Yonago Acta medica 2010;53:9–16

NKG2D+CD4+ T Cells with Immune Suppressive Property
Increase in Patients with Colorectal Cancer
Manabu Yamamoto, Hiroaki Saito, Kyoichi Kihara, Kuniyuki Katano and Masahide
Ikeguchi
Division of Surgical Oncology, Department of Surgery, School of Medicine, Tottori University Faculty of Medicine, Yonago 683-8504, Japan

Some studies suggest that small populations of CD4+ T cells with activation-independent,
constitutive, NKG2D expression are found in normal peripheral blood and have immune
suppressive properties. The present study was designed to investigate NKG2D expression on CD4+ T lymphocytes and its relationship to immune evasion in colorectal cancer patients. We examined NKG2D expression on both circulating and tumor infiltrating CD4+ and CD8+ T cells or NK cells and evaluated it by multicolor flow cytometry.
Furthermore, intracellular cytokine staining was carried out to determine the cytokine
profile of NKG2D+CD4+ T cells in colorectal cancer patients. As a result, NKG2D expression on circulating and tumor-infiltrating CD8+ T cells and NK cells was downregulated in colorectal cancer patients. On the other hand, circulating and tumor-infiltrating
NKG2D+CD4+ T cells increased in colorectal cancer patients. NKG2D+CD4+ T cells
produced more immune suppressive cytokines, such as interleukin-10 and transforming
growth factor- 1, than did NKG2D-CD4+ T cells. Increased NKG2D+CD4+ T cells aswell as decreased NKG2D expression on CD8+ T cells and NK cells may be one of the key
mechanisms responsible for immune evasion by tumors in colorectal cancer.
Key words: CD4+ T lymphocyte; colorectal cancer; NKG2D

T cells play an essential role in the immunosurveillance and destruction of tumor cells. Accumulating evidence indicates that specific T-cell immune
responses can be raised against many tumors (Boonet al., 1994; Sahin et al., 1997). Nonetheless, attempts to translate this knowledge into clinicallyeffective immunotherapies have met with only limited success (Dunn et al., 2004; Rosenberg et al.,2004), because tumors develop mechanisms thatallow them to escape host immune responses (Dunnet al., 2002; Whiteside, 2003). One of the majormechanisms is the activity of T cells with negative
immune regulatory function at tumor sites, which
can markedly suppress immune responses and induce immune tolerance (Woo et al., 2001; Liyanage

et al., 2002; Curiel et al., 2004; Wang et al., 2004,
2005). Negative immune regulatory activity at tumor sites has typically been attributed to regulatory
T (Treg) cells. A high density of tumor-infiltrating
Foxp3+ Tregs has been associated with poor outcomes in various solid tumors, including ovarian(Curiel et al., 2004; Sato et al., 2005), pancreatic(Hiraoka et al., 2006), and hepatocellular carcinomas (Gao et al., 2007; Kobayashi et al., 2007).
Therefore, it is essential to understand the detailed
mechanisms of immune cells with regulatory function in cancer patients to develop more effective
immunotherapy.
Recent findings suggest that other T cellsubsets can function as suppressors of antitumor

Abbreviations: NK, natural killer; PBMC, peripheral blood mononuclear cell; TIL, tumor infiltrating lymphocyte; Treg,
regulatory T

9M. Yamamoto et al.

immune responses (Shevach, 2002; Wang, 2006).
One such T cell might be the NKG2D+CD4+ T
cell. NKG2D is a type II C-lectin-like protein
encoded by a gene located next to the NKG2A,
NKG2C and NKG2E genes within the naturalkiller (NK) gene complex on human chromosome
12p12-p13 and mouse chromosome 6 (Glienke etal., 1998). NKG2D is an activating cell surface
receptor expressed by NK cells, gamma-delta T
cells, some cytolytic CD8+ alpha-beta T cells and
NKT cells (Bauer et al., 1999; Vivier et al., 2002;
Raulet, 2003; Watzl, 2003). In cancer patients, tumor infiltrating and systemic NK cells and CD8+
T cells often express little NKG2D and are functionally compromised (Groh et al., 2002). On theother hand, small populations of CD4+ T cellswith activation-independent, constitutive, NKG2D
expression occur in normal peripheral blood (Baueret al., 1999; Groh et al., 2006; Allez et al., 2007;
Sundstrom et al., 2007). Groh et al. (2006) recently
demonstrated that increased populations of CD4+
T cells among tumor infiltrating lymphocytes (TILs)
and in peripheral blood were positive for NKG2D
in cancer patients with breast, lung, colon andovarian carcinomas and melanomas. They also
showed that NKG2D+CD4+ T cells observed incancer patients appeared biased toward an IL-10and TGF-dominated cytokine profile, indicatingthat NKG2D+CD4+ T cells had immune suppressive properties. However, little is known about
NKG2D+CD4+ T cells in cancer patients thus far.
Colorectal cancer is the fourth most commonly diagnosed malignancy, with an estimated1,023,000 new cases and 529,000 deaths each year
(Parkin et al., 2005). Considering the importantfunction of immune cells with regulatory function,such as Tregs, in tumor progression and prognosis,
it is extremely important to determine the presenceof NKG2D+CD4+ T cells with immune suppressive function in colorectal cancer patients. Furthermore, NKG2D expression on CD8+ T cells and NK
cells has not yet been determined in colorectal cancer. In the present study, we determined NKG2D
expression on CD4+ and CD8+ T cells and NK
cells obtained from colorectal cancer patients. We

also analyzed the function of NKG2D+CD4+ T
cells to investigate one of the mechanisms responsible for immune evasion in patients with colorectal
cancer.

Materials and Methods
Patients and normal donors
Forty-two patients (22 males and 20 females), treated at Tottori University Hospital and pathologicallydiagnosed with colorectal cancer, were enrolled
in this study. None of the patients received radiotherapy, chemotherapy or any other medical intervention before donating blood. Informed consent
for blood donation was obtained from all individuals. Patient characteristics are shown in Table 1.
Healthy controls (n = 24; 18 males and 6 females)
were age-matched (62.5 ± 8.9 years for the controls
versus 65.3 ± 9.1 years for the patients), and eachexperiment was performed in parallel. The clinicopathological findings were determined according
to the Japanese Classification of Colorectal Carcinoma (Japanese Society for Cancer of the Colonand Rectum, 2009).
Preparation of peripheral blood mononuclear cells (PBMCs)
An amount (40 mL) of peripheral blood was drawn
from each of the controls and patients before surgery or chemotherapy and PBMCs were separated
by centrifugation over a Ficoll-Paque (Pharmacia,
Uppsala, Sweden) gradient.
Isolation of TILs
Freshly excised tumor tissues were minced andincubated in 1.5 mg/mL of collagenase D (Wako
Pure Chemical Industries, Osaka, Japan). Cellsuspensions were then filtered through a mesh filter(BD Falcon, Franklin Lakes, NJ).

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NKG2D+CD4+ T cells in colorectal cancer

Flow cytometry analysis
Flow cytometry analysis was performed on a FACSCalibur (Becton Dickinson, Franklin Lakes, NJ),
using the following antibodies: anti-CD3-PE-Cy5(Biolegend, San Diego, CA), anti-CD4-FITC, antiCD4-PE-Cy5, anti-CD8-FITC, anti-CD56-FITC
and anti-NKG2D-PE (BD Pharmingen, Franklin
Lakes, NJ). For intracellular cytokine staining,
PBMCs were cultured in the presence of either
Leukocyte Activation Cocktail (BD Pharmingen)or lipopolysaccharide (Calbiochem, Darmstadt,
Germany). Anti-cytokine antibodies were antiIFN- -FITC (BD Pharmingen), anti-IL-10-Alexa
fluor 488 (eBioscience, San Diego, CA) and antiLAP-TGF- 1-PerCP (R&D systems, Minneapolis,
MN). For the staining of IFN- and IL-10, cells
were fixed and permeabilized with BD Cytofix/
Cytoperm solution.
Media
Culture medium consisted of RPMI 1640 (Cambrex
Bio Science Walkersville, Walkersville, MD), 1%
penicillin/streptomycin (Invitrogen, Carlsbad, CA)
and 10% heat-inactivated human serum AB (Gemini Bio-Products, Woodlands, CA).
Statistical analysis
To determine statistical differences between the 2groups, either paired t-tests or Mann-Whitney Utests were used. The accepted level of significancewas P < 0.05. GraphPad Prism software (GraphPad
Software, La Jolla, CA) was used for all statisticalanalyses.

