Distribution of Th17 cells and FoxP3(+) regulatory
T cells in tumor-infiltrating lymphocytes,
tumor-draining lymph nodes and peripheral blood
lymphocytes in patients with gastric cancer
Takanori Maruyama, Koji Kono,1 Yoshiki Mizukami, Yoshihiko Kawaguchi, Kousaku Mimura, Mitsuaki Watanabe,
Shinichirou Izawa and Hideki Fujii
First Department of Surgery, University of Yamanashi, Yamanashi, Japan
(Received March 26, 2010 Revised April 30, 2010 Accepted May 6, 2010 Accepted manuscript online May 19, 2010 Article first published online June 10,
2010)
Although Th17 cells reportedly play critical roles in the develop-
ment of autoimmunity and allergic reactions, information on Th17
cells in cancer-bearing hosts is still limited. In the present study,
we investigated the distribution of Th17 cells in relation to regula-
tory T cells (Treg) in the tumor-infiltrating lymphocytes (TILs),
regional lymph node lymphocytes, and peripheral blood lympho-
cytes of gastric cancer patients. Interleukin (IL)-17-producing
CD4(+) cells as Th17 cells and CD4(+)CD25(+)FoxP3(+) cells as Treg
were evaluated by flow cytometry and expressed as a percentage
of the total CD4+ cells, in addition to performing a Th1 Th2 bal-
ance assay. Moreover, immunohistochemical staining for IL-17 and
FoxP3 were performed. In TILs from patients with early disease
(n = 27), the frequency of Th17 cells was significantly higher than
that in the normal gastric mucosa (23.7 8.9 vs 4.5 3.1%). In TILs
from patients with advanced disease (n = 28), the frequency of
Th17 cells was also significantly higher, but lower compared to
early disease, than that in the normal gastric mucosa (15.1 6.2 vs
4.0 2.0%). This observation for Th17 cell-distribution was also
confirmed by immunohistochemistry. When the ratio of Th17 Treg
in TILs was evaluated in individual cases, it was more markedly
increased in early than in advanced disease. In conclusion, the
accumulation of Th17 cells as well as Treg in the tumor microenvi-
ronment of gastric cancer occurred in early disease and then the
infiltration of Th17 cells gradually decreased according to the dis-
ease progression, in contrast to increased Treg. (Cancer Sci 2010;
101: 19471954)
G astric cancer is one of the most common cancers through-
out the world. Despite efforts to introduce new treatment
modalities such as surgery combined with chemotherapy(1) or
chemo-radiotherapy(2), the control of gastric cancer at an
advanced stage remains difficult. It has been shown that the
infiltrating grade of CD3(+) tumor-infiltrating lymphocytes
(TILs) was correlated with a favorable outcome in patients with
several types of cancer, including gastric cancer.(3,4) Thus, it is
important to understand immunoregulation in gastric cancer, in
order to develop novel treatment strategies or improve the effi-
cacy of standard therapy.
It has been reported that nave CD4(+) helper T cells can
develop into four types of T cells: Th1, Th2, Th17, and regula-
tory T cells (Treg), based on the local cytokine milieu, and the
balance between these four types of helper T cell is important
for the regulation of immune responses in several mouse
models,(57) although information in humans is still limited and
controversial. Th17 cells are characterized as interleukin (IL)-
17-producing CD4(+) T cells which also produce IL-21, IL-22,
doi: 10.1111/j.1349-7006.2010.01624.x
2010 Japanese Cancer Association
and IL-26.(57) It has been reported that retinoid orphan nuclear
receptor (RORC) is a key regulator of Th17 cell lineage differ-
entiation,(8,9) and that Th17 cells play critical roles in the devel-
opment of autoimmunity and allergic reactions.(10,11) However,
information on Th17 cells in cancer-bearing hosts is still limited,
although there are a few reports that Th17 cells may play active
roles in antitumor immunity.(1215)
Regulatory T cells (Treg) play important roles in immunolog-
ical self-tolerance, and are functionally immunosuppressive
subsets of T cells.(16,17) There is accumulating evidence that an
increased population of Treg in TILs or PBMCs is one of the
reasons for impaired antitumor immunity in cancer-bearing
hosts.(1820) We have reported that the frequency of Treg among
TILs, lymphocytes derived from tumor-draining regional lymph
nodes, and PBMCs was higher in patients with gastric cancer in
comparison to their normal counterparts.(21) It has been shown
that the balance between Treg and Th17 cells is a key factor that
regulates helper T-cell function relating to the Th1 Th2 shift in
autoimmune disease and graft versus host disease (GVHD).(22)
However, there is limited information on the balance between
Treg and Th17 cells in cancer patients.(14)
In the present study, we investigated the distribution of
Th17 cells in relation to Treg in TILs, regional lymph node
lymphocytes (RLNLs), and PBMCs of patients with gastric
cancer.
Materials and Methods
Patients. Fifty-five consecutive patients with gastric cancer,
who were operated on in the University of Yamanashi Hospital
from 2009 to 2008, were enrolled in the present study. The char-
acteristics of the patients are summarized in Table 1. Patients
with gastric cancer were divided into two groups: those with
early disease (n = 27) corresponding to stage I according to the
TNM classification for gastric cancer (International Union
Against Cancer, UICC), and those with advanced disease corre-
sponding to stages II, III, and IV (n = 28). Regional lymph
nodes in the stomach were classified into N1 regional
lymph nodes adjacent to the gastric tumor and N2 regional
lymph nodes marginally distant from the tumor, according to the
Japanese Classification of Gastric Carcinoma.(23) Mesenteric
lymph nodes, which were not regional lymph nodes of the stom-
ach, were used as control lymph nodes.
None of the patients received radiotherapy, chemotherapy, or
other medical interventions before the study. Written informed
1To whom correspondence should be addressed.
E-mail: kojikono@yamanashi.ac.jp
Cancer Sci | September 2010 | vol. 101 | no. 9 | 19471954
Table 1. Characteristics of patients with gastric cancer Flow cytometric analysis of Treg. To analyze the prevalence
of T-regs, CD4+CD25+FoxP3+ cells were evaluated using the
Early Advanced Human Regulatory T-cell Staining Kit (eBioscience) according
disease disease to the manufacturers protocol. Briefly, the single-cell suspen-
(n = 27) (n = 28) sion was stained using a cocktail of anti-CD4-FITC mAb and
Age (mean SD) (years) 71.5 9.0 70.3 11.4 anti-CD25-APC mAb, and, then, after permeabilization, samples
Sex were blocked by normal rat serum, and stained using anti-
Male 15 19 FoxP3-phycoerythrin (PE) mAb or PE-conjugated rat IgG2a
Female 12 9 used as an isotype control. Subsequently, the number of FoxP3-
Stage positive cells on the gating of CD4+ cells was evaluated, and the
I 27 0 frequency of FoxP3+ T-regs was expressed as a percentage of
II 12 the total CD4+ cells.
III 9 Flow cytometric analysis of Th1 Th2. Lymphocytes were
IV 7 incubated in X-VIVO15 medium in the presence of PMA
Histology (50 mg mL; Sigma), Ionomycin (800 mg mL; Sigma), and Bre-
Differentiated adenocarinoma 15 12 feldin A (3 mg mL; eBioscience) for 4 h at 37C. Then, cells
Undifferentiated adenocarcinoma 12 16 were stained with CD4-APC (R&D Systems) for 30 min at 4C
Tumor which was followed by treatment with Fixation Permeabiliza-
T1 27 3 tion Reagent (eBioscience) for 45 min at 4C. For the analysis
T2 10 of Th1, lymphocytes were stained with IFN-c-PE mAb (R&D
T3 10 Systems) in combination with IgG2b mAb (R&D Systems) as
T4 5 an isotype control for 30 min at 4C. For the analysis of Th2,
Lymph node metastasis lymphocytes were stained with IL-4-PE (R&D Systems) in com-
N0 27 6 bination with mouse IgG1 (R&D Systems) as an isotype control
N1 14 for 30 min at 4C. Then, the percentage of IFN-c-positive or
N2 4 IL-4-positive cells on the gating of CD4+ cells was evaluated.
N3 4 Cytokine production by T cells. Peripheral blood mononuclear
Lymphatic invasion cells (PBMCs) (5.0 106 cells) or regional lymph node lym-
