OncoImmunology

ISSN: (Print) 2162-402X (Online) Journal homepage: http://www.tandfonline.com/loi/koni20

Natural killer T cell activation overcomes

immunosuppression to enhance clearance of
postsurgical breast cancer metastasis in mice
Simon Gebremeskel, Daniel R. Clattenburg, Drew Slauenwhite, Lynnea
Lobert & Brent Johnston
To cite this article: Simon Gebremeskel, Daniel R. Clattenburg, Drew Slauenwhite, Lynnea
Lobert & Brent Johnston (2015) Natural killer T cell activation overcomes immunosuppression
to enhance clearance of postsurgical breast cancer metastasis in mice, OncoImmunology, 4:3,
e995562, DOI: 10.1080/2162402X.2014.995562
To link to this article: http://dx.doi.org/10.1080/2162402X.2014.995562

Accepted author version posted online: 22

Jan 2015.
Published online: 22 Jan 2015.
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Date: 21 December 2016, At: 07:36

ORIGINAL RESEARCH
OncoImmunology 4:3, e995562; March 2015; 2015 Taylor & Francis Group, LLC

Natural killer T cell activation overcomes

immunosuppression to enhance clearance of
postsurgical breast cancer metastasis in mice
Simon Gebremeskel1,4, Daniel R. Clattenburg1,4, Drew Slauenwhite1, Lynnea Lobert1,4, and Brent Johnston1,2,3,4,*
1

Department of Microbiology & Immunology; Dalhousie University; Halifax, Nova Scotia, Canada; 2Department of Pediatrics; Dalhousie University; Halifax, Nova Scotia, Canada;
3
Department of Pathology; Dalhousie University; Halifax, Nova Scotia, Canada; 4Beatrice Hunter Cancer Research Institute; Halifax, Nova Scotia, Canada

Keywords: dendritic cells, metastatic breast cancer, myeloid derived suppressor cells, natural killer T cells, tumor immunotherapy
Abbreviations: a-GalCer, a-galacotosylceramide; ALT, alanine aminotransferase; DC, dendritic cell; FBS, fetal bovine serum;
GM-CSF, granulocyte-macrophage colony-stimulating factor; IFNg, interferon-g; IL, interleukin; i.p., intraperitoneal;
i.v., intravenous; MDSC, myeloid derived suppressor cell; NK cell, natural killer cell; NKT cell, natural killer T cell; RPMI-1640,
Roswell Park Memorial Institute medium-1640; TCR, T cell receptor; Th, T helper.

Metastatic lesions are responsible for over 90% of breast cancer associated deaths. Therefore, strategies that target
metastasis are of particular interest. This study examined the efcacy of natural killer T (NKT) cell activation as a postsurgical immunotherapy in a mouse model of metastatic breast cancer. Following surgical resection of orthotopic 4T1
mammary carcinoma tumors, BALB/c mice were treated with NKT cell activating glycolipid antigens (a-GalCer, a-CGalCer or OCH) or a-GalCer-loaded dendritic cells (DCs). Low doses of glycolipids transiently reduced metastasis but did
not increase survival. A high dose of a-GalCer enhanced overall survival, but was associated with increased toxicity and
mortality at early time points. Treatment with a-GalCer-loaded DCs limited tumor metastasis, prolonged survival, and
provided curative outcomes in 45% of mice. However, survival was not increased further by additional DC treatments
or co-transfer of expanded NKT cells. NKT cell activation via glycolipid-loaded DCs decreased the frequency and
immunosuppressive activity of myeloid derived suppressor cells (MDSCs) in tumor-resected mice. In vitro, NKT cells
were resistant to the immunosuppressive effects of MDSCs and were able to reverse the inhibitory effects of MDSCs on
T cell proliferation. NKT cell activation enhanced antitumor immunity in tumor-resected mice, increasing 4T1-specic
cytotoxic responses and IFNg production from natural killer (NK) cells and CD8C T cells. Consistent with increased
tumor immunity, mice surviving to day 150 were resistant to a second tumor challenge. This work provides a clear
rationale for manipulating NKT cells to target metastatic disease.

Introduction
NKT cells are a subset of immunoregulatory T cells that
bridge innate and adaptive immune responses.1,2 NKT cells
express an invariant T cell receptor (TCR)-a chain rearrangement (Va14-Ja18 in mice and Va24-Ja18 in humans) paired
with a restricted repertoire of TCR-b chains (Vb8.2/7/2 in mice
and Vb11 in humans).3 Unlike conventional T cells which recognize peptide antigens, NKT cells recognize glycolipid antigens
presented via the MHC class I-like molecule CD1d.4 Following
activation, NKT cells can produce a wide array of immunoregulatory cytokines, including interferon-g (IFNg), tumor necrosis
factor, interleukin-2 (IL-2), IL-4, IL-10, IL-13, IL-17, IL-21,
and granulocyte-macrophage colony-stimulating factor (GMCSF).5-7 Rapid cytokine production enables NKT cells to influence downstream immune responses, making them a promising
therapeutic target for immune modulation therapy.

