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Blockade of Myeloid-Derived Suppressor
Cells after Induction of Lymphopenia
Improves Adoptive T Cell Therapy in a
Murine Model of Melanoma
Krithika N. Kodumudi, Amy Weber, Amod A. Sarnaik and
Shari Pilon-Thomas
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2012 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2012; 189:5147-5154; Prepublished online 24
October 2012;
doi: 10.4049/jimmunol.1200274
http://www.jimmunol.org/content/189/11/5147
The Journal of Immunology
Blockade of Myeloid-Derived Suppressor Cells after
Induction of Lymphopenia Improves Adoptive T Cell
Therapy in a Murine Model of Melanoma
Krithika N. Kodumudi, Amy Weber, Amod A. Sarnaik, and Shari Pilon-Thomas
M
elanoma is a leading cause of cancer mortality in the
United States, resulting in one American dying every
60 min. Currently, there are few nonsurgical options for
the treatment of metastatic melanoma (1, 2). Cancer immunotherapy, which focuses on the induction of immunity against tumor cells, is a promising approach for the treatment of melanoma.
A major hurdle in the development of effective immunotherapy is
tumor-induced suppression, including factors such as regulatory
T cells (Tregs) and myeloid-derived suppressor cells (MDSC) that
can limit the effectiveness of tumor-specific T cells (3, 4). Tregs
have been shown to suppress antitumor activity and ablation of
this population has led to tumor eradication (5). In preclinical
melanoma models as well as in cancer patients, depletion of Tregs
followed by dendritic cell (DC)‑based vaccination has led to enhanced vaccine-mediated tumor-specific T cell responses (3, 6, 7).
MDSC are implicated in the maintenance of immune tolerance to
tumors (4, 8, 9). MDSC are a phenotypically heterogeneous cell
population consisting of myeloid progenitor cells and immature
Immunology Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa,
FL 33612
Received for publication January 19, 2012. Accepted for publication September 24,
2012.
This work was supported by National Institutes of Health Grant R00 CA128825-01
(to S.P.-T.).
Address correspondence and reprint requests to Dr. Shari Pilon-Thomas, Department
of Immunology, H. Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, Stabile Research Building-2, Tampa, FL 33612. E-mail address: Shari.
[email protected]
Abbreviations used in this article: CM, complete medium; DC, dendritic cell; DCFDA,
29,79-dichlorofluorescein diacetate; DTX, docetaxel; MDSC, myeloid-derived suppressor cell; TBI, total body irradiation; Treg, T regulatory cell.
Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1200274
myeloid cells. In mice, these cells are broadly defined as CD11b+
Gr-1+ cells. Recent reports have shown that MDSC by themselves
can induce tumor-specific Tregs (10). Increased levels of circulating MDSC have been correlated with advanced disease stage
and extensive metastatic tumor burden in cancer patients (11).
There have been several reports showing that lymphopenia induced by nonmyeloablative chemotherapeutic treatments or total
body irradiation (TBI) can modulate immune responses and increase antitumor immunity (12–14). These treatments induce a
severe lymphopenic condition in the host and leads to the subsequent expansion of T cells in the periphery in a process known as
homeostatic proliferation. Homeostatic proliferation can lead to
the proliferation and activation of T cells specific for self-Ags (15,
16). Studies have shown increased antitumor responses following
irradiation and adoptive T cell transfer in preclinical murine
models (17, 18). Adoptive transfer of autologous tumor-infiltrating
lymphocytes after lymphodepleting chemotherapy has resulted in
objective responses in melanoma patients (12, 19). However, only
one-fifth of the treated patients demonstrated complete tumor
regressions. Although it has been shown that administration of cyclophosphamide and fludarabine or TBI in mice before adoptive
transfer reduces or eliminates immunosuppressive populations (13),
little is known about the effects of TBI on reconstitution of Tregs
and MDSC. Hence, it is important to understand the role of these
suppressor populations after the induction of lymphopenia to design effective immunotherapeutic treatments to achieve complete
tumor regression.
