Expression of indoleamine 2,3-dioxygenase in nasopharyngeal carcinoma impairs the cytolytic function of peripheral blood lymphocytes
© Liu et al; licensee BioMed Central Ltd. 2009
Received: 27 April 2009
Accepted: 30 November 2009
Published: 30 November 2009
Tumor-specific cytotoxic T cells and infiltrating lymphocytes are frequently found in tumor tissues in patients with nasopharyngeal carcinoma (NPC). Most patients with NPC, however, especially those with advanced stages, have a poor clinical prognosis despite conventional immunotherapy. The aim of this work was to examine the effect of indoleamine 2,3-dioxygenase (IDO), an immunosuppressive enzyme, on the lymphocyte function in NPC.
The NPC cell line CNE2 was treated by interferon-γ (IFNγ) and the levels of IDO expression was analyzed by Western blotting and reverse phase high-performance liquid chromatography (HPLC). Lymphocytes from health human exposed to the milieu created by IDO-positive CNE2 cells and the lymphocyte cytotoxicity to target tumor cells was analyzed by standard lactate dehydrogenase (LDH) release assay. Additionally, expression of IDO was determined by Immunohistochemical assay in the tumor tissues form clinically evaluated NPC.
IDO expression was acutely induced in the NPC cell line CNE2 by low dose interferon-γ (IFNγ) or by co-incubation with activated lymphocytes. Exposure to the milieu created by IDO-positive CNE2 cells did not promote lymphocyte death, but lymphocyte cytotoxicity against target tumor cells was impaired. The suppression of lymphocyte cytotoxic function was fully restored when the conditioned medium was replaced by fresh medium for 24 h. In additionally, the IDO-positive cells were found scattered in the tumor tissues from patients with NPC.
Altogether, these findings suggest that IDO-mediated immunosuppression may be involved in the tumor immune evasion, and that blocking IDO activity in tumor cells may help to re-establish an effective anti-tumor T cell response in NPC.
Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)-associated malignancy with high prevalence in Southern China and Southeast Asia . Guangdong province, also called Canton, has the highest prevalence, earning NPC the name of 'Canton tumor'. Due to the non-specific nature of the nasal and aural symptoms and the difficulty of making a clinical examination of the nasopharynx, most patients with the disease are diagnosed only when the tumor has reached an advanced stage (stages III and IV) . Radiotherapy is the main treatment for this disease, but patients with intermediate and advanced stages who only receive radiotherapy have a 5-10-year survival rate of only 40%. Hence, novel approaches to the treatment of NPC are needed to improve the prognosis of patients with NPC.
Immunotherapeutic strategies aimed at boosting anti-tumor immunity are promising candidates for the treatment of NPC. A few studies have focused on reversing the impaired immune response to NPC tumors . Determination of the mechanisms behind the dysfunction of cytotoxic T lymphocytes in patients with NPC would undoubtedly be of help in the development of optimal immunotherapeutic strategies for NPC. It has been reported that cytokine expression in tumor infiltrating lymphocytes (TILs) in NPC patients is comparable to that in healthy controls. Interferon-γ (IFNγ) is one of the prominent cytokines associated with immune activation and immunosuppression .
IFNγ, also called type II interferon or immune interferon, is mainly produced by activated T cells and NK cells, and acts as an important mediator of the immune system, involving activities such as immuno-modulation, lymphocyte recruitment and activation, anti-pathogen and anti-tumor activity . Although IFNγ was first used to treat patients with NPC in 1987 , there was no further report on IFNγ therapy for NPC since 1993 due to some cases were shown to be unresponsive. In most cases of NPC, the dense infiltration of lymphocytes is observed in the tumor site, and EBV-associated viral antigens in tumor cells are presented for lymphocyte recognition, nevertheless IFNγ fails to exert its intended anti-viral and anti-tumor effects in the patients with NPC [7, 8].
