IMMUNOLOGY
ORIGINAL ARTICLE
S100A9 a new marker for monocytic human myeloid-derived
suppressor cells
Fei Zhao,1 Bastian Hoechst,2 Austin
Duffy,1 Jaba Gamrekelashvili,1
Suzanne Fioravanti,1 Michael P.
Manns,3 Tim F. Greten1 and
Firouzeh Korangy1
1
National Institutes of Health, National
Cancer Institute, Medical Oncology Branch,
Bethesda, MD, USA, 2Institutes of Molecular
Medicine and Experimental Immunology,
University of Bonn, Bonn, Germany, and
3
Departments of Gastroenterology, Hepatology
and Endocrinology, Hannover Medical School,
Hannover, Germany
doi:10.1111/j.1365-2567.2012.03566.x
Received 8 September 2011; revised 18
January 2012; accepted 27 January 2012.
Correspondence: F. Korangy and T. F.
Greten, National Institutes of Health,
National Cancer Institute, Building
10/12N226, 9000 Rockville Pike, Bethesda,
MD 20892, USA. Emails: firouzeh.korangy@
nih.gov, [email protected]
Senior author: Tim F. Greten and Firouzeh
Korangy.
Summary
Myeloid-derived suppressor cells (MDSC) are a heterogeneous population
of cells that negatively regulate the immune response during tumour progression, inflammation and infection. Only limited data are available on
human MDSC because of the lack of specific markers. We have identified
members of the S100 protein family – S100A8, S100A9 and S100A12 –
specifically expressed in CD14+ HLA-DR)/low MDSC. S100A9 staining in
combination with anti-CD14 could be used to identify MDSC in whole
blood from patients with colon cancer. An increase in the population of
CD14+ S100A9high MDSC was observed in the peripheral blood from
colon cancer patients in comparison with healthy controls. Finally, nitric
oxide synthase expression, a hallmark of MDSC, was induced in
CD14+ S100A9high upon lipopolysaccharide/interferon-c stimulation. We
propose S100 proteins as useful markers for the analysis and further characterization of human MDSC.
Keywords: cancer; flow cytometry/FACS; immune response; tolerance;
tumour immunology
Introduction
Myeloid-derived suppressor cells (MDSC) have been characterized as a population of cells that can negatively regulate T-cell function. They are a heterogeneous population
of myeloid origin including macrophages, granulocytes
and other cells, which suppress immune responses in vivo
as well as in vitro. An increase in the frequency of MDSC
in the peripheral blood of patients with different types of
cancers has been demonstrated.1,2 Murine MDSC are characterized by co-expression of Gr-1 and CD11b, and can be
further subdivided into two major groups: CD11b+
Gr-1high granulocytic MDSC (which can also be identified
as CD11b+ Ly-6G+ Ly6Clow MDSC) and CD11b+ Gr-1low
monocytic MDSC (which can also be identified as
CD11b+ Ly-6G) Ly6Chigh MDSC). We have previously
identified CD49d as another marker to distinguish these
176
two murine cell populations from each other.3 We could
demonstrate that CD11b+ CD49d+ monocytic MDSC were
more potent suppressors of antigen-specific T cells in vitro
than CD11b+ CD49d) granulocytic MDSC.
S100A9 has recently been reported to be essential for
MDSC accumulation in tumour-bearing mice. It was also
shown that S100A9 inhibits dendritic cell differentiation by
up-regulation of reactive oxygen species. Finally, no increase
in the frequency of MDSC was observed in S100A9 knockout mice, which also showed strong anti-tumour immune
responses and rejection of implanted tumours,4 indicating
the relevance of S100A9+ MDSC in tumour settings.
In contrast to murine MDSC, human MDSC are not so
clearly defined because of the lack of specific markers.
Human MDSC have been shown to be CD11b+, CD33+
and HLA-DR)/low. In addition, interleukin-4 receptor a,
vascular endothelial growth factor receptor, CD15 and
Published 2012. This article is a U.S. Government work and is in the public domain in the USA., 136, 176–183
S100 a new MDSC marker
CD66b have been suggested as more specific markers for
human MDSC. However, these markers can only be found
on some MDSC subsets.5 It has been suggested that monocytic MDSC are CD14+ 2,6 and granulocytic MDSC express
CD15,7,8 whereas both groups of MDSC are HLA-DR)/low
and CD33+. The heterogeneous expression of these markers
suggests that multiple subsets of human MDSC can exist.