Conventional anticancer agents have been explored for their
ability to target suppressor populations (20–24). Gemcitabine, a
nucleoside analog, has been shown to reduce splenic MDSC in
mice bearing large tumors without affecting T cells, NK cells, macrophages, or B cells (25). Serafini et al. (26) showed that sildenafil
downregulates arginase activity and NO synthase expression, thereby
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
Administration of nonmyeloablative chemotherapeutic agents or total body irradiation (TBI) prior to adoptive transfer of tumorspecific T cells may reduce or eliminate immunosuppressive populations such as T regulatory cells (Tregs) and myeloid-derived
suppressor cells (MDSC). Little is known about these populations during immune reconstitution. This study was designed to understand the reconstitution rate and function of these populations post TBI in melanoma tumor‑bearing mice. Reconstitution rate
and suppressive activity of CD4+CD25+Foxp3+ Tregs and CD11b+Gr1+ MDSC following TBI-induced lymphopenia was measured
in B16 melanoma tumor‑bearing mice. To ablate the rapid reconstitution of suppressive populations, we treated mice with
docetaxel, a known chemotherapeutic agent that targets MDSC, in combination with adoptive T cell transfer and dendritic cell
immunotherapy. Both Treg and MDSC populations exhibited rapid reconstitution after TBI-induced lymphopenia. Although
reconstituted Tregs were just as suppressive as Tregs from untreated mice, MDSC demonstrated enhanced suppressive activity of
CD8+ T cell proliferation compared with endogenous MDSC from tumor-bearing mice. TBI-induced lymphopenia followed by
docetaxel treatment improved the efficacy of adoptive T cell transfer and dendritic cell immunotherapy in melanoma-bearing
mice, inducing a significant reduction in tumor growth and enhancing survival. Tumor regression correlated with increased CTL
activity and persistence of adoptively transferred T cells. Overall, these findings suggest that TBI-induced MDSC are highly
immunosuppressive and blocking their rapid reconstitution may improve the efficacy of vaccination strategies and adoptive
immunotherapy. The Journal of Immunology, 2012, 189: 5147–5154.
5148
reducing the suppressive function of MDSC. Antimicrotubule agents,
such as docetaxel (DTX) and paclitaxel, have immune-enhancing
properties when used alone or in combination with immunotherapy
(22, 27, 28). A recent study has shown that paclitaxel promotes
differentiation of MDSC into DC in vitro in a TLR4-independent
manner (29). Paclitaxel has also been shown to reduce Tregs and
their inhibitory function (28). DTX has been shown to decrease
splenic MDSC in mice bearing mammary tumors, leading to an
increased antitumor response (22). Although recent reports have
shown the effect of DTX on MDSC, it has not been used for specific
inhibition of MDSC in the setting of lymphopenia for the treatment
of melanoma. Hence, in this study, we investigated the reconstitution of suppressor populations (Tregs and MDSC) after lymphodepletion. Furthermore, we investigated whether blockade of reconstituting MDSC leads to persistence of adoptively transferred
T cells and enhances the efficacy of adoptive T cell transfer and
vaccination in a murine model of melanoma.
Materials and Methods
Six- to 8-wk-old C57BL/6 mice were purchased from Harlan Laboratories
(Indianapolis, IN). OT-I mice were purchased from The Jackson Laboratory
(Bar Harbor, ME). Mice were housed at the Animal Research Facility of the
H. Lee Moffitt Cancer Center and Research Institute. All animal protocols
were reviewed and approved by the Institutional Animal Care and Use
Committee at the University of South Florida.
Cell lines
B16 melanoma was maintained by serial in vitro passages in complete
medium (CM) consisting of RPMI 1640 medium supplemented with 10%
heat-inactivated FCS, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 2 mM fresh L-glutamine, 100 mg/ml streptomycin, 100 U/ml penicillin,
50 mg/ml gentamicin, 0.5 mg/ml fungizone (all from Life Technologies,
Rockville, MD), and 0.05 mM 2-ME (Sigma-Aldrich, St. Louis, MO). B16
expressing OVA (M05) cells were maintained by serial in vitro passages in
CM supplemented with 0.8 mg/ml G418.
Reagents
For analysis of immune cell populations, the following anti-mouse Abs were
purchased from BD Biosciences (San Diego, CA): anti-Ly6G and -Ly6C
biotinylated Abs, anti-CD11b PerCP-Cy5.5, anti-CD11b allophcocyanin,
anti–Gr-1 PE, anti–Gr-1 PE Cy7, anti-CD4 Pacific blue, anti-CD8 Alexa
Fluor 780, and anti-CD3 FITC. The CD4 + CD25 + Treg isolation kit and
streptavidin microbeads were purchased from Miltenyi Biotec (Auburn,
CA). The anti-mouse Foxp3 T regulatory staining kit was purchased from
eBioscience (San Diego, CA). Clinical grade DTX (Taxotere; Sanofi-Aventis)
was used in this study.
Lymphopenia model
A total of 1 3 105 B16 or 3 3 105 M05 tumor cells were injected s.c. in the
left flank of C57BL/6 or Ly5.2 mice. Three days later, mice received a
sublethal dose (600 cGy) of TBI administered by a [137Cs] gamma radiation source.
