The Journal of Immunology
Role of gd T Cells in a-Galactosylceramide–Mediated
Christophe Paget,*,1Melvyn T. Chow,*,†,‡,1Helene Duret,* Stephen R. Mattarollo,*,xand
Mark J. Smyth*,†,‡
Attempts to harness mouse type I NKT cells in different therapeutic settings including cancer, infection, and autoimmunity have
proven fruitful using the CD1d-binding glycolipid a-galactosylceramide (a-GalCer). In these different models, the effects of
a-GalCer mainly relied on the establishment of a type I NKT cell-dependent immune cascade involving dendritic cell, NK cell,
B cell, or conventional CD4+and CD8+T cell activation/regulation as well as immunomodulatory cytokine production. In this
study, we showed that gd T cells, another population of innate-like T lymphocytes, displayed a phenotype of activated cells
(cytokine production and cytotoxic properties) and were required to achieve an optimal a-GalCer–induced immune response.
Using gene-targeted mice and recombinant cytokines, a critical need for IL-12 and IL-18 has been shown in the a-GalCer–induced
IFN-g production by gd T cells. Moreover, this cytokine production occurred downstream of type I NKT cell response, suggesting
their bystander effect on gd T cells. In line with this, gd T cells failed to directly recognize the CD1d/a-GalCer complex. We also
provided evidence that gd T cells increase their cytotoxic properties after a-GalCer injection, resulting in an increase in killing of
tumor cell targets. Moreover, using cancer models, we demonstrated that gd T cells were required for an optimal a-GalCer–
mediated anti-tumor activity. Finally, we reported that immunization of wild-type mice with a-GalCer enhanced the adaptive
immune response elicited by OVA, and this effect was strongly mediated by gd T cells. We conclude that gd T cells amplify the
innate and acquired response to a-GalCer, with possibly important outcomes for the therapeutic effects of this compound.
Journal of Immunology, 2012, 188: 3928–3939.
cally activate type I NKT cells through TCR ligation (2). Type I
NKT cells (hereafter referred to as NKT cells) are T lymphocytes
carrying a semi-invariant TCR composed by the canonical Va14-
Ja18 TCRa-chain (Va24-Ja18 in humans) combined with a lim-
ited array of TCRb-chains (Vb8, Vb7, or Vb2 in mice, Vb11 in
humans). In response to a-GalCer, NKT cells rapidly and vigor-
ously produce Th1, Th2, and Th17 cytokines (reviewed in Refs.
3–5), which in turn amplify or regulate innate/adaptive immune
responses by inducing the maturation of dendritic cells (DC) (6, 7)
and by influencing the functions of NK cells (8), macrophages (9),
-Galactosylceramide (a-GalCer) is a marine sponge-
derived glycolipid Ag (1) that binds CD1d, a MHC
class I-like molecule, expressed by APCs to specifi-
conventional CD4+and CD8+T lymphocytes (10, 11), and
B lymphocytes (12). Consequently, a-GalCer exerts a potent ad-
juvant activity in vivo, rendering it a powerful candidate for
clinical therapies (reviewed in Refs. 3, 4, 13, 14). Indeed, a large
number of studies in mice confirmed the beneficial effect of
a-GalCer in various mouse experimental models, predominantly
in prophylactic settings including infectious diseases, autoimmu-
nity, allergic reactions, and cancer. For example, the protective
anti-tumor effect of a-GalCer has been shown against melanomas,
carcinomas, hematopoietic malignancies, and their metastases.
This effect is mainly due to the synthesis of IFN-g by NKT cells
and to the bystander activation of effector cells, including NK and
Similar to NKT cells, gd T cells are unconventional
T lymphocytes with innate-like cell hallmarks (reviewed in Refs.
19, 20). Their preactivated phenotype allows them to be one of the
earliest responders during stress/inflammation, and so they can
rapidly produce large amounts of cytokines to regulate immune
responses (21). Cross-talk between NKT and gd T cells has been
recently reported. For example, activation/accumulation of gd
T cells during TLR3 agonist-induced liver inflammation can be
resolved by NKT cells (22). Conversely, IL-17–producing (Vg4+)
gd T cells have been shown to negatively regulate NKT cell
activation in a model of acute hepatitis (23). Finally, airway
hyperresponsiveness can be enhanced through a synergistic ac-
tivity of NKT and Vg1+gd T cells (24). Thus, gd T cells are able
to either positively or negatively regulate NKT cell response and
vice versa according to the tissue studied and the subset of gd
T cells activated. However, surprisingly, no studies have yet in-
vestigated the potential contribution of gd T cells in the immune
responses triggered by a-GalCer. Additionally, in recent studies it
was shown that gd T cells directly responded to cytokines without
any TCR engagement (25–28), suggesting that the cytokine cas-
*Cancer Immunology Program, Peter MacCallum Cancer Centre, East Melbourne,
Victoria 3002, Australia;†Sir Peter MacCallum Department of Oncology, University
of Melbourne, Parkville, Victoria 3010, Australia;‡Department of Pathology, Uni-
versity of Melbourne, Parkville, Victoria 3010, Australia; andxUniversity of Queens-
land Diamantina Institute, Princess Alexandra Hospital, Woolloongabba, Queensland
1C.P. and M.T.C. contributed equally to this work.
Received for publication December 14, 2011. Accepted for publication February 13,
Address correspondence and reprint requests to Prof. Mark J. Smyth, Cancer Im-
munology Program, Peter MacCallum Cancer Centre, East Melbourne, VIC 3002,
Australia. E-mail address: email@example.com
This work was supported by a National Health and Medical Research Council of
Australia Australia Fellowship and Program Grant 454569 (to M.J.S.). C.P. was
supported by a postdoctoral fellowship from the U.S. Department of Defense (Grant
10752705). M.T.C. was supported by a Cancer Research Institute Ph.D. scholarship.
S.R.M. was supported by a Balzan Foundation fellowship.
The online version of this article contains supplemental material.
Abbreviations used in this article: asGM1, asialo GM1; CBA, cytometric bead array;
DC, dendritic cell; FasL, Fas ligand; a-GalCer, a-galactosylceramide; WT, wild-
cade elicited by a-GalCer could be strong enough to lead to gd
T cell activation. Moreover, a-GalCer treatment has been recently
shown to alleviate murine listeriosis, an effect partially lost after
gd T cell depletion, suggesting a potential role of this population
to achieve an optimal therapeutic effect of this lipid (29). Despite
this finding, little is known about the precise functions of gd
T cells as well as the mechanisms leading to their activation in the
context of a-GalCer. Our report demonstrates the novel finding
that gd T cells produce regulatory cytokines in a-GalCer–medi-
ated immune responses that in turn amplify innate and acquired
responses to this lipid.
Materials and Methods
C57BL/6J wild-type mice were purchased from the Walter and Eliza Hall
Institute of Medical Research. C57BL/6 TCRd cell-deficient (TCRd2/2)
mice, C57BL/6 Ja18-deficient (Ja182/2) mice (30), C57BL/6 IL-12p35–
deficient (IL-12p352/2) mice, and C57BL/6 IL-18–deficient (IL-182/2)
mice (31) were bred in house at the Peter MacCallum Cancer Centre. All
mice were backcrossed to C57BL/6J at least 10 times. Mice were used at
the ages of 8–10 wk. All experiments were performed in accordance with
the animal ethics guidelines ascribed by the National Health and Medical
Research Council of Australia. All experiments were approved by the Peter
MacCallum Cancer Centre Animal Ethics Committee.
Reagents and Abs
a-GalCer was from Alexis Biochemical (San Diego, CA). Anti-asialo
GM1 (anti-asGM1) was purchased from Wako Chemicals (Richmond,
VA). mAbs against mouse CD3 (17A2; Pacific Blue-conjugated), NK1.1
(PK136; PE-Cy7– or FITC-conjugated), TCRgd (GL3; allophycocyanin-
conjugated), CD69 (FN50; PE-conjugated), IFN-g (XMG1.2; PE-
conjugated), IL-17A (TC11-18H10; PE-conjugated), CD27 (LG.7F9; PE-
Cy5–conjugated), TCRb (H57-597; PE-conjugated), CD19 (1D3; Pacific
Blue- or PE-conjugated), F4/80 (BM8; PE-conjugated), Vg1 (2.11; FITC-
conjugated), granzyme B (NGZB; PE-conjugated), NKG2D (C7; PE-
conjugated), Fas ligand (FasL, MFL3; PE-conjugated), CD107a (1D4B;
PE-conjugated), IL-12Rb2 (305719; PE-conjugated), and IL-18Ra
(112614; allophycocyanin-conjugated), as well as isotype controls, were
purchased from BD Biosciences (San Diego, CA), BioLegend (San Diego,
CA), R&D Systems (Minneapolis, MN), or eBioscience (San Diego, CA).
Recombinant mouse IL-12p70 and IL-18 were from R&D Systems. PMA
and ionomycin were from Sigma-Aldrich (St. Louis, MO).
Preparation of splenic and liver cells
Splenic and hepatic mononuclear cells from vehicle- or a-GalCer–treated
mice were prepared as described previously (32). Briefly, livers were
perfused with PBS, excised, and finely minced, followed by enzymatic
digestion for 30 min at 37˚C in PBS containing 1 mg/ml collagenase type
IV and 1 mg/ml DNase type I (Roche). After washing, liver homogenates
were resuspended in a 35% Percoll gradient, carefully layered onto 70%
Percoll, and centrifuged at 2300 rpm at 22˚C for 30 min. The layer at the
interface between the two Percoll concentrations was carefully aspirated
and washed in PBS containing 2% FCS. RBCs were removed with ACK
Mice were injected i.p. with vehicle or a-GalCer (2 mg/mouse). In some
cases, NK cells were specifically depleted using 200 mg i.p. rabbit anti-
asGM1 Ab on days 22 and 0 prior to a-GalCer injection. Saline-perfused
livers and spleens were harvested at different time points and mononuclear
cells were prepared as described above. Then, GolgiPlug (for IFN-g de-
tection) or GolgiPlug plus GolgiStop (for IL-17A detection) (BD Bio-
sciences) was added for 2 h. Cell suspensions were blocked in the presence
of 2.4G2 prior to staining with appropriate dilutions of allophycocyanin-
conjugated TCRgd, Pacific Blue-labeled anti-CD3, and PE-Cy7–conju-
gated NK1.1 for 30 min in PBS containing 2% FCS and 0.01% NaN3.
Cells were then fixed and permeabilized using the BD Cytofix/Cytoperm
fixation/permeabilization kit and incubated with PE-conjugated mAb
against IFN-g, IL-17A, or control isotype mAb in permeabilization buffer.
Cells were acquired and analyzed on a LSR-II cytometer (BD Bio-
sciences). FACS analysis was performed with FlowJo (Tree Star, Ashland,
Detection of cytokines
Cytokines were detected using the BD cytometric bead array (CBA) system
(BD Biosciences) according to the manufacturer’s instructions. Acquisi-
tion was performed on an LSR-II (BD Biosciences). A total of 300 bead
events for each cytokine were collected. Analysis of CBA data was per-
formed using the FCAP array software (Soft Flow, St. Louis, MO). IL-18
quantification was determined using the ELISA kit from MBL Interna-
tional (Woburn, MA).
Isolation of gd T and NK cells and cytotoxicity assays
Spleens were harvested from vehicle- or a-GalCer–treated mice. RBCs
were lysed with ACK lysis buffer prior to gd T and NK cell enrichment
with autoMACS (depletion of TCR-b+, CD19+, and F4/80+cells). Then,
gd T- and NK-enriched splenic cells were sorted and purity was always
.95%. For stimulation assays, purified cells were cultured for 20 h in
complete RPMI 1640 (10% FCS, 10 U/ml penicillin/streptomycin, L-glu-
tamine) containing recombinant mouse IL-12p70 (50 pg/ml) and/or IL-18
(1 ng/ml). For killing assays, cells were cocultured with targets labeled
with [51Cr] for a period of 20 h at an E:T ratio of 20:1. In some cases, cells
were cultured with YAC-1 for 4 h and checked for cytotoxic marker ex-
pression using anti-NKG2D, anti-FasL, anti-granzyme B, or anti-CD107a
and appropriated isotype controls.
Experimental lung metastasis and B cell lymphoma models
Lung metastasis model. B16F10 melanoma and 3LL Lewis lung carcinoma
cells were maintained as described previously (33). Wild-type (WT) or
TCRd2/2mice received B16F10 or 3LL cells by i.v. injection. Three hours
before, mice were injected i.p. with saline or a-GalCer (2 mg/mouse). Mice
were killed on day 14, and surface lung metastases were counted with the
aid of a dissecting microscope. NK cells were specifically depleted in mice
using 200 mg i.p. rabbit anti-asGM1 Ab on days 0, 1, and 7 after tumor
inoculation as described (34).
B cell lymphoma model. GFP+299/3.2 cells (5 3 104) generated from
Em2myc transgenic mice were i.v. injected into WT or TCRd2/2mice.
Three hours prior, mice were injected i.p. with saline or a-GalCer (2 mg/
mouse). On day 11 after tumor inoculation, mice were bled and the tumor
burden was assessed by flow cytometry. Concentration of WBCs in total
blood was measured using the automated hematology analyzer Advia 120
Immunization of mice and analysis of the CD8+T cell response
WT or TCRd2/2mice were immunized with OVA (50 mg/mouse) by i.p.
injection in the presence or absence of a-GalCer (2 mg/mouse). Spleens
were harvested 10 d after, and cells were restimulated with OVA (100 mg/
ml) at 37˚C for 3 d. Cytokine production was measured by the CBA
Results are expressed as the means 6 SD or means 6 SEM. The statistical
significance of differences between experimental groups was calculated by
a one way-ANOVAwith a Bonferroni post test or an unpaired Student t test
(GraphPad Prism 5 Software, San Diego, CA). The possibility of using
these parametric tests was assessed by checking whether the population
was Gaussian and the variance was equal (Bartlett test). Results with a p
value of ,0.05 were considered significant.
