Yonago Acta medica 2011;54:1–10

Increased B7-H1 and B7-H4 Expressions on Circulating
Monocytes and Tumor-Associated Macrophages are Involvedin Immune Evasion in Patients with Gastric Cancer
Tomoyuki Matsunaga, Hiroaki Saito and Masahide Ikeguchi
Division of Surgical Oncology, Department of Surgery, School of Medicine, Tottori University
Faculty of Medicine, Yonago 683-8504, Japan

Expression of the costimulatory molecule B7-H4, a member of the inhibitory B7 family,
has been reported to be upregulated on tumor-associated macrophages, and this overexpression may be involved in immune evasion in cancer patients. The present study was
designed to investigate B7-H4 expression on monocytes/macrophages and its relationshipwith immune evasion in gastric cancer patients. B7-H4 expression on circulating monocytes and tumor-associated macrophages was evaluated by multicolor flow cytometry.
Carboxyfluorescein succinimidyl ester proliferation assays and quantitative interferongamma enzyme-linked immunosorbent assays were carried out to determine the inhibitory
effect of B7-H4+ monocytes on CD4+ T cells. B7-H4 expression on circulating monocytes
was upregulated and these B7-H4+ monocytes showed immunosuppressive properties. B7H4 expression was closely related to the depth of invasion, as well as the presence of lymphatic and venous invasion. There were significant correlations between B7-H4 expression
and B7-H1 or HLA-DR expression on monocytes/macrophages in gastric cancer patients.
B7-H4 expression was remarkably upregulated in gastric cancer tissues compared with peripheral blood samples. Complete surgical removal of the tumor decreased B7-H4 expression on circulating monocytes from gastric cancer patients. Cocultures of gastric cancer
cell lines and monocytes led to upregulation of B7-H4 expression on monocytes. Increased
B7-H4 expression on circulating monocytes and tumor-associated macrophages may be
one of the key mechanisms responsible for immune evasion by tumors in gastric cancer.
Key words: B7-H1; B7-H4; gastric cancer; macrophage; monocyte

The last 2 decades of research in the cancer immunology field have revealed that the majority of cancer cells express antigens that can be recognized by
T cells of the host immune system. However, despite the expression of these antigens, spontaneous
immune-mediated rejection of cancer cells seems
to be a rare event. The failure of spontaneous
immune-mediated tumor elimination can generally
be explained by poor T-cell priming or inadequate

execution of the effector phase of the immune response. In each of these contexts, potential roles
for T-cell costimulatory and coinhibitory receptors
have been suggested.
T lymphocytes are thought to play an important role in the control of tumors as a result of theircytotoxic activity and by releasing soluble factors.
Optimal activation of T cells requires at least 2
signals, namely antigen recognition and costimula-

Abbreviations: CFSE, carboxyfluorescein succinimidyl ester; ELISA, enzyme-linked immunosorbent assay; FACS, fluorescence-activated cell sorting; IFN, interferon; IL, interleukin; MHC, major histocompatibility complex; NF, nuclear
factor; PBMC, peripheral blood mononuclear cell; TAA, tumor-associated antigen; Treg cell, regulatory T cell1

T. Matsunaga et al.

small-cell lung cancer (Sun et al., 2006), ductal andlobular breast cancer (Salceda et al., 2005; Tringler
et al., 2005) and renal cell carcinoma (Krambeck etal., 2006). Moreover, Kryczek et al. demonstrated
that human ovarian cancer-associated B7-H4+
macrophages inhibited TAA-specific T-cell immunity, in part through B7-H4 (Kryczek et al., 2006).
These accumulating lines of evidence suggest thepossibility that inhibitory B7 family members,
including B7-H1 and B7-H4, are closely related toimmune evasion in cancer patients.
Gastric cancer is one of the most common
cancers in Asia and its mortality rate still ranks
second among all cancer deaths worldwide (Parkin
et al., 2005). Despite the expression of tumor rejection antigens such as MAGE 1–3 (Inoue et al.,1995) and the presence of tumor-specific cytotoxic
T cells (Hoshino et al., 1997), the immune systemfails to exhibit immune responses against gastriccarcinoma cells, similar to the case for other cancer
cells. However, the mechanisms for how gastriccancer cells overcome the antitumor immunological responses are poorly understood. Therefore, we
undertook the present study to determine the expression of B7-H4 as well as B7-H1 on circulating
monocytes and tumor-associated macrophages toclarify one of the possible mechanisms responsiblefor immune evasion in gastric cancer patients.