Isolation of MDSC and MDSC suppressor assay
MDSC were isolated from the spleens of naive, B16 tumor bearing mice, or
B16 tumor-bearing mice treated with TBI. Splenocytes were depleted of
RBCs using ammonium-chloride-potassium chloride lysis buffer and washed
twice with cold MACS buffer (1% BSA in PBS with 2 mmol/l EDTA).
Washed cells were resuspended at 2 3 108 cells in 1 ml MACS buffer and
incubated with 100 ml biotinylated anti-Ly6C and Ly6G (Gr-1) Abs
(Miltenyi Biotec) for 20 min at 4˚C. Labeled splenocytes were then incubated at 4˚C with 100 ml streptavidin microbeads (Miltenyi Biotec) for
15 min. Cells were washed, resuspended in 5 ml MACS buffer, and applied
to a MACS column for positive selection, according to the manufacturer’s
instructions (Miltenyi Biotec). The purity of cell populations as analyzed
by flow cytometery was .95%. For functional assays, splenocytes from
OT-I mice were used as responder cells. CD8+ T cells from these mice have
a transgenic TCR that recognize the OVA 257–264 peptide. MDSC from naive,
tumor-bearing mice with or with out TBI treatment were cultured at different
ratios with 2 3 105 splenocytes from OT-I mice in the presence of control or
specific peptides. Cell proliferation was measured by [3H]thymidine uptake.
All experiments were performed in triplicate.
Functional assays
To test the function of Tregs from TBI-treated or untreated tumor-bearing
mice, a CD4 +CD25+ regulatory T cell isolation kit was used to isolate
Tregs on day 21 after B16 injection. For stimulator cells, T cell-depleted
splenic cells from C57BL/6 mice were used and cultured at 5 3 104 cells/
well in a 96 well plate coated with 0.25 mg anti-CD3. Naive CD4+CD252
T cells were cocultured at 5 3 104cells/well. CD4+CD25+ T cells from B16bearing mice treated with or without TBI were added at 5 3 104cells/well.
Naive T cells alone, stimulators alone, and inhibitory cells alone served as
controls. After 72 h of culture, proliferation was analyzed by the incorporation of [3H]thymidine during the final 6 h of culture. All experiments were
performed in triplicate.
Reactive oxygen species production
The oxidation-sensitive dye 29,79-dichlorofluorescein diacetate (DCFDA)
(Molecular Probes/Invitrogen, Eugene, OR) was used for the measurement
of reactive oxygen species production by MDSC (30). Cells were incubated
at room temperature in serum-free RPMI 1640 medium in the presence
of 3 mmol/l DCFDA with or without 300 nmol/l PMA for 30 min, washed
with PBS, and then labeled with anti-CD11b and Gr-1 Abs. After incubation
on ice for 20 min, cells were washed with PBS and analyzed using flow
cytometry.
Arginase activity
Arginase activity was measured in cell lysates as described previously (31).
Briefly, cells were lysed for 30 min with 100 ml 0.1% Triton X-100.
Subsequently, 100 ml 25 mM Tris-HCl and 10 ml 10 mM MnCl2 were added,
and the enzyme was activated by heating for 10 min at 56˚C. Arginine hydrolysis was conducted by incubating the lysate with 100 ml 0.5 M L-arginine (pH 9.7) at 37˚C for 2 h. The reaction was stopped with 900 ml H2SO4
(96%)/H3PO4 (85%)/H2O (1/3/7, v/v/v). Urea concentration was measured at
540 nm after addition of 40 ml of a-isonitrosopropiophenone (dissolved in
100% ethanol), followed by heating at 95˚C for 30 min.
NO production
Equal volumes of culture supernatants (100 ml) were mixed with Greiss
reagent (1% sulfanilamide in 5% phosphoric acid and 0.1% N-1-naphthylethylenediamine dihydrochloride in double-distilled water). After 10 min
of incubation at room temperature, the absorbance at 550 nm was measured
using a microplate plate reader (Bio-Rad, Hercules, CA). Nitrite concentrations were determined by comparing the absorbance values for the
test samples to a standard curve generated by a serial dilution of 0.25 mM
sodium nitrite.
Flow cytometry
Spleens were harvested under sterile conditions. Single-cell suspensions
were prepared, and red cells were removed using ammonium-chloridepotassium chloride lysing buffer. One million cells (splenocytes or MDSC)
were incubated for 30 min on ice in staining medium with relevant Abs for the
surface expression analysis according to the standard protocols. After
washing, the samples were analyzed using an LSRII (BD Biosciences), and
the data were analyzed using FlowJo software (Tree Star).