a-GalCer induces splenic and hepatic gd T cell activation to
To investigate whether gd T cells participate in the immune cas-
cade elicited by a-GalCer, we investigated in a kinetic manner the
activation status of splenic and hepatic gd T cells. As shown in
Fig. 1A, these cells expressed higher levels of CD69 as early as
4 h after a-GalCer injection. We next investigated by intracellular
staining the IFN-g production of gd T cells. Interestingly, we
observed that gd T cells produced IFN-g 8 h after a-GalCer ad-
ministration and sustained this secretion at least for an extra 16 h
(Fig. 1B). As a control, we observed that NK cells were activated
with similar kinetics to those of gd T cells. Investigation of other
cytokines demonstrated that gd T cells can also produce IL-17A,
but not TNF (Fig. 1C and data not shown). A lack of IFN-g
The Journal of Immunology3929
production by either gd Tor NK cells in a-GalCer–treated Ja182/2
mice revealed that these cells are likely to be activated in a re-
sponse downstream of type I NKT cells (Fig. 1D, Supplemental
Fig. 1). Additionally, when cocultured with a-GalCer–pulsed DC,
sorted gd T cells did not produce cytokines or enhanced CD69
expression (Supplemental Fig. 1). Similarly, use of a-GalCer/
CD1d tetramer failed to stain gd T cells (Supplemental Fig. 1).
Overall, a-GalCer administration leads to IFN-g production by gd
T cells probably through a bystander effect involving type I
T cells produce IFN-g and IL-17A
in vivo after injection of a-GalCer.
WT mice were injected i.p. with ve-
hicle or a-GalCer (2 mg/mouse) and
were sacrificed at different time points.
(A) Cells were analyzed by flow
cytometry and gated gd T (CD3+
TCRgd+) or NK (CD32NK1.1+) cells
were screened for surface CD69 ex-
pression. The mean fluorescence in-
tensity is indicated. One representative
experiment (liver) out of three is
shown (upper panel). The average 6
SEM of three independent experiments
is shown in the right panel (n = 9
mice). (B and C) Spleen and liver cells
were treated with GolgiPlug (or Gol-
giPlug plus GolgiStop for IL-17A de-
tection) for another 2 h and were
labeled with CD3, TCRgd, and NK1.1
mAbs, fixed, and permeabilized for
intracellular cytokine staining. Cells
were analyzed by flow cytometry and
screened for intracellular IFN-g and
IL-17A production. Gates were set
based on the isotype control. The per-
centages of cells positive for IFN-g (B)
or IL-17A (C) are represented. One
representative experiment of three is
shown. (B) The average 6 SEM of
shown in the lower panel (n = 9 mice).
(C) The average 6 SEM of three in-
dependent experiments is shown in the
right panel (n = 9). Differences in
mean were analyzed using the two-
tailed Student t test. **p , 0.01, ***p ,
0.001. (D) Sera of WTor Ja182/2mice
were collected at different time points
levels of IFN-g were quantified using
CBA. Data represent the average 6 SD
of two independent experiments (n = 8
Hepatic and splenic gd
gd T CELLS IN a-GalCer–MEDIATED IMMUNITY
a-GalCer–mediated IFN-g production by gd T cells is not
restricted to a particular subset
Recent studies have demonstrated that the gd T cell subsets have
diverse functional specializations. This includes the spectrum of
cytokines produced, which is regulated by TCR-dependent and
-independent mechanisms and the organ studied (reviewed in
Refs. 19, 35). For example, the CD27 molecule could be con-
sidered as a determinant of gd T cell differentiation in which the
CD27+subset is mainly associated with a Th1 profile (36). Based
on this, we checked IFN-g production by gd T cell subsets dis-
tinguished by CD27 expression. Surprisingly, we observed that
both subsets were able to produce IFN-g (Fig. 2A), although more
CD27+gd T cells from spleen secreted IFN-g compared with their
CD272counterparts. Similarly, the type of TCR expressed by gd
T cells appears to determine the properties of these cells (35).
Thus Vg1+cells produce Th1- and Th2-type cytokines and Vg4+
and liver cells were treated with GolgiPlug for another 2 h and were labeled with CD3, TCRgd, and CD27 (A) or Vg1 (B) mAbs, fixed, and permeabilized.
Gated CD3+TCRgd+CD27+/2(A) or CD3+TCRgd+Vg1+/2(B) cells were analyzed for intracellular IFN-g production. (A) Percentages of either hepatic
CD27+or CD272gd T cells positive for IFN-g are represented (upper panel). The average 6 SEM of three experiments is shown in the lower panel (n = 9
mice). (B) Percentages of hepatic Vg1+or Vg12gd T cells positive for IFN-g are represented (upper panel). The average 6 SEM of three independent
experiments is shown in the lower panel (n = 9).
IFN-g production by gd T cell subsets. (A and B) WT mice were injected i.p. with a-GalCer (2 mg/mouse) and sacrificed 12 h later. Spleen
The Journal of Immunology 3931
cells produce Th17-type cytokines. As shown in Fig. 2B, we ob-
served a preference for IFN-g production by Vg1+gd T cells in
response to a-GalCer in an organ-dependent manner with .80%
of IFN-g+gd T cells in the liver are Vg1+, whereas only 50% of
Vg1+cells were the source of IFN-g in the spleen. This suggests
that a-GalCer–induced IFN-g production by gd T cells is not
entirely restricted to a particular subset bearing a particular Vg-
chain and perhaps some gd T cell activation is mediated through
a TCR-independent mechanism.
IFN-g production by gd T cells is fully IL-12p35–dependent
and partially IL-18–dependent
The kinetics of gd T cell activation suggested that these cells are
activated downstream of the type I NKT cells. It is well estab-
or IL-182/2mice were injected i.p. with a-GalCer (2 mg/mouse) and sacrificed 12 h later. Spleen and liver cells were treated with GolgiPlug for another 2 h.
Gated CD3+TCRgd+cells from vehicle- or a-GalCer–treated WTor IL-12p352/2mice were analyzed for intracellular IFN-g production. The percentages of
hepatic gd T cells positive for IFN-g are represented (upper panel). (A) One experiment of two is shown. (B) One experiment of three is shown. The average 6
SD of IFN-g+splenic and hepatic CD3+TCRgd+cells is shown in the lower panel (n = $8). Differences in mean were analyzed using the two-tailed Student
t test. **p , 0.01, ***p , 0.001. (C) Expression of IL-12Rb2 and IL-18Ra has been assessed by flow cytometry on hepatic and splenic gd T cells. (D) Cell-
sorted splenic gd T cells (CD3+TCRgd+) were incubated with recombinant mouse IL-12 (50 pg/ml) and/or IL-18 (1 ng/ml) proteins. Twenty hours later,
cytokine production was measured by CBA. Data represent the mean 6 SEM of two independent experiments performed in triplicate.
IFN-g production by gd T cells in response to a-GalCer requires IL-12p35 and IL-18, but not TCR engagement. (A and B) WT, IL-12p352/2,
gd T CELLS IN a-GalCer–MEDIATED IMMUNITY
lished that APC maturation, including DC, is a critical determi-
nant of the immune response elicited by a-GalCer (innate and
acquired) (7, 37). Indeed, DC maturation leads to the release of an
array of activating cytokines, including IL-12p70 and IL-18, two
cytokines known to participate in gd T cell activation in particular
contexts (26, 38). Thus, we next investigated the potential re-
quirement of these cytokines in a-GalCer–induced gd T cell ac-
tivation. To address this possibility, we assessed IFN-g production
by splenic and hepatic gd T cells using IL-12p352/2and IL-182/2
mice. As depicted in Fig. 3A (left panel), IL-12p35 deficiency
results in a complete abrogation of gd T cell activation. Moreover,
the lack of IL-18 also significantly reduced splenic and hepatic gd
T cell activation by 38 and 55%, respectively (Fig. 3A, right
panel). Of note, IFN-g production by NK cells was also abrogated
in IL-12p352/2mice and reduced in IL-182/2mice (not shown).
Flow cytometric analysis of gd T cells showed that both hepatic
and splenic gd T cells expressed IL-12Rb2 and IL-18Ra (Fig. 3B).
Finally, to further investigate the involvement of these two cyto-
kines, sorted gd T cells were treated with recombinant IL-12p70
and/or IL-18. As shown in Fig. 2C, IL-12p70 or IL-18 individually
failed to promote IFN-g synthesis by splenic gd T cells. In con-
trast, combined addition of both cytokines induced IFN-g pro-
duction by gd T cells. Taken together, these results indicated the
critical role of IL-12p70 and IL-18 in a-GalCer–mediated gd
T cell activation, and that these cytokines were necessary and
sufficient to activate gd T cells.
a-GalCer enhanced gd T cell cytotoxicity
We have previously shown that a-GalCer can increase cytotoxic
properties of NK cells (17, 39), so we tested whether this effect
can also be observed for gd T cells by assessing the expression
of different cytolytic effector markers. Interestingly, we observed
that, along with NK cells, splenic gd T cells from a-GalCer–
treated mice triggered CD107a degranulation as well as FasL,
NKG2D, and granzyme B upregulation when cultured with YAC-
1, suggesting increased cytotoxic properties of these cells (Fig.
4A, Supplemental Fig. 2). Of note, in vitro priming of spleen cells
with a-GalCer also resulted in an increase expression of cytotoxic
mouse) and were sacrificed 24 h after. Spleen cells (E) from vehicle- or a-GalCer–treated mice were cocultured with YAC-1 cells (T) at an E:T ratio of 15:1
for 4 h. Surface (CD107a, FasL, and NKG2D) or intracellular (granzyme B) expression of cytotoxic markers was evaluated on/in gated gd T (CD3+TCRgd+)
and NK (CD32NK1.1+) cells. The average 6 SEM of positive cells (FasL, CD107a, and granzyme B) or mean of fluorescence intensity (NKG2D) for the
indicated markers is depicted (n = 6 mice). Differences in mean were analyzed using the two-tailed Student t test. *p , 0.05, **p , 0.01, ***p , 0.001. (B)
Purified splenic gd T cells (CD3+TCRgd+) from vehicle- or a-GalCer–treated mice were tested for ex vivo cytotoxicity against YAC-1 (left panel) or P815
(right panel) targets in a standard 200 h [51Cr] release assay at an E:T ratio of 20:1. Each bar is the mean of triplicate wells 6 SD of three independent
experiments. Differences in mean were analyzed using the two-tailed Student t test. **p , 0.01.
a-GalCer treatment in vivo increases cytotoxic properties of gd T cells. (A) WT mice were injected i.p. with vehicle or a-GalCer (2 mg/
The Journal of Immunology 3933
markers in/on gd T cells (Supplemental Fig. 3). To directly test
this, we next employed a [51Cr] release assay, using YAC-1 and
P815 cells as target cells. We demonstrated that sorted splenic gd
T cells from a-GalCer–treated mice more effectively killed target
cells compared with those sorted from control mice (Fig. 4B). A
consistent increase in killing activity was observed against these
two cell lines (from 2–3 to 12–18%). Taken together, these results
demonstrate that a-GalCer induced overexpression of cytotoxic
molecules on gd T cells resulting in an increased ability to kill
Cross-talk between NK cells and gd T cells
Because NK and gd T cells displayed a similar behavior (IFN-g
production and cytotoxic properties) after a-GalCer injection, we
investigated the potential cross-talk between these two cell pop-
ulations. Examination of IFN-g production by hepatic and splenic
NK cells in TCRd2/2mice demonstrated no alterations in the
ability of these NK cells to produce cytokines compared with the
WT mice (Fig. 5A). In contrast, NK cell depletion prior to
a-GalCer treatment significantly reduced IFN-g production by gd
T cells underlying a role of this population in the bystander ac-
tivation of gd T cells (Fig. 5B). Of note, NK cell depletion was
consistently .95% (Fig. 5B) and did not affect NKTand gd T cell
compartments (Fig. 5B and data not shown). Because NK cells
have been demonstrated to cross-talk with DC, we studied whether
the decrease in IFN-g production by gd T cells could be due to
a reduced DC activation in absence of NK cells. Consistent with
this, we observed that levels of IL-12p70 (Fig. 5C, left panel) and
IL-18 (Fig. 5C, right panel) in the sera of NK cell-depleted mice
were significantly decreased compared with untreated mice.
gd T cells partially contribute to the anti-tumoricidal activity
a-GalCer induces a strong cytokine burst resulting in the secretion
of an array of regulatory cytokines. To investigate the potential
contribution of gd T cells in this cytokine cascade, we quantified
the level of different cytokines potentially produced by gd T cells
in the serum of a-GalCer–treated TCRd2/2mice compared with
their WT counterparts. As depicted in Fig. 6A, the absence of gd
T cells significantly affects the overall IFN-g, but not IL-4, pro-
duction elicited by a-GalCer. Of note, although we could not
detect the presence of IL-17A in the serum of a-GalCer–treated
mice, in vitro stimulation of spleen cells with a-GalCer led to IL-
17A secretion in a gd T cell-dependent manner (Supplemental
Fig. 4). The protective anti-tumor effect of a-GalCer is mainly due
to the rapid synthesis of IFN-g by type I NKT cells and the by-
stander activation of both NK and CD8+CTL (15, 16). Thus, we
addressed the possibility that the tumoricidal effect of a-GalCer
could partially depend on gd T cells. Using B16F10 melanoma
and 3LL Lewis lung carcinoma, we compared the efficacy of
prophylactic administration of a-GalCer on pulmonary metastases
development in mice lacking gd T cells compared with control
mice. As depicted in Fig. 6B, the anti-metastatic effect of
a-GalCer was significantly reduced in the absence of gd T cells in
both models. Of note, in concert with our previous study (17), NK
cell depletion completely abrogated the anti-metastatic effect of
the a-GalCer. In parallel, we have also addressed this question
using a model of hematological malignancy by transplanting
a GFP+-B cell lymphoma cell line (299/3.2 clone) generated from
Em-myc transgenic mice, a model mimicking human non-Hodg-
kin’s lymphomas (40). Interestingly, pretreatment of mice with
a-GalCer substantially delayed the development of B cell lym-
phoma in control mice (Fig. 6C). Once again, this effect was
dependent on gd T cells as the tumor burden was significantly less
well controlled in a-GalCer-treated TCRd2/2mice. Taken to-
gether these data demonstrate that gd T cells are not critical but
are required for an optimal tumoricidal effect of a-GalCer.