tion. T cell receptor complexes, which recognize
antigenic peptides presented by major histocompatibility complex (MHC) molecules, are criticalfor maintaining the specificity of the immune response. Costimulation is an antigen-independent
signal required for sustained cell proliferation,effector/memory cell generation and prevention ofanergy or apoptosis, and can be either positive or
inhibitory. Positive costimulation can enhance T
and B cell activation, while inhibitory costimulation can just as strongly attenuate these responses.
The final outcome of an immune response is likely
to depend on the balance between these positive
and negative signals (Driessens et al., 2009). The
expression of costimulatory molecules is restricted
to professional antigen-presenting cells, such as
dendritic cells, macrophages and monocytes. The
B7 family comprises such costimulatory moleculesand consists of both stimulatory and inhibitory
molecules (Carreno and Collins, 2002; Greenwald
et al., 2002; Liang and Sha, 2002; Chen, 2004;
Driessens et al., 2009). Signals mediated by theB7 family members result in T-cell suppression
or activation in different settings. B7-1 and B7-2
are 2 identified stimulatory B7 family members,
while B7-H1 and B7-H4 are 2 identified inhibitory
B7 family members (Chen, 2004). Since the final
outcome of an immune response is likely to dependon the balance between these positive and negative
signals, it is possible that overexpression of inhibitory B7 family members induces immunosuppression through the suppression of T cell function. In
fact, previous studies have demonstrated that bothovarian cancer and ovarian cancer-associated myeloid dendritic cells express B7-H1 and that these
B7-H1+ cancer cells and myeloid dendritic cells
reduce tumor-associated antigen (TAA)-specific Tcell immunity through distinct mechanisms (Donget al., 2002; Curiel et al., 2003). With regard to
B7-H4, several research groups have shown thathuman ovarian cancers express high levels of B7H4 protein (Choi et al., 2003; Salceda et al., 2005;
Bignotti et al., 2006; Kryczek, Zou et al., 2006;
Simon et al., 2006; Tringler et al., 2006). In addition, high levels of B7-H4 were detected in non-Materials and Methods
Patients and normal donors
Forty-three patients (29 males and 14 females) who
were treated at Tottori University Hospital andpathologically diagnosed as gastric cancer were enrolled in this study. None of the patients received
radiotherapy, chemotherapy or other medical interventions before surgery. The patients’ characteristics are shown in Table 1. Healthy controls (n = 18;
13 males and 5 females) who were treated at Tottori
University Hospital and diagnosed as inguinal hernia or cholelithiasis were age-matched (67.9 ± 10.9
years for the controls versus 67.6 ± 8.9 years for the2

B7-H4 on CD14+ cells in gastric cancer

Table 1. B7-H4 expression on circulating monocytes and clinicopathologic characteristics ingastric cancer patients
Number B7-H4
of patients		
Histopathology*
DifferentiatedUndifferentiatedDepth of invasion†
T1 (early)
T2/T3/T4 (advanced)
Lymph node metastasis
AbsentPresent
Lymphatic invasion
AbsentPresent
Vascular invasion
AbsentPresent
Stage
I
II/ III/ IV

Isolation of tumor-infiltrating lymphocytes
Freshly excised tumor tissues were minced andthen digested with 1.5 mg/mL of collagenase D(Wako Pure Chemical Industries, Osaka, Japan).
The resulting cell suspensions were filtered througha mesh filter (BD Falcon, Franklin Lakes, NJ).