We have previously shown direct ex vivo isolation of a
new subset of MDSC that are significantly increased in
the peripheral blood and tumours of patients with hepatocellular carcinoma. These cells express CD14, have low
or no expression of HLA-DR and have high arginase
activity. CD14+ HLA-DR)/low cells not only suppress the
proliferation of and interferon-c secretion by autologous
T cells, but also induce CD25+ Foxp3+ regulatory T cells
that are suppressive in vitro.9 Others have been able to
detect CD14+ cells with suppressor activity in the peripheral blood from patients with other malignancies such as
melanoma, colon cancer and head and neck cancer.8,10
We have been able to demonstrate their suppressor activity in patients with colon cancer (data not shown).
Although many studies have shown the presence of
human MDSC in different pathological conditions,
understanding their biology in human cancer requires
further characterization of these cells.
In this study, we searched for specific markers present on
human MDSC by performing a differential display analysis
on CD14+ HLA-DR+ monocytes and CD14+ HLA-DR)/low
MDSC. S100A12 was expressed more strongly in
CD14+ HLA-DR)/low MDSC than in CD14+ HLA-DR+
monocytes. Based on these results we analysed the expression of S100A8, S100A9 and S100A12 in CD14+ HLA-DR)/
low
MDSC in both whole blood and peripheral blood mononuclear cells (PBMC) from healthy volunteers and patients
with cancer. We demonstrated that the frequency of S100A9
MDSC correlated with the frequency of CD14+ HLA-DR)/
low
MDSC and we found an increase in the frequency of
CD14+ S100A9high MDSC in the peripheral blood from
patients with cancer. Finally, we demonstrate that CD14+
S100A9high cells expressed high levels of nitric oxide synthase (NOS2), which is one of the proposed mediators of
the inhibitory properties of MDSC. We therefore propose
S100A9 as an additional useful marker for human MDSC.
The PBMC were isolated as described above. CD14+ HLADR)/low and CD14+ HLA-DR+ cells were isolated using
CD14-MicroBeads (Miltenyi, Bergisch-Gladbach, Germany) followed by FACS sorting using a BD FACS Aria II
cell sorter (Becton-Dickinson). RNA extraction was performed using NucleoSpin RNA II (Macherey-Nagel,
Düren, Germany) followed by Linear T7-based amplification of the RNA. Gene expression analysis was performed
using a PIQOR Immunology Microarray (Miltenyi). RNA
isolation, amplification and Microarray were performed
by Miltenyi-Biotec. Microarray data were deposited in the
GEO database and the accession number is GSE32001.
Materials and methods
Flow cytometry analysis
Blood samples
Blood samples were collected from patients with colon
cancer and healthy controls. None of the patients were
receiving chemotherapy at the time of blood collection. All
patients gave written informed consent for research testing
under protocols approved by the Institutional Review
Board of the National Cancer Institute, National Institutes
of Health. Patient information is summarized in Table 1.
Table 1. Patient characteristics
Characteristics
Value
Total number
Average age, years
Male/female
Average age, years (male/female)
Stage IV (male/female)
14
614
9/5
618/608
8/5
Cell isolation and sorting
Human PBMC were isolated from freshly obtained blood
by Ficoll density gradient centrifugation (Lonza, Walkersville, MD). Whole blood lysate was obtained by lysing
whole blood with ACK Lysing Buffer (Quality Biological,
Gaithersburg, MD) as the manual indicated. MDSC
(CD14+ HLA-DR)/low) and control monocytes (CD14+
HLA-DR+) were sorted from PBMC using BD FACSAria
II cell sorter (Becton-Dickinson, Mountain View, CA).
The gating strategy is shown in Supplementary material,
Fig. S1. CD4, CD8, B cells and dendritic cells were sorted
by CD3+ CD4+, CD3+ CD8+, B220+ and CD11c+ (BD
Biosciences, San Jose, CA) markers, respectively. The purity of the cells after sorting was > 95%. Granulocytes for
the Western blotting were obtained by lysing the red
blood cell pellet after the Ficoll density gradient centrifugation with ACK Lysing buffer.