gd T cells are required in the promotion of the CD8+T cell
response triggered by a-GalCer
a-GalCer has been demonstrated to promote the development of
strong Ag-specific responses by enhancing CD4+and CD8+T cell
functions as well as B cell maturation in the context of a coad-
ministered protein (12, 41, 42). In this study, we investigated
whether gd T cells could play a part in the a-GalCer–specific
enhancement of adaptive immune responses. For that purpose, we
immunized WT or TCRd2/2mice with OVA in presence or ab-
sence of a-GalCer. After 10 d, spleen cells from immunized mice
were restimulated in an Ag-specific manner and cytokine pro-
duction was assessed. As expected, OVA restimulation of spleen
cells from a-GalCer–treated mice resulted in an enhanced level of
both Th1 (IFN-g) and Th2 (IL-4, IL-5, and IL-13) cytokines
compared with mice immunized with OVA alone, even if the pro-
Th1 was far more pronounced than the pro-Th2 effect (Fig. 7 and
data not shown). Of note, we were unable to detect IL-17A after
antigenic restimulation. Interestingly, the Th1-, but not Th2-,
promoting effect of a-GalCer was partially decreased in TCRd2/2
mice. Thus, these results indicate that gd T cells are mandatory in
the optimal Th1-promoting effect of a-GalCer on the development
of an adaptive immune response.
a-GalCer exerts powerful type I NKT cell-dependent immuno-
modulatory activities that are currently being tested in therapy
against different pathologies such as cancer, infections, autoim-
munity, or allergy (reviewed in Refs. 3, 43–45). Our data dem-
onstrate the functional importance of gd T cells in a-GalCer–
mediated immune responses and by extension of its protective
First, we show that i.p. administration of a-GalCer leads to
splenic and hepatic gd T cell activation (CD69 overexpression),
resulting in IFN-g production by these cells. Kinetic analysis
demonstrates that this cytokine production starts only 4 h after
a-GalCer injection and peaks around 8–12 h. Numerous studies
have highlighted that individual subsets within the gd T cell
population have more specialized effector functions (reviewed in
Refts. 19, 35). However, our analysis failed to precisely identify
a specific subset of gd T cells involved in IFN-g production. For
example, the nature of the TCR expressed, especially the Vg-
chain, is responsible for the functional properties of the gd T cells
in mice (35). We observed that both Vg1+and Vg12(including
a large proportion of Vg2+gd T cells; data not shown) produced
IFN-g, although Vg1+preferentially produced the cytokine in the
liver. Similarly, a new classification of thymic and peripheral gd
T cell subsets has been recently proposed (36) in which the TNFR
family member CD27 molecule could be considered as a marker
in the Th1/Th17 balance. In this study, the CD27+subset is mainly
associated with a Th1 profile, and CD272has a Th17 profile. In
concert, a rough analysis of IFN-g production by gd T cells re-
garding the expression of CD27 indicates that ∼75% of this cy-
tokine was produced by the CD27+subset. A differential analysis
of each subset demonstrates that both hepatic CD27+and CD272
gd T cells produced IFN-g, indicating no intrinsic properties of
the CD27+subset to secrete IFN-g in this setting. However, results
from the spleen indicated that CD27+gd T cells are more capable
producers of IFN-g compared with their CD272counterpart.
Overall, the absence of a clear preference for CD27+gd T cells
after a-GalCer could be explained by the fact that we failed to
gd T CELLS IN a-GalCer–MEDIATED IMMUNITY
for intracellular IFN-g staining. Plots represent the percentage of IFN-g1NK cells in liver and spleen of a-GalCer–treated animals (upper panel) of one
representative experiment out of three. The average 6 SEM of IFN-g+NK cells is shown in the lower panel (n = 9). (B) Control or NK cell-depleted micewere
treated as in (A). Plots represent the percentage of IFN-g+gd T (CD3+TCRgd+) cells in liver and spleen of a-GalCer–treated animals (upper panel) of one
representative experiment out of two. The average 6 SD of two independent experiments is shown in the lower panel (n = 6–8). Differences in mean were
analyzedusing the two-tailedStudent t test. *p, 0.05.Ofnote, anti-asGM1treatment depletedefficiently liverand splenicNKcells(upper right panelanddata
not shown). (C) Sera of control or NK cell-depleted mice were collected 12 h after a-GalCer administration and levels of IL-12p70 and IL-18 were quantified
using CBA and ELISA kits, respectively. Data represent the average 6 SD of two independent experiments (n = 6–8). Differences in mean were analyzed using
the two-tailed Student t test. **p , 0.01, ***p , 0.001.
NK cells are required for a-GalCer–induced gd T cell activation. (A) WTor TCRd2/2mice were injected i.p. with vehicle or a-GalCer (2 mg/
The Journal of Immunology 3935
detect any modulation of CD70 (CD27 ligand) by flow cytometry
on both DC and macrophages after a-GalCer administration (data
not shown). This contrasts with upregulation of CD70 mRNA
transcripts in CD8a+, but not CD8a2, DC reported using the same
Using gene-targeted mice, we demonstrated that host IL-12 and
IL-18 were important for an optimal IFN-g production by gd
T cells. Similar to NK cells, it is probable that IL-12 induces IFN-
g production by gd T cells and IL-18 potentiates the effect of IL-
12 by upregulating expression of IL-12R on gd T cells (46, 47).
Moreover, given the observation that gd T cells expressing diverse
TCR produced IFN-g, we suggest that these cells are probably not
able to directly recognize the a-GalCer/CD1d complex. In line
with this, we failed to stain gd T cells with the a-GalCer/CD1d
tetramer. When cocultured with a-GalCer–loaded DC, purified gd
T cells could not produce cytokine or enhance activation marker
(CD69). IFN-g production by gd T cells approximately fits the
kinetics of NK cell activation, suggesting that this cytokine pro-
duction occurs downstream of type I NKT cells. In concert, ad-
ministration of a-GalCer in Ja182/2mice fully abrogated IFN-g
production by gd T cells regardless of the time point analyzed.
However, in the absence of commercially available neutralizing
Ab against pan-gd TCR, we cannot definitely rule out the possi-
bility that TCR engagement is required in our model. Nonetheless,
a-GalCer administration and levels of IFN-g and IL-4 were evaluated by CBA. Data represent the average 6 SEM of three independent experiments (n =
9). (B) WT or TCRd2/2mice were treated i.p. with vehicle or a-GalCer at day 0. Three hours later, mice were inoculated i.v. with 5 3 105B16F10 cells
(left panel) or 5 3 1053LL cells (right panel). Fourteen days after tumor inoculation, lungs were harvested and B16F10 or 3LL lung colonies were counted
and recorded. Data represent the mean 6 SEM of two independent experiments pooled (n = 12) (B16F10) or the mean 6 SD of one representative ex-
periment (3LL). Differences in mean were analyzed using a one-way ANOVA. *p , 0.05, **p , 0.01, ***p , 0.001. (C) WT or TCRd2/2mice were
treated i.p. with vehicle or a-GalCer at day 0. Three hours later, mice were inoculated i.v. with 5 3 104B cell lymphoma cells (clone 299/3.2 GFP+)
generated from Em-myc Tg mice. Eleven days after inoculation, mice were bled and tumor burden was assessed by flow cytometry (GFP+CD19+) and are
represented as number of tumor cells per volume of blood (3109/L). Data represent the mean 6 SD of one representative experiment out of two (n = 5
mice). Differences in mean were analyzed using a one-way ANOVA. *p , 0.05.
gd T cells participate in a-GalCer–mediated anti-tumor responses. (A) Sera of WTor TCRd2/2mice were collected in a kinetic manner after
gd T CELLS IN a-GalCer–MEDIATED IMMUNITY
our findings suggest that gd T cells can secrete IFN-g in response
to IL-12 and IL-18 without any TCR signaling. Of note, recent
studies have also demonstrated that IL-18 (or IL-1b) and IL-23
can lead to Th17 cytokine secretion by gd T cells without TCR
engagement (26, 28). Moreover, it is still possible that along with
these two cytokines, other TCR-independent factors, including
others cytokines, TLR agonists, Ig, or TNFR superfamily core-
ceptors (48, 49), could influence IFN-g production by gd T cells.
