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.
28), we sought to restore protection in J
treated with MCA-1 sarcoma by adoptive transfer of NK T
cells. In initial experiments, total liver lymphocytes were
transferred, since NK T cells are highly abundant within
this population of cells, and we could avoid using mAb to
engage cell surface molecules for purification. Over 80% of
281 mice treated with WT liver
lymphocytes were able to completely inhibit the growth of
MCA-1 (Fig. 2 A), while the rest had significantly smaller
tumors (approximately one-fifth the size, measured in cm
0.01) than PBS-injected controls (unpublished data).
This was similar to the tumor incidence observed in WT
mice, the majority of which remained tumor free. An addi-
tional control group of MCA-1–injected J
were treated with J
which have all populations except NK T cells. These mice
remained susceptible to tumor growth and were indistin-
-derived liver lymphocytes,
guishable from PBS-injected controls. Protection was also
achieved when transferring WT liver lymphocytes into
TCRJ?281?/? mice that had been inoculated with either
MCA-3 or MCA-4 tumor cells (unpublished data). This
data strongly suggested that inhibition of growth was due
to the transfer of CD1d-restricted NK T cells present
among the WT liver lymphocytes.
Having established that liver lymphocytes, probably NK
T cells, could protect in this system in the absence of cell
surface molecule engagement by mAb, we sought to for-
mally define which liver lymphocyte subset was respons-
ible for protection. NK1.1? ?? TCR? (NK T cells),
NK1.1??? TCR? (T cells), and NK1.1??? TCR? (NK
cells) were purified by FACS® and adoptively transferred
intravenously into MCA-1–injected J?281?/? mice (Fig. 2
B). Mice that received NK T cells (2.5 ? 105) were able to
inhibit the growth of MCA-1, while those receiving the
from WT and J?281?/? mice and labeled with CD1/?-GalCer tetramer and mAb specific for ?? TCR to identify NK T cells. (B) Groups of WT and
J?281?/? mice were injected subcutaneously (hind flank) with 105 MCA-1, -3, -4 or CD1.1 sarcoma cell lines. Results were recorded every other day as
the mean tumor size (in cm2) ? SEM. Three mice per group were used. Significant difference from the PBS-treated J?281?/? group was determined us-
ing a Mann-Whitney U test when n ? 4. *P ? 0.05.
NK T cell–deficient (J?281?/?) mice are more susceptible to growth of MCA-induced sarcoma lines. (A) Liver lymphocytes were isolated
cells protects J?281?/? mice against
MCA-1 tumor growth. Groups of WT
and J?281?/? mice were inoculated sub-
cutaneously (hind flank) with 105 MCA-1
cells. (A) Subgroups of J?281?/? mice
then received between 1 and 3 ? 106
WT or 3 ? 106 J?281?/? liver lympho-
cytes, 200 ?L 2% NMS.PBS, or (B) 2.5 ?
105 purified NK1.1??? TCR? (NK T),
NK1.1??? TCR? (NK), NK1.1? ??
TCR? (T) cells, or 200 ?L 2%
NMS.PBS, via intravenous adoptive
Adoptive transfer of NK T
transfer. NK T cells typically constituted between 30–35% of WT liver lymphocytes, and sorted populations were at least 97, 93, and 94% pure, respec-
tively. Results represent pooled data from 10 (A) or 4 (B) independent experiments (with exception of J?281?/? liver lymphocytes [A] and purified NK
cell [B] transfers from one experiment only, and purified T cell transfers [B] from two experiments). Between three and six mice per group per experi-
ment were used. Total number of mice in each group that developed tumors is shown in parentheses. Significant difference from the PBS-treated
J?281?/? groups was determined using a Fisher’s exact test. *P ? 0.05; **P ? 0.01.
NK T Cells and Tumor Immunosurveillance
same number of T cells or NK cells could not, specifically
defining NK T cells as the population responsible for re-
Dose Response for NK T Cell–mediated Protection Against
To determine the threshold number of NK T
cells required to restore protection, several liver lympho-
cyte doses (3 ? 106, 106, 0.33 ? 106, and 0.11 ? 106) (Fig.
3 A), and purified NK T cell doses (2.5 ? 105, 105, and
5 ? 104) (Fig. 3 B) were tested. Inhibition of tumor growth
was clearly dose–dependent, with 106 liver lymphocytes
(?3.0 ? 105 NK T cells) and 2.5 ? 105 purified NK T
cells required for optimal prevention of tumor growth. As
the total number of NK T cells in the protective dose of
liver lymphocytes was very similar to that of purified NK T
cells, it was unlikely that mAb-binding altered the func-
tional status of transferred NK T cells. Although lower
doses of NK T cells failed to completely prevent tumor
growth, partial protection was observed with smaller and
slower growing tumors corresponding to the number of
NK T cells injected.
Factors that Are Critical for NK T Cell–mediated Protec-
Having established that transfer of NK T cells effec-
tively protected J?281?/? mice from tumor growth, we
sought to determine which NK T cell molecules were me-
diating this protection. When liver lymphocytes from IFN-
??/? mice were used as a source of NK T cells, they were
unable to prevent tumor growth (Fig. 4 A), despite the
ability of endogenous cells in these J?281?/? recipients to
produce IFN-?, indicating an essential role for NK T cell–
derived IFN-?. In contrast, pfp?/? liver lymphocytes re-
tained the ability to inhibit tumor growth, indicating that
direct, pfp-dependent, tumor lysis by NK T cells was not
an essential mechanism in this model.
As it was unlikely that NK T cells were acting alone, we
further investigated what other cells and/or molecules were
important downstream of, or in conjunction with, NK T
cell activation. Purified WT NK T cells were transferred
into various gene-targeted and/or mAb-depleted mice
(Fig. 4 B i–vii). Similar to J?281?/? mice (Fig. 4 B i),
CD1d?/? mice were susceptible to MCA-1 growth (Fig. 4
B ii). However, in contrast to J?281?/? recipients, NK T
cell transfer failed to prevent tumor growth in CD1d?/?
mice (Fig. 4 B ii), indicating that CD1d expression by cells
other than the tumor, such as APCs, was critical for NK T
cell–mediated immunosurveillance. CD1d expression by
the tumor itself was not essential; MCA-1 is close to, if not
completely, negative for CD1d expression (unpublished
data) and, as shown above, a CD1d?/? tumor line grew in
J?281?/? but not WT mice (Fig. 1 B).
IFN-??/? recipients were also susceptible to MCA-1
growth, and transfer of (IFN-? sufficient) WT NK T cells
did not restore complete protection (Fig. 4 B ii), although
growth was slower when compared with PBS-injected
IFN-??/? mice. It is possible that transferred WT NK T
cells may provide sufficient IFN-? to confer initial protec-
tion, however, IFN-? production by downstream non-NK
T cells is clearly critical for effective eradication of the tu-
mor. Pfp-deficient recipients were susceptible to MCA-1
growth (Fig. 4 B iv), and WT NK T cells were unable to
protect in these mice, although again, the tumor grew at a
slower rate when these cells were transferred. This indi-
cated that, although NK T cell–derived pfp was not critical
(Fig. 4 A), pfp-expressing NK T cells may be partially ef-
fective at controlling this tumor. However, pfp production
by other cells, possibly NK or T cells, is clearly important
for efficient NK T cell–mediated tumor immunosurveil-
lance (Fig. 4 B iv). It was also noteworthy that both PBS-
treated IFN-??/? and pfp?/? mice had smaller and slower
growing tumors when compared with PBS-injected
J?281?/? mice (Fig. 4 B i, iii, and iv; P ? 0.01 and P ?
0.05, respectively). This suggests that NK T cells present
within IFN-??/? and pfp?/? mice may still be able to use
other, IFN-?/pfp-independent, pathways/mechanisms to
protect against MCA-1 tumor growth, although clearly,
NK T cell–derived and non-NK T cell–derived IFN-?,
and pfp-expression by downstream effector cells appear to
be critical in providing optimal protection against this tu-
mor. Transfer of NK T cells into MCA-1–injected, NK or
CD8-depleted J?281?/? mice, or RAG-1?/? mice, re-
vealed that NK, CD8?, and T cells are important for a
clear protective response induced by NK T cells (Fig. 4 B
v–vii). Tumor growth in PBS-injected, NK, or CD8-
depleted J?281?/?, or RAG-1?/? mice (Fig. 4 B v–vii)
was not significantly different from that of PBS-injected
MCA-1. Groups of WT and J?281?/? mice were injected subcutane-
ously (hind flank) with 105 MCA-1 cells. (A) Subgroups of J?281?/?
mice then received either 3 ? 106, 106, 0.33 ? 106, 0.11 ? 106 liver lym-
phocytes or 200 ?L 2% NMS.PBS or (B) 2.5 ? 105, 105, 5 ? 104 purified
NK1.1??? TCR? (NK T) cells or 200 ?L 2% NMS.PBS, via intrave-
nous adoptive transfer. NK T cells constituted 31% of the liver lympho-
cyte population and sorted NK T cells were enriched to 98%. Results
were recorded as the mean tumor size (cm2) ? SEM. Between three and
five mice per group were used. Significant difference from the PBS-
treated J?281?/? groups was determined using a Mann-Whitney U test
when n ? 4. *P ? 0.05.
Dose response for NK T cell–mediated protection against
Crowe et al.
NK T Cells Are Required Early in the Response Against
To investigate whether NK T cells could also
protect against established tumors, J?281?/? mice were
treated with WT liver lymphocytes 7 d after MCA-1 injec-
tion (Fig. 5). Delayed transfer of WT cells had no effect on
tumor growth, indicating that NK T cells are required ei-
ther earlier in the response, or in significantly greater num-
bers for effective inhibition of established tumors.
MCA-1 Does Not Induce ?-GalCer–like Activation of NK
We have previously shown that ?-GalCer can
activate NK T cells that have been adoptively transferred
into B16F10-inoculated NK T cell–deficient mice, to ini-
tiate potent tumor rejection (20). To investigate whether
MCA-1 tumor activates NK T cells in a manner similar to
?-GalCer, possibly by providing CD1d-binding glycolipid
antigens, WT mice were simultaneously injected with
B16F10 and MCA-1. Mice were injected subcutaneously
on the hind flank with varying doses of B16F10, and some
mice were also injected on the same flank, in an adjacent
region, with MCA-1 (Fig. 6). As expected, MCA-1
growth was inhibited, presumably due to the presence of
NK T cells, however, this response had no effect on the
adjacent growth of B16F10 tumor. Similar results were
achieved when B16F10 was injected intravenously into
WT mice, some of which were also injected subcutaneous
with MCA-1 (unpublished data). Since B16F10 growth
and metastasis is profoundly inhibited after ?-GalCer treat-
ment of J?281?/? mice (20) that have been injected with
WT NK T cells, these data suggest that MCA-1 sarcoma
does not activate NK T cells to act in a manner similar to
that following ?-GalCer stimulation.
ated protection. (A) WT or J?281?/? mice were injected
subcutaneously (hind flank) with 105 MCA-1 cells. Sub-
groups of J?281?/? mice were simultaneously treated with
either PBS, or 3 ? 105 NKT cells, as part of a liver lym-
phocyte population, from either WT, IFN-??/?, or pfp?/?
donor mice via intravenous adoptive transfer. Significant
difference from the PBS-treated J?281?/? group was de-
termined by a Fisher’s exact test. *P ? 0.05; **P ? 0.01.
(B) Groups of (i) J?281?/?, (ii) CD1d?/?, (iii) IFN-??/?,
(iv) pfp?/?, (v) J?281?/? mice treated with depleting anti-
asGM1 Ab, (vi) J?281?/? mice treated with depleting
anti-CD8, and (vii) RAG-1?/? mice, were injected subcu-
taneously (hind flank) with 105 MCA-1 cells. One-half of
each group of mice were simultaneously injected with 2.5 ?
105 sorted WT NK T cells via intravenous adoptive
transfer, while the other half were injected intravenously
with 2% NMS.PBS. Sorted NK T cells were always at least
97% pure as determined by FACS®. (B) i, ii, and iii repre-
sent pooled data from two independent experiments, iv
represents data from one of these experiments, v, vi, and
vii represent data from one independent experiment with
three to six mice per group per experiment. Significant
difference from the PBS-treated control group of each ex-
periment was determined using a Mann-Whitney U test.
*P ? 0.05; **P ? 0.01.