P value

1825

3.5 ± 2.2
6.1 ± 5.7

0.255

23
20

2.9 ± 2.1
7.4 ± 5.7

0.000830
134.4 ± 4.6
6.3 ± 4.9

0.098

9
34

2.4 ± 1.4
5.7 ± 5.1

0.03

12
31

2.4 ± 1.3
6.1 ± 5.2

0.015

31
12

5.2 ± 4.8
4.4 ± 4.9

0.44

Flow cytometry analysis
Fluorescence-activated cell sorting (FACS) analysis was performed using a FACSCalibur (Becton
Dickinson, Franklin Lakes, NJ). The following antibodies were used to classify the cells:
anti-CD3-PE-Cy5, anti-CD14-FITC, anti-HLADE-PE, anti-B7-H1-PE and anti-B7-H4-PE (BD
PharMingen, Franklin Lakes, NJ).
Measurement of IFN-gamma concentrations* Differentiated, papillary or tubular adenocarcinoma;
undifferentiated, poorly differentiated, mucinous adenocarcinoma or signet-ring cell carcinoma.
† T1, tumor has invaded the lamina propria or submucosa;
T2, tumor has invaded the muscularis propria or subserosa; T3, tumor has penetrated the serosa; T4, tumor has
invaded adjacent organs.

The interferon (IFN)-gamma concentrations were
measured by enzyme-linked immunosorbent assay(ELISA) using a Human IFN-gamma ELISA Kit(R&D Systems, Minneapolis, MN).
Media
A complete culture medium consisting of RPMI
1640 (Gibco-BRL, Grand Island, NY), 1% lglutamine (Invitrogen, Carlsbad, CA), 1% penicillin/streptomycin (Invitrogen), 2.5% HEPES buffer
solution (Gibco-BRL), 1% sodium pyruvate (SigmaAldrich, St. Louis, MO), 1% essential amino acids(Sigma-Aldrich) and 10% heat-inactivated fetal
calf serum (Thermo Trace, Melbourne, Australia)
was used. For T-cell cultures, fetal calf serum was
replaced with 10% human AB serum (Gemini BioProducts, Woodlands, CA).

patients), and each experiment was performed inparallel for the patients and healthy controls. The
clinicopathological findings were determined according to the Japanese Classification of Gastric
Carcinoma (Japanese Gastric Cancer Association,1998).
Preparation of PBMCs
Peripheral blood mononuclear cells (PBMCs) wereprepared by drawing a 30-mL peripheral blood
sample from each of the controls or from the patients before surgery and centrifuged through a
Ficoll-Paque (Pharmacia, Uppsala, Sweden) gradient. Informed consent was obtained from all thesubjects before blood donation.

Cell lines
The gastric cancer cell lines MKN-45 and MKN74 were purchased from the RIKEN Cell Bank
(Tsukuba, Japan).
3T. Matsunaga et al.

CSFE labeling and proliferation assay
Cell proliferation was determined using carboxyfluorescein succinimidyl ester (CFSE) dilution assays. CD4+ T cells were positively selected with
anti-CD4 microbeads. For staining with CFSE
(Invitrogen, Carlsbad, CA), CD4+ T cells at 1 × 106
cells/mL in phosphate-buffered solution were incubated with 2 μM CFSE for 10 min at 37˚C. Staining was terminated by adding RPMI 1640 containing 10% human AB serum (Gemini Bio-Products)
at 4˚C, followed by 1 wash with phosphate-buffered
solution. The CFSE-labeled CD4+ T cells wereseeded in anti-CD3 antibody-coated 96-well plates(3 × 105 cells/well; Nunc A/S, Roskilde, Denmark).
In other experiments, CD14+ cells were depleted ofother cells using anti-CD3, anti-CD16, anti-CD19,
anti-CD56 and anti-glycophorin A microbeads(Miltenyi, Gladbach, Germany) and sorted basedon their CD14+B7-H4+ or CD14+B7-H4 – phenotypes. Next, either B7-H4+ or B7-H4– monocytes
were added to CD4+ T cells at a ratio of 1:5. The
CD4+ T cells were profiled by CFSE labeling after
5 days of incubation. Unstained cells were included in all experiments and were used to set thecompensations on the flow cytometer.