IFNγ has the specific ability to induce indoleamine 2,3-dioxygenase (IDO) expression in various kinds of tumors . IDO is responsible for initiating the first, rate-limiting step in tryptophan metabolism in the kynurenine (Kyn) pathway . Growing evidence suggests that IDO-mediated tryptophan metabolism in antigen presenting cells and tumor cells represent a vital mechanism for potential T cell suppression during tumor growth. Localized tryptophan deficiency and the accumulation of toxic metabolites in tumor-draining lymph nodes and the tumor microenvironment could contribute to the growth arrest, inactivation, and even death of T cells [11–13]. IDO has been investigated in cervical, colorectal, hepatocellular, ovarian carcinoma, endometrial cancer and thyroid cancer [14–18], but to our knowledge, no detailed studies have investigated the expression of IDO in NPC.
We therefore aimed to examine the roles of IFNγ and IDO in NPC, in order to throw new light on the mechanism by which immune evasion affects therapeutic treatment of NPC.
The human nasopharyngeal carcinoma cell line CNE2 was established at Hunan Medical College, China [19, 20]. CNE2 cells used in this study were maintained in our laboratory in RPMI 1640 medium (Gibco-BRL, MD) supplemented with 10% fetal calf serum (FCS, PAA Laboratories, MA) and antibiotics (100 U/ml penicillin, 100 μg/ml streptomycin). When the monolayer of CNE2 cells reached 60% confluency, IFNγ (China National Biotec Group, Shanghai, China) was added to the medium at 0-500 U/ml, for the indicated time.
Human peripheral blood was donated by healthy volunteers, following appropriate informed consent. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation through Ficoll-Hypaque (Tian Jin Hao Yang Biological Manufacture Co., TianJin, China) according to standard procedures. Monocytes and phagocytes were removed by adherence to plastic by culturing PBMCs in RPMI 1640 containing 50 U/ml exogenous interleukin-2 (IL-2, Kexing Biotech Co., Shenzhen, China) for 4-5 h. The non-adherent peripheral blood lymphocyte (PBL) fraction was harvested and cultured at a density of 2 × 106 cells/ml in 24-well plates with complete RPMI 1640 medium (containing 10% FCS, 100 U/ml penicillin and 100 μg/ml streptomycin). IL-2 100 U/ml was added to the medium every other day, for an incubation period of at least 4 days.
Conditioned medium treatment
CNE2 cells were cultured in 6-well plates (3 × 105 cells/well) in the absence or presence of 50 U/ml IFNγ for 12 h, and the medium was then replaced by fresh medium, with or without 100 μM 1-methyl-D/L-tryptophan (1 MT, Sigma-Aldrich, MO). Twenty-four hours after medium replacement, culture medium was harvested as conditioned medium (CM) from CNE2 cells (CNE2-CM), and used for the incubation of PBLs.
To prepare the CM from activated PBLs for treating CNE2 cells, IL-2-treated PBLs were suspended in complete RPMI 1640 medium or CNE2-CM, and all media were supplemented with 100 U/ml IL-2. After culture for 24 h, PBLs were harvested for cytotoxicity assays and the supernatant was collected and used for incubating CNE2 cells. CNE2 cells were first cultured in complete RPMI 1640 medium for 18 h, and the medium was then replaced by the above PBL CM and the CNE2 cells were incubated for 24 h.
Formalin-fixed and paraffin-embedded NPC samples (n = 9) were selected from archival material from the Department of Pathology in Sun Yat-sen University of Medical Sciences. The sections were deparaffinized in xylene and rehydrated through graded alcohol washes to distilled water. Endogenous peroxidase was blocked with 0.3% H2O2 for 30 min and non-specific staining was prevented by incubation in commercial goat serum (Wuhan Boster Biological Technology Ltd, Wuhan, China). Sections were stained using the streptavidin-peroxidase (SP) staining method by incubation with rabbit anti-human IDO polyclonal antibody (1:500, generated in our laboratory ) at room temperature for 1 h. After washing in phosphate buffered saline (PBS) containing 0.05% Tween-20 (PBS-T), the sections were incubated with secondary antibodies from the EnVision™ Detection Kit (Gene Tech, Shanghai, China). Slides were stained in the absence of primary antibodies to evaluate non-specific secondary antibody reactions and were counterstained with hematoxylin.