Factors that are critical for NK T cell–medi-
Groups of WT and J?281?/? mice were inoculated subcutaneously (hind
flank) with 105 MCA-1 cells (d0, ?, ?, ?). 7 d later, some J?281?/?
mice were injected with 106 WT liver lymphocytes via intravenous adop-
tive transfer (?). As a control to show the WT liver lymphocytes (NK T
cells) were protective, other J?281?/? mice were inoculated with MCA-1
on the same day as lymphocyte transfer (?). NK T cells constituted 27%
of the whole liver lymphocyte population as determined by FACS®. Re-
sults were recorded as the mean tumor size (in cm2) ? SEM. Four mice
per group were used.
NK T cells are required early in the response against MCA-1.
NK T Cells and Tumor Immunosurveillance
It is well established that NK T cells can be induced by
either IL-12 or ?-GalCer to mediate potent antitumor ef-
fects both in vitro and in vivo (4, 15, 16), however, this
does not necessarily indicate that NK T cells play a natural/
physiological role in tumor recognition and immunity. We
recently published that Ja281?/? mice are more susceptible
to development of MCA-induced fibrosarcomas when
compared with WT mice, suggesting that NK T cells
might be important in the prevention of tumor growth in
this model (21). Herein, using adoptive transfer techniques
and gene-targeted mice, we have clearly demonstrated a
critical role for NKT cells in immunosurveillance against
MCA-induced fibrosarcomas, and have also identified the
key cells and molecules that support this process.
Although NK T cell activation in this system does not
require treatment with an exogenous CD1d-binding ligand
such as ?-GalCer, CD1d expression by recipient cells, pos-
sibly APCs, was nonetheless found to be important, indi-
cating that NK T cells were responding to these tumors in
a TCR-dependent fashion. What remains to be deter-
mined is whether NK T cell activation is caused by the
presentation of tumor-derived Ag in the context of CD1d,
or rather, whether the presence of CD1d is simply required
in addition to other, CD1d-independent, tumor-induced
‘danger signals.’ It is conceivable that NK T cells were be-
ing activated by a CD1d-binding endogenous ligand, mim-
icking the effects of exogenously added ?-GalCer, how-
ever, the downstream effector functions after NK T cell
activation by MCA-1 were clearly distinct from those fol-
lowing NK T stimulation by ?-GalCer. In contrast to
?-GalCer–induced tumor rejection (18, 20), NK T cell–
mediated tumor immunosurveillance in this model was pfp
dependent, involved both NK and CD8? T cells, and most
importantly, did not mimic the effect of ?-GalCer in its
ability to invoke NK T cell–mediated B16F10 rejection.
We previously published that NK T cells can kill MCA-1
in vitro in a pfp-dependent manner when induced by IL-2
and IL-12 (21). This suggested that the critical contribu-
tion of NK T cells in vivo was direct tumor cell lysis.
However, transferred pfp?/? NK T cells retained their
ability to prevent MCA-1 growth, indicating NK T cell–
derived pfp was clearly not critical for protection. WT NK
T cells transferred into pfp?/? mice slowed the growth of
MCA-1, indicating that, although not essential, NK T cell–
derived pfp may make a minor contribution to MCA-1 re-
jection. On the other hand, pfp-expression by recipient
cells was critical, supporting the idea that NK T cell–medi-
ated tumor immunosurveillance relies on downstream pfp-
expressing effector cells. These may include NK and CD8?
T cells, both of which were required for optimal preven-
tion of sarcoma growth.
NK cells are known to be activated downstream of NK
T cells after ?-GalCer stimulation (29, 30). This is clearly
necessary, although apparently not sufficient, for NK T
cell–mediated immunosurveillance in this sarcoma model.
These findings are in concert with our previous study
showing that NK depletion by anti-asGM1 abrogates pro-
tection against primary MCA-induced sarcomas in WT
mice (25). The involvement of both NK and CD1d-depen-
dent NK T cells has also been shown to be important in
the cyclophosphamide/IL-12–induced regression of a large
MCA-induced sarcoma (31). In contrast the role for CD8?
T cells in ?-GalCer–mediated tumor rejection is contro-
versial (19, 20), although we have now shown that they
play a critical role in immunosurveillance of MCA-induced
tumor cell lines. Although the susceptibility of CD8-
depleted mice to MCA-1 supports previous findings in-
volving other MCA-induced sarcoma lines (32), our earlier
findings did not reveal a role for CD8? cells in NK T cell–
mediated protection against primary MCA-induction of
sarcomas (21). At least two possibilities may explain this.