Microarray analysis
The following antibodies were used in the FACS staining:
CD14-Vioblue (Miltenyi Biotec GmbH, Bergisch
Gladbach, Germany), HLA-DR-allophycocyanin (BD Biosciences), S100A9-FITC (Biolegend, San Diego, CA),
NOS2-phycoerythrin (Santa Cruz Biotechnology, Santa
Cruz, CA). Freshly obtained PBMC or whole blood lysate
were resuspended in flow cytometry buffer and surfacestained with antibodies for 15 min at 4°C. Samples for
intracellular staining were additionally fixed and permeabi-
Published 2012. This article is a U.S. Government work and is in the public domain in the USA., 136, 176–183
177
F. Zhao et al.
lized using BD Cytofix/Cytoperm Fixation/Permeabilisation Kit (BD Biosciences) according to the manufacturer’s
instructions. FACS acquisition was performed on LSR-II
(Becton-Dickinson) and results were analysed using
FLOWJO software (TreeStar Inc, Ashland, OR).
Determination of mRNA expression by quantitative PCR
RNA was isolated using an RNeasy Micro Kit (Qiagen,
Hilden, Germany). Complementary DNA synthesis was
carried out with an iScript Kit (Bio-Rad, Munich, Germany) and quantitative PCR was performed using the following primers: S100A12: forward primer 50 -CAC ATT
CCT GTG CAT TGA GG-30 , reverse primer 50 -TGC AAG
CTC CTT TGT AAG CA-30 ; S100A8: forward primer 50 TGT CTC TTG TCA GCT GTC TTT CA-30 , reverse primer 50 -CCT GTA GAC GGC ATG GAA AT-30 ; S100A9:
forward primer 50 -GGA ATT CAA AGA GCT GGT GC30 , reverse primer 50 -TCA GCA TGA TGA ACT CCT CG30 ; cyclophilin A: forward primer 50 -ATG CTC AAC CCC
ACC GTG T-30 , reverse primer 50 -TCT GCT GTC TTT
GGG ACC TTG TC-30 . Reactions were performed in triplicate using iQ SYBR Green Supermix (Bio-Rad) and normalized to endogenous cyclophilin A mRNA level using
the DDCt method.
CD14+ S100A9high cells correspond to CD14+
HLA-DR)/low MDSC
Western blot analysis
Lysates from FACS sorted CD14+ HLA-DR)/low MDSC
and CD14+ HLA-DR+ monocytes were denatured at 95°
for 5 min and subjected to SDS–PAGE. The gel was blotted onto nitrocellulose membrane followed by incubation
with anti-S100A12 antibody (Abcam, Cambridge, UK) or
a control anti-glyceraldehyde 3-phosphate dehydrogenase
antibody (Sigma, St Louis, MO). Binding of the antibodies was visualized using horseradish peroxidase-conjugated rabbit anti-mouse IgG (Abcam). Western blot
imaging and quantitative analysis were performed using
FluorChem HD2 Multiplex Fluorescent Imaging System
(Cell Biosciences Inc., Santa Clara, CA).
Statistical analysis
All the statistical analyses were based on two-tailed Student’s t-test. All P-values < 005 were considered to be
significant.
Results
S100A8/A9/A12 is differentially expressed in
CD14+ HLA-DR)/low MDSC and CD14+ HLA-DR+
monocytes
Differential gene expression analysis was performed to
identify genes expressed in CD14+ HLA-DR)/low MDSC
178
but not in CD14+ HLA-DR+ monocytes. Using PIQOR
Immunology Microarrays (Miltenyi), we found that
S100A12 was 40-fold more strongly expressed in MDSC
than in monocytes (GEO database accession no.
GSE32001). Real time PCR was performed on FACS-sorted
MDSC (CD14+ HLA-DR)/low) and monocytes (CD14+
HLA-DR+) from peripheral blood to confirm these results.