Fig. 1. NKG2D expression on circulating CD8+ T cells
or NK cells.
a: Representative result of NKG2D expression on circulating CD8+ T cells in either normal controls orcolorectal cancer patients by FACS.
b: NKG2D expression on circulating CD8+ T cells incolorectal cancer patients was significantly lower
than in normal controls (P = 0.01).
c: Representative result of NKG2D expression on
circulating NK cells in either normal controls orcolorectal cancer patients by FACS.
d: NKG2D expression on circulating NK cells in colorectal cancer patients was significantly lower thanin normal controls (P = 0.037).

Results
NKG2D expression on CD8+ T lymphocytes
and NK cells in patients with colorectal cancer
NKG2D expression on circulating CD8+ T lymphocytes and NK cells is downregulated in various
11

M. Yamamoto et al.

types of cancer patients, but this has not yet been
determined in colorectal cancer. Therefore, wefirst determined NKG2D expression on circulating
CD8+ T lymphocytes and NK cells in both normal
controls and colorectal cancer patients. NKG2D
expression of circulating CD8+ T lymphocytes incolorectal cancer patients (73.6 ± 19.6%) was significantly lower than that in normal controls (84.5± 16.0%; P = 0.01; Figs. 1a and b). Furthermore,
NKG2D expression of circulating NK cells in colorectal cancer patients (86.3 ± 7.3%) was significantly lower than those in normal controls (90.0 ±
6.2%; P = 0.037; Figs. 1c and d).
Fig. 2. NKG2D expression on tumor infiltrating CD8+ T
cells or NK cells.
a: NKG2D expression on CD8+ T cells in tissue ofcolorectal cancer was significantly lower than thaton circulating CD8+ T cells (P = 0.0079).
b: NKG2D expression on NK cells in tissue of colorectal cancer was significantly lower than on circulating NK cells (P < 0.0001).

NKG2D expression on CD8+ T cells and NK
cells in the tissue of colorectal cancer
We then determined NKG2D expression on CD8+
T cells and NK cells obtained from colorectal cancer tissue. NKG2D expression on CD8+ T cells in
the tissue of colorectal cancer (40.0 ± 14.9%) was
significantly lower than that of circulating CD8+ T

Fig. 3. NKG2D expression on CD4+ T cells.
a: Representative result of NKG2D expression on circulating CD4+ T cells ineither normal controls or colorectal cancer patients by FACS.
b: The frequency of NKG2D+CD4+ T cells in colorectal cancer patients was
significantly higher than in normal controls (P = 0.048).
c: The frequency of NKG2D+CD4+ T cells in tissue of colorectal cancer was
significantly higher than in circulating CD4+ T cells (P = 0.034).

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NKG2D+CD4+ T cells in colorectal cancer
Table 1. The frequency of NKG2D+CD4+ T cells
and clinicopathological characteristics in colorectal cancer patients

cells (67.7 ± 22.0%; P = 0.0079; Fig. 2a). NKG2D
expression on NK cells in the tissue of colorectal
cancers (34.8 ± 15.1%) was also significantly lower
than that of circulating NK cells (86.6 ± 8.9%; P <0.0001; Fig. 2b).

Number NKG2D+CD4+ P
of patients
T cells (%)
value
Depth of invasion*
M/SM/MP6
SS/SE/SI (A/AI)
25
Lymph node metastasis
Absent17
Present
14
Lymphatic invasion†
ly0/1
14
ly2/3
17
Vascular invasion‡
v0/1
14
v2/3
17
Liver metastasis
Absent28
Present
3
Stage of disease
Stage I/II
17
Stage III/IV
14

Increased NKG2D+CD4+ T cells among
TILs and in peripheral blood in colorectal
patients
CD4+ T cells in peripheral blood were significantly
more positive for NKG2D in colorectal cancer patients (5.0 ± 4.6%) than that in normal controls (3.0
± 2.2%; P = 0.048; Figs. 3a and b). There were significantly more NKG2D+CD4+ T cells in the tissue of colorectal cancers (16.5 ± 16.6%) than thosein peripheral blood (4.1 ± 1.7%; P = 0.034; Fig. 3c).
Table 1 shows the correlation between the frequency of NKG2D+CD4+ T cells and various clinicopathological factors. No significant differencesin the frequency of NKG2D+CD4+ T cells wereobserved in terms of depth of invasion, lymph nodemetastasis, liver metastasis and stage of disease.

7.4 ± 6.9
3.9 ± 1.90.22

5.0 ± 4.4
4.1 ± 2.3

0.71

5.7 ± 4.9
3.7 ± 1.7

0.24

5.6 ± 4.8
3.7 ± 2.0

0.21

4.6 ± 3.7
5.0 ± 2.60.53

5.0 ± 4.4
4.1 ± 2.3

0.71

* M, tumor invasion of mucosa; SM, tumor invasion of submucosa; MP, tumor invasion of muscularis propria; SS,tumor invasion of subserosa; SE, tumor invasion of serosal; SI, direct tumor invasion of other organs or structures;A, tumor invasion through muscularis propria; AI, direct
tumor invasion of other organs or structures.
† Lymphatic invasion: ly0–ly3, grade of lymphatic vessel
invasion.
‡ Vascular invasion: v0–v3, grade of vascular invasion.

Cytokine profile of NKG2D+CD4+ T cells inpatients with colorectal cancer
Finally, we determined the cytokine profile of
NKG2D+CD4+ T cells to show their immune
suppressive function. NKG2D+CD4+ T cells produced significantly less IFN- than NKG2D-CD4+
T cells (Fig. 4a). On the other hand, expressionof the immune suppressive cytokines, IL-10 and

TGF- 1, by NKG2D+CD4+ T cells was significantly increased compared to NKG2D-CD4+ T
cells (Figs. 4b and c).

Fig. 4. Cytokine profile ofCD4+ T cells.
a: IFN-gamma.
b: IL-10.
c: TGF-beta 1.
MFI, mean fluorescence intensity.

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M. Yamamoto et al.

NKG2D can be induced on human CD4+ T
cells by TCR-CD3 complex stimulation (Groh etal., 2003). A previous study demonstrated that increased populations of CD4+ T cells among TILs
and in peripheral blood were positive for NKG2D
in cancer patients with breast, lung, colon andovarian carcinomas and melanomas (Groh et al.,2006). In the present study, we have also demonstrated that the frequency of NKG2D+CD4+ T
cells in patients with colorectal cancer is significantly more than in normal controls. Furthermore,
the increased population of NKG2D+CD4+ T
cells in the tissue of colorectal cancer was morestriking than that in peripheral blood. Those increased NKG2D+CD4+ T cells produced less IFNthan NKG2D-CD4+ T cells. On the other hand,
NKG2D+CD4+ T cells produced more immunosuppressive cytokines, such as IL-10 and TGF- 1
than NKG2D-CD4+ T cells, indicating that theincreased number of NKG2D+CD4+ T cells weobserved in colorectal cancer patients in the present study had immunosuppressive properties andmight correlate with immune evasion in colorectal
cancer patients. To the best of our knowledge, thisstudy is the first to demonstrate decreased NKG2D
expression on CD8+ and NK cells and an increased
population of NKG2D+CD4+ T cell with immune
suppressive function in colorectal cancer.
The role of increased NKG2D+CD4+ T cells
we observed in the present study in clinical settings
remains unclear. In fact, the increased frequency
of NKG2D+CD4+ T cells in peripheral blood was
not correlated with disease progression, indicating that an increase in NKG2D+CD4+ T cells inperipheral blood might be an early event in the
progression of colorectal cancer. On the other
hand, some paper demonstrated that the presenceof immune cells with immunosuppressive properties, such as Regulatory T cells which were characterized by CD4+Foxp3+ T cells, in the tissue ofcarcinoma was associated with poor outcome invarious solid tumors, including ovarian (Curiel etal., 2004; Sato et al., 2005), pancreatic (Hiraoka etal., 2006), and hepatocellular carcinoma (Kobayashi
et al., 2007; Gao et al., 2007). In fact, the increased

Discussion
CD8+ T cells and NK cells are thought to play animportant role in the control of tumors as a resultof their cytotoxic activity and by releasing soluble
factors. It has been reported that the functions of
CD8+ T cells and NK cells are impaired in cancer patients, which is related to immune evasion
by cancer. Although the detailed mechanisms
responsible for impaired function of CD8+ T cells
and NK cells remain unclear, recent studies demonstrated that decreased NKG2D expression on
CD8+ T cells and NK cells was closely related
to this phenomenon (Groh et al., 2002; Wu et al.,2004). In the present study, we demonstrated that
the NKG2D expression of circulating CD8+ T cells
and NK cells in colorectal cancer patients was
significantly lower than that in normal controls.
Furthermore, the reduction in NKG2D expression
on CD8+ T cells and NK cells in the tissue of colorectal cancer was more striking than in peripheral
blood. We have previously shown that NKG2D
expression significantly correlated with IFN- production in CD8+ T cells in patients with gastriccancer, indicating that downregulated NKG2D expression is closely related to the low responsivenessof CD8+ T cells to cancers (Osaki et al., 2007).
Moreover, Groh et al. (2002) reported that NKG2D-low MART-1 specific CD8+ T cells isolated
from TILs from a MIC-positive melanoma showed
no or little induction of IFN- after stimulation with
MART-1 peptide, while a substantial proportion ofidentically treated NKG2D high MART-1-specific
T cells from a MIC-negative melanoma produced astrong IFN- response. With regard to the functionof NK cells, it has been demonstrated that downregulated NKG2D expression correlated with areduction in cytotoxic activity by NK cells (Wu etal., 2004). Therefore, the downregulated NKG2D
expression of CD8+ T cells and NK cells observedin cancer tissue and peripheral blood in the present study might be one of the key mechanisms bywhich colorectal cancer impairs the function of immune cells such as CD8+ T cells and NK cells.
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NKG2D+CD4+ T cells in colorectal cancer
population of NKG2D+CD4+ T cells in the tissueof colorectal cancer was more striking than that inperipheral blood in the present study. Although wewere not able to determine the prognostic significance of tumor-infiltrating NKG2D+CD4+ T cells
due to small number of data, it is likely that tumorinfiltrating NKG2D+CD4+ T cells is associatedwith poor prognosis in colorectal cancer. Further
investigation to show the prognostic significance ofincreased tumor-infiltrating NKG2D+CD4+ T cells
is imperative.
The mechanisms by which NKG2D+CD4+ T
cells increase remains unclear in the present study.
In this regard, Groh et al. (2006) demonstrated that
expansion of the NKG2D+CD4+ T cell population
was dependent on the presence of tumor-associated
MICA and correlates with serum concentrationsof sMICA in cancer patients. In the present study,
however, there was no difference in the concentration of sMICA between normal controls and colorectal cancer patients (data not shown). We are
currently planning to perform some experiments
to investigate the detailed mechanisms that induce
expansion of NKG2D+CD4+ T cells in colorectal
cancer patients.
In conclusion, our data demonstrate decreased
NKG2D expression on both CD8+ T cells andNK cells and an increase in the NKG2D+CD4+
T cell population in colorectal cancer patients.
NKG2D+CD4+ T cells exhibit immune suppressivefunction by producing cytokines, such as IL-10 and
TGF- 1. Therefore, the MIC-NKG2D system mightmore exclusively contribute to immunosuppression by tumors in colorectal cancer. On the other
hand, the mechanisms by which NKG2D+CD4+ T
cells increase are still unknown. Further investigation to show the mechanisms leading to increased
NKG2D+CD4+ T cells is urgently required.

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Received December 21, 2009; accepted January 6, 2010
Corresponding author: Manabu Yamamoto

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