It is now well established that NKT cells play important roles

in tumor immunosurveillance and antitumor immunity in the
absence of exogenous stimulation.8,9 This is likely mediated by
recognition of tumor-associated glycolipid antigens,10 stress
induced glycolipid self-antigens,11,12 or inflammatory cytokines.13,14 Preclinical and clinical studies have shown that NKT
cell activation with exogenous glycolipids provides significant
protection from tumor progression.1520 Although several studies
have examined the role of NKT cell activation on primary mammary carcinoma growth,21,22 the potential therapeutic benefits of
NKT cell activation in metastatic breast cancer have not been
explored in detail. The aim of this work was to examine the role
of NKT cell activation in a postsurgical breast cancer metastasis
model.
Despite recent advances in the development of cancer vaccines
and immunotherapies, tumor tolerance and immune suppression
remain formidable obstacles. Tumor-derived factors disrupt

*Correspondence to: Brent Johnston; Email: Brent.Johnston@dal.ca

Submitted: 09/19/2014; Revised: 11/28/2014; Accepted: 12/02/2014
http://dx.doi.org/10.1080/2162402X.2014.995562

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normal myeloid differentiation, leading to the accumulation of

heterogeneous populations of undifferentiated myeloid cells
referred to collectively as MDSCs.23 As the tumor burden
increases, MDSCs accumulate in a variety of tissues, where they
act to enhance tumor progression and metastasis.24 MDSCs can
directly suppress CD4C and CD8C T cell responses, and indirectly promote immune suppression through the induction of
FoxP3C regulatory T cells.2528 Therefore, immunotherapeutic
strategies that target both tumor cells and MDSCs are of particular interest. Given that NKT cells have been shown to regulate
MDSC-mediated immune suppression during viral infection,29
the role of NKT cell activation on cancer-associated immunosuppression requires further investigation.
Using a postsurgical breast cancer metastasis model, we
found that NKT cell activation using a-galactosylceramide
(a-GalCer)-loaded DCs reduced metastatic disease and significantly enhanced survival outcomes. Enhanced survival was associated with reductions in MDSC frequency and activity, and
the induction of robust antitumor responses by NK cells and
CD8C T cells. These results demonstrate that NKT cell activation therapy is a promising therapeutic strategy to target metastatic disease.

Results
Glycolipid treatments reduce metastasis
Free glycolipids have been shown to have protective effects in
cancer models.7,16,30 We investigated whether a-GalCer, a Th1polarizing analog (a-C-GalCer),31 or a Th2 polarizing analog
(OCH)32 could protect mice from 4T1 breast cancer metastasis.
In our postsurgical breast cancer model (Fig. 1A), the glycolipid
analogs stimulated increases in serum IFNg and IL-4 levels that
were consistent with their reported cytokine polarizing bias
(Fig. 1B, C). Consistent with an important role for IFNg in
tumor control,33,34 there were significant reductions in lung
metastasis one week (Day 21) following treatment with a-GalCer
or a-C-GalCer, but not with OCH (Fig. 1D).
Given that a-GalCer administration yielded optimal protection from tumor metastasis and is currently the lead compound
for investigation in clinical trials,15,35 we focused on a-GalCer
mediated antitumor responses. Although treatment with 4 mg
a-GalCer resulted in a significant reduction in tumor metastasis
at early time points, this protection was lost by day 28
(Fig. 1E). Consistent with this, there was no significant increase
in the survival of mice treated with free glycolipid (Fig. 1F). To
improve tumor control, we hypothesized that higher doses of
glycolipid or multiple treatments would be required to achieve
clinical success.
As repeated NKT cell stimulation with free glycolipid is
known to induce anergy,3638 we performed a dose response
study using single injections of a-GalCer. A significant increase
in overall survival time was observed with the highest dose (i.p.
20 mg); however, this was associated with increased mortality
within 48 h of treatment (Fig. 1G). Increased mortality is likely
linked to increased liver toxicity as indicated by high levels of

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alanine aminotransferase (ALT) activity39 (Fig. 1H). The level of