Higher S100A12 expression was seen in MDSC than in
monocytes (Fig. 1a). S100 is a family of proteins including
21 calcium-binding proteins.11 Among them, S100A8,
S100A9 and S100A12 are closely related. We focused on
these three proteins because monoclonal antibodies for
FACS and Western blotting were available for them. First,
we analysed the expression of S100A8 and S100A9 genes in
the PBMC of healthy donors. Both S100A8 and S100A9
were about 10-fold to 15-fold more expressed in MDSC
than in monocytes (Fig. 1a). CD4+ and CD8+ T cells, as
well as B cells and dendritic cells, were used as controls
and no relevant expression of S100A8, S100A9 or S100A12
was found in these cells. The higher expression of S100 in
MDSC was further confirmed by Western blot analysis in
which S100A12 expression was seen in MDSC from several
healthy donors and in patients with colon cancer but not
in monocytes (Fig. 1b–e).
Next, we analysed S100A9 and HLA-DR expression in
CD14+ cells in PBMC or whole blood of healthy controls.
CD14+ S100A9high and CD14+ S100A9low cells from whole
blood and PBMC were analysed for HLA-DR expression.
As shown in Fig. 2(a), S100A9 expression was higher in
CD14+ HLA-DR)/low MDSC than in CD14+ HLA-DR+
monocytes. Correspondingly, CD14+ S100A9high cells
expressed less HLA-DR than CD14+ S100A9low cells
(Fig. 2b). Mean fluorescence intensity (MFI) of S100A9
or HLA-DR was also analysed. Both PBMC and whole
blood lysate showed higher S100A9 expression in
CD14+ HLA-DR)/low MDSC (MFI 5736 ± 1525 in whole
blood and 17236 ± 3171 in PBMC; P < 005) than in
CD14+ HLA-DR+ monocytes (MFI 1728 ± 289 in whole
blood and 11420 ± 2014 in PBMC; Fig. 2c). This difference was statistically significant when cells were analysed
from whole blood. Next, we also compared HLA-DR
expression on CD14+ S100A9low and CD14+ S100A9high
cells from whole blood. HLA-DR MFI was lower on
CD14+ S100A9high than on CD14+ S100A9low cells (MFI
1875 ± 158 versus 5947 ± 1019; P < 0001). Similar
results were seen when HLA-DR expression was tested on
CD14+ S100A9high or CD14+ S100A9low PBMC (2030 ±
291 versus 4231 ± 727; P < 005; Fig. 2d).
As MDSC are increased in patients with different types
of cancer, we next tested PBMC and whole blood from
patients with colon cancer. Peripheral blood from 14 ran-
Published 2012. This article is a U.S. Government work and is in the public domain in the USA., 136, 176–183
S100 a new MDSC marker
Donor 2
Donor 3
D
BM 14C
SC
M
D
C
o
M
on
SC
M
D
M
on
o
SC
M
D
M
on
o
S100A12
2
ra
n
Donor 1
**
G
(b)
3
S100A12
10 k
GAPDH
37 k
1
ra
n
G
o
on
M
M
D
ra
n
G
o
on
M
SC
D
M
20
Pat. 2
SC
Pat. 1
C
D
PB 14
M C
(c)
S100A8
**
C
PB D14
M C
0
30
S100A12
10 k
GAPDH
37 k
10
0
**
(d)
S100A9
S100A12
50
25
2·0
(e)
1·5
*
1·5
S100A12
75
1·0
0·5
1·0
0·5
C
D
8
B
4
D
C
D
C
on
M
D
M
o
0
SC
Relative mRNA level
Relative mRNA level
Relative mRNA level
(a)
0
Mono MDSC
CD14PBMC
0
Gran
Mono
MDSC
Pat. 1
CD14PBMC
Gran
Pat. 2
Figure 1. Myeloid-derived suppressor cells (MDSC) express high amounts of S100A8, S100A9 and S100A12. (a) CD14+ HLA-DR)/low MDSC,
CD14+ HLA-DR+ monocytes (mono) and control cell populations (CD4+ T cells, CD8+ T cells, B cells and dendritic cells) were sorted from
peripheral blood mononuclear cells (PBMC) of healthy donors. Expression level of S100A8, S100A9 and S100A12 are examined by quantitative
real-time PCR. Expression is set relative to cyclophilin A mRNA. Cumulative results of four independent experiments are shown (**P < 0001).