Fig. 1. B7-H4 expression on circulating monocytes.
a: Representative FACS data for B7-H4 expression on
circulating monocytes in the normal controls andgastric cancer patients.
b: B7-H4 expression is significantly higher on circulating monocytes from the gastric cancer patients thanon those from the normal controls (P = 0.013).
[ ], number of subjects. FACS, fluorescence-activatedcell sorting.

The GraphPad Prism software (GraphPad Software, La Jolla, CA) was used for all statisticalanalyses.

Results

Regulation of B7-H4 expression
PBMCs (5 × 106) were cultured for different times
with cancer cells, cell-free malignant ascites or serum obtained from gastric cancer patients in 6-wellplates. After each period of coculture (12, 24 and48 h), PBMCs were harvested and analyzed fortheir B7-H4 expression and supernatants were obtained after centrifuging at 300 × g for 5 min.

B7-H4 expression on circulating monocytes
and tumor-associated macrophages in patients with gastric cancer
First, we determined the B7-H4 expression on
circulating monocytes from the normal controls(n = 18) and gastric cancer patients (n = 43). The
B7-H4 expression was significantly higher on circulating monocytes from the gastric cancer patients
than on those from the normal controls (4.9 ± 4.8%
versus 2.0 ± 1.4%, P = 0.013; Fig. 1). Table 1 shows
the correlations between the B7-H4 expression on
circulating monocytes and various clinicopathologic factors. The circulating monocytes from advanced gastric cancer patients exhibited higher B7-

Statistical analysis
The statistical significance of differences between
2 groups was determined by the paired t-test or
Mann-Whitney U-test. Correlations were analyzed
by the Spearman rank correlation coefficient test.
The accepted level of significance was P < 0.05.
4

B7-H4 on CD14+ cells in gastric cancer

Fig. 2. B7-H4 expression on
tumor-associated macrophages is significantly higher ingastric cancer tissues than on
circulating monocytes (P =0.0011).

H4 expression than those from early gastric cancer
patients (7.4 ± 5.7% versus 2.9 ± 2.1%, P = 0.0008).
The B7-H4 expression was significantly higher inpatients with lymphatic invasion than in patients
without lymphatic invasion (5.7 ± 5.1% versus 2.4 ±
1.4%, P = 0.03). Moreover, significantly higher B7H4 expression was observed in patients with venous
invasion than in patients without venous invasion(6.1 ± 5.2% versus 2.4 ± 1.3%, P = 0.015). Thesefindings indicate that increased B7-H4 expression
on circulating monocytes is closely correlated withdisease progression.
Next, we determined the B7-H4 expression
on tumor-associated macrophages obtained fromgastric cancer tissues. The B7-H4 expression was
significantly higher on tumor-associated macrophages in the gastric cancer tissues than on circulating monocytes (13.3 ± 6.8% versus 2.5 ± 2.7%,
P = 0.0011; Fig. 2).

Fig. 3. B7-H1 expression on circulating monocytes.
a: Representative FACS data for B7-H1 expression on
circulating monocytes from the normal controls andgastric cancer patients.
b: B7-H1 expression is significantly higher on circulating monocytes from the gastric cancer patients thanon those from the normal controls (P < 0.0001).
[ ], number of subjects. FACS, fluorescence-activatedcell sorting.

sus 4.7 ± 2.3%, P < 0.0001; Fig. 3). Furthermore,
the B7-H1 expression was significantly higher ontumor-associated macrophages in the gastric cancer
tissues than on circulating monocytes (18.4 ± 7.6%
versus 9.5 ± 6.6%, P = 0.0042; Fig. 4). Next, weanalyzed the correlation between B7-H1 expression
and B7-H4 expression and found that B7-H4 expression was closely correlated with B7-H1 expression in both the peripheral blood samples (r = 0.62,
P < 0.00001; Fig. 5a) and gastric cancer tissues(r = 0.65, P = 0.049; Fig. 5b).