The role of CD8? cells may differ in primary responses to
MCA-induced sarcomas when compared with transplanted
sarcoma lines; or, as the current experiments involve re-
population of NK T cell–deficient mice with relatively low
numbers of NK T cells, it may be possible that in the ab-
sence of normal NK T cell numbers, the role for other cells
such as CD8? T cells increases.
IFN-? is a critical factor in ?-GalCer–mediated tumor
rejection (18–20). This was also the case for the natural tu-
mor surveillance model described herein. Importantly, sim-
ilar to the ?-GalCer model (20), both NK T cell–derived
and non-NK T cell–derived IFN-? was required, suggest-
ing it may function at multiple stages in this process. This
cytokine appears to be important for the activation of the
downstream effector cells (29, 30, 33), which would ex-
plain why it must be initially produced by NK T cells. Ad-
ditionally, IFN-? production by recipient effector cells
might act to amplify the immune response, or may have
antitumor effects of its own (34–36).
The in vivo migratory and homing behavior of NK T
cells is very poorly understood, as is the impact that a tu-
mor may have on this process. Using immunohistology,
we have failed to detect a significant population of NK T
cells at the tumor site in WT mice (unpublished data), nor
have we been able to detect these cells, at the doses used
for protection, after adoptive transfer into tumor-treated,
and nontumor-treated, J?281?/? mice (unpublished data).
cells. Groups of WT mice were injected subcutaneously (hind flank) with
either 5 ? 106, 106, or 5 ? 105 B16F10 cells. Some mice were then in-
jected on the same hind flank with 105 MCA-1 cells. Results were re-
corded as the mean tumor size (in cm2) ? SEM with five mice per group.
MCA-1 does not induce ?-GalCer–like activation of NK T
Crowe et al.
This is probably due to the fact that we are transferring
very low numbers of NK T cells, combined with the possi-
bility that they die shortly after being activated in response
to the tumor, as has been reported after ?-GalCer stimula-
tion (28, 30, 37, 38). The model we favor is that NK T
cells are rapidly stimulated by the presence of the tumor
cell glycolipids, leading to potent NK T cell–derived IFN-?
production and early activation of effector cells, including
NK and CD8? T cells. These effector cells then directly
attack the tumor through direct pfp-dependent lysis and
IFN-? secretion. The early recognition of the tumor ap-
pears to be key, as delayed transfer of NK T cells provided
no detectable protection.
A role for NK T cells in tumor rejection is usually only
observed after stimulation with exogenous therapeutic fac-
tors such as IL-12 and ?-GalCer. In the absence of these
factors, some tumors such as B16F10 melanoma and 3LL
lung carcinoma grow readily, despite the presence of NK T
cells in these mice. Nonetheless, these earlier studies hinted
that with the right environment, NK T cells had the po-
tential to mediate tumor immunosurveillance. An obvious
question relates to why NK T cells require exogenous
stimulation to respond to some tumors, such as B16F10,
but not for others, such as the sarcomas used in this study.
This may be related to the type of CD1d-binding gly-
colipid Ag expressed by these tumors, which may be spe-
cific for sarcomas, MCA-induced tumors, or other as yet
unidentified molecules, that might be differentially ex-
pressed by various tumors, such as activating ligands (e.g.
NKG2D ligands; reference 39). Another possibility is that
resistance of some tumors to NK T cells may relate to the
length of time the tumor has been cultured in vitro. In sup-
port of this, we found that the MCA-1 line became more
resistant to NK T cell–mediated tumor immunosurveil-
lance with time in culture (unpublished data).
Clearly, the influence of NK T cells in tumor immunity
may vary ranging from suppression (12) to promotion of
tumor rejection (this study). Therefore, it will not only be
important to determine the signals that activate NK T cells
in response to various tumors, but also the signals that regu-
late the outcome of the NK T cell function (suppressive or
aggressive; reference 40). These differential signals may be
derived from different APCs (DC versus B cells, macro-
phages) or tumor cells themselves, in conjunction with dis-
tinct microenvironmental factors. These factors may
include cytokines such as IL-12 (14, 41, 42) and IL-7 (43–
45), which are thought to promote proinflammatory (via
increased IFN-? production), and antiinflammatory (via in-
creased IL-4 and IL-13 production) responses, respectively.