(b, c) S100A12 expression was tested by Western blot analysis on 105 sorted MDSC and monocytes (Mono) of healthy donors (b) or colon cancer patients (c). CD14) PBMC were used as negative and granulocytes (Gran) as positive controls, respectively. (d, e) Quantitative bioluminescence analysis for S100A12 expression. S100A12 protein concentration is shown as a ratio of S100A12 to glyceraldehyde 3-phosphate
dehydrogenase. The S100 expression in cells derived from healthy donors (n = 3) (d) and colon cancer patients (n = 2) (e) is shown (*P < 005).
domly selected patients with colon cancer (Table 1) was
analysed. Similarly, CD14+ HLA-DR)/low MDSC showed
higher S100A9 expression than CD14+ HLA-DR+ monocytes both in whole blood lysate (3350 ± 398 versus
2097 ± 228; P < 005) and PBMC (34355 ± 9520 versus 21137 ± 6175; Fig. 3a). The CD14+ S100A9high cells
showed lower HLA-DR expression than CD14+ S100A9low
cells (2382 ± 233 versus 4303 ± 702 for whole blood
and 1532 ± 268 versus 3116 ± 619 for PBMC; P < 005
for both; Fig. 3b).
Next, we analysed whether the frequency of
CD14+ S100A9high cells in the peripheral blood of healthy
donors and cancer patients correlates with the frequency
of CD14+ HLA-DR)/low MDSC. We have previously
shown that CD14+ HLA-DR)/low cells are significantly
increased in the peripheral blood and tumours of patients
with cancer.9 As shown in Fig. 4, the frequency of CD14+
S100A9high cells correlated with that of CD14+ HLA-DR)/
low
cells in both healthy donors and cancer patients. Similar to the increase in CD14+ HLA-DR)/low cells, there was
also a significant increase in CD14+ S100A9high cells in
the peripheral blood of cancer patients as compared with
healthy donors.
NOS2 expression in CD14+ S100A9high cells
As S100A9 is an intracellular protein, it was not possible
to sort these cells to test their suppressor function. However, it has been shown that MDSC suppress T-cell function by Arginase-1 and NOS2-dependent mechanisms.
We therefore tested CD14+ S100A9high cells for expression
of NOS2 in cancer patients. Whole blood lysate was stimulated with lipopolysaccharide and interferon-c before
expression of NOS2 was analysed. Upon lipopolysaccharide and interferon-c stimulation, a significant induction
of NOS2 was observed both in CD14+ HLA-DR)/low as
well as in CD14+ S100A9high cells (Fig. 5a,b). The MFI of
NOS2 was increased in both CD14+ S100A9high and
CD14+ S100A9low cells (10037 ± 2363 versus 2097 ±
128; P < 005) and CD14+ HLA-DR)/low MDSC versus
CD14+ HLA-DR+ monocytes (6300 ± 500 versus 2220
± 250; P < 005; Fig. 5c,d).
Published 2012. This article is a U.S. Government work and is in the public domain in the USA., 136, 176–183
179
F. Zhao et al.
(a)
(b)
HLA-DR
HLA-DR
WB
PBMC
S100A9
(d) 800
*
2000
HLA-DR (MFI)
S100A9 (MFI)
(c) 2500
S100A9
1500
1000
500
0
PBMC
CD14+HLA-DR+
CD14+HLA-DR–/low
HLA-DR
**
*
600
400
200
0
WB
S100A9
WB
PBMC
CD14+S100A9low
CD14+S100A9high
Figure 2. Combined S100A9, CD14 and HLA-DR analysis on peripheral blood mononuclear cells (PBMC) and whole blood cells from healthy
donors. Freshly isolated PBMC or whole blood lysate (WB) from healthy donors were stained with CD14, HLA-DR and S100A9. Gates were set
on the upper/lower 10% of S100A9/HLA-DR cells. (a, c) S100A9 expression compared between CD14+ HLA-DR)/low myeloid-derived suppressor
cells (MDSC) and CD14+ HLA-DR+ monocytes. Representative FACS data (a) and average mean fluorescence intensity (MFI) analysis from nine
healthy controls (c) are shown. *P < 005. (b, d) HLA-DR expression is compared between CD14+ S100A9high and CD14+ S100A9low cells. Representative FACS data (a) and average MFI analysis (c) are shown. *P < 005, **P < 0001.
Discussion
Numerous studies have shown the existence of counterregulatory immune mechanisms in patients with cancer.
One of the recently identified mechanisms involves the
recruitment of the heterogeneous population of MDSC.
These cells have been widely studied in different mouse
and human cancer models.12 We have previously reported
the accumulation of CD14+ HLA-DR)/low MDSC in
patients with hepatocellular carcinoma. These cells suppressed T cells and natural killer cells directly and could
also suppress T-cell responses indirectly by inducing regulatory T cells.9,13,14 However, their heterogeneous nature
and lack of a specific marker that clearly defines these
cells limits the full understanding of the biology of
MDSC.
Murine MDSC have been divided into two major
groups: CD11b+ Gr-1high granulocytic MDSC (also
CD11b+ Ly-6G+ Ly6Clow MDSC) and CD11b+ Gr-1low
monocytic MDSC (which can also be identified as CD11b+
Ly-6GLy6Chigh MDSC).15,16 We have previously identified
CD49d as another marker on murine MDSC, which distinguishes these two cell populations from each other. We
have also shown that monocytic CD11b+ CD49d+ MDSC
were more potent suppressors of antigen-specific T cells in
vitro than CD11b+ CD49d) granulocytic MDSC and sup180
pressed T-cell responses through a nitric oxide-mediated
mechanism.3
Limited data are available on the biology of MDSC in
human diseases and their interpretation is complicated by
the different markers that have been used to analyse
human MDSC subtypes in various clinical settings.17
Most studies concur with the observation that MDSC
express CD11b and CD33 but lack the expression of
markers of mature myeloid cells such as CD40, CD80,
CD83 and HLA-DR. Both CD14+ HLA-DR)/low and
CD14) CD15+ HLA-DR)/low MDSC have been described5
and molecules such as interleukin-4 receptor-a and vascular endothelial growth factor receptor have been used as
additional markers.18 However, these markers cannot be
used to distinguish HLA-DR)/low MDSC from HLA-DR+
monocytes.
Differential expression analysis of CD14+ HLA-DR)/low
MDSC and CD14+ HLA-DR+ monocytes revealed
S100A8, S100A9 and S100A12 as new markers in MDSC.
The over-expression of S100 in MDSC was confirmed by
real-time PCR as well as by Western blot and FACS analysis. Members of the S100 family of calcium-binding proteins play essential roles in epithelial tissues and
participate in a wide range of cellular processes, including
transcription, proliferation and differentiation.19,20
S100A8, S100A9 and S100A12 are specifically linked to
Published 2012. This article is a U.S. Government work and is in the public domain in the USA., 136, 176–183
S100 a new MDSC marker
*
*
3500
25
2000
400
20
A9
00
S1
HLA-DR (MFI)
400
200
0
WB
PBMC
+
high
CD14 S100A9
CD14+S100A9low
Figure 3. Combined S100A9, CD14 and HLA-DR analysis on
peripheral blood mononuclear cells (PBMC) and whole blood cells
from patients with colon cancer. Fresh isolated PBMC or whole
blood lysate from 10 colon cancer patients are stained with CD14,
HLA-DR and S100A9. Gates are set on the upper/lower 10% of
S100A9/HLA-DR cells. (a) S100A9 expression is compared between
CD14+ HLA-DR)/low MDSC (grey bar) and CD14+ HLA-DR+
monocytes (white bar). *P < 005. (b) HLA-DR expression is compared between CD14+ S100A9high (grey bar) and CD14+ S100A9low
(white bar) cells. *P < 005.
innate immune functions by their expression in cells of
myeloid origin.21 S100A8 and S100A9 are found in granulocytes, monocytes and the early differentiation stages of
macrophages. Their expression can also be induced in
keratinocytes and epithelial cells under inflammatory conditions. In contrast, S100A12 is restricted more to granulocytes.22 S100 proteins are related to pro-inflammatory
mechanisms and a significant over-expression can be
found at sites of inflammation.23 These proteins have
been shown to exert their pro-inflammatory activity
through receptor for advanced glycation end products
(RAGE).24 Interestingly, Gebhardt et al. have demonstrated that RAGE-deficient mice are resistant to DMBA
(7,12-dimethyl-benz[a]anthracene)/TPA (12-O-Tetradecanoylphorbol-13-Acetate)-induced skin carcinogenesis and
exhibit a severe defect in sustaining inflammation during
the promotion phase, indicating a pivotal role for S100/
RAGE in promoting inflammation-induced carcinogenesis.25 S100A8 and S100A9 are reportedly up-regulated in
many cancers, including colon cancer,26 and have been
implicated in the regulation of tumour cell proliferation
w
+
14
CD
–/
lo
+
+
w
14 –/lo
14
CD DR
CD
LA
H
Healthy
DR
*
*
5
A-
CD14+HLA-DR+
CD14+HLA-DR–/low
10
HL
PBMC
WB
gh
0
15
hi
200
(b) 600
*
30
% MDSC
S100A9 (MFI)
(a) 5000
+
h
14 hig
CD A9
00
S1
Cancer patients
Figure 4. Frequency of CD14+ S100A9high cells corresponds to the
myeloid-derived suppressor cell (MDSC) frequency defined by
CD14+ HLA-DR)/low cells in whole blood lysate. Frequencies of
CD14+ S100A9high cells and CD14+ HLA-DR)/low MDSC were analysed and calculated in whole blood lysate from healthy donors and
colon cancer patients. Each pair of dots indicates the frequency of
CD14+ S100A9high cells and CD14+ HLA-DR)/low MDSC from the
same blood donor. The granulocytes were excluded based on forward
and side scatter analysis. *P < 005.
and metastasis. Murine MDSC have been shown to
secrete S100A8/A9 proteins and blocking of the binding
of S100A8/A9 to MDSC reduces MDSC levels in blood.27
Multiple suppressor functions still remain as the major
hallmark of MDSC. NOS2 and arginase-1 are both strongly
expressed in MDSC and have been shown to be responsible
for immune suppression. Because S100 is an intracellular
protein we could not use this marker for direct isolation of
cells followed by functional analysis. Instead, we were able
to demonstrate that CD14+ S100A9high but not
CD14+ S100A9low cells expressed NOS2 in response to
lipopolysaccharide and interferon-c stimulation, suggesting
that S100A9 can specifically identify MDSC and distinguish
them from CD14+ HLA-DR+ monocytes.
It should be noted that S100 family members are all
intracellular proteins, which makes it impossible to use
this marker to isolate MDSC and use them in functional
studies. Therefore, we were able to provide indirect data
indicating that CD14+ S100A9high cells are MDSC. In
addition, our data clearly demonstrated a better discrimination between MDSC and non-MDSC when MDSC in
whole blood were analysed for S100A9 expression. Therefore, we would suggest using this marker when whole
blood is available for the analysis of MDSC.
In summary, we describe S100A9, as a new marker in
MDSC that can be used to identify CD14+ MDSC.
S100A9 can be used instead of or in combination with
HLA-DR staining. The latter has never been a precise
Published 2012. This article is a U.S. Government work and is in the public domain in the USA., 136, 176–183
181
F. Zhao et al.
(a)
(b)
LPS
+
S100A9
HLA-DR
IFN-γ
NO stim
NOS2
NOS2
(d) 800
*
NOS2 (MFI)
NOS2 (MFI)
(c) 1500
1000
500
0
NOS2
CD14+
S100A9high
CD14+
S100A9low
*
600
400
200
0
CD14+
HLA-DR–/low
marker for the identification of MDSC. Future studies
are needed to examine the role of S100A8, S100A9 and
S100A12 in other human MDSC subtypes with the aim
of further characterization of these cells. This will help
further our understanding of their mechanism of action
and help to target them for immunotherapeutic
approaches.
Acknowledgements
This research was supported (in part) by the Intramural
Research Program of the National institutes of Health,
National Cancer Institute, Center for Cancer Research.
This work was supported by a grant to MPM from the
Initiative and Networking Fund of the Helmholtz Association within the Helmholtz Alliance on Immunotherapy
of Cancer. We would like to thank the Experimental
Transplantation and Immunology Branch cell sorting
facility for technical assistance with cell sorting.
Disclosures
None of the authors have any financial conflict of interest.
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Additional Supporting Information may be found in the
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Figure S1. Peripheral blood mononuclear cells were
isolated by Ficoll density gradient and stained for CD14
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Please note: Wiley-Blackwell is not responsible for the
content or functionality of any supporting materials supplied by the authors. Any queries (other than missing
material) should be directed to the corresponding author
for the article.
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