B7-H1 expression on circulating monocytes and tumor-associated macrophages
and its relationship with B7-H4 expression
in gastric cancer patients
B7-H1 is another inhibitory member of the B7
family. To clarify the correlation between B7-H1
expression and B7-H4 expression on circulating
monocytes and tumor-associated macrophages,
we determined the B7-H1 expression in the gastriccancer patients and normal controls. The B7-H1
expression was significantly higher on circulating
monocytes from the gastric cancer patients than on
those from the normal controls (15.2 ± 10.0% ver-Fig. 4. B7-H1 expression issignificantly higher on tumorassociated macrophages ingastric cancer tissues than on
circulating monocytes (P =0.0042).

5

T. Matsunaga et al.

B7-H4 expression and HLA-DR expression
on monocytes/macrophages in gastric cancer patients
Kuang et al. observed that monocytes in the peritumoral stroma had an activated phenotype with increased expressions of HLA-DR, CD80 and CD86
(Kuang et al., 2009). Furthermore, they foundsignificant correlations between the numbers of B7H1+ and HLA-DRhigh monocytes in hepatocellular
carcinoma tissues. Therefore, we determined the
HLA-DR expression on monocytes/macrophages toinvestigate whether the same phenomenon was observed in gastric cancer patients. Significant correlations between B7-H4 expression and HLA-DR
expression were found on both circulating monocytes (Fig. 6a) and tumor-associated macrophages(Fig. 6b).

Fig. 5. B7-H4 expression is closely correlated with B7H1 expression in both peripheral blood samples (r = 0.62,
P < 0.0001) and gastric cancer tissues (r = 0.65, P = 0.049).

B7-H4+ monocytes/macrophages exhibit an
immunosuppressive phenotype

Fig. 6. There are significant correlations between B7-H4
expression and HLA-DR expression on circulating monocytes (r = 0.54, P = 0.0008) and tumor-associated macrophages (r = 0.88, P = 0.0072). MFI, mean fluorescenceintensity.

To examine the immunosuppressive function of B7H4+ monocytes/macrophages, we sorted B7-H4+
monocytes and cocultured the isolated cells with
CD4+ T cells in anti-CD3 antibody-coated wells.
CFSE assays revealed that B7-H4+ monocytes suppressed the proliferation of CD4+ T cells comparedwith B7-H4 – monocytes (Fig. 7a). Furthermore,
B7-H4+ monocytes significantly suppressed IFNgamma secretion by CD4+ T cells compared with
B7-H4– monocytes (750 ± 623 ng/mL versus 996 ±689 ng/mL) (Fig. 7b).
Decreased B7-H4 expression on circulating
monocytes from gastric cancer patients after complete surgical removal of the tumor

Fig. 7. Immunosuppressive effects of B7-H4+ monocytes
on CD4+ T cells.
a: B7-H4 + monocytes suppress the proliferation of
CD4+ T cells compared with B7-H4 – monocytes, as
evaluated by CFSE assays.
b: B7-H4 + monocytes significantly suppress IFNgamma secretion by CD4+ T cells compared with
B7-H4 – monocytes (P = 0.023).
CFSE, carboxyfluorescein succinimidyl ester.

B7-H4 expression was detected on 7.9 ± 6.9% and
2.8 ± 1.3% of monocytes (n = 12) before surgery
and after surgery (1 month later), respectively,
and this difference was statistically significant(P = 0.029; Fig. 8).

6

B7-H4 on CD14+ cells in gastric cancer

Fig. 8. Increased B7-H4 expression on circulating monocytes from gastric cancer patients is downregulated after
complete surgical removal of
the tumor (P = 0.029).

