotoxicity
mediated by NK cells. We and others have previously shown that NK
cell-mediated rejection of RMA-S tumor cells is pfp-dependent
(18, 29). The growth of RMA-S-m tumor cells was pfp and,
to a lesser extent, IFN--dependent (Fig. 3d).
Importantly, NK cell-mediated rejection of RMA-S-CD80 in WT mice was
not observed in either pfp-/- or
IFN--/- mice, indicating the critical
requirement of these molecules in CD80-stimulated tumor rejection by NK
cells (Fig. 3d).
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Given primary rejection of RMA-S tumor cells that express CD80, we
next determined whether the mice could resist secondary tumor
challenge. WT mice initially rejecting RMA-S-CD80 were rechallenged in
the opposite flank with RMA (1 x 106, 12 wk
after primary inoculation) and coincidently depleted of NK cells,
CD8+ T cells, or CD4+ T
cells. The growth of RMA cells was similar in all untreated and
lymphocyte-depleted naive WT mice (Fig. 4a). As demonstrated in Fig. 4a (right panel), WT mice previously inoculated
with RMA-S-CD80 cells were able to effectively reject 1 x
106 RMA tumor cells. Similarly, WT mice depleted
of NK cells were also able to effectively reject secondary challenge
with RMA tumor cells, suggesting that NK cells were not playing a key
role in the secondary RMA tumor rejection. Depletion of either
CD8+ or CD4+ T cells
prevented the secondary rejection of RMA tumor cells, although
depletion of CD4+ T cells was not as effective,
suggesting that CD8+ T cells could mount some
response independently of CD4+ T cells (Fig. 4a).
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). All tumors inoculated
s.c. grew avidly in naive B6 mice at the doses chosen. Strikingly, of
the mice that had undergone a primary NK cell-mediated rejection of
RMA-S-CD80, only the group receiving B16F10 (1 x
106 s.c.) developed tumors. By contrast, the
growth of RMA-S-m, RMA, and EL-4 were not detected in these mice. These
data indicated that the initial NK cell-mediated rejection triggered by
tumor CD80 expression had not simply caused prolonged NK cell activity,
but had promoted strong immunity to parental-related tumors that lacked
CD80 expression.
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). The CTL response against
leukemias induced by MuLV of the Moloney (MBL-2), and Rauscher (RMA,
RMA-S) types in B6 mice is predominantly H-2Db
restricted (22, 30). A peptide derived from the gag leader
sequence was identified as the major target epitope (murine leukemia
virus gagL aa 75–83, gagL75–83)
(31). A subdominant H-2Kb-restricted
response to an envelope-derived peptide (murine leukemia virus env aa
189–196, env189–196) (22) and one
potent MuLV env-derived Th epitope (32) have been
described. Although EL-4 is a dimethylbenzanthracene-induced thymoma
(and not MuLV-induced), EL-4 and MBL-2 also share a recently described
nonviral Ag with RMA, and indeed RMA and EL-4 may share a common origin
(23). Therefore, we monitored responses to each of these
Ags in the spleens of mice challenged 6 wk earlier with 5 x
105 live RMA-S-CD80 tumor cells,
107 irradiated RMA-S-CD80 tumor cells, or
107 irradiated RMA-S-m tumor cells. Initially,
CD8+ T cells from these mice were isolated and
stimulated in vitro as bulk cultures with irradiated RMA tumor cells.
The CTL assay suggested that most specificity and activity generated by
RMA-S-CD80 priming in vivo were directed at the
gagL75–83 epitope (Fig. 6, a and
b). Notably, irradiated RMA-S-CD80 tumor cells could also
stimulate the CTL response, albeit to a lesser extent (Fig. 6, c and d), whereas irradiated RMA-S-m tumor cells
did not stimulate detectable CTL activity (Fig. 6, e and
f). The CTL activity against the
gagL75–83 epitope (Fig. 6) was consistent with
the ability of RMA-S-CD80-primed mice to successfully reject RMA, but
not B16F10 tumors that do not express the MuLV Ags (Fig. 5
). The
ability of immunized mice to reject EL-4 and RMA-S tumors (Fig. 5) was
supported by the CTL response to EL-4 or RMA-S alone or to B16F10
pulsed with a common nonviral Ag shared between EL-4 and RMA (Fig. 6).
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). CD4+ T cells
from mice primed with live RMA-S-CD80 tumor cells displayed
proliferative capacity in response to env-H19, but not control Th
peptide OVA-H (Fig. 7a). These cultures also produced high
levels of IFN- (Fig. 7b). Similarly, primary challenge
with irradiated RMA-S-CD80 tumor cells provoked detectable
CD4+ T cell proliferation (Fig. 7c)
and IFN- production (Fig. 7d) in response to env-H19. We
did not detect a Th1 response in the spleens of mice that had been
primed with irradiated RMA-S-m tumor cells (Fig. 7, e and
f). Thus, both specific CTL and Th1 responses to MuLV Ags
were detected in the mice that had previously rejected RMA-S-CD80 tumor
cells.
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In agreement with CTL and Th1 responses, WT mice that had been
initially primed with 107 irradiated RMA-S-CD80
tumor cells were resistant to a secondary RMA tumor challenge with up
to 5 x 105 live tumor cells (Fig. 8a), whereas those primed with
107 irradiated RMA-S-m tumor cells were not
(Fig. 8a). The RMA tumor rejection following irradiated
RMA-S-CD80 tumor cell priming was as avid as that generated by
immunization with live RMA-S-CD80 tumor cells (data not shown), as
expected from the similar CTL and Th1 responses (Figs. 6 and 7). To
determine the contribution of NK cells and IFN- to the priming of
secondary rejection, WT mice were primed with 107
irradiated RMA-S-CD80 tumor cells and coincidently depleted of NK cells
and/or IFN- for 2 wk after tumor inoculation. Five weeks after the
primary tumor inoculation, the mice were challenged with
106 RMA tumor cells. Tumors were only rejected in
untreated WT mice and WT mice receiving control Ig, whereas mice
depleted of CD4+ or CD8+
cells displayed no resistance to tumor challenge. The mice depleted of
NK cells and/or IFN- were unable to completely reject the challenge
with an equivalent dose of RMA tumor cells (Fig. 8b). These
results indicated that NK cells and IFN- were key factors in the
development of adaptive T cell immunity following NK cell-mediated
innate immune responses against tumors.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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secretion,
proliferation, and cytotoxicity. Both pfp and IFN- were critical for
NK cell-mediated rejection of RMA-S-CD80 tumors. RMA-S-CD80 tumor cells
did not augment the IFN- secretion, proliferation, and cytotoxicity
of NK cells from CD28-/- mice, and the growth
of these tumor cells was not inhibited in
CD28-/- mice, suggesting that CD80 mediated its
effect exclusively via CD28 (data not shown). Cooperation existed
between innate and adaptive immune responses against tumors, because NK
cell activation by CD80 on MHC class I- and II-deficient RMA-S tumor
cells was key in priming optimal T cell immunity against subsequent
tumor challenge. Importantly, IFN- produced during the priming phase
was also vital in the creation of secondary immunity to the parental
tumors lacking CD80 expression.
Both CTL and Th1 immunity were induced against MuLV and other epitopes
expressed by the RMA-S-CD80 tumor cells. The priming stimulus helped
establish T cell memory that prevented the growth of parental RMA
tumors at least 6 wk after the primary challenge. Interestingly,
following RMA-S-CD80 vaccination, a previous study reported the
generation of MHC class I-restricted CD8+ CTLs
that required target cell TAP deficiency for efficient recognition
(33). We too detected such CTL following priming with
RMA-S-CD80 tumor cells, because RMA-S tumor cells were rejected upon
secondary challenge (Fig. 5), and CTL reactive with RMA-S were detected
in bulk CD8+ T cell cultures restimulated from
immunized mice (Fig. 6a). In similar experiments with CD70,
a weaker costimulator of NK cells, we did not detect CTL reactive with
RMA-S (34).
NK cells, unlike T cells, are activated in the absence of self-MHC
class I molecules, and they are particularly important in the earliest
stages of immune responses, where they exert cytotoxicity and/or
cytokine production. Several studies have shown that NK cells can
regulate CTL responses, but only a few have suggested that IFN-
produced by NK cells (10, 35) might induce Th1- and
CTL-mediated immunity. Of note, IFN- production by activated NK
cells has also been shown to promote Th1 responses and enhance B
cell-mediated autoimmunity (36). Nevertheless, the idea
that NK cell IFN- promotes T cell immunity has not been widely
accepted. Another NK cell-activating receptor constitutively expressed
by the majority of NK cells, CD27, also primes significant T cell
memory (37). Unlike CD27, which only stimulates NK cell
IFN- production and proliferation (38), CD28 also
triggers NK cell cytolytic function (11, 12). In this
study, RMA-S-CD80 tumor cells were more sensitive to NK cytotoxicity in
vitro, and the potent in vivo rejection of RMA-S-CD80 tumor cells by NK
cells was pfp dependent. However, priming of secondary immunity did not
require live tumor cells, suggesting that ligation of CD28 on NK cells
and IFN- production was sufficient. Recently, it was shown that
STAT-1-deficient mice were incapable of rejecting RMA-S tumor doses
that were rejected in IFN-R-deficient mice (39). These
data suggest that a STAT-1-dependent pathway, independent of IFN-,
may also be critical for effective NK cell-mediated tumor rejection. It
remains to be determined whether this STAT-1 pathway is also required
in enhanced NK cell-mediated rejection primed by ligation of
CD80.