An alternative possibility was that the tumor rejection
observed in this model was due to minor transplantation
antigens, expressed by the MCA-induced sarcoma lines,
that may differ between the J?281?/? donor mice from
which they were originally derived, and the C57BL/6 re-
cipients. We believe this to be highly unlikely for the fol-
lowing reasons. (a) The J?281?/? mice (originally 129
strain) were backcrossed to C57BL/6 mice nine times be-
fore the induction of the sarcomas. Furthermore, we found
a similar pattern of susceptibility in MCA-1–treated
CD1d?/? mice, which had also backcrossed to C57BL/6
mice 10 times. Given the number of times each separate
line had been backcrossed to C57BL/6 mice, CD1d?/? re-
cipient mice would be unlikely to carry (and therefore be
tolerant to) the same minor transplantation antigens that
would be present on the J?281?/? mouse-derived MCA-1
sarcoma line. Therefore, if the response was directed
against minor transplantation antigens, we would have pre-
dicted that MCA-1 would have been rejected in CD1d?/?
mice. (b) MCA-1, MCA-3, and MCA-4 tumor lines were
also injected into (129 ? C57BL/6)F1 recipient mice,
which would express any of the potential minor transplan-
tation antigens that may be expressed by the tumors.
Whereas all these tumors grew as expected in J?281?/? re-
cipients, they were rejected in F1 mice in an identical fash-
ion to that in C57BL/6 mice (unpublished data). (c) Adop-
tive transfer of purified, C57BL/6-derived, NK T cells was
sufficient to mediate tumor rejection, whereas C57BL/6-
derived, conventional NK1.1? T cells had no effect on tu-
mor growth (Fig. 2 B). (d) The difference in tumor growth
between C57BL/6 and J?281?/? recipient mice was al-
ready apparent by day 3, which would represent a remark-
ably rapid response against a minor transplantation antigen.
Taken together, these points indicate that this model in-
volves a CD1d-dependent, NK T cell–mediated antitumor
response, rather than a more conventional immune re-
sponse against minor transplantation antigens.
In summary, this study clearly demonstrates a critical role
for NK T cells in immunosurveillance of MCA-induced fi-
brosarcomas, in the absence of exogenous NK T cell stim-
ulation. This is most likely mediated by their acting as an
early warning system that initiates an antitumor response
subsequently performed by dedicated effectors such as NK
cells and/or CTLs. From a clinical viewpoint, this study
suggests that maintenance and/or restoration of normal
numbers of NK T cells, particularly after immune-deplet-
ing conditions, is an important consideration for the pre-
vention of tumor growth and metastasis. Future research
should now be aimed at determining whether this role for
NK T cells in immunosurveillance extends to other tumor
types, as well as gaining a more thorough understanding of
the exact molecular mechanisms required for activation and
regulation of NK T cell function in the absence of exoge-
nous factors. In particular it will be important to attempt to
isolate tumor-derived CD1d-binding ligands that may be
key to this response.
The authors wish to thank Dr. Alan Baxter for critically reviewing
the manuscript, Prof. Hideo Yagita for helpful discussions, Prof.
Mitchell Kronenberg for provision of CD1d/?-GalCer tetramers,
Prof. Masaru Tanigachi for provision of J?281?/? mice, Prof. Luc
Vankaer for provision of CD1d?/? mice, Dr. Phillip Darcy for assis-
tance with tumor measurements, Daniel Pellicci and Konstantinos
Kyparissoudis for technical assistance, Dr. Elise Randle-Barrett and
Gerard Tarulli for operating the Flow Cytometric Cell Sorter, Chris-
tine Hall and the staff at Peter MacCallum Cancer Institute for their
maintenance and care of the mice used in these studies, and Shayna
Street for providing the J?281?/?-derived MCA-induced sarcomas.
NK T Cells and Tumor Immunosurveillance
N.Y. Crowe is supported by an Australian Postgraduate Re-
search Award. M.J. Smyth and D.I. Godfrey are supported by
National Health and Medical Research Council of Australia
(NHMRC) Research Fellowships and D.I. Godfrey was supported
by an ADCORP/Diabetes Australia Fellowship. The project was
supported by the Human Frontier Science Program and donations
from Rothschild Australia.
Submitted: 17 January 2002
Revised: 18 March 2002
Accepted: 24 April 2002
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