serum IFNg increased and the level of serum IL-4 decreased with
increasing dose of a-GalCer (Fig. 1I and J), suggesting a dosedependent polarization toward a stronger Th1 response.
a-GalCer-loaded DCs provide significant protection from
tumor metastasis
Compared to free glycolipid administration, transfer of
a-GalCer loaded DCs mediates a more Th1 skewed cytokine
response and does not induce NKT cell anergy.38 A single adoptive transfer of a-GalCer-loaded DCs into tumor-resected mice
resulted in a significant reduction in tumor metastasis and a significant increase in survival (Fig. 2A and B). Administration of
a-GalCer-loaded DCs was not associated with increased liver
toxicity (peak ALT levels at 12 h: 498 80 IU/L with
a-GalCer-loaded DCs vs. 2725 340 IU/L in mice receiving
20 mg of a-GalCer; p < 0.05). Mice that survived to day 150
had no detectable tumor cells by clonogenic assay, suggesting
that they were cured of metastatic disease. Surprisingly, an additional treatment with a-GalCer-loaded DCs on day 34 did not
enhance survival compared to the single treatment (Fig. 2B).
This was not due to NKT cell anergy as the second administration of a-GalCer-loaded DCs induced serum cytokine responses
that were comparable to both the initial stimulation on day 13
and primary cytokine responses in non-tumor bearing mice
(Fig. 2C and D).
As NKT cell numbers and function are reduced in many cancer patients,40,41 including those with breast cancer,41 the effectiveness of endogenous NKT activation could be limited.
Therefore, we examined whether adoptive transfer of expanded
NKT cells in combination with NKT cell activation would
enhance therapeutic outcome in our mice. The adoptive transfer
of expanded NKT cells alone or in combination with free
a-GalCer did not enhance survival of tumor-resected mice
(Table 1). Similarly, adoptive transfer of NKT cells with
a-GalCer-loaded DCs also failed to enhance survival compared
to treatment with a-GalCer-loaded DCs alone (Table 1).
Blood MDSC levels as a prognostic marker for NKT cell
based immunotherapy
Immunosuppressive MDSCs have been shown to accumulate
in cancer patients and in the 4T1 tumor model.24,42,43 However,
the relationship between NKT cells and MDSCs is unclear as
NKT cells have been shown to suppress or promote MDSC accumulation and activity in different settings.29,37,44-46 We prepared
hematoxylin and eosin-stained cytospins of peripheral blood
samples to characterize the leukocyte populations. Compared to
nave controls, tumor bearing mice had significantly higher numbers of granulocytic cells with few lymphocytes and monocytes
(Fig. 3A). NKT cell activation was associated with a decrease in
the accumulation of granulocytic cells. However, there were no
morphological distinctions between the granulocytes observed in
the nave and diseased mice. Consistent with the cytospin analysis, the frequency of CD11bC Gr-1C cells in nave blood was low
(Fig. 3B and C), but this population expanded with progressive
tumor growth. Consistent with previous reports,4749 the

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Figure 1. Treatment with free glycolipid following primary 4T1 tumor resection provides transient protection from lung metastases. (A) Schematic of the
postsurgical NKT cell activation model. Mice were inoculated with 2 105 4T1 mammary carcinoma cells and primary tumors were resected on day 12.
A single dose of free glycolipid was delivered i.p. on day 13. Serum levels of (B) IFNg and (C) IL-4 were measured 2, 6, and 24 h following administration
of a-GalCer, a-C-GalCer, or OCH (i.p. 4 mg) (n D 36 per group). *p < 0.05 compared to a-GalCer. (D) Number of 4T1 colony forming units (CFU) present
in lung cell suspensions isolated at day 21 from mice treated with a-GalCer, a-C-GalCer, OCH (i.p. 4 mg), or saline vehicle (n D 1225 per group). (E) Number of 4T1 CFU present in lung cell suspensions isolated at day 21, 28, or 35 after treatment with a-GalCer (i.p. 4 mg) (n D 712 per group). (F, G) Survival
was assessed following treatment with a-GalCer (i.p. 1 mg, 4 mg or 20 mg) (n D 510 per group). *p < 0.05 compared to saline control. (H) Serum ALT levels were measured following administration of 1 mg, 4 mg or 20 mg a-GalCer (n D 3 per group). *p < 0.05 compared to 4 mg, yp < 0.05 compared to
1 mg. The dotted line indicates baseline ALT levels in nave mice. Serum levels of (I) IFNg and (J) IL-4 were measured 24 h following treatment with
a-GalCer (i.p. 1 mg, 4 mg or 20 mg) (n D 36 per group). *p < 0.05 compared to 4 mg a-GalCer.

CD11bC Gr-1C population declined following tumor resection.

However, the CD11bC Gr-1C population later surged as metastatic lesions developed in the mice receiving no additional treatment (data not shown), mice treated with unloaded DCs, and a
subset of mice that relapsed following NKT cell activation therapy (Fig. 3C). Mice with circulating levels of CD11bC Gr-1C
cells exceeding 50% succumbed to tumor metastasis. Surviving
mice that responded well to NKT cell activation therapy maintained low levels of CD11bC Gr-1C cells (Fig. 3B and C),

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suggesting that CD11bC Gr-1C levels could be used as a prognostic marker in NKT cell based immunotherapy.
To determine whether the expansion of blood CD11bC GrC
1 cells correlated with immunosuppression, T cell proliferation
assays were performed in the presence of blood leukocytes from
nave mice, and tumor-resected mice treated with unloaded DCs
or a-GalCer-loaded DCs. Blood leukocytes isolated from the
unloaded DC treatment group or relapsed mice significantly suppressed proliferation of nave T cells, whereas blood leukocytes