Generation of bone marrow-derived DC and Ag pulsing
Erythrocyte-depleted mouse bone marrow cells were cultured in CM supplemented with 20 ng/ml GM-CSF and 10 ng/ml IL-4 (R&D Systems,
Minneapolis, MN) as described previously (18). On day 5, cells were harvested by gentle pipetting and layered onto an Optiprep gradient (AxisShield, Oslo, Norway). The low-density cell interface was collected and
washed twice. Resulting DC were washed one time and resuspended at 1 3
106 cells/ml CM containing 20 ng/ml GM-CSF and 20 ng/ml IL-4. DC
were pulsed overnight with 10 mcg/ml of OVA 257–264 peptide (Invitrogen,
Carlsbad, CA).
Immunization schedule
MDSC blockade in TBI-treated mice in combination immunotherapy. A
total of 3 3 105 M05 tumor cells were injected s.c. in the left flank of
C57BL/6 mice. Three days later, mice received a sublethal dose (600 cGy)
of TBI administered by a [137Cs] gamma radiation source. Mice received
5 3 106 OT-I T cells/mouse on day 4 and two injections of DC pulsed with
OVA 257–264 peptide (1 3 106/ mouse/ vaccination) on days 4 and 10. Four
doses of DTX at 16 mg/kg were given i.p., (i.p.), every 3 d starting on day 4.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
Mice
TBI-INDUCED MDSC AND Tregs
The Journal of Immunology
5149
Control groups received T cells alone, DC alone, DTX alone, or PBS. Tumor
size was measured and recorded every 2 d. In another set of experiments,
splenocytes were harvested from these mice 1 wk after the final immunization for in vitro assays.
[51Cr] release cytotoxicity assay
A [51Cr] release assay was done as described previously (18). M05 cells
were used as targets. Spleens of treated mice were harvested on day 21,
and purified T cells were used as effector cells. T cell purity was measured
by flow cytometry and cells were 95% positive for CD90 (data not shown).
Briefly, target M05 cells were labeled with 100 mCi [51Cr] (Amersham
Biosciences) at 37˚C in a 5% CO2 atmosphere for 1.5 h. The labeled tumor
cells were washed three times and added to the effector cells in triplicate
wells of 96-well round-bottom microplates at 100:1, 50:1, and 25:1 E:T
ratios. After 5 h, the percentage of specific [51Cr] release was determined
by the following equation: ([experimental cpm 2 spontaneous cpm]/[total
cpm incorporated 2 spontaneous cpm]) 3 100. All determinations were
done in triplicate, and the SE of all assays was calculated and was typically
5% of the mean or less.
Cytometric bead array
Statistical analysis
A Mann–Whitney U test (unpaired) or a Student t test was used to compare
differences between two treatment groups. All statistical evaluations of
data were performed using GraphPad Prism software. Statistical significance
was achieved at p , 0.05.
Results
Reconstitution of immune subsets after lymphopenia
To evaluate the reconstitution of immune subsets after induction of
lymphopenia, naive and B16 tumor-bearing mice received 600 rad
TBI. We observed lymphopenia and leukopenia in both naive and
B16 tumor-bearing mice, evidenced by total splenocyte counts
post-TBI (data not shown). As shown in Fig. 1A, CD4+ and CD8+
T cells were detected on day 7 in the wild-type mice, with 60% of
CD4+ T cells reconstituted (percentage of normal) and 40% of
CD8+ T cells reconstituted by day 21. In B16 tumor-bearing mice,
30% reconstitution of CD4+ T cells and 20% CD8+ T cells was
observed (Fig. 1B). CD4+CD25+Foxp3+ Tregs were reconstituted to
70% in wild-type and 100% in B16 tumor-bearing mice by day 10.
Although, CD11b+Gr-1+ MDSC were reconstituted to 100% by day
10 in both wild-type and B16 tumor-bearing mice. Alternatively, the
cell numbers of splenic immune subsets per million of splenocytes
were calculated post-TBI are shown as mean 6 SD (n = 4) in Table I.
These findings demonstrate that in a lymphopenic environment,
MDSC and Tregs recover from lymphodepletion faster than CD4+
and CD8+ T cells, and this difference is further accentuated in tumorbearing mice, with proliferation resulting in higher inhibitory cell
frequency compared with basline by 21 d.
Repopulating Tregs are suppressive after TBI
Because Tregs and MDSC undergo rapid reconstitution after TBI,
we sought to determine the suppressive function on T cell proliferation. Tregs were purified from the spleens of B16 tumor-bearing
mice with or without TBI on day 21 posttumor injection. Inhibitor
alone, stimulator alone or responder alone served as controls. As
shown in Fig. 2A, CD4+CD25+ Tregs from the spleens of TBI-treated
B16 tumor-bearing mice were as suppressive as the CD4+CD25+
Tregs from tumor-bearing mice with no TBI treatment, whereas
FIGURE 1. Reconstitution of splenic immune subsets after TBI. Mice
received 1 3 105 B16 tumor cells on day 0, followed by 600 rad TBI on day
3. Spleen cells from wild-type (A) and B16 tumor-bearing (B) mice were
analyzed for CD4+, CD8+, CD11b+Gr-1+ MDSC, and CD4+CD25+Foxp3+
Tregs on days 3, 7, 10, 14, and 21. Data represent the percentage of normal
compared with splenocytes from mice not treated with TBI. Data are representative of three independent experiments (n = 4).
CD4+CD252 cells in the absence of CD4+CD25+ Tregs had significant proliferation (p , 0.01). This result shows that reconstituting
Tregs post-TBI are suppressive and behave similarly to endogenous
Tregs from tumor-bearing mice.
Repopulating MDSC are highly immunosuppressive after TBI
Next, we evaluated the function of TBI-induced MDSC. We purified Gr-1+ cells from the spleens of naive and tumor-bearing
mice that received TBI on day 21 posttumor injection. B16 tumorbearing mice with no TBI treatment were used as a control. MDSC
were cocultured with OT-I splenocytes at various ratios in the
presence of OVA257–264 peptide, and T cell proliferation was evaluated. OT-I cells alone and MDSC alone served as controls. We
observed that TBI-induced MDSC from tumor-bearing mice were
significantly more suppressive compared to the endogenous MDSC
from tumor-bearing mice that did not receive TBI treatment (Fig.
2B). Even at the lowest ratio of 1 MDSC:12 T cells, there was
significant (p , 0.01) suppressive activity, whereas the endogenous
MDSC from tumor-bearing mice were not able to inhibit T cell
proliferation. MDSC derived from TBI-treated naive mice (not
bearing tumor) did not show any suppressive activity compared with
control OT-I cells pulsed with peptide alone. To further analyze
suppressive activity, MDSC were added at various ratios to OT-I
splenocytes in the presence of OVA257–264 peptide, and IFN-g
production was evaluated. We observed significantly suppressed
IFN-g production by OT-I cells when cocultured with MDSC from
tumor-bearing mice with or without TBI at 1:3 ratios (p , 0.01;
Fig. 2C). However, at both 1:6 and 1:12 ratios, no suppressive activity was observed in MDSC from non–TBI-treated tumor-bearing
mice, whereas a significant reduction of IFN-g production was
observed in OT-I cells when cocultured with MDSC from TBItreated tumor-bearing mice. Collectively, these data show that repopulating MDSC post-TBI has enhanced immunosuppressive activity
of MDSC on a per cell basis.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
Briefly, splenocytes were plated at 2 3 106, cocultured with 2 3 105 irradiated M05 melanoma tumor cells, and incubated for 48 h. Culture
supernatants were analyzed for IFN-g production using commercially available cytometry bead array kit (BD Biosciences). Briefly, 25 ml mixed capture
beads were incubated with 25 ml culture supernatant and 25 ml PE detection
reagent for 2 h at room temperature. The immunocomplexes were then
washed and analyzed using a FACSCalibur affixed with a 488-nm laser (BD
Biosciences), according to the manufacturer’s protocol.
5150
TBI-INDUCED MDSC AND Tregs
Table I. Cell number (3104) per million of splenocytes
CD4
Naive
Pre-TBI
Post-TBI
Day3
Day 7
Day 10
Day 14
Day 21
CD8
B16
Naive
25 6 2.3
0.5
2.2
9.4
10.1
16.5
6
6
6
6
6
0.01
0.03
0.80
1.21
0.34
CD11bGr-1
B16
16.3 6 1.52
0.2
2.3
7.8
11.3
12.5
6
6
6
6
6
0.04
0.12
0.61
1.10
0.20
0.8
1.9
5.8
6.7
7.8
6
6
6
6
6
0.01
0.02
0.05
0.11
0.21
Naive
CD4CD25Foxp3
B16
2.5 6 0.31
0.6
3.5
6.5
4.5
4.9
6
6
6
6
6
0.02
0.01
0.21
0.80
0.12
0.1
0.9
2.5
2.9
3.8
6
6
6
6
6
0.02
0.01
0.31
0.21
1.14
Naive
B16
1.2 6 0.03
0.2
0.9
2.6
3.5
6.3
6
6
6
6
6
0.01
0.05
0.26
0.97
0.03
0.1
0.4
0.9
1.1
1.4
6
6
6
6
6
0.01
0.02
0.1
0.32
0.66
0.3
0.4
1.3
1.5
1.8
6
6
6
6
6
0.01
0.02
0.11
0.21
0.05
n = 4/group. Data compiled from three independent experiments.
DTX reduces reconstitution rate of MDSC post-TBI
Because MDSC from TBI-treated tumor-bearing mice demonstrated
enhanced suppressive activity, we wanted to evaluate whether we
FIGURE 2. The suppressive activity of reconstituted Tregs/MDSC (A) CD4+CD25+ cells were
purified on day 20 from B16 tumor-bearing mice
treated with or without TBI. T cell-depleted
splenic cells from C57BL/6 mice were used as
stimulators and were cultured at 5 3 104 cells/
well in a 96-well plate coated with 0.25 mg antiCD3. Naive CD4+CD252 T cells were cocultured
at 5 3 104 cells/well. CD4+CD25+ T cells were
added at 5 3 104 cells/well. After 72 h of culture,
proliferation was analyzed by the addition of
[3 H]thymidine during the final 6 h of culture.
All experiments were performed in triplicate. (B)
CD11b+Gr-1+ MDSC were purified using a MACs
column and were cultured at various ratios with
2 3 105 splenocytes from OT-I mice in the presence of OVA257–264 peptide. After 72 h of culture,
proliferation was analyzed by the addition of
[3H]thymidine during the final 6 h of culture. (C)
IFN-g production was measured in the supernatants after 48 h of coculture with a cytometric
bead array assay as described in Materials and
Methods. *p , 0.01.
could abrogate their reconstitution with DTX treatment. To evaluate the effect of DTX on reconstituting MDSC post-TBI, C57BL/6
mice were injected s.c. with B16 cells on day 0 and received 600 rad
TBI on day 3. Mice received four doses of DTX injected i.p. at
3 d or weekly intervals. Spleens were harvested on days 3, 7, 10, 14,
and 21 posttumor injection, and immune subsets were analyzed by
flow cytometry. As shown in Fig. 4A, B16 tumor-bearing mice that
received DTX had a significant reduction in TBI-induced MDSC,
regardless of whether they received the treatment on a weekly basis
or every 3 d (p , 0.05). By day 10, we observed a reduction in TBIinduced MDSC reconstitution in DTX-treated mice compared with
the TBI-treated tumor-bearing mice that did not receive DTX. By
day 21, mice that received DTX every 3 d had a significant reduction
in their MDSC reconstitution, up to 50% (p , 0.002) compared with
the untreated mice (250%), while we observed 100% reconstitution
in mice receiving the treatment on a weekly interval (p , 0.01).
Previous studies have shown that DTX treatment can increase
the number of CD4+ and CD8+ T cells (22, 27). To determine whether
DTX, given every 3 d, had an effect on reconstituting T cells, we
analyzed the CD4+ and CD8+ T cell reconstitution in TBI-treated
tumor-bearing mice after DTX treatment. As shown in Fig. 4B and
4C, we did not see any significant difference in reconstitution of
CD4+ and CD8+ on days 7, 10, 14, and 21 in mice that received DTX.
These data show that DTX given at 3-d intervals significantly reduced the MDSC reconstitution post-TBI without modulating T cell
populations.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
Next, we sought to understand the mechanism for the increased
suppressive activity. Several factors have been implicated in
MDSC-mediated immune suppression, including the production of
arginase, NO, and ROS (30–32). We compared the production of
these factors in MDSC from B16 tumor-bearing mice treated with
or without TBI. Gr-1+ cells were purified from the spleens of TBItreated or untreated tumor-bearing mice on day 21 posttumor injection. OT-I splenocytes were cultured at different ratios of MDSC
in the presence of OVA257–264 peptide for 48h. OT-I splenocytes
alone and MDSC alone served as controls. OT-I splenocytes cultured with different ratios of MDSC in the presence of OVA257–264
peptide had a significant increase in the NO production compared
with the endogenous MDSC from the tumor bearing mice (p ,
0.05; Fig. 3A). The oxidation-sensitive dye DCFDA was used for
the measurement of ROS production by MDSC and analyzed by
flow cytometry. As shown in Fig. 3B, there was an increased ROS
production in TBI-induced MDSC compared with endogenous
MDSC from tumor-bearing mice. Next, arginase activity in cell
lysates was also measured. We observed a similar trend as that seen
in the NO and ROS levels, with increased arginase activity in the
TBI-induced MDSC compared with the endogenous MDSC (p ,
0.01; Fig. 3C).
The Journal of Immunology
5151
Blockade of TBI-induced MDSC with DTX improves the
antitumor efficacy of adoptive T cell transfer and DC
immunotherapy
Next, we evaluated whether blocking TBI-induced MDSC with
DTX would improve the efficacy of adoptive T cell transfer and DC
immunotherapy. C57BL/6 mice were injected s.c. with M05 cells on
day 0 and received 600 rad TBI on day 3. Mice received 5 3 106 OT-I
T cells on day 4 and were vaccinated with DC pulsed with OVA257–264
peptide (DCOVA) on days 4 and 10. DTX was given every 3 d starting
on day 4 for a total of four doses. As shown in Fig. 5A, after four
doses of DTX treatment, a significant reduction in tumor size was
observed in mice that received OT-I T cells and DCOVA immunotherapy compared with mice that received only T cells and DCOVA
vaccination (p , 0.001). However, mice that received DTX treat-
ment with DCOVA or T cells alone had a significant reduction in
tumor growth compared with the untreated groups (p , 0.001;
Fig. 5B). These data suggest that blockade of TBI-induced MDSC
can improve the efficacy of adoptive T cell transfer and DC vaccination.
Blockade of TBI-induced MDSC enhances the persistence of
adoptively transferred T cells and improves CTL function
We next examined whether adoptively transferred T cells persisted
for a longer duration in mice treated with DTX. Ly5.2 mice were
injected with 3 3 105 M05 cells followed by TBI on day 3. Mice
received 5 3 106 OT-I (Ly5.1 mice) T cells on day 4. DCOVA vaccinations were given on days 4 and 10. Four doses of DTX were
given every 2–3 d beginning on day 4. Splenocytes were collected at
FIGURE 4. DTX treatment in combination with TBI reduces MDSC in tumorbearing mice. Mice were injected with
B16 tumor cells on day 0, followed by
TBI on day 3. Mice received DTX treatment on a weekly basis or every 3 d
starting on day 4. Spleen cells from mice
that received 600 rad alone or 600 rad +
DTX were analyzed for CD11b+Gr-1+
MDSC (A), CD4+ T cells (B), and CD8+
T cells (C) on days 3, 7, 10, 14, and 21
posttumor injection. Graphs represents
the percent of normal of CD11b+Gr-1+
MDSC, CD4+, and CD8+ T cells (n = 4/
group). *p , 0.01.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
FIGURE 3. TBI-induced MDSC express
high levels of NO, ROS, and arginase. (A) NO
production (data represent the mean 6 SD of
triplicates; *p , 0.05). OT-I cells (1 3 105
cells/well) were stimulated in triplicates for
48 h with OVA257–264 peptide in the presence
of different ratios of MDSC, and NO levels
were measured. (B) The levels of ROS production in MDSC from untreated or TBItreated mice bearing B16 tumor was measured
using DCFDA staining and flow cytometry.
*p , 0.05. Data represent one of the three independent experiments. (C) Arginase activity
was measured in untreated and TBI-treated
mice bearing B16 tumors. Mean 6 SD of two
independent experiments is shown. *p , 0.01.
5152
TBI-INDUCED MDSC AND Tregs
FIGURE 5. DTX treatment in combination with TBI
enhances the immunotherapeutic efficacy of adoptive
T cell transfer and DC vaccination. Mice received 3 3
105 M05 tumor cells on day 0, followed by TBI on day
3. On day 4, 5 3 106 OT-I T cells were adoptively
transferred followed by two doses of DC pulsed with
OVA 257–264 peptide on days 4 and 10. Four doses of
DTX treatment were given every 3 d starting from day
4. (A) Tumor growth. (B) Survival curve. *p , 0.001.
IFN-g production as a measure of T cell function. As shown in
Fig. 6B, restimulation with OVA peptide for 48 h resulted in higher
IFN-g production by splenocytes from mice that received DTX
treatment in combination with OT-I T cells and DCOVA vaccination
(980 6 35.4 pg/ml) compared with splenocytes from mice that
received OT-I T cells and DCOVA vaccination alone (597 6 30.4 pg/ml;
p , 0.01). These data suggest that blockade of TBI-induced MDSC
FIGURE 6. Addition of DTX treatment in combination with adoptive T cell transfer and DC vaccination increases the persistence OT-I T cells in mice.
Ly5.2 mice were injected with 3 3 105 M05 cells, followed by TBI on day 3. Mice received 5 3 106 OT-I (Ly5.1 mice) T cells on day 4. DCOVA vaccinations were given on days 4 and 10. Four doses of DTX were given every 2–3 d beginning on day 4. (A) The percentage of CD8+ and OVA tetramerpositive cells was measured by flow cytometry. (B) IFN-g production was measured in the splenocytes of treated mice. Splenocytes were plated at 2 3 106,
cocultured with 2 3 105 irradiated M05 cells, and incubated for 48 h. Culture supernatants were analyzed for IFN-g production using commercially
available cytometry bead array kit. Data shows the mean 6 SD of triplicates. *p , 0.01. (C) A 5-h [51Cr] release assay was performed using M05 tumor
cells as targets. Spleens of treated mice were harvested on day 21, and purified T cells were used as effector cells. Data show the mean 6 SE of triplicates
from a chromium release assay. *p , 0.05.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
various time points, and the percentage of OT-I T cells was measured by flow cytometry. Mice that received therapy with OT-I
T cells and DCOVA in combination with DTX had a higher percentage of OVA tetramer specific T cells on days 8, 15, and 20. As
shown in Fig. 6A, a higher percentage of CD8+ OVA-specific T cells
were observed in mice that received DTX treatment compared with
the mice that received immunotherapy alone. We next evaluated
The Journal of Immunology
with DTX enhanced the persistence and activity of adoptively
transferred T cells.
Next, we examined whether reduction of TBI-induced MDSC by
DTX enhances the function of CD8+ T cells. Splenic T cells from
mice receiving T cells and DCOVA, treated with or without DTX,
were purified on day 21 and cocultured with 51Cr-labeled M05
melanoma tumor cells at E:T ratios of 100:1, 50:1, or 25:1. T cells
from mice that received DCOVA and OT-I T cells in combination
with DTX treatment exhibited higher cytotoxicity against M05
melanoma tumor cells at a 100:1 ratio compared with mice that did
not receive DTX treatment (p , 0.05; Fig. 6C). However, no significant differences were measured at lower ratios. These data
suggest that blockade of TBI-induced MDSC with DTX improves
the CTL function in vaccinated mice against M05 melanoma tumor
cells.
Discussion
little ROS but high levels of NO (31). These suppressive factors have
been shown to have a direct effect on the inhibition of T cell
function (41–44). Although it has been shown in several studies that
MDSC use multiple factors such as NO, ROS, and arginase for their
suppressive function on T cells, this study shows that after the induction of lymphopenia, MDSC have increased production of these
factors compared with the endogenous MDSC from non–TBItreated tumor-bearing mice. It has been shown that various tumorderived factors define the expansion of these MDSC subsets.
However, in our study, we observed elevated levels of all of these
suppressive factors. It is possible that both monocytic and granulocytic MDSC are being reconstituted following lymphodepletion
and that each population contributes to increased immunosuppressive function. Studies to define the factors responsible for the rapid
expansion of these MDSC subsets in a lymphopenic environment
are ongoing.
MDSC frequency is increased in melanoma patients and is associated with disease progression (45, 46). Strategies to target
MDSC include inhibition of expansion or promotion of differentiation into mature cells that no longer possess suppressive activity
(21, 47). All-trans-retinoic acid has been shown to induce MDSC
differentiation that leads to neutralization of ROS production in
both mice and cancer patients (23, 48). Several studies have also
shown that MDSC can be directly eliminated using chemotherapeutic drugs such as gemcitabine, DTX, suntinib, or flurouracil
(20, 22, 23, 26, 28). Treatment of mice bearing large tumors with
chemotherapeutic drugs has been shown to result in dramatic reductions in the number of splenic MDSC and a marked improvement in immunotherapeutic efficacy (24, 25). To block rapidly
reconstituting MDSC in our model, we used DTX, a chemotherapeutic drug previously shown to block MDSC expansion (22). In
melanoma-bearing lymphopenic mice, DTX treatment decreased
MDSC reconstitution and increased the persistence of adoptively
transferred T cells, resulting in delayed tumor growth and enhanced
survival. These are the key findings that can potentially improve the
design of clinical trials in patients with melanoma.
Although lymphodepletion strategies are an attractive tool for
adoptive immunotherapy protocols, our results demonstrate that
MDSC and Tregs are rapidly reconstituted and are highly immunosuppressive after the induction of lymphopenia. It is possible that
the rapid reconstitution and suppressive functions of TBI-induced
MDSC and Tregs may limit the effectiveness of immunotherapy to
induce antitumor T cell responses, which in turn contribute to failure
at inducing complete tumor regressions. Blocking the reconstitution
of TBI-induced MDSC or Tregs enhanced antitumor immunity in
a murine model and may improve current adoptive therapy strategies
for the treatment of metastatic melanoma.
Acknowledgments
We thank Dr. Dmitry Gabrilovich for valuable comments during the preparation of this manuscript. We also thank the Flow Cytometry Core Facility
at the H. Lee Moffitt Cancer Center and Research Institute for contributions to this work.
Disclosures
The authors have no financial conflicts of interest.
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TBI-INDUCED MDSC AND Tregs