In order to detect the expression of IDO in CNE2 cells they were immunohistochemically stained using the same SP staining method as for the clinical specimens.
Western blot analysis
The treated CNE2 cells were lysed in cell lysis buffer (50 mmol/l Tris-Cl, pH = 8.0; 150 mmol/l NaCl; 0.02% NaN3; 0.1% sodium dodecyl sulfate (SDS); 1% NP-40; 0.5% sodium deoxycholate; 1 mol/l EDTA). The lysates were cleared by centrifugation and denatured by boiling in Laemmli buffer. Equal amounts of protein (20 μg/well) were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Bio-Rad, CA). After blocking in Tris-buffered saline containing 0.1% Tween-20 and 5% nonfat milk, the membrane was incubated with the rabbit anti-human IDO polyclonal antibody, followed by incubation of horseradish peroxidase-conjugated anti-rabbit IgG (Wuhan Boster Biological Technology Ltd). Protein was visualized by enhanced chemiluminescence (ECL, Pierce, IL), For the loading control, β-actin (1:10,000, Beijing Biosynthesis Biotechnology Co., Beijing, China) was probed with the primary antibodies after the membrane was stripped of the IDO antibodies using stripping buffer.
Measurement of IDO activity
The culture medium for IDO activity analysis was mixed with 1/4 volume of 30% trichloroacetic acid and then centrifuged at maximum speed for 20 min at 4°C to precipitate the protein. The concentration of Kyn in the culture medium was measured by reverse phase high-performance liquid chromatography (HPLC), using a Waters Alliance HPLC system (Waters, MA). Kyn was detected by the UV detector at 340 nm and the results were processed in Breeze version 3.30 SPA. IDO activity was determined by the specific Kyn production after subtracting the non-specific Kyn concentration in the RPMI 1640 medium with 10% FCS.
Flow cytometric analysis
PBLs untreated and pre-treated with IL-2 were plated into 24-well plates in complete RPMI 1640 medium, or CNE2-CM without 1 MT (described in 2.2). After 48 h incubation, cells were harvested, washed with PBS, and resuspended in annexin V-binding buffer. Fluorescein isothiocyanate-conjugated annexin V (Trevigen, MD) was added to a final concentration of 100 ng/ml and cells were incubated in the dark for 15 min at room temperature. Propidium iodide (PI, Trevigen) was added to each sample so that the final concentration of PI was 50 mg/ml before flow cytometric analysis.
The expression of IDO in the NPC cell line CNE2 is highly sensitive to IFNγ stimulation
Enzymatic activity of IDO is up-regulated by IFNγ stimulation in the NPC cell line CNE2
Activated PBLs induce expression of IDO in the NPC cell lineCNE2
IDO expression in NPC cells would alter the microenvironment by depletion of tryptophan and production of Kyn, so potentially affecting the ability of PBLs to induce IDO expression. To explore this possibility, PBLs were stimulated with 100 U/ml IL-2 for 96 h and washed to remove early generated cytokines. After further incubation with IL-2-supplemented CNE2-CM from CNE2 cells treated or untreated with IFNγ for 24 h, the PBL supernatants were collected and used to incubate CNE2 cells for 24 h. Western blot analysis showed that, as with CM prepared by incubating PBLs with CNE2-CM derived from untreated CNE2 cells, CM from PBLs which had been cultured in CNE2-CM derived from IFNγ-treated CNE2 cells was also able to induce IDO expression (Fig. 3C).
Exposure to the microenvironment created by IDO-positive CNE2 cells does not severely reduce PBL survival
Exposure to conