Yonago Acta medica 2012;55:1–9

Upregulation of TIM-3 and PD-1 on CD4+ and CD8+ T Cells
Associated with Dysfunction of Cell-Mediated Immunity
after Colorectal Cancer Operation
Yosuke Arai, Hiroaki Saito and Masahide Ikeguchi
Division of Surgical Oncology, Department of Surgery, School of Medicine, Tottori University Faculty of Medicine, Yonago 683-8504, Japan

Surgery has been reported to suppress cell-mediated immunity; however, the detailed
mechanisms responsible for this remain unclear. We obtained information on expressionsof T-cell immunoglobulin domain and mucin domain 3 (TIM-3) and programmed cell
death 1 (PD-1) on both CD4+ and CD8+ T cells postoperatively from colorectal cancer
patients and evaluated by multicolor flow cytometry. The functions of CD4+ and CD8+ T
cells based on TIM-3 and PD-1 expressions were determined by quantitating the concentration of interferon- (IFN- ) by enzyme-linked immunosorbent assay. The study demonstrated a rapid and significant decrease in number of total lymphocytes, CD4+ T cells
and CD8+ T cells. These immune cells reached the minimum on day 1 and then increased,
but still significantly decreased on days 3 and 7. The frequency of PD-1+CD4+ T cells andTIM-3+CD4+ T cells significantly increased after colorectal surgery. IFN- secretion by
PD-1+TIM-3+CD4+ T cells was significantly decreased compared to secretion by either
PD-1+TIM3-CD4+ T cells or PD-1−TIM-3−CD4+ T cells. Furthermore, IFN- secretionby PD-1+TIM-3+CD8+ T cells was significantly decreased compared to secretion by either
PD-1−TIM-3+CD8+ T cells or PD-1−TIM-3−CD8+ T cells. The expression of PD-1 andTIM-3 was closely related to impaired cell-mediated immunity that was observed after
surgery for colorectal cancer. New treatment targeting TIM-3 and PD-1 on CD4+ andCD8+ T cells during the perioperative period may provide a breakthrough in the treatment of colorectal cancer patients.
Key words: colorectal cancer; programmed cell death 1; surgical trauma; T-cell immunoglobulin
domain and mucin domain 3

Colorectal cancer is one of the most prevalentsolid organ cancers in both men and women. Approximately one million cases are recorded every
year worldwide, and over half a million patientsdie of this disease yearly (Parkin et al., 2005).
Surgical removal of the primary tumor provides
the best chance of long-term disease-free survival

for patients with colorectal cancer. Unfortunately,a significant number of colorectal cancer patientstypically develop metastases, even after successful
resection of the primary tumor. It is now becoming
clear that surgery itself contributes to developmentof both local recurrences and distant metastases.
Surgical trauma and the concomitant wound-heal-

Abbreviations: ELISA, enzyme-linked immunosorbent assay; FACS, fluorescence-activated cell sorting; HCV, hepatitis
C virus; HIV, human immunodeficiency virus; IFN- , interferon- ; PD-1, programmed cell death 1; PD-L, PD-1-ligand;
Tc1, type 1 CD8+ cytotoxic T cell; Th, helper T lymphocyte; TIM-3, T-cell immunoglobulin domain and mucin domain 3;
TIM-3L, TIM-3 ligand1

Y. Arai et al.

ing processes induce local and systemic changes,
including impairment of tissue integrity and production of inflammatory mediators and angiogenic
factors. Subsequently, this can lead to immune
suppression and enhanced growth or adhesion oftumor cells, all of which increase the chance of
exfoliated tumor cells developing into secondary
malignancies (van der Bij et al., 2009).
Surgery has been reported to suppress cellmediated immunity, which plays a key role in hostdefense against bacterial and viral infections (Gupta
and Watson, 2001; Jacobi et al., 2002). T cells area critical component of cellular immunity that are
involved in the recruitment and activation of effector cells, as well as amplification of the specificimmune response to pathogens and cancer cells.
T-cell dysfunctions are characterized by diminished cytokine production, impaired killing andhypoproliferation. According to Wichmann et al.
(2005), there is a significant decrease in cell number of lymphocyte subpopulations, such as helper
T lymphocytes (Th), cytotoxic T lymphocytes andnatural killer cells after surgery. After conventionalcholecystectomy, T cells produce significantly lessinterferon- (IFN- ), tumor necrosis factor- andinterleukin-2 (Brune et al., 1999). Surgical stress isknown to induce a shift in the Th1/Th2 balance toward Th2, which suggests downregulation of cellmediated immunity and upregulation of antibodymediated immunity after surgery (Decker et al.,1996). However, the detailed mechanisms responsible for the dysfunction of T cells after surgery
remain unclear.
Recent studies have demonstrated that programmed cell death 1 (PD-1) and T-cell immunoglobulin domain and mucin domain 3 (TIM-3)
expression on CD4+ and CD8+ T cells is closely
associated with T-cell dysfunction in human immunodeficiency virus (HIV), hepatitis C virus(HCV) and cancer patients. PD-1 (CD279) was
identified as a gene that was upregulated in a T-cellhybridoma undergoing apoptotic cell death, andwas thus named PD-1 (Okazaki and Honjo, 2006).
PD-1 is inducibly expressed on CD4+ T cells,
CD8+ T cells, natural killer-like T cells, B cells
and monocytes upon activation (Greenwald et al.,2005; Okazaki and Honjo, 2006). Recent studies
have shown that PD-1 is highly expressed by CD8+
T cells during chronic lymphocytic choriomeningitis virus infection, and that the PD-1–PD-1-ligand(PD-L) pathway plays a major role in regulating
T-cell exhaustion during infection (Barber et al.,2006). Furthermore, several new studies suggest arole for the PD-1–PD-L pathway in exhaustion ofvirus-specific CD8+ T cells during HIV infection.
Several reports show that PD-1 expression is elevated on HIV-specific CD8+ T cells and that blocking
the PD-1–PD-L pathway leads to increased T-cellproliferation and effector cytokine production (Dayet al., 2006; Petrovas et al., 2006; Trautmann etal., 2006). A previous work has shown that PD-L
is upregulated in HIV infection (Trabattoni et al.,2003). Collectively, these observations suggest that
the PD-1–PD-L pathway may indeed be operating
during chronic HIV infection.
TIM-3 is a transmembrane protein constitutively expressed on type 1 CD8+ cytotoxic T cells(Tc1) in mice and humans (Monney et al., 2002).
Several lines of evidence support the role of TIM-3
as an inhibitory molecule that downregulates effector Th1/Tc1 cell responses. In mice, blocking theTIM-3–TIM-3 ligand (TIM-3L) pathway resulted in
hyperproliferation of Th1-type cells and abrogated
the induction of peripheral and transplantation
tolerance (Sabatos et al., 2003; Sanchez-Fueyo etal., 2003). TIM-3 interacts with its ligand galectin-9 to induce cell death in Th1 cells (Zhu et al.,2005). In humans, TIM-3 expression is defective
in CD4+ T cells producing high levels of IFN- ,as well as those isolated from the cerebrospinal
fluid of patients with multiple sclerosis (Koguchi etal., 2006). Recently, TIM-3 upregulation has beenreported in HIV-specific and HCV-specific CD8+
T cells in patients with progressive HIV infection
and chronic hepatitis C, respectively (Jones et al.,2008; Golden-Mason et al., 2009). TIM-3+ HIVand HCV-specific CD8+ T cells were distinct fromthe PD-1+ CD8+ T cells and exhibited T-cell dysfunction. In addition to virus infection, PD-1 andTIM-3 expression on CD4+ and CD8+ T cells arealso related to T-cell exhaustion in cancer patients(Fourcade et al., 2010; Sakuishi et al., 2010).
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Immunosuppression after colorectal surgery

Given that PD-1 and TIM-3 expressions on
CD4+ and CD8+ T cells are closely associatedwith T-cell dysfunction in HIV, HCV and cancer
patients, it is likely that these might be related tosuppression of cell-mediated immunity induced
after surgery. In the present study, therefore, weinvestigated the expressions of PD-1 and TIM-3 onlymphocytes to determine the role of these molecules in the immune-suppression that occurs after
operation for colorectal cancer.

Materials and Methods
Patients and normal donors
Thirty-six patients (20 males and 16 females),treated at the Tottori University Hospital and pathologically diagnosed with colorectal cancer, wereenrolled in this study. None of the patients received
radiotherapy, chemotherapy or other medical interventions before surgery. Informed consent forblood donations was obtained from all individuals.
The clinicopathological findings were determinedaccording to the Japanese Classification of Colorectal Carcinoma (Japanese Society for Cancer of theColon and Rectum, 2009). The clinicopathologicalcharacteristics of the patients enrolled in the present study are shown in Table 1.

Table 1. Clinicopathologic characteristics incolorectal cancer patients in the present study
Clinicopathologic factor
GenderAge (yr)
Procedure
Cancer site

Depth of invasion*
Lymph node metastasis
Lymphatic invasion
Vascular invasion
Liver metastasis
Lung metastasis
Stage of disease

n (%)
Male
Female66.8 ± 3.1
OpenLaparoscopic
Cecum
AscendingTransverse
Descending
Sigmoid
RectumM/SM/MP
SS/SE/SI (A1/A2/AI)
AbsentPresent
AbsentPresent
AbsentPresent
AbsentPresent
AbsentPresent
0
I
II
III
IV

20 (56)16 (44)
11 (30)25 (70)2 (5)6 (16)
5 (14)1 (3)
6 (16)
17 (47)16 (44)20 (56)26 (72)10 (28)7 (19)
29 (81)8 (22)
28 (78)35 (97)1 (3)34 (94)2 (6)2 (6)12 (33)
11 (30)8 (22)
3 (8)Preparation of peripheral blood mononuclear cells
Approximately 30 mL of peripheral blood was
drawn from the patients before surgery as wellas on days 1, 3, 7 and 30 after surgery, and blood
samples were prepared by centrifugation over a Ficoll-Paque (Pharmacia, Uppsala, Sweden) density
gradient.
Flow cytometry analysis
Fluorescence-activated cell sorting (FACS) analysis was performed using an FACS Calibur (Becton
Dickinson, Franklin Lakes, NJ). The following
antibodies were used to determine cell phenotype:anti-CD4-FITC, anti-CD8-FITC, anti-PD-1-PE(BD PharMingen, Franklin Lakes, NJ), anti-TIM3-PE (R&D Systems, Minneapolis, MN) and antiCD3-PE-Cy5 (Biolegend, San Diego, CA).

* 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 serosa;SI, direct tumor invasion of other organs or structures;
A1, tumor invasion through muscularis propria into a nonperitonealized part;A2, tumor invasion of non-peritonealized, pericolic or perirectal tissues;AI, direct tumor invasion of other organs or structures.

Measurement of IFN- concentrations
CD4+ or CD8+ T cells were sorted based on TIM3−PD-1−, TIM-3+PD-1−, TIM-3−PD-1+ and TIM3+PD-1+ phenotype. Each population was seededin anti-CD3 antibody-coated 96-well plates (1 ×3

Y. Arai et al.

Comparison with
the Pre level:*** P < 0.001.

Fig. 1. Changes in number of total leukocytes (a), neutrophil count (b) and serum concentration of C-reactive protein (c)after surgery. CRP, C-reactive protein; POD, postoperative day; Pre, preoperation.

10 4 cells/well; Nunc A/S, Roskilde, Denmark).
After 48 h incubation, supernatants were collected.
The IFN- concentrations of the supernatants were
measured by enzyme-linked immunosorbent assay(ELISA) using a human IFN- ELISA Kit (R&D
Systems).

Results
Absolute leukocyte and lymphocyte counts
and C-reactive protein levels after colorectal
cancer operation
The number of total leukocytes increased postoperatively and reached a maximum on day 1(Fig. 1a). The number of neutrophils also increased
postoperatively, reached a maximum on day 1 andthen decreased, but still significantly elevated on
day 3 (Fig. 1b). Further, a significant increase in Creactive protein levels was also observed on days
1, 3 and 7 compared with preoperative values (Fig.
1c). On the other hand, a rapid and significant decrease in number of total lymphocytes (Fig. 2a),CD4+ T cells (Fig. 2b) and CD8+ T cells (Fig. 2c)
was also observed. These cells reached a minimumon day 1 and then increased, but still significantly
decreased on days 3 and 7.

Media
Complete culture medium consisting of RPMI
1640 (Invitrogen, Carlsbad, CA), 1% L -glutamine (Invitrogen), 1% penicillin/streptomycin (Invitrogen), 2.5% HEPES buffer solution(Invitrogen), 1% sodium pyruvate (Sigma-Aldrich,
St. Louis, MO), 1% essential amino acids (SigmaAldrich) and 20% fetal bovine serum (SigmaAldrich) was used.
Statistical analysis
All values are expressed as mean ± SD. To determine statistical differences between 2 groups,
either paired or unpaired t-tests were used. The
accepted level of significance was P < 0.05. The
GraphPad Prism software (GraphPad Software, La
Jolla, CA) was used for all statistical analyses.

