J. Exp. Med.
Volume 196, Number 1, July 1, 2002 119–127
The Rockefeller University Press • 0022-1007/2002/07/119/9 $5.00
A Critical Role for Natural Killer
T Cells in Immunosurveillance of
Nadine Y. Crowe,
Mark J. Smyth,
and Dale I. Godfrey
Department of Pathology and Immunology, Monash University Medical School, Melbourne,
Victoria 3181, Australia
Cancer Immunology, Trescowthick Laboratories, Peter MacCallum Cancer Institute, Melbourne,
Victoria 3002, Australia
Natural killer (NK) T cells initiate potent antitumor responses when stimulated by exogenous
factors such as interleukin (IL)-12 or
whether this reflects a physiological role for these cells in tumor immunity. Through adoptive
transfer of NK T cells from wild-type to NK T cell–deficient (T cell receptor [TCR] J
mice, we demonstrate a critical role for NK T cells in immunosurveillance of methylcholan-
threne (MCA)-induced fibrosarcomas, in the absence of exogenous stimulatory factors. Using
the same approach with gene-targeted and/or antibody-depleted donor or recipient mice, we
have shown that this effect depends on CD1d recognition and requires the additional involve-
ment of both NK and CD8
T cells. Interferon-
stream, non-NK T cells, is essential for protection, and perforin production by effector cells,
but not NK T cells, is also critical. The protective mechanisms in this more physiologically rel-
evant system are distinct from those associated with
tumor rejection. This study demonstrates that, in addition to their importance in tumor immu-
notherapy induced by IL-12 or
-GalCer, NK T cells can play a critical role in tumor immuno-
surveillance, at least against MCA-induced sarcomas, in the absence of exogenous stimulation.
-GalCer), however, it is not clear
production by both NK T cells and down-
-GalCer–induced, NK T cell–mediated,
Key words: rodent • NK T cells • NK cells • tumor immunity • methylcholanthrene
NK T cells are a specialized subset of T cells that, in mice,
are commonly characterized by the coexpression of NK1.1,
and a heavily biased
TCR, with the great majority ex-
pressing an invariant V
8.2, 7 or 2 TCR-
chains (for a review, see refer-
ences 1 and 2). This TCR specifically recognizes gly-
colipid-Ag in conjunction with the MHC class I–like mol-
ecule, CD1d (for a review, see references 2 and 3).
Disruption of either the invariant TCR-
results in a selective deficiency of NK T cells, while all
other lymphocyte subsets remain intact (4–7). Upon TCR
stimulation, NK T cells rapidly produce both proinflamma-
tory cytokines, (e.g. IFN-
tory cytokines (e.g. IL-4, IL-10, IL-13), suggesting an im-
chain, or CD1d,
) and antiinflamma-
portant role for these cells in immunoregulation. Several
studies have shown that NK T cells can suppress cell-medi-
ated immunity and prevent self-tissue destruction. For ex-
ample, NK T cells prevent pancreatic islet
tion in NOD mice in an IL-4–/IL-10–dependent manner
(8), mediate systemic suppression associated with anterior
chamber-associated immune deviation via IL-10 produc-
tion (9), induce allograft tolerance (10), and prevent graft-
versus-host disease in mice (11). NK T cells have also been
shown to suppress tumor antigen-specific, CTL-mediated
tumor rejection in an IL-13–dependent manner (12).
In apparent contrast with the above studies, NK T cells
promote potent tumor rejection in response to exogenous
factors such as IL-12 (4, 13, 14) and
-GalCer–induced NK T cell–
dependent antitumor activity is dependent on IFN-
M.J. Smyth and D.I. Godfrey are co-chief investigators.
Address correspondence to Dale Godfrey, Monash University Medical
School, Department of Pathology and Immunology, Commercial Rd.,
Prahran 3181, Victoria, Australia. Phone: 613-9903-0075; Fax: 613-9903-
0018; E-mail: firstname.lastname@example.org
methylcholanthrene; NMS, normal mouse serum; pfp, perforin; RAG,
recombination activation gene; WT, wild-type.
Abbreviations used in this paper:
NK T Cells and Tumor Immunosurveillance
tion, and requires NK cells, (18–20), while the role for
T cells is controversial (19, 20). Despite the clear
ability of NK T cells to initiate potent antitumor responses
in response to exogenous immunotherapeutic stimuli,
whether this represents a physiological role for NK T cells
in tumor rejection remains unclear, with two recent studies
suggesting an opposing role for NK T cells in tumor immu-
nity. Terabe et al. (12) reported that NK T cells were re-
sponsible for incomplete tumor regression by IL-13–medi-
ated inhibition of tumor-specific CTL, suggesting that NK
T cells may normally inhibit tumor immunity, perhaps re-
flecting their immunosuppressive role in autoimmune dis-
ease. In contrast, we previously reported that NK T cell–
deficient (TCR J
281 ) mice were more susceptible to
methylcholanthrene (MCA)-induced fibrosarcoma, suggest-
ing an aggressive role for NK T cells in preventing growth
of these tumors (21). A disadvantage of the latter study was
that it provided little insight into the role of NK T cells and
NK T cell–derived factors in this model. It was not clear if
NK T cells were directly killing the tumor in a perforin
(pfp)-dependent fashion, as suggested by our in vitro studies,
(21) or if they were acting indirectly via the production of
factors which lead to the activation of downstream effectors
such as NK cells as has been demonstrated in
induced systems (18–20). It was also unknown whether the
NK T cell response, in the absence of
pendent on an interaction between TCR and CD1d, which
might suggest recognition of tumor-derived glycolipid anti-
gen. In this study, using adoptive transfer of NK T cells
from wild-type (WT) or various gene-targeted mice, we
demonstrate unequivocally that NK T cells can play a criti-
cal role in immunosurveillance of MCA-induced fibrosar-
comas, and reveal the mechanisms behind this process.
-GalCer, was de-
Materials and Methods
Inbred C57BL/6 WT mice were purchased from The
Walter and Eliza Hall Institute of Medical Research, Melbourne,
Australia, and Clear Japan, Inc. The following gene-targeted mice
were bred at the Peter MacCallum Cancer Institute: C57BL/6-
Chiba University Graduate School of Medicine, Chiba, Japan and
backcrossed to C57BL/6 for nine generations) (4); C57BL/6 pfp-
) (from G. Karupiah, John Curtin School of
Medical Research, Canberra, Australia and derived from C57BL/
6 ES cells) (22); C57BL/6 IFN-
nentech, Inc. and backcrossed to C57BL/6 for 10 generations)
(23); C57BL/6 CD1d deficient (CD1d
Kaer, Vanderbilt University School of Medicine, Nashville, TN
and backcrossed to C57BL/6 for 10 generations) (6); and
C57BL/6 recombination activation gene (RAG)-1 deficient
; from L. Corcoran, The Walter and Eliza Hall Insti-
tute of Medical Research, Melbourne, Australia and backcrossed
to C57BL/6 for 10 generations). Mice of 6–14 wk of age were
used in all experiments that were performed according to animal
experimental ethics committee guidelines.
Isolation of Liver Lymphocyte Subsets.
lated from the liver as described previously (24). To avoid non-
specific binding of antibodies to FcR-
with anti–mouse CD16/32 (2.4G2) mAb (grown in-house) be-
) (provided by M. Taniguchi,
; from Ge-
; provided by L. van
Lymphocytes were iso-
, cells were preincubated
fore staining with FITC-conjugated anti-
597) and PE-conjugated anti-NK1.1 (clone PK-136). All flow
cytometry reagents were purchased from BD PharMingen, unless
otherwise indicated. Cells were gated and sorted as described pre-
viously (24). After washing twice with PBS, the stained cells were
analyzed on a FACStar
™ (Becton Dickinson) and the data
processed by the CELLQuest™ program (Becton Dickinson).
Tumor Models In Vivo.
MCA-1, -3, -4 and CD1.1 were de-
rived from J
281 and CD1d
g MCA. B16F10 mouse melanoma and MCA-induced
sarcoma cell lines were maintained as described previously
(20, 21). For growth of MCA-induced sarcoma lines, groups of
WT and gene targeted and/or Ab-depleted mice were injected
subcutaneously (right hind leg) with 10
same day, some mice received liver lymphocytes from WT or
gene-targeted mice or FACS
-purified liver lymphocyte popula-
tions from WT mice, or 2% normal mouse serum (NMS) in PBS
(2% NMS.PBS) via intravenous adoptive transfer. In the delayed
transfer experiments, some mice received liver lymphocytes 7 d
after tumor inoculation. All mice were observed every other day
and tumor growth was measured with a caliper square as the
product of two diameters. Results were recorded as the mean tu-
mor size (cm
SEM. Significant difference in the number of
mice which remained susceptible to tumor development com-
pared with PBS-treated J
Fisher’s exact test (
in tumor growth rate, compared with PBS-treated control
groups, was determined using a Mann-Whitney U test (
In the B16F10 subcutaneous growth model, groups of WT mice
were injected subcutaneously (hind leg) with 10
and varying doses of B16F10 were injected subcutaneously
within the same region at a distinct site. All mice were observed
every other day and B16F10 subcutaneous tumor growth was
measured with a caliper square as the product of two diameters.
Results were recorded as the mean tumor size (cm
AsialoGM1 and CD8 Depletion.
depleted in J
281 mice after intraperitoneal injection of 20
g rabbit antiasialoGM1 Ab (Wako Chemicals) on days
and 7 (day 0 being day of tumor inoculation) as described previ-
ously (25). CD8
cells were specifically depleted using anti-
CD8-depleting Ab (clone 53–6.7) (grown in-house) using the
TCR (clone H57–
mice, respectively, injected
sarcoma cells. On the
groups was determined using a
0.01). Significant difference
NK cells were specifically
NK T Cell–deficient Mice Are Susceptible To Sarcoma
We have previously shown that NK T cell–defi-
cient, TCR J
281 , mice (Fig. 1 A) are more susceptible
to MCA-induced fibrosarcomas and a fibrosarcoma line
(MCA-1) when compared with WT mice, suggesting an
important role for NK T cells in preventing the growth of
these tumors (21). This phenotype can be demonstrated us-
ing several distinct fibrosarcoma lines (Fig. 1 B), including
some that were generated from MCA-treated CD1d
mice. These data are consistent with previous observations
that sarcomas derived from RAG-2
entially in RAG-2
mice over syngenic WT mice (26).
Adoptive Transfer of NK T Cells into J
stores Protection Against MCA-1 Sarcoma.
vious difference between WT and J
presence of CD1d-restricted NK T cells (Fig. 1 A) (4, 27,
mice grow prefer-
As the most ob-
281 mice is the
Crowe et al.
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