Gastric cancer cell lines induce B7-H4 expression on monocytes
Fig. 9. Cocultures of gastric cancer cell lines (MKN-45
and MKN-74) and PBMCs exhibit a remarkable increasein B7-H4 expression on monocytes after 24 h. Seen aremean fluorescence intensity (MFI) (mean ± SD). Pre, be-Finally, we carried out experiments using gastriccancer cell lines to examine the effects of cancer
cells on B7-H4 expression. We used 2 gastric cancer cell lines, MKN-45 and MKN-74. As shownin Fig. 9, cocultures of the gastric cancer cell lines
and PBMCs resulted in remarkable increases in B7H4 expression on monocytes. This phenomenon
was not observed when PBMCs were cultured in
the presence of supernatants from the 2 gastric cancer cell lines. Moreover, upregulation of B7-H4 expression was not observed in the presence of serumor ascites obtained from the gastric cancer patients.
These results indicate that direct contacts between
cancer cells and monocytes, but not soluble factors, are indispensable for B7-H4 overexpression on
monocytes.

fore coculture.

et al., 2007) and ovarian cancer patients (Curiel etal., 2004). Furthermore, myeloid-derived suppressor cells (Ochoa et al., 2007; Hoechst et al., 2008)
and CD4+NKG2D+ T cells (Groh et al., 2006) candiminish immunity against cancer cells in cancer
patients. These accumulating lines of evidence indicate that immunosuppressive cells play importantroles in tumor progression.
In the present study, we determined B7-H4
expression on circulating monocytes and tumorassociated macrophages in gastric cancer patientsto clarify one of the key mechanisms responsiblefor immune evasion in gastric cancer. B7-H4 expression was significantly higher on circulating
monocytes from the gastric cancer patients thanon those from the normal controls. B7-H4 expression was closely related to the depth of invasion,as well as the presence of lymphatic and venous
invasion. Furthermore, B7-H4 expression was significantly higher on tumor-associated macrophages
in the gastric cancer tissues than on circulating
monocytes. The B7-H4+ circulating monocytes
exhibited an immunosuppressive phenotype thatsuppressed CD4 + T-cell proliferation and IFNgamma secretion by CD4+ T cells. Therefore, B7-

Discussion
Immune suppressor cells are currently an area ofactive research interest. The best-studied immunosuppressive cell population is the well-known, yetstill relatively newly described, CD4+CD25+ regulatory T cells (Treg cells) (Sakaguchi et al., 2001;
Shevach, 2002; von Herrath and Harrison, 2003).
Human Treg cells (Woo et al., 2002; Curiel et al.,2004) disable TAA-specific T-cell immunity (Zou,2005, 2006). It has been shown that increased
numbers of Treg cells are correlated with poor
survival in hepatocellular carcinoma patients (Fu7T. Matsunaga et al.