PD-1 and TIM-3 expressions on CD4+ andCD8+ T cells after colorectal cancer operation
Figure 3a shows representative results of PD-1 andTIM-3 expressions on CD4+ T cells in peripheral
blood obtained from colorectal cancer patients on
postoperative day 3 by FACS. PD-1 and TIM-3
expressions on both CD4+ and CD8+ T cells was
4Immunosuppression after colorectal surgery

Comparison with
the Pre level:*** P < 0.001.
** P < 0.01.

Fig. 2. Changes in number of total lymphocytes (a), CD4+ T cells (b) and CD8+ T cells (c) after surgery. POD, postoperative day; Pre, preoperation.

examined and the frequency of PD-1+CD4+ T cells(Fig. 3b) and TIM-3+CD4+ T cells (Fig. 3c) significantly increased after colorectal surgery. Figure
4a shows representative results of PD-1 and TIM3 expressions on CD8+ T cells in peripheral bloodobtained from colorectal cancer patients on postoperative day 3 by FACS. Moreover, a significant
increase in the frequency of PD-1+CD8+ T cells(Fig. 4b) and TIM-3+CD8+ T cells (Fig. 4c) was
also observed after colorectal cancer operation.

Functional differences of CD4+ and CD8+
T cells depending on PD-1 and TIM-3 expressions
Each phenotype of CD4+ and CD8+ T cells basedon PD-1 and TM-3 expressions was sorted, andthe function of each phenotype was determinedby measuring IFN- secretion. IFN- secretion by
PD-1+TIM-3+CD4+ T cells was significantly lessthan that by either PD-1+TIM-3–CD4+ T cells or

Comparison with
the Pre level:*** P < 0.001.
** P < 0.01.
*P < 0.05.

Fig. 3. FACS analysis of leukocytes obtained from patients after surgery.
a Representative results of PD-1 and TIM-3 expressions on CD4+ T cells in peripheral blood obtained from colorectal cancer patients on postoperative day 3 by FACS.
b Changes in PD-1 expression on CD4+ T cells after surgery.
c Changes in TIM-3 expression on CD4+ T cells after surgery.
FACS, fluorescence-activated cell sorting; PD-1, programmed cell death 1; TIM-3, T-cell immunoglobulin domain andmucin domain 3; POD, postoperative day; Pre, preoperation.

5

Y. Arai et al.

Comparison with
the Pre level:*** P < 0.001.
** P < 0.01.
*P < 0.05.

Fig. 4. FACS analysis of leukocytes obtained after surgery.
a Representative results of PD-1 and TIM-3 expressions on CD8+ T cells in peripheral blood obtained from colorectal cancer patients on postoperative day 3 by FACS.
b Changes in PD-1 expression on CD8+ T cells after surgery.
c Changes in TIM-3 expression on CD8+ T cells after surgery.
FACS, fluorescence-activated cell sorting; PD-1, programmed cell death 1; TIM-3, T-cell immunoglobulin domain andmucin domain 3; POD, postoperative day; Pre, preoperation.

PD-1–TIM-3–CD4+ T cells (Fig. 5a). Furthermore,
IFN- secretion by PD-1+TIM-3+CD8+ T cells was
significantly less than that by either PD-1–TIM3+CD8+ T cells or PD-1–TIM-3–CD8+ T cells (Fig.
5b).

Discussion
During the perioperative and postoperative periods, a complex biologic response takes place in
response to surgical stress. This response is intended to restore homeostasis as one aspect of hostdefenses against surgical stress. Thus, it is a very
important response for the host. On the other hand,
surgical stress induces suppression of cell-mediatedimmunity that is harmful to patients with cancer
who underwent operation, since it may diminish apatient’s ability to prevent postoperative infections.
Of importance is that surgical stress-induced suppression of cell-mediated immunity may place apatient at higher risk for tumor recurrence. To assess postoperative cell-mediated immune functionin the present study, the absolute number of circu-

Comparison with the PD-1+TIM-3+ level: *P < 0.05.

Fig. 5. IFN- productions by CD4+ T cells (a) and CD8+
T cells (b) based on PD-1 and TIM-3 expressions. IFN- ,interferon- ; PD-1, programmed cell death 1; TIM-3, Tcell immunoglobulin domain and mucin domain 3.

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Immunosuppression after colorectal surgery

ity levels and significantly shorter hospitalization.
With regard to prognosis, some prospective randomized clinical trials have suggested that there
were no differences between laparoscopic andopen surgery (Guillou et al., 2005; Veldkamp et al.,2005). Of importance is that a randomized clinical
trial demonstrated the prognosis of patients who
underwent laparoscopic surgery is significantly better than those who underwent open surgery (Lacyet al., 2008). This might be due to well preserved
cell-mediated immunity in patients who underwent
laparoscopic surgery. In fact, our preliminary data
indicated that TIM-3 expression on CD4+ andCD8+ T cells obtained from patients who underwent laparoscopic surgery was significantly lower
than that obtained from patients who underwent
open surgery, indicating that cell-medicated immunity is well preserved in patients who underwent
laparoscopic surgery compared with those who
underwent open surgery. Further investigations are
required to clearly demonstrate this matter.
Surgery induces tumor cell dissemination,and handling of the tumor during resection can
result in the spilling of tumor cells, as free floating tumor cells in the peritoneum, circulation andliver increase (Weitz et al., 1999; Conzelmann etal., 2005). Therefore, suppression of cell-mediatedimmunity after operation for colorectal cancer may
place the patient at higher risk for tumor recurrence. In fact, according to several clinical studies
that analyzed peri- and post-operatively obtained
blood, tumor cell presence is a strong predictor ofcolorectal cancer recurrence (Koch et al., 2005;
Allen-Mersh et al., 2007). Our data clearly demonstrated that both TIM-3 and PD-1 expressions wererelated to impaired function of T cells after operation for colorectal cancer. Therefore, PD-1 andTIM-3 might be good targets to restore impaired
cell-mediated immunity after operation for colorectal cancer. In fact, blockade of both the TIM-3–
TIM-3L and PD-1–PD-L pathways reverses thefunction of T cells, and is effective in controlling
tumor growth.
In conclusion, PD-1 and TIM-3 expressions
are closely related to the impaired cell-mediatedlating immunocompetent cells (CD4+ and CD8+ T
cells) was measured, since the absolute number ofcirculating immunocompetent cells have been considered good indicators of the individual patient’s
cell-mediated immune function. In the present
patients, the absolute number of total lymphocytes,
CD4+ T cells and CD8+ T cells significantly decreased after colorectal cancer operation. These results showed evidence of impaired function of cellmediated immunity after operation, consistently
with a previous report by Wichmann et al. (2005).
Although there are numerous reports demonstrating inadequate cellular immunity after surgery,most studying the effect of surgical trauma on immune function have generally assessed differencesin cell number and activity of immune cell subsets,
as well as determined levels of circulating factors
and mRNA expression of select genes (Hamid etal., 1984; Hansbrough et al., 1984; Faist et al., 1986;
Ogawa et al., 2000; Kalff et al., 2003). However,the detailed mechanisms responsible for these defects in cellular immunity remain unclear. Therefore, we determined PD-1 and TIM-3 expressions
on CD4+ and CD8+ T cells to demonstrate one
mechanism likely responsible for impaired cell-mediated immunity after colorectal surgery. Our dataclearly demonstrated that PD-1 expression on both
CD4+ and CD8+ T cells significantly increased
after operation. Furthermore, TIM-3 expression
on both CD4+ and CD8+ T cells also significantly
increased after operation. Of importance is that
IFN- production by CD4+ and CD8+ T cells was
related to TIM-3 and PD-1 expressions on CD4+
and CD8+ T cells in the present study, indicatingthat TIM-3+ PD-1+ CD4+ and CD8+ T cells arehighly dysfunctional compared with other CD4+
and CD8+ T cells. These data indicate that both
TIM-3 and PD-1 expressions on CD4+ and CD8+
T cells is related to impaired function of these cells
after operation.
Laparoscopic-assisted colectomy has gained
acceptance for the treatment of colorectal cancer
patients. The main advantage of laparoscopic surgery over open surgery is decreased invasivenessthat results in a rapid return to preoperative activ7

Y. Arai et al.

immunity that is observed after surgery -forcolorectal cancer, and the development of specifictreatments targeting TIM-3 and PD-1 on CD4+ andCD8+ T cells during the perioperative period may
provide a breakthrough in the treatment of colorectal cancer patients.

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11 Golden-Mason L, Palmer BE, Kassam N, TownshendBulson L, Livingston S, McMahon BJ, et al. Negative
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in hepatitis C virus infection and its blockade rescues dysfunctional CD4+ and CD8+ T cells. J Virol
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Received December 22, 2011; accepted January 4, 2012
Corresponding author: Yosuke Arai

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