Interestingly, we have also highlighted the ability of gd T cells
to produce IL-17A in response to a-GalCer. The role of IL-17A
in host defense against pathogens including bacteria, fungus, and
parasites is well documented essentially through the ability of this
cytokine to induce neutrophil recruitment (50, 51). However, the
potential contribution of this cytokine in the beneficial role of
a-GalCer is poorly understood. For instance, a protective role of
host IL-17 has been shown in a-GalCer–induced acute hepatitis
(52). As suggested by our in vitro results, it is still possible that
along with NKT cells, IL-17A–producing gd T cells significantly
participate in this cytokine production and by extension in the
beneficial effect of this cytokine in this setting. However, addi-
tional investigations will be required to address this point.
Our analysis of gd T cell activation indicates these cells behave
like NK cells (mechanisms of activation, IFN-g production, and
increase cytotoxicity) after a-GalCer administration. Neverthe-
less, we consistently observed that IFN-g production by NK cells
peaked slightly earlier than gd T cells. To investigate whether this
difference could be explained by an influence of NK cells on gd
T cell activation, we studied IFN-g production of gd T cells in
absence of NK cells. Interestingly, we observed that both splenic
and hepatic gd T cells from anti–asGM1-treated mice produced
far less IFN-g compared with controls. Because NK cells have
been proven to participate in DC maturation (46), the slightly
earlier production of IFN-g by these cells could amplify DC
maturation, including cytokine production, initially engaged
through their interaction with type I NKT cells and in turn be part
of gd T cell transactivation in our setting. In line with this, levels
of IL-12p70 and IL-18 in sera of anti–asGM1-treated mice were
significantly reduced compared with control mice. Nevertheless,
NK cell depletion only partially reduced IFN-g production by gd
T cells, indicating that DC maturation engaged in cross-talk with
type I NKT cells was sufficient to activate gd T cells, and sub-
sequently NK cell-enhanced cytokine production (e.g., IL-12 and
IL-18) by DC led to optimal IFN-g secretion. In agreement with
this proposed scenario of activation kinetics (type I NKT/NK/gd
T cells), the absence of gd T cells did not modulate the ability of
NK cells to produce IFN-g, suggesting no feedback loop to NK
We have already demonstrated the capacity of a-GalCer to
increase NK cell cytotoxicity (17). In this study, we have also
shown that a-GalCer treatment leads to an increase in the cyto-
toxicity mediated by splenic gd T cells. Indeed, when cocultured
with target cells, gd T cells from a-GalCer–treated mice modu-
lated their phenotype by increasing NKG2D, FasL, CD107a, and
granzyme B, whereas those from vehicle-treated mice failed to do
so. This is a feature also observed after in vitro gd T cell priming
with a-GalCer. Even if gd T cells are less capable of exerting
cytotoxicity compared with NK cells, this observation, combined
with their ability to produce IFN-g, led us to investigate the po-
tential role of gd T cells in the anti-tumor effect of a-GalCer.
Using mouse models of lung metastases and B cell lymphoma, we
show that gd T cells are required for the full anti-tumor activity of
Finally, while confirming that a-GalCer enhanced adaptive Th1
immunity to a coadministered protein (7, 17, 42), we have also
highlighted the pivotal role of gd T cells in the Th1 arming.
Despite a key role for DC, CD40, IFN-g, and TNF-a being pro-
posed after a-GalCer (7, 41), the contribution of other cellular
components of the immune system have never been studied. The
reasons why gd T cells can contribute to the development of a
strong Th1 adaptive immune response remain elusive. Because
IFN-g can enhance MHC class I Ag processing and presentation
via JAK/STAT1 signaling and so in turn potentiate APC functions
(53), the early IFN-g production by gd T cells could, along with
type I NKT and NK cells, potentially have an impact at this level.
Furthermore, past studies have shown that activated human and
mouse gd T cells acquired Ag-presenting functions, including
MHC class II and CD40 expression (25, 54). This conversion into
APC could also partially explain our results. The potential in-
volvement of gd T cells in the Ab response downstream of
a-GalCer has not been addressed in our study. Nevertheless, IFN-
g has been proposed to be a key factor for IgG2a, but not IgG1,
production in mice immunized with a-GalCer and proteins (12).
In this context, we can speculate that gd T cells may play a role in
B cell response triggered by a-GalCer and coinjected proteins.
This work underlies the role of gd T cells in a-GalCer–mediated
immune responses and highlights how the NKT/NK/gd T cell axis
is important in the development and regulation of innate and ac-
quired immune responses.
cell-dependent manner. Spleen cells from WT or TCRd2/2mice immu-
nized with OVA (50 mg/mouse) in presence or absence of a-GalCer (2 mg/
mouse) were restimulated in an Ag-specific manner with OVA (100 mg/ml)
for 72 h. Cytokine profile (Th1 versus Th2) was evaluated by CBA. One
representative experiment of two is shown (n = 5 mice). A one-way
ANOVA has been used to analyze the variance followed by a Bonferroni
multiple comparison test to compare all groups of mice. **p , 0.01.
a-GalCer promotes OVA-specific Th1 response in a gd T
The Journal of Immunology 3937
We thank Qerime Mundrea, Ben Venville, and Shellee Brown for maintain-
ing and caring for the mice. We thank Andrea Newbold for producing the
299/3.2 GFP+lymphoma. We thank the Peter MacCallum flow cytometry
core facility for technical assistance. Prof. Dale I. Godfrey and Dr. Daniel
M. Andrews are acknowledged for helpful discussions and critical reading
of the manuscript.
The authors have no financial conflicts of interest.
1. Morita, M., K. Motoki, K. Akimoto, T. Natori, T. Sakai, E. Sawa, K. Yamaji,
Y. Koezuka, E. Kobayashi, and H. Fukushima. 1995. Structure-activity rela-
tionship of a-galactosylceramides against B16-bearing mice. J. Med. Chem. 38:
2. Kawano, T., J. Cui, Y. Koezuka, I. Toura, Y. Kaneko, K. Motoki, H. Ueno,
R. Nakagawa, H. Sato, E. Kondo, et al. 1997. CD1d-restricted and TCR-
mediated activation of va14 NKT cells by glycosylceramides. Science 278:
3. Bendelac, A., P. B. Savage, and L. Teyton. 2007. The biology of NKT cells.
Annu. Rev. Immunol. 25: 297–336.