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inoculation). While NKT

cell activation did not affect
size of the primary tumor
(data not shown), it
resulted in a modest but
significant reduction in the
levels
of
circulating
MDSCs in tumor bearing
mice (Fig. 4A). To further
support the direct role for
NKT cells in reducing
MDSC frequency and suppressive activity, we added
NKT cells to co-cultures of
MDSCs and nave T cells.
MDSCs had no effect on
NKT cell proliferation in
response to anti-CD3/28coated activator beads, and
activated NKT cells were
able to overcome the suppressive activity of MDSCs
to rescue the proliferation
of nave T cells (Fig. 4B
and C).
Effect of NKT cell
activation on antitumor
immunity
To examine the effect of
NKT cell activation on
antitumor immunity, we
examined IFNg production
and cytotoxic activity of
purified NK cells and
CD8C T cells isolated from the spleens of nave mice or tumorresected mice that had received treatment with vehicle- or
a-GalCer-loaded DCs. Compared to cells from nave or vehicletreated mice, NK cells and CD8C T cells isolated from mice
treated with a-GalCer-loaded DCs exhibited enhanced cytotoxic
activity against 4T1 cells (Fig. 5A and B). Similarly, NK cells
and CD8C T cells from mice treated with a-GalCer-loaded DCs
released greater quantities of IFNg into the culture media
(Fig. 5C and D). Cells isolated from mice that received two

Figure 2. Treatment with a-GalCer-loaded DCs following primary 4T1 tumor resection confers long-term protection
from lung metastasis. Mice were inoculated with 2 105 4T1 mammary carcinoma cells and primary tumors were
resected on day 12. Mice were treated on day 13, or days 13 and 34, with unloaded DCs or a-GalCer-loaded DCs (i.v.
2 105 cells). (A) Number of 4T1 CFU present in lung cell suspensions isolated at day 21, 28, and 35 post-4T1 injection (n D 68 per group). *p < 0.05 compared to unloaded DCs. (B) Survival was assessed following treatment with
unloaded DCs, a single treatment with a-GalCer-loaded DCs, or two treatments with a-GalCer-loaded DCs (n D 911
per group). *p < 0.016 via Bonferroni threshold analysis. Serum levels of (C) IFNg and (D) IL-4 were measured 2, 6,
and 24 h following the rst (day 13) or second (day 34) treatment with a-GalCer-loaded DCs in tumor resected mice
and in nave mice treated with a-GalCer-loaded DCs (n D 36 per group).

from complete responders did not suppress T cell proliferation

(Fig. 3D).
Given that the primary tumor is responsible for the initial elevation in MDSC levels,47 it was not clear whether the sustained
reduction in circulating MDSCs following NKT cell activation
was due to direct effects of NKT cells on MDSCs or due to
maintenance of a reduced tumor burden. To determine whether
NKT cells directly altered levels of circulating MDSCs, we activated NKT cells in unresected mice (day 7 post-tumor

Table 1. Survival outcomes in mice receiving NKT cell adoptive transfer in combination with free a-GalCer or a-GalCer-loaded DCs
Treatment group
Unloaded DCs
25 105 a-GalCer DCs
0.51 106 NKT cells C 25 105 a-GalCer DCs
0.51 106 NKT cells C a-GalCer
0.21 106 NKT cells

No response ( 50 days)a

Partial response (50150 days)

Complete response (150C days)

9/9b (3237)
6/21 (3750)
12/21 (3246)
8/9 (4247)
8/9 (3841)

0
7/21 (54, 61, 63, 67, 68, 75, 83)
5/21 (53, 56,59, 62, 72)
1/9 (94)
1/9 (58)

0
8/21 (150)
4/21 (150)
0
0

Survival outcomes in mice were stratied into three categories: non-responders survived  50 d post-4T1 cell injection, partial responders survived > 50 d
but < 150 d post-4T1 injection, and complete responders were healthy at 150 d post-4T1 cell injection. bThe number of mice in each response category out
of the total number of mice in treatment group. The range or individual day(s) of sacrice are indicated in brackets.
a

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treatments with a-GalCerloaded DCs exhibited

equivalent cytotoxic activity
and IFNg release to cells
isolated from mice that
received one treatment.
This suggests that one treatment induces a maximal
immune response, consistent with the inability of
the second treatment to
enhance survival further
(Fig. 2B).
Consistent with the
enhanced immune responses
observed in in vitro assays,
mice surviving to day 150
had significantly decreased
tumor growth following rechallenge with 4T1 cells
(Fig. 5E).