In studies first demonstrating that CD80-expressing tumors were
rejected in an NK cell-mediated fashion, CD80 facilitated rejection
appeared to occur through a cross-presentation mechanism (40, 41). Given that RMA-S alone was not immunogenic in our study, it
is possible that RMA-S-CD80 tumor cells triggered IFN- production
and/or tumor cell death that was sufficient to enhance cross-priming.
In this study, we demonstrated that IFN- aids the development of Th1
and CTL function, and thus the IFN- produced by CD28-stimulated NK
cells may promote local APC activity and/or directly enhance CTL and
Th1 development. Recent studies have indicated that activated NK cells
can lyse target cells and surrounding immature dendritic cells (DCs)
that have phagocytosed and processed foreign Ag, thereby providing
antigenic cellular debris that can be internalized by maturing DCs that
present to T cells in the lymph nodes (42). Costimulatory
molecules such as CD80 are also typically expressed on APC, such as DC,
and recently cell-to-cell contact between DC and resting NK cells was
demonstrated to result in a substantial increase in both NK cell
cytolytic activity and IFN- production (43). Lysis of
autologous DC by NK cells in vitro has been shown to be dependent on
granule exocytosis; however, induction of maturation of DC, including
up-regulation of MHC class I and II molecules and costimulatory
molecules, renders DC less susceptible to lysis by NK cells
(44). Pfp-mediated cytotoxicity can eliminate tumor cells
directly (29, 28, 45), but at the same time it may also
limit the number of immature DC available for maturation by Ags, thus
minimizing CTL activity and potential immunopathology (46, 47). Importantly, receptors mediating DC or target cell
recognition by NK cells (including CD28) do not seem to be involved in
DC-mediated NK cell priming (42). CD28 alone does not
control NK cell-mediated lysis and IFN- production. Therefore,
understanding the balance between various activation and inhibitory
molecules on NK cells and whether they trigger cytotoxicity and/or
IFN- production will be critical to elucidating the role of NK cells
in innate and adaptive responses to tumors and thus in designing better
immunotherapies.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Mark J. Smyth, Cancer Immunology Program, Peter MacCallum Cancer Institute, Locked Bag 1, A’Beckett Street, 8006 Victoria, Australia. E-mail address: m.smyth{at}pmci.unimelb.edu.au
3 Abbreviations used in this paper: pfp, perforin; DC, dendritic cell; env, envelope; gagL, gag leader; GFP, green fluorescence protein; -m, vector alone-infected controls; MuLV, murine leukemia virus; WT, wild type.
Received for publication December 21, 2001. Accepted for publication February 27, 2002.
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J. A. Westwood, J. M. Kelly, J. E. Tanner, M. H. Kershaw, M. J. Smyth, and Y. Hayakawa Cutting Edge: Novel Priming of Tumor-Specific Immunity by NKG2D-Triggered NK Cell-Mediated Tumor Rejection and Th1-Independent CD4+ T Cell Pathway J. Immunol., January 15, 2004; 172(2): 757 - 761. [Abstract] [Full Text] [PDF]
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 S. E. Johansson, H. Hall, J. Bjorklund, and P. Hoglund Broadly impaired NK cell function in non-obese diabetic mice is partially restored by NK cell activation in vivo and by IL-12/IL-18 in vitro Int. Immunol., January 1, 2004; 16(1): 1 - 11. [Abstract] [Full Text] [PDF]
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V. Douin-Echinard, J.-M. Peron, V. Lauwers-Cances, G. Favre, and B. Couderc Involvement of CD70 and CD80 intracytoplasmic domains in the co-stimulatory signal required to provide an antitumor immune response Int. Immunol., March 1, 2003; 15(3): 359 - 372. [Abstract] [Full Text] [PDF]
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Y. Hayakawa, J. M. Kelly, J. A. Westwood, P. K. Darcy, A. Diefenbach, D. Raulet, and M. J. Smyth Cutting Edge: Tumor Rejection Mediated by NKG2D Receptor-Ligand Interaction Is Dependent upon Perforin J. Immunol., November 15, 2002; 169(10): 5377 - 5381. [Abstract] [Full Text] [PDF]
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), CD8+
cells (•), or CD4+ cells (
). Naive mice (naive) were
treated similarly to control mice. Tumor growth was followed every
second day, and data are expressed as the mean ± SE of five mice
in each group.
), 107 irradiated (irr) RMA-S-CD80 tumor cells
(
). b, Coincidently upon priming
with irradiated RMA-S-CD80 tumor cells, some mice were depleted of
CD4+ cells (
, solid lines), CD8+ cells (