4. Cerundolo, V., J. D. Silk, S. H. Masri, and M. Salio. 2009. Harnessing invariant
NKT cells in vaccination strategies. Nat. Rev. Immunol. 9: 28–38.
5. Godfrey, D. I., and M. Kronenberg. 2004. Going both ways: immune regulation
via CD1d-dependent NKT cells. J. Clin. Invest. 114: 1379–1388.
6. Chung, Y., W. S. Chang, S. Kim, and C. Y. Kang. 2004. NKT cell ligand
a-galactosylceramide blocks the induction of oral tolerance by triggering den-
dritic cell maturation. Eur. J. Immunol. 34: 2471–2479.
7. Fujii, S., K. Shimizu, C. Smith, L. Bonifaz, and R. M. Steinman. 2003. Acti-
vation of natural killer T cells by a-galactosylceramide rapidly induces the full
maturation of dendritic cells in vivo and thereby acts as an adjuvant for com-
bined CD4 and CD8 T cell immunity to a coadministered protein. J. Exp. Med.
8. Carnaud, C., D. Lee, O. Donnars, S. H. Park, A. Beavis, Y. Koezuka, and
A. Bendelac. 1999. Cutting edge: cross-talk between cells of the innate immune
system: NKT cells rapidly activate NK cells. J. Immunol. 163: 4647–4650.
9. Nieuwenhuis, E. E., T. Matsumoto, M. Exley, R. A. Schleipman, J. Glickman,
D. T. Bailey, N. Corazza, S. P. Colgan, A. B. Onderdonk, and R. S. Blumberg.
2002. CD1d-dependent macrophage-mediated clearance of Pseudomonas aeru-
ginosa from lung. Nat. Med. 8: 588–593.
10. Singh, N., S. Hong, D. C. Scherer, I. Serizawa, N. Burdin, M. Kronenberg,
Y. Koezuka, and L. Van Kaer. 1999. Cutting edge: activation of NK T cells by
CD1d and a-galactosylceramide directs conventional T cells to the acquisition of
a Th2 phenotype. J. Immunol. 163: 2373–2377.
11. Stober, D., I. Jomantaite, R. Schirmbeck, and J. Reimann. 2003. NKT cells
provide help for dendritic cell-dependent priming of MHC class I-restricted
CD8+T cells in vivo. J. Immunol. 170: 2540–2548.
12. Galli, G., P. Pittoni, E. Tonti, C. Malzone, Y. Uematsu, M. Tortoli, D. Maione,
G. Volpini, O. Finco, S. Nuti, et al. 2007. Invariant NKT cells sustain specific
B cell responses and memory. Proc. Natl. Acad. Sci. USA 104: 3984–3989.
13. Fujii, S., K. Shimizu, H. Hemmi, and R. M. Steinman. 2007. Innate Va14+
natural killer T cells mature dendritic cells, leading to strong adaptive immunity.
Immunol. Rev. 220: 183–198.
14. Van Kaer, L., and S. Joyce. 2005. Innate immunity: NKT cells in the spotlight.
Curr. Biol. 15: R429–R431.
15. Hayakawa, Y., K. Takeda, H. Yagita, S. Kakuta, Y. Iwakura, L. Van Kaer,
I. Saiki, and K. Okumura. 2001. Critical contribution of IFN-ga and NK cells,
but not perforin-mediated cytotoxicity, to anti-metastatic effect of a-gal-
actosylceramide. Eur. J. Immunol. 31: 1720–1727.
16. Nishimura, T., H. Kitamura, K. Iwakabe, T. Yahata, A. Ohta, M. Sato, K. Takeda,
K. Okumura, L. Van Kaer, T. Kawano, et al. 2000. The interface between innate
and acquired immunity: glycolipid antigen presentation by CD1d-expressing
dendritic cells to NKT cells induces the differentiation of antigen-specific cy-
totoxic T lymphocytes. Int. Immunol. 12: 987–994.
17. Smyth, M. J., N. Y. Crowe, D. G. Pellicci, K. Kyparissoudis, J. M. Kelly,
K. Takeda, H. Yagita, and D. I. Godfrey. 2002. Sequential production of inter-
feron-g by NK1.1+T cells and natural killer cells is essential for the anti-
metastatic effect of a-galactosylceramide. Blood 99: 1259–1266.
18. Takeda, K., Y. Hayakawa, M. Atsuta, S. Hong, L. Van Kaer, K. Kobayashi,
M. Ito, H. Yagita, and K. Okumura. 2000. Relative contribution of NK and
NKT cells to the anti-metastatic activities of IL-12. Int. Immunol. 12: 909–914.
19. Bonneville, M., R. L. O’Brien, and W. K. Born. 2010. gd T cell effector func-
tions: a blend of innate programming and acquired plasticity. Nat. Rev. Immunol.
20. Born, W. K., Z. Yin, Y. S. Hahn, D. Sun, and R. L. O’Brien. 2010. Analysis of
gamma delta T cell functions in the mouse. J. Immunol. 184: 4055–4061.
21. Hayday, A. C. 2009. gd T cells and the lymphoid stress-surveillance response.
Immunity 31: 184–196.
22. Gardner, T. R., Q. Chen, Y. Jin, and M. N. Ajuebor. 2010. Toll-like receptor 3
ligand dampens liver inflammation by stimulating Va14 invariant natural killer
T cells to negatively regulate gd T cells. Am. J. Pathol. 176: 1779–1789.
23. Zhao, N., J. Hao, Y. Ni, W. Luo, R. Liang, G. Cao, Y. Zhao, P. Wang, L. Zhao,
Z. Tian, et al. 2011. Vg4 gd T cell-derived IL-17A negatively regulates
NKT cell function in Con A-induced fulminant hepatitis. J. Immunol. 187: 5007–
24. Jin, N., N. Miyahara, C. L. Roark, J. D. French, M. K. Aydintug, J. L. Matsuda,
L. Gapin, R. L. O’Brien, E. W. Gelfand, and W. K. Born. 2007. Airway
hyperresponsiveness through synergy of gammadelta T cells and NKT cells. J.
Immunol. 179: 2961–2968.
25. Cheng, L., Y. Cui, H. Shao, G. Han, L. Zhu, Y. Huang, R. L. O’Brien,
W. K. Born, H. J. Kaplan, and D. Sun. 2008. Mouse gd T cells are capable of
expressing MHC class II molecules, and of functioning as antigen-presenting
cells. J. Neuroimmunol. 203: 3–11.
26. Lalor, S. J., L. S. Dungan, C. E. Sutton, S. A. Basdeo, J. M. Fletcher, and
K. H. Mills. 2011. Caspase-1-processed cytokines IL-1b and IL-18 promote IL-
17 production by gd and CD4 T cells that mediate autoimmunity. J. Immunol.
27. Martin, B., K. Hirota, D. J. Cua, B. Stockinger, and M. Veldhoen. 2009.
Interleukin-17-producing gd T cells selectively expand in response to pathogen
products and environmental signals. Immunity 31: 321–330.
28. Sutton, C. E., S. J. Lalor, C. M. Sweeney, C. F. Brereton, E. C. Lavelle, and
K. H. Mills. 2009. Interleukin-1 and IL-23 induce innate IL-17 production from
gd T cells, amplifying Th17 responses and autoimmunity. Immunity 31: 331–
29. Emoto, M., Y. Emoto, I. Yoshizawa, E. Kita, T. Shimizu, R. Hurwitz,
V. Brinkmann, and S. H. Kaufmann. 2010. a-GalCer ameliorates listeriosis by
accelerating infiltration of Gr-1+cells into the liver. Eur. J. Immunol. 40: 1328–
30. Cui, J., T. Shin, T. Kawano, H. Sato, E. Kondo, I. Toura, Y. Kaneko, H. Koseki,
M. Kanno, and M. Taniguchi. 1997. Requirement for Va14 NKT cells in IL-12-
mediated rejection of tumors. Science 278: 1623–1626.
31. Takeda, K., H. Tsutsui, T. Yoshimoto, O. Adachi, N. Yoshida, T. Kishimoto,
H. Okamura, K. Nakanishi, and S. Akira. 1998. Defective NK cell activity and
Th1 response in IL-18-deficient mice. Immunity 8: 383–390.
32. Paget, C., T. Mallevaey, A. O. Speak, D. Torres, J. Fontaine, K. C. Sheehan,
M. Capron, B. Ryffel, C. Faveeuw, M. Leite de Moraes, et al. 2007. Activation of
invariant NKT cells by Toll-like receptor 9-stimulated dendritic cells requires
type I interferon and charged glycosphingolipids. Immunity 27: 597–609.
33. Smyth, M. J., M. Taniguchi, and S. E. Street. 2000. The anti-tumor activity of IL-
12: mechanisms of innate immunity that are model and dose dependent. J.
Immunol. 165: 2665–2670.
34. Smyth, M. J., N. Y. Crowe, and D. I. Godfrey. 2001. NK cells and NKT cells
collaborate in host protection from methylcholanthrene-induced fibrosarcoma.
Int. Immunol. 13: 459–463.
35. O’Brien, R. L., and W. K. Born. 2010. gd T cell subsets: a link between TCR and
function? Semin. Immunol. 22: 193–198.
36. Ribot, J. C., A. deBarros, D. J. Pang, J. F. Neves, V. Peperzak, S. J. Roberts,
M. Girardi, J. Borst, A. C. Hayday, D. J. Pennington, and B. Silva-Santos. 2009.
CD27 is a thymic determinant of the balance between interferon-g- and inter-
leukin 17-producing gd T cell subsets. Nat. Immunol. 10: 427–436.
37. Kitamura, H., K. Iwakabe, T. Yahata, S. Nishimura, A. Ohta, Y. Ohmi, M. Sato,
K. Takeda, K. Okumura, L. Van Kaer, et al. 1999. The natural killer T (NKT) cell
ligand a-galactosylceramide demonstrates its immunopotentiating effect by in-
ducing interleukin (IL)-12 production by dendritic cells and IL-12 receptor ex-
pression on NKT cells. J. Exp. Med. 189: 1121–1128.
38. Das, H., M. Sugita, and M. B. Brenner. 2004. Mechanisms of Vd1 gd T cell
activation by microbial components. J. Immunol. 172: 6578–6586.
39. Smyth, M. J., M. E. Wallace, S. L. Nutt, H. Yagita, D. I. Godfrey, and
Y. Hayakawa. 2005. Sequential activation of NKT cells and NK cells provides
effective innate immunotherapy of cancer. J. Exp. Med. 201: 1973–1985.
40. Adams, J. M., A. W. Harris, C. A. Pinkert, L. M. Corcoran, W. S. Alexander,
S. Cory, R. D. Palmiter, and R. L. Brinster. 1985. The c-myc oncogene driven by
immunoglobulin enhancers induces lymphoid malignancy in transgenic mice.
Nature 318: 533–538.
41. Fujii, S., K. Liu, C. Smith, A. J. Bonito, and R. M. Steinman. 2004. The linkage
of innate to adaptive immunity via maturing dendritic cells in vivo requires
CD40 ligation in addition to antigen presentation and CD80/86 costimulation. J.
Exp. Med. 199: 1607–1618.
42. Hermans, I. F., J. D. Silk, U. Gileadi, M. Salio, B. Mathew, G. Ritter, R. Schmidt,
A. L. Harris, L. Old, and V. Cerundolo. 2003. NKT cells enhance CD4+and
CD8+T cell responses to soluble antigen in vivo through direct interaction with
dendritic cells. J. Immunol. 171: 5140–5147.
43. Tessmer, M. S., A. Fatima, C. Paget, F. Trottein, and L. Brossay. 2009. NKT cell
immune responses to viral infection. Expert Opin. Ther. Targets 13: 153–
44. Tupin, E., Y. Kinjo, and M. Kronenberg. 2007. The unique role of natural
killer T cells in the response to microorganisms. Nat. Rev. Microbiol. 5: 405–
45. Wu, L., and L. Van Kaer. 2009. Natural killer T cells and autoimmune disease.
Curr. Mol. Med. 9: 4–14.
46. Cooper, M. A., T. A. Fehniger, A. Fuchs, M. Colonna, and M. A. Caligiuri. 2004.
NK cell and DC interactions. Trends Immunol. 25: 47–52.
47. Lauwerys, B. R., N. Garot, J. C. Renauld, and F. A. Houssiau. 2000. Cytokine
production and killer activity of NK/T-NK cells derived with IL-2, IL-15, or the
combination of IL-12 and IL-18. J. Immunol. 165: 1847–1853.
48. Ribot, J. C., A. debarros, and B. Silva-Santos. 2011. Searching for “signal 2”:
costimulation requirements of gd T cells. Cell. Mol. Life Sci. 68: 2345–
gd T CELLS IN a-GalCer–MEDIATED IMMUNITY
49. Wesch, D., C. Peters, H. H. Oberg, K. Pietschmann, and D. Kabelitz. 2011. Mod- Download full-text
ulation of gd T cell responses by TLR ligands. Cell. Mol. Life Sci. 68: 2357–2370.
50. Curtis, M. M., and S. S. Way. 2009. Interleukin-17 in host defence against
bacterial, mycobacterial and fungal pathogens. Immunology 126: 177–185.
51. Iwakura, Y., S. Nakae, S. Saijo, and H. Ishigame. 2008. The roles of IL-17A in
inflammatory immune responses and host defense against pathogens. Immunol.
Rev. 226: 57–79.
52. Wondimu, Z., T. Santodomingo-Garzon, T. Le, and M. G. Swain. 2010. Pro-
tective role of interleukin-17 in murine NKT cell-driven acute experimental
hepatitis. Am. J. Pathol. 177: 2334–2346.
53. Zhou, F. 2009. Molecular mechanisms of IFN-gamma to up-regulate MHC class
I antigen processing and presentation. Int. Rev. Immunol. 28: 239–260.
54. Brandes, M., K. Willimann, and B. Moser. 2005. Professional antigen-
presentation function by human gammadelta T Cells. Science 309: 264–268.
The Journal of Immunology3939