Discussion
Whereas decreases in
NKT cell number and/or
function have been linked to
increased tumor incidence
and progression,8,9,41 NKT
cell activation has been
shown to enhance antitumor responses.7,15,16,21,30,31
Although clinical trials examining NKT cell activation
therapies in patients with
advanced/recurrent disease
have reported few cases of
objective tumor regression,
tumors tended to remain
stable without the appear- Figure 3. Postsurgical administration of a-GalCer-loaded DCs reduces the frequency and suppressive activity of
ance of new metastatic blood MDSCs. (A) Representative images of hematoxylin and eosin-stained cytospins of blood leukocytes isolated
foci.15,1720,35 This suggests from nave mice, and tumor resected mice treated with unloaded or a-GalCer-loaded DCs (day 35 and day 150). (B)
from naive mice and tumor
that NKT cell activation Representative ow cytometry plots of Gr-1 and CD11b staining of peripheral blood cells
resected mice treated with unloaded or a-GalCer-loaded DCs. (C) Frequency of Gr-1CCD11bC cells in the blood of
therapy might be more mice treated with unloaded or a-GalCer-loaded DCs. Treated mice were separated into groups exhibiting transient
effective at targeting meta- (relapse) or complete responses to treatment (n  6 mice per group). (D) Proliferation of nave T cells stimulated with
static disease than primary anti-CD3/anti-CD28-coated beads was examined by dilution of Oregon Green. Cells were incubated in the absence
tumors. Since primary or presence of peripheral blood leukocytes from nave mice, tumor resected mice receiving unloaded DCs, tumor
breast
cancer
tumors resected mice responding to a-GalCer-loaded DC therapy, or tumor resected mice that relapsed following a-GalCerloaded DC therapy. Values indicate frequency of proliferating nave T cells (n D 511 per group). *p < 0.05 compared
are effectively treated by to naive blood; yp < 0.05 compared to mice treated with unloaded DCs.
surgical resection, and
disseminated metastasis remains the primary cause of mortal- stimulated antitumor immune function, and increased
ity,50 NKT cell activation therapy presents a promising thera- survival.
peutic avenue. Using a mouse model of metastatic breast
While several studies have examined the role of NKT cells and
cancer, we found that postsurgical NKT cell activation via the therapeutic effects of NKT cell activation in breast cancer
a-GalCer-loaded DCs significantly reduced tumor metastasis, models, the findings have been inconsistent. Terabe et al.51

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Figure 4. NKT cell activation reduces the frequency and suppressive

activity of MDSCs. (A) Frequency of circulating Gr-1CCD11bC cells in 4T1
tumor-bearing mice treated with unloaded or a-GalCer-loaded DCs on
day 7 post 4T1 injection (n D 4 per group). *p < 0.05 compared to treatment with unloaded DCs. (B) Proliferation of NKT cells was measured by
eFluor 670 dilution within NKT cells stimulated with of anti-CD3/antiCD28 beads alone or in the presence of MDSCs derived from the blood
of 4T1 tumor-bearing mice (n D 5 per group). (C) Proliferation of Oregon
green-labeled naive T cells was measured in response to anti-CD3/antiCD28-coated beads in the absence or presence of NKT cells and MDSCs
derived from blood of 4T1 tumor-bearing mice (n D 5 per group). *p <
0.05 compared to bead stimulation alone; yp < 0.05 compared to bead
stimulation in the presence of MDSCs.

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showed that NKT cell deficient Ja18/ mice had significantly

lower metastasis and improved survival in the 4T1 model, implicating an enhanced antitumor response in the absence of NKT
cells. This finding is consistent with another report revealing
enhanced antitumor CD8C T cell responses in Ja18/ mice following treatment with local radiation and CTLA-4 blockade.52
These studies suggest that NKT cells play suppressive roles in
antitumor immunity. However, Teng et al. 21 showed that NKT
cell activation therapy, in combination with antibodies targeting
CD262 (DR-5) and CD137, dose-dependently protected mice
from primary tumor growth and metastases, suggesting an antitumor effect of glycolipid-mediated NKT cell activation. Chen and
Ross 53 reported that DCs loaded with a-GalCer and retinoic
acid tended to reduce growth and metastasis of orthotopic 4T1
tumors, but their results did not reach statistical significance.
Our studies show that NKT cell activation using a larger number
of a-GalCer-loaded DCs significantly reduced postsurgical
tumor metastasis and increased survival. The differences in the
role of NKT cells in these studies may relate to activation of
NKT cells by endogenous vs. exogenous stimuli. NKT cell activation with a-GalCer-loaded DCs induces a robust IFNg
response which promotes antitumor immunity, while endogenous tumor glycolipids have been shown to induce Th2-polarized responses or suppress NKT cell activation.10,54
Administration of a-GalCer or the Th1 skewing analog a-CGalCer significantly reduced tumor metastasis at early timepoints, whereas the Th2 skewing analog OCH had no significant
effect. While Th1 skewing is important for tumor control in
mouse models,31,33,34 and elevated IFNg production is associated with prolonged survival in clinical trials,20,35 we previously
found that OCH treatment effectively controlled metastasis of
B16 melanoma and Lewis lung carcinoma cells to the liver in
C57BL/6 mice.7 It is likely that the ratio of Th1:Th2 cytokines
ultimately impacts tumor control, and differences in immune
skewing between BALB/c and C57BL/6 mice could influence the
effect of OCH. We focused on a-GalCer in this study because it
has been used extensively in human clinical trials where it has
been shown to be safe and well tolerated.17,18,20
The ability of free a-GalCer to protect from metastasis was
transient, suggesting that multiple treatments or higher doses
would be needed therapeutically. However, these strategies are
limited by the induction of anergy and toxicity, respectively.
Anergy is likely induced by the glycolipid presentation via B-cells
or other CD1d-expressing cells that poorly present the antigen
and/or lack appropriate co-stimulatory signals.38,55 In contrast,
glycolipid delivery via DCs induces potent NKT cell activation
without induction of anergy.38 We show that immunotherapy
with a-GalCer-loaded DCs protected mice from 4T1 tumor
metastasis and significantly increased survival. Importantly, mice
surviving to day 150 were free of metastasis, suggesting they had
been cured of disease.
Despite promising results with a single treatment of a-GalCerloaded DCs, a second treatment 3 weeks later did not enhance
immune function further or significantly improve the survival outcome. Similarly, we observed no additional survival benefit
by adoptively transferring NKT cells in our model of metastasis.