H4+ circulating monocytes and tumor-associatedmacrophages may be related to immune evasion in
gastric cancer patients.
In addition to B7-H4, another member of theB7 family, B7-H1, is also selectively expressed byvarious cellular components in the tumor microenvironment and has been reported to be related toimmune evasion in cancer patients (Dong et al.,
2002; Curiel et al., 2003). In the present study, B7H1 expression was also significantly higher on circulating monocytes from the gastric cancer patients
than on those from the normal controls. Furthermore, B7-H1 expression was significantly higher
on tumor-associated macrophages in the gastriccancer tissues than on circulating monocytes. The
B7-H1 expression on circulating monocytes andtumor-associated macrophages was correlatedwith their HLA-DR expression, indicating that B7H1+ circulating monocytes and tumor-associatedmacrophages show activated phenotypes (data notshown). These findings are consistent with those ina previous report (Kuang et al., 2009). Of interestis that B7-H4 expression on circulating monocytes
and tumor-associated macrophages was also correlated with their HLA-DR expression. Moreover,B7-H4 expression was closely correlated with B7H1 expression in both peripheral blood samples
and gastric cancer tissues. These findings indicatethat circulating monocytes and tumor-associatedmacrophages were activated and that both B7H1 and B7H4 expressions were upregulated as aresult of this activation in gastric cancer patients.
To the best of our knowledge, this is the first study
to demonstrate overexpression of B7-H1 and B7H4 on circulating monocytes and tumor-associatedmacrophages in gastric cancer patients.
Previous reports have demonstrated that
upregulation of B7-H1 and B7-H4 is related tosoluble factors, such as IL-6, IL-10 and TNF-alpha(Kryczek et al., 2006; Kuang et al., 2009). However, serum and ascites obtained from the gastriccancer patients did not affect B7-H4 expression on
monocytes in the present study (data not shown).
Furthermore, supernatants obtained from 2 gastriccancer cell lines also did not affect B7-H4 expres-sion on monocytes (data not shown). These findings indicate that soluble factors are not related tothe upregulation of B7-H4 observed in the present
study. On the other hand, Kryczek et al. showedthat upregulation of B7-H4 expression was correlated with the presence of Treg cells (Kryczek et al.,2007). Therefore, we determined the frequencies
of Foxp3+ Treg cells in our blood and cancer tissue samples and found no correlation between B7H4 expression on monocytes/macrophages and thefrequency of Foxp3+ Treg cells (data not shown).
In the present study, we observed remarkable upregulation of B7-H4 expression on macrophages
in cancer tissues compared with peripheral blood
samples. Furthermore, a significant decrease in
B7-H4 expression on circulating monocytes fromgastric cancer patients was observed after complete
surgical removal of the tumor. These findings suggest the possibility that cancer cells themselves
may affect the B7-H4 overexpression on monocytes and macrophages in gastric cancer patients.
In fact, cocultures of gastric cancer cell lines andmonocytes induced upregulation of B7-H4 expression on monocytes. Since the supernatants fromthe 2 gastric cancer cell lines did not affect B7-H4
expression, it is likely that direct contacts between
cancer cells and monocytes/macrophages are indispensable for the upregulation of B7-H4 expression
on monocytes/macrophages. Further investigations to clarify which molecules on tumor cells are
related to the upregulation of B7-H4 expression on
monocytes/macrophages are urgently required.
Since monocytes/macrophages are closely
related to immune evasion, they are important
targets for cancer immunotherapy. In this regard,
Hagemann et al. showed that macrophages arepolarized via IL-1R and MyD88 to an immunosuppressive “alternative” phenotype that requires
IkappaB kinase beta-mediated nuclear factor(NF)-kappa B activation (Hagemann et al., 2008).
When NF-kappa B signaling is specifically inhibited in tumor-associated macrophages, the cells
become cytotoxic toward tumor cells and switch
to a “classically” activated phenotype of interleukin (IL)-12high and MHC IIhigh, but IL-10low and8

B7-H4 on CD14+ cells in gastric cancer

arginase-1low. Targeting NF-kappa B signaling intumor-associated macrophages also promotes theregression of advanced tumors in vivo by inducing
macrophage tumoricidal activity and activating theantitumor activity through IL-12-dependent NK
cell recruitment. Furthermore, Luo et al. demonstrated that DNA vaccines targeting legumain, amember of the asparaginyl endopeptidase family
that functions as a stress protein, induce a robust
CD8+ T-cell response against tumor-associatedmacrophages, which dramatically reduced theirdensity in tumor tissues and markedly suppressedtumor growth and metastasis in a murine model(Luo et al., 2006). These findings show that targeting of tumor-associated macrophages is attractive
and effective for anticancer strategies. Since B7H1+ and B7-H4 + monocytes/macrophages withimmunosuppressive properties are increased in
gastric cancer patients, targeting B7-H1+ and B7H4+ monocytes/macrophages may provide a breakthrough in the treatment of gastric cancer.
In conclusion, B7-H1+ and B7-H4+ circulating monocytes and tumor-associated macrophages
with immunosuppressive activity were increased in
gastric cancer patients. This phenomenon may be
one of the key mechanisms responsible for immune
evasion in gastric cancer patients. Therefore, targeting B7-H1+ and B7-H4+ monocytes/macrophages may provide a breakthrough in the treatmentof gastric cancer. Further investigations to clarify
which molecules are related to the upregulation of
B7-H4 expression on monocytes/macrophages areurgently required.

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Received December 17, 2010; accepted December 27, 2010
Corresponding author: Hiroaki Saito, MD

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