ly0 23 2 phocytes (RLNLs; 5.0 106 cells) were incubated with 1 mL of
ly1 3 10 X-VIVO15 medium for 72 h in a 48-well plate (Coaster, Cam-
ly2 1 11 bridge, MA, USA) pre-coated with anti-CD3 mAb (2.5 lg mL,
ly3 0 5 OKT3; Miltenyi Biotec, Auburn, CA, USA) plus anti-CD28
Venous invasion mAb (1.25 lg mL; 15E8, Miltenyi Biotec) to induce cytokine
v0 21 6 production. The obtained supernatants were stored at )70C and
v1 5 11 their contents were determined using ELISA kits (Human
v2 1 8 IL-17A, Human IL-21, and Human IL-23 [p19 p40] Kits; eBio-
v3 0 3 science), according to the manufacturers recommendations.
Immunohistochemistry. Interleukin (IL)-17-staining of tissue
Early disease, stage I; advanced disease, stages II, III, and IV. sections with gastric cancer was conducted using the Cell & Tis-
Tumor-node-metastasis (TNM) classification of gastric cancer sue Staining Kit (HRP-DAB system; R&D Systems) on paraf-
(International Union Against Cancer, UICC). Japanese Classification
of Gastric Carcinoma.
fin-embedded 4-lm-thick sections. Briefly, each paraffin section
was deparaffinized, followed by antigen retrieval with Epitope
Retrieval Solution (DakoCytomation, Glostrup, Denmark) in a
consent was obtained from all individuals. All samples were preheated water bath (98C, 40 min). Endogenous peroxidase
taken in a blinded manner for clinical information. was blocked, and antihuman IL-17 antibody (10 mg mL; R&D
Cell preparation. Peripheral blood mononuclear cells Systems) was applied overnight at 4C. Thereafter, the sections
(PBMCs) were isolated using Ficoll-Paque Plus (Amersham were incubated with secondary Abs as per the Cell & Tissue
Biosciences, Uppsala, Sweden) density gradient solution. Tumor Staining Kit, and counter-stained with hematoxylin. Negative
tissue, normal gastric mucosa, and regional lymph nodes from control staining was performed with goat IgG mAb (R&D Sys-
gastric cancer were isolated during surgery and homogenized by tems) instead of the specific primary antibody.
mechanical mincing with X-VIVO15 medium (Cambrex, East FoxP3-staining was conducted using the avidinbiotinperox-
Rutherford, NJ, USA). Subsequently, the single-cell suspension idase complex method (LSAB+ System-HRP; Dako, Glostrup,
was purified by centrifugation with Ficoll-Paque Plus. Denmark). Briefly, each paraffin section was dewaxed, followed
Flow cytometric analysis of Th17 cells. To analyze the preva- by antigen retrieval with Target Retrieval Solution (10 mmol
lence of Th17 cells, IL-17-producing CD4(+) lymphocytes were citrate buffer [pH 9.0]; Dako) in a preheated water bath (98C,
evaluated. Lymphocytes were incubated in X-VIVO15 medium 40 min), and endogenous peroxidase was blocked by Chemmate
in the presence of PMA (50 mg mL; Sigma-Aldrich, St. Louis, Peroxidase Blocking Solution (Dako). Then, biotinylated antihu-
MO, USA), Ionomycin (800 mg mL; Sigma), and Monensin man FoxP3 antibody (diluted by PBS, 1:20; eBioscience) was
(2 mM; eBioscience, San Diego, CA, USA) for 4 h at 37C. applied for 40 min at room temperature. Thereafter, the sections
Then, cells were stained with CD4-APC (R&D Systems, Minne- were incubated with streptavidin-conjugated horseradish peroxi-
apolis, MN, USA) for 30 min at 4C, followed by treatment dase (Dako) for 10 min, followed by development with 3,
with Fixation Permeabilization Reagent (eBioscience) for 3-diaminobenzidine (Dako) for 5 min, and counter-staining
45 min at 4C, and stained with IL-17A-PE mAb (eBio64- with hematoxylin. Negative control staining was performed with
DEC17; eBioscience) in combination with mouse IgG1 k mAb isotype control, mouse IgG2a (Dako), instead of the primary
(eBioscience) as an isotype control for 30 min at 4C. Then, the antibody.
number of IL-17-positive cells on the gating of CD4+ cells was Statistical analysis. The non-paired Students t-test was used
evaluated, and the frequency of Th17 cells was expressed as a to examine differences between the groups. Correlations between
percentage of the total CD4+ cells. values were evaluated using non-parametric Spearmans rank

1948 doi: 10.1111/j.1349-7006.2010.01624.x

2010 Japanese Cancer Association
correlation. Findings were considered significant when P-values
were <0.05.

Results
Distribution of Th17 cells in gastric cancer. Representative
flow cytometric data in TILs from an early disease case showed
that IL-17-producing CD4(+) cells could be quantitatively eval-
uated by intracellular staining with flow cytometry (Fig. 1a). In
TILs from patients with early disease, the frequency of Th17
cells was significantly higher than that in intra-epithelial lym-
phocytes (IELs) of normal gastric mucosa in the same cohorts
(23.7 8.9 vs 4.5 3.1%) (Fig. 1b). Also, in TILs from patients
with advanced disease, the frequency of Th17 cells was signifi-
cantly higher than that in IELs of normal gastric mucosa in the
same cohorts (15.1 6.2 vs 4.0 2.0%). These results indicated
that Th17 cells more strongly accumulated in TILs in compari-
son to the normal mucosa in gastric cancer. Of note, the fre-
quency of Th17 cells in TILs from patients with advanced
disease was significantly decreased compared to that in those
with early disease (15.1 6.2 vs 23.7 8.9%). Furthermore,
there was no significant difference regarding the prevalence of
Th17 cells in IELs of normal gastric mucosa between early and
advanced disease, indicating that the accumulation of Th17 cells
in the background mucosa did not differ among patients. In
addition, we did not observe any correlation between Th17 prev-
alence in TILs and Helicobacter pylori (H. pylori) infection
(data not shown).
In addition, immunohistochemical staining indicated IL-17-
positive lymphocytes were frequently observed in early gastric
cancer in comparison with advanced gastric cancer, as shown in
the representative staining (Fig. 2). Summarized data with quan-
titative analysis of immunohistochemistry showed that the fre-
quency of Th17 cells in TILs from patients with advanced
disease was significantly decreased compared to that in those
with early disease (Fig. 2). Thus, regarding the tumor microen-
vironment, the accumulation of Th17 cells occurred at a rela-
tively early stage of the disease, and their levels of infiltration
gradually decreased according to disease progression.
In PBMCs from patients with advanced disease, the frequency
of Th17 cells was significantly lower than that in healthy donors
(6.0 2.0 vs 11.0 3.9%, Fig. 1c). However, there was no sig-
nificant difference in the frequency of Th17 cells between
patients with early disease and healthy donors (9.2 4.0 vs
11.0 3.9%, Fig. 1c).
In RLNLs, the frequency of Th17 cells in N1 regional lymph
nodes of all cases was significantly higher than that in non-
regional control lymph nodes (Nc) (19.5 9.8 vs 11.2 7.9%,
Fig. 1d). There were no significant differences in the frequency
of Th17 cells in N1, N2, or Nc between early and advanced dis-
ease (data not shown). Fig. 1. Prevalence of Th17 cells in gastric cancer. (a) Representative
Distribution of Treg in gastric cancer. To analyze the preva- flow cytometric data showing interleukin (IL)-17-producing CD4(+)
lence of Treg, CD4+CD25+FoxP3+ cells were evaluated by flow cells. (b) The frequencies of Th17 cells (%Th17) in tumor-infiltrating
lymphocytes (TILs) in comparison to intra-epithelial lymphocytes (IELs)
cytometry and expressed as a percentage of the total CD4+ cells, of normal gastric mucosa are shown. The patients were divided into
consistent with our previous report.(21) Representative flow cyto- early disease (early) corresponding to stage I and advanced disease
metric data in TILs from an advanced disease case was shown (advanced) corresponding to stages II, III, and IV. (c) The frequencies
in Figure 3(a). In TILs from patients with advanced disease, the of Th17 cells (%Th17) in peripheral blood mononuclear cells (PBMCs)
frequency of Treg in TILs was significantly higher than that in in comparison with healthy donors (Healthy). (d) Regional lymph
IELs of normal gastric mucosa (26.9 7.1 vs 5.6 3.2%, nodes in the stomach were classified into N1 regional lymph nodes
adjacent to the gastric tumor and N2 regional lymph nodes
Fig. 3b). Furthermore, the frequency of Treg in TILs from marginally distant from the tumor, according to the Japanese
patients with early disease was also significantly increased com- Classification of Gastric Carcinoma. Mesenteric lymph nodes were
pared to that in IELs of normal gastric mucosa (17.5 5.9 vs used as control lymph nodes (Nc).
4.1 2.8%). These observations were further confirmed with
immunohistochemical analysis, as shown in Fig. 4. Thus, the
accumulation of Treg had already occurred at a relatively early In PBMCs from patients with advanced disease, the frequency
stage of the disease and increased according to disease progres- of Treg significantly increased compared to that in healthy
sion, in agreement with our previous report.(21) donors (5.9 2.1 vs 2.5 1.0%, Fig. 3c), and there was a

Maruyama et al. Cancer Sci | September 2010 | vol. 101 | no. 9 | 1949
2010 Japanese Cancer Association
 Fig. 2. Immunohistochemical staining for inter-
leukin (IL)-17 in gastric cancer. Representative
immunostaining for IL-17 is shown for advanced
and early gastric cancer in addition to normal
gastric mucosa and negative control. Summarized
data with quantitative analysis indicated that the
frequency of Th17 cells in tumor-infiltrating
lymphocytes (TILs) from patients with advanced
disease was significantly decreased compared to
that in those with early disease.

Fig. 3. Prevalence of regulatory T cells in gastric cancer. (a) Representative flow cytometric data showing CD4(+)CD25(+)FoxP3(+) cells. (b) The
frequencies of regulatory T cells (%FoxP3) in tumor-infiltrating lymphocytes (TILs) in comparison to intra-epithelial lymphocytes (IELs) of normal
gastric mucosa are shown. (c) The frequencies of regulatory T cells (%FoxP3) in peripheral blood mononuclear cells (PBMCs) in comparison with
healthy donors (healthy). (d) Prevalence of regulatory T cells in lymph nodes of gastric cancer.

significant difference in the frequency of Treg in PBMCs
between early disease cases and healthy donors (4.3 1.0 vs
2.5 1.0%).
In RLNLs, the frequency of Treg in N1 regional lymph nodes
of all cases was significantly higher than that in non-regional
control lymph nodes (Nc) (18.9 6.9 vs 12.1 6.0%, Fig. 3d).
There was a significant difference in the frequency of Treg
between N1 and N2 lymph nodes (18.9 6.9 vs 13.9 5.5%).
Balance of Th17 Treg according to the progression of
gastric cancer. When the ratio of Th17 Treg was evaluated in

1950 doi: 10.1111/j.1349-7006.2010.01624.x

2010 Japanese Cancer Association
Fig. 4. Immunohistochemical staining for FoxP3(+)
regulatory T cells in gastric cancer. Representative
immuno-staining for FoxP3 were shown in
advanced and early stage of gastric cancer in
addition to normal gastric mucosa and negative
control.
individual cases, the ratio of Th17 Treg in TILs was signifi-
cantly elevated in early compared to that in advanced disease,
while the ratio in IELs of normal gastric mucosa did not differ
between early and advanced disease (Fig. 5a). When each value
of Th17 and Treg in TILs in individual cases was plotted
(Fig. 5b), the patients with early disease showed a higher Th17
cell level with lower Treg in comparison to patients with
advanced disease. Thus, the balance of Th17 Treg in tumor mic-
roenvironments shifted from Th17-dominant to Treg-dominant
according to disease progression.
Cytokine productions by T cells. In order to confirm IL-17
production by the T cells tested, supernatants produced by
PBMCs or RLNLs in response to CD3 plus CD28 stimulation
were evaluated using ELISA. In Figure 6(a,b), the level of IL-
17 production was significantly correlated with the prevalence
of Th17 cells evaluated by FACS analysis, confirming the pres-
ence of Th17 cells and quantitative accuracy of FACS analysis.
In addition, IL-21 and IL-23 production was also measured,
since IL-21 and IL-23 were reported to be related to the regula-
tion of IL-17.(6) There was no significant correlation between
the prevalence of Th17 cells and production of IL-21 or IL-23
(Fig. 6a,b).
Th1 Th2 balance in gastric cancer. In order to further analyze
the helper T-cell balance in gastric cancer, Th1-type cells
(IFN-c-producing cells among CD4[+]) and Th2-type cells
(IL-4-producing cells among CD4[+]) were evaluated (Table 2).
Fig. 5. Balance of Th17 regulatory T cells (Treg) according to the progression of gastric cancer. (a) The ratio of Th17 Treg was evaluated in
individual cases. (b) Each value of the prevalence of Th17 (%Th17) and regulatory T cells (%FoxP3) in tumor-infiltrating lymphocytes (TILs) was
plotted.
Maruyama et al.
Tumor-infiltrating lymphocytes (TILs) in both early and
advanced disease revealed that there were increased Th2 cells
and decreased Th1 cells in comparison to IELs of normal gastric
mucosa, indicating that the balance of Th1 Th2 in TILs was
Th2-shifted in comparison to that in IEL.
In RLNLs, there were no significant differences in the
Th1 Th2 balance in N1 or N2 in comparison to that in control
lymph nodes (Table 2).
Discussion
The present study provided comprehensive data regarding the
Th17 Treg balance relating to disease progression in gastric
cancer. Importantly, the accumulation of Th17 cells as well as
Treg in the tumor microenvironment occurred at an early stage
of the disease and then the infiltration of Th17 cells gradually
decreased according to disease progression, in contrast to
increased Treg.
There have been several reports describing Th17 cells in
human cancer, in which the accumulation of Th17 cells was
observed in TILs in prostate,(15,24) myeloma,(25) renal cancer,(26)
and ovarian cancer.(14) In the present study, we performed a
quantitative and comprehensive analysis of Th17 cells in TILs,
RLNLs, and PBMCs in gastric cancer. As a result, the accumu-
lation of Th17 cells was observed in TILs at an early stage of
gastric cancer, and the infiltration of Th17 cells gradually
Cancer Sci | September 2010 | vol. 101 | no. 9 | 1951
2010 Japanese Cancer Association
Fig. 6. Cytokine productions of T cells. Supernatant produced by peripheral blood mononuclear cells (PBMCs) or N1 regional lymph node
lymphocytes in response to CD3 plus CD28 stimulation was evaluated using ELISA for interleukin (IL)-17, IL-21, and IL-23. In Figure 6(a,b), the
levels of IL-17 production were significantly correlated with the prevalence of Th17 cells evaluated by FACS analysis.

Table 2. Th1 Th2 balance in patients with gastric cancer (n = 55) reached maximal levels in advanced tumors in mouse mod-
els.(13) This discrepancy may be explained by the difference in
Th1 (%) Th2 (%) tumor biology between mice and humans, whereby transplanta-
TILs Early disease 34.5 13.4 *** 11.5 6.1 * ble tumors were implanted and grew rapidly in murine tumor

IELs
LNs
Advanced disease

N1
28.2
66.0
54.1



9.1 **
]
18.8
20.1
] 15.9
6.0
4.1



9.9 *
]
4.9
2.9
] models, while human tumors gradually developed and grew
slowly. Thus, it is likely that the cytokine profile in the tumor
microenvironment differs between mouse and human cancer. In
N2 51.9 20.4 3.1 1.7 fact, it has recently been shown that the biology of human can-
Nc 62.0 21.9 2.9 2.0 cer may reflect chronic or recurrent inflammation, different from
murine models.(27)
*P < 0.01, **P < 0.05. IELs, intra-epithelial lymphocytes; TILs, tumor- Interleukin (IL)-17 and IL-23 have been reported to be related
infiltrating lymphocytes. Interferon (IFN)-c-producing and interleukin to H. pylori infection(28) in the stomach. In the present study, we
(IL)-4-producing cells were expressed as a percentage of the total
CD4(+) cells. Early disease (n = 27) corresponds to stage I according
did not observe any correlation regarding the prevalence of
to the TNM classification. Advanced disease (n = 28) corresponds to Th17 within the tumor with H. pylori infection. Moreover, we
stages II, III, and IV according to the TNM classification. Regional did not identify a significant difference in the accumulation of
lymph nodes in gastric cancer were classified as follows: N1, lymph Th17 cells in the normal gastric mucosa as a background among
nodes adjacent to the gastric tumor; N2, lymph nodes marginally all patients. Hence, H. pylori infection did not affect Th17-
distant from the tumor. Mesenteric lymph nodes were used as control skewing in TILs of gastric cancer.
lymph nodes (Nc).
Recent data on human Th17 cells has indicated that IL-23 is
an important inducer of the differentiation of human Th17
cells,(29) and that Th17 polarization is induced by IL-1b,
decreased according to disease progression. This observation possibly enhanced by IL-6, and may be suppressed by
was confirmed by both flow cytometric analysis and immunohis- transforming growth factor-b (TGF-b).(29,30) Also, it was shown
tochemical staining in the present study. This finding is consis- that Th17 cells related to gut inflammation were regulated by
tent with a previous report on prostate cancer in which an IL-21.(31) Thus, the fact that there was an increase in Th17 cells
inverse correlation was noted between Th17-skewing and the in early disease and a gradual decrease in advanced disease in
tumor grade.(24) Thus, it is likely that Th17 cells accumulated in the present study may reflect the local cytokine milieu including
the tumor at an early cancer stage and the tumor microenviron- IL-21, IL-23, IL-1b, or TGF-b within the tumor microenviron-
ment shifted away from Th17-skewing according to tumor ment of gastric cancer. However, in the present study, we did
progression. not observe a direct correlation between the prevalence of Th17
In contrast to the present human study, it was demonstrated cells and IL-21 production, or between the prevalence of Th17
that Th17 cells increased following tumor development and cells and IL-23 production. Furthermore, it has been recently