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Compared to DC treatment alone, there was a

trend for decreased survival
benefit in mice receiving
NKT cell adoptive transfer
in combination with DC
treatment. However, this
was not statistically significant (p D 0.0831). Our
expanded NKT cells are
functional as adoptive
transfer of expanded NKT
cells protected NKT celldeficient mice from metastasis of intrasplenic B16
melanoma inoculations
(unpublished observations). This suggests that
the dose and/or timing
interval of NKT cell transfer relative to surgery and
DC treatment requires further optimization in the
4T1 model. In contrast to
our results, co-treatment
with expanded NKT cells
and glycolipid-loaded DCs
has been reported to be
beneficial in human cancer
trials.19,56 Differences in
treatment schedules and
time course of disease may
account for the differences
in efficacy of NKT cell
adoptive transfer in clinical
studies vs. our experimental model. As mice have a
higher frequency of NKT
cells, it is also possible that
additional NKT cells are
more useful in human
patients where NKT cell
number and/or function
are limited.
Several clinical studies
have reported the use of
peripheral blood MDSCs
as biomarkers of disease.24,42 In particular, levels of MDSCs have been
directly correlated with
clinical stage and metastatic burden in breast
cancer patients.24,42 Furthermore, MDSCs have
previously been shown to

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Figure 5. Enhanced tumor-specic responses and protection from tumor re-challenge in mice treated with a-GalCerloaded DCs. Cell mediated cytotoxicity of sorted (A) NK cells (CD49bC TCRb-) and (B) CD8 T cells (CD8C TCRbC) isolated from the spleens of nave mice or tumor resected mice treated with unloaded DCs or a-GalCer-loaded DCs (n D
4 per group). DCs were administered once (day 13) or twice (days 13 and 34) and responses were measured 3 weeks
later. Cytotoxicity was measured via annexin V and 7-amino-actinomycin D staining of 4T1 cells. Supernatant IFNg levels were measured from sorted (C) NK cells and (D) CD8C T cells after 4 h or 18 h in culture with 4T1 cells (n D 4 per
group). (E) Tumor resected mice that survived to day 150 after treatment with a-GalCer-loaded DCs were re-challenged in the ank with 4T1 cells. Tumor volume was compared to tumors grown in nave mice (n D 5 per group). *p
< 0.05 compared to nave mice; yp < 0.05 compared to mice treated with a-GalCer-loaded DCs.

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reduce the efficacy of immunotherapies by impairing antigen presentation and T cell activation.26-28 We found that MDSCs were
a good biomarker of metastatic disease and presented a potent
prognostic marker for NKT cell activation therapy. Based on circulating levels of MDSCs, we could track responses to NKT cell
activation therapy and identify mice that relapsed. This allows
identification of individuals that could benefit from re-treatment
or combination therapies with other agents.
The effects of NKT cell stimulation on the frequency and
activity of MDSCs remains unclear. NKT cell activation in a
mouse model of multiple sclerosis was reported to provide protection via the induction of MDSCs,44 and administration of
free a-GalCer has been shown to increase MDSCs in a hepatotoxicity model.37 In contrast, NKT cells suppresses MDSC accumulation during viral infection,29 and activated NKT cells can
induce MDSC differentiation into DCs.29,46,57,58 In expanded
memory T cell cultures, an increased number of mouse CD49bC
T cells or human CD161C T cells reduced the suppressive effects
of MDSCs, leading to enhanced IFNg production in response to
tumor antigens.45,58 Although these studies did not use markers
that allow definitive identification of NKT cells, they are consistent with our findings that activated NKT cells rescued T cell
proliferation from MDSC mediated suppression. It is likely that
the ability of NKT cells to induce or inhibit MDSC function
depends on the context of the NKT cell stimulation, the nature
of the antigen presenting cell, and stromal signals. Pathways
involving CD1d, CD40L, and NKG2D have been implicated in
the regulatory effects of NKT cells on MDSC activity.29,58,59
We show that NKT cell activation resulted in robust CD8C
T cell and NK cell mediated antitumor responses. While NKT
cells can directly lyse tumor cells, this mechanism of antitumor
immunity in vivo is largely associated with hematological
tumors.60,61 However, cytokine production from activated NKT
cells activates and matures DCs and facilitates downstream effector functions of NK cells, CD8C cytotoxic T cells, and Th1
CD4C T cells.1 Consistent with this, there were increased cytotoxic and IFNg responses by NK cells and tumor-specific CD8C
T cells in mice treated with NKT cell activation therapy. Mice
that survived long term (150 days) maintained durable immunity
that limited growth of 4T1 tumors following re-challenge.
In conclusion, we show that NKT cell activation using
a-GalCer-loaded DCs was effective in targeting metastatic breast
cancer lesions in mice and significantly enhanced survival. This
was associated with reduced expansion of MDSCs and induction
of potent antitumor responses by NK cells and CD8C T cells.
Our findings provide preclinical evidence supporting the development of therapeutic NKT cell activation strategies to target
metastatic breast cancer.

Materials and Methods

Mice
Female BALB/c mice were acquired form Charles River Laboratories. Mice were maintained in the Carleton Animal Care
Facility at Dalhousie University and used at 812 weeks of age.

e995562-8

All experimental procedures were approved by the University

Committee on Laboratory Animals following the guidelines of
the Canadian Council on Animal Care.
Flow cytometry
The following antibodies were obtained from eBioscience or
BioLegend: purified CD16/32 (clone 97); fluorescein isothiocyanate-labeled TCRb (clone H57-597); phycoerythrin-labeled Gr1 (clone RB6-8C5), CD49b (clone DX5) and CD8a (clone 536.7); allophycocyanin-labeled CD11b (clone M1/70), CD8a
(clone 53-6.7), and TCRb (clone H57-597). Allophycocyaninlabeled CD1d tetramers loaded with the synthetic glycolipid
PBS57 were obtained from the NIH Tetramer Core Facility.
Prior to staining, all cell samples were pre-incubated with antiCD16/32 antibody to block non-specific binding. Cells were
incubated 20 min at 4 C with antibody panels to stain cell subsets, washed, and fixed in 2% paraformaldehyde (Fisher Scientific). Unloaded CD1d tetramers and isotype matched control
antibodies were used to establish gating and quadrants. Analysis
was performed using a two laser FACSCalibur with CellQuest
Pro software (BD Biosciences). A two-laser FACSAria cell sorter
with FACSDiva software (BD Biosciences) was used to sort cell
populations (>95% purity) for adoptive transfers and in vitro
cytotoxicity assays.
Primary mammary tumor resection model
4T1 mammary carcinoma cells were cultured at 37 C, 5%
CO2 in Dulbeccos modified Eagles medium supplemented with
10% fetal bovine serum (FBS), 100 mg/mL streptomycin, and
100 units/mL penicillin (Fisher-Hyclone). Cells were harvested
in the logarithmic growth phase using trypsin-ethylenediaminetetraacetic acid (SigmaAldrich). Cells were resuspended in saline
and 2 105 cells (50 mL volume) were injected subcutaneously
into the fourth mammary fat pad. Primary mammary tumors
were resected 12 d after tumor cell injection when the primary
tumors were 200 mm3 in size.
Glycolipid mediated protection from tumor metastasis
One day following primary tumor resection (day 13), mice
received an intraperitoneal (i.p.) injection of saline vehicle or
4 mg of the NKT cell-activating glycolipid antigens a-GalCer
((2S,3S,4R)-1-O-(a-D-galactipyranosyl)-2-(N-hexacosanoylamino)-1,3,4-octadecanetriol; Toronto Research Chemicals), the
Th1 skewing analog a-C-GalCer ((2S,3S,4R)-1-CH2-(a -D-galactopyranosyl)-2-(N-hexacosanoylamino)-1,3,4-octadecanetriol;
NIH Tetramer Core Facility), or the Th2 skewing analog OCH
((2S,3S,4R)-1-(a-D-galactopyranosyl)-2-tetracosanoylaminononan-3,4-diol; NIH Tetramer Core Facility). To examine
immune activation, serum cytokine levels were collected at various time points following stimulation. IFNg and IL-4 levels were
determined via enzyme-linked immunosorbent assays (eBioscience). Lung metastasis was examined using a clonogenic plating assay.62 Lungs were dissociated by mechanical dispersion
through a sterile wire mesh and cultured on petri plates in the
presence of 60 mM 6-thioguanine. After 14 d, plates were fixed
with methanol and stained with 0.03% methylene blue to

OncoImmunology

Volume 4 Issue 3

enumerate metastatic 4T1 colonies. In some experiments the

time course of lung metastasis (day 21, 28, and 35) was examined
and in separate experiments the effect of different doses of
a-GalCer (i.p. 1 mg, 4 mg, or 20 mg) on survival was assessed.
To examine liver toxicity, serum samples were collected at different time points and ALT activity was quantified by NADH oxidation assay (Cayman Chemicals).39
DC based NKT cell immunotherapy
To generate bone marrow derived DCs, bone marrow cells
from mouse femurs were cultured in 6 well plates with complete
RPMI-1640 (containing 10% FBS, 50 mM 2-mercaptoethanol,
2 mM L-glutamine, 100 mg/mL streptomycin, and 100 units/
mL penicillin) supplemented with 40 ng/mL GM-CSF (Peprotech). Cells received fresh media containing GM-CSF on day 3.
On day 6, non-adherent cells were collected and loaded overnight
in complete RPMI-1640 containing 0.4 mg/mL a-GalCer and
20 ng/mL GM-CSF. a-GalCer-loaded DCs were harvested the
next day and 2 105 DCs were injected intravenously (i.v.) into
mice to induce NKT cell activation. Mice received treatment on
day 13 (one day following tumor resection) or on days 13 and
34. Serum cytokine responses were measured at several time
points after treatment. To examine long-term immunity, mice
that survived to day 150 were re-challenged in the right flank
with 2 105 4T1 cells. Tumor growth was monitored using a
digital caliper.
NKT cell adoptive transfer in combination with NKT cell
activation therapy
NKT cells were expanded in vivo by administering a-GalCerloaded DCs (i.v. 6 105 cells) to nave BALB/c mice. After
72 h, liver tissue was dispersed through a sterile wire mesh. Lymphocytes were obtained by centrifugation through a 33% percoll
gradient (GE Healthcare). TCRbC CD1d tetramerC NKT cells
were purified by sorting and 2 105 or 1 106 NKT cells were
adoptively transferred into tumor resected mice (day 13). NKT
cells were activated 2 h after transfer using a-GalCer (i.p. 4 mg)
or a-GalCer-loaded DCs (i.v. 2 105 cells).
MDSC isolation and characterization
Blood was obtained at different time points via submandibular venipuncture: 70 mL of blood was collected in 10 mL of heparin sulfate (10,000 U/mL; SigmaAldrich). Red blood cell lysis
was performed and cells were used for cytospin preparations,
flow cytometry, or T cell suppression assays. For cytospin preparations, cells were resuspended in 100 mL of phosphate buffered
saline containing 2% FBS and loaded onto microscope slides via
centrifugation. Slides were stained with hematoxylin and eosin
and cell morphology was examined using a Zeiss Axiovert 200M
microscope with a Hamamatsu Orca R2 Camera. To analyze
MDSCs by flow cytometry, cells were stained using anti-Gr-1
and anti-CD11b antibodies.
To determine whether activated NKT cells directly influence
MDSC frequency, mice were inoculated with 2 105 4T1 cells
in the mammary fat pad. Once tumors were palpable (day 7),
a-GalCer-loaded DCs were administered to activate NKT cells.

www.tandfonline.com

Primary tumor growth and blood MDSC frequency were

monitored.
T cell suppression assay
To test immunosuppressive activity of blood MDSCs, 70 mL
of blood was drawn from nave mice and tumor-resected mice
that received 2 105 unloaded or a-GalCer-loaded DCs. Cells
were resuspended in 200 mL RPMI-1640 following red blood
cell lysis, and 50 mL was added to each well in a round bottomed
96 well plate. Responder splenocytes were isolated from the
spleens of nave mice by mechanical dispersion through wire
mesh followed by red blood cell lysis. Responder cells were
labeled with 5 mM Oregon green (Life Technologies) and resuspended in RPMI-1640 (supplemented with 100 mg/mL streptomycin, and 100 units/mL penicillin, 10% FBS and 1% HEPES).
Responder cells (2 105) were combined in wells with blood
leukocytes from tumor-resected animals. Proliferation was
induced by adding T-Activator anti-CD3/28 Dynabeads (Life
Technologies) in a ratio of 1 bead:2 splenocytes. Samples were
incubated at 37 C 5% CO2 for 72 h. Responder cell proliferation was measured by flow cytometry, gating on TCRbC cells
and assessing cell division by Oregon green dilution.
To examine the direct effects of NKT cell activation on
MDSC suppressive function and the ability of MDSCs to inhibit
proliferation of activated NKT cells, 1 105 NKT cells labeled
with eFlour 670 (eBioscience) were added to the T cell suppression assay outlined above. Both T cell and NKT cell proliferation
were simultaneously monitored via Oregon green and eFlour
670 dilution, respectively.
Immune function assays
To characterize whether NKT cell activation induced CD8C
T cell and/or NK cell mediated antitumor immunity, CD8C
TCRbC T cells and CD49bC TCRb NK cells were isolated
and cultured at a 1:1 ratio with Oregon green-labeled 4T1 target
cells. Cells were cultured in complete RPMI-1640 for 4 h or
18 h. Following incubation, supernatants were collected for analysis of IFNg levels. Cytotoxicity against Oregon green-labeled
4T1 cells was determined by flow cytometry via staining with
allophycocyanin-labeled annexin V and 7-amino-actinomycin D
(eBioscience).
Statistical analysis
Data are expressed as mean SEM unless otherwise stated. A
non-parametric two-tailed MannWhitney U-test was used to
compare between two data groups. Comparisons between more
than two data groups were made using a KruskalWallis nonparametric analysis of variance with Dunns post-test. Survival
data were analyzed by log-rank (MantelCox) significance test.
Statistical significance was set at p < 0.05. For survival data comparing multiple pairs of treatment groups, the statistical significance level was set using the Bonferroni corrected threshold (p <
(0.05/K), where K is the number of comparisons being performed. Statistical computations were carried out using GraphPad Instat 3.02 and GraphPad Prism 5.

OncoImmunology

e995562-9

Disclosure of Potential Conflicts of Interest

Funding

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Robyn Cullen and Dr. Linnea Veinotte for their

technical assistance with this project.
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