1952 doi: 10.1111/j.1349-7006.2010.01624.x

2010 Japanese Cancer Association
shown that regulated on activation, normal T cell expressed and
secreted (RANTES) and monocyte chemotactic protein-1
(MCP-1) secreted by tumor cells and tumor-derived fibroblasts
within microenvironment mediate the recruitment of Th17
cells.(32) Further study to clarify which factors regulate Th17
cells within tumor microenvironment will be needed.
We confirmed that the frequency of Treg in TILs was
significantly higher than that in normal tissue in patients
with gastric cancer, and that Treg are already present in
small localized tumors at an early stage of gastric cancer, in
agreement with our previous reports.(21,33) Taken together,
the accumulation of Th17 cells as well as Treg in the tumor
microenvironment occurred at an early stage and then the
infiltration of Th17 cells gradually decreased according to
disease progression, resulting in Treg dominance in the
Th17 Treg balance at an advanced stage of gastric cancer.
These observations at an advanced stage of cancer are con-
sistent with a recent report on ovarian cancer, wherein the
proportions of Th17 cells and Treg were inversely corre-
lated.(14) The mechanisms behind the accumulation of Th17
cells Treg in the tumor microenvironment are still under
investigation. For example, we and others reported that CC
chemokine ligand (CCL)22 and CCL17 within the tumor
microenvironment are related to the accumulation of Treg
through CCR4.(20,21) Furthermore, it has been shown that
tumor-associated macrophages are capable of inducing
Th17 cell development in vitro, (14) and that RANTES and
MCP-1 secreted by tumor cells and tumor-derived fibroblasts
within the microenvironment mediate the recruitment of
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Th17 cells.(32) These dynamic alterations in the Th17 Treg
balance may be caused by the cytokine and chemokine
profiles according to the progression of cancer. Although
the fact that Th17 cells were reciprocally related to Treg
suggested an important role of Th17 in antitumor immunity,
it remains controversial whether Th17 cells promote tumor
growth or regulate antitumor response. Alternatively,
increased Th17 at early stage may reflect cytokine profile in
acute inflammation, while decreased Th17 at advanced
stage may reflect cytokine profile in chronic and recurrent
inflammation.
Although in general, Th1 Th2 balance is thought to be shifted
towards a Th2-type in malignancy, there is still controversy
regarding the Th1 Th2 balance in the TILs, lymph nodes, and
peripheral blood lymphocytes (PBLs) of patients with gastric
cancer.(34) In the present study, we showed that Th2 shift was
significantly observed in TILs in comparison to normal gastric
mucosa; however, there was no significant difference in
Th1 Th2 balances between early and advanced disease of
gastric cancer. These results suggested that subsets of
Th17 Treg might be affected more dynamically according to
disease progression than Th1 Th2 balance.
In conclusion, the accumulation of Th17 cells as well as Treg
in the tumor microenvironment occurred at an early stage of
the disease and then the infiltration of Th17 cells gradually
decreased according to disease progression, in contrast to
increased Treg. A better understanding of the underlying
mechanism regulating the Th17 Treg balance may lead to a
novel therapeutic strategy for gastric cancer.
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2010 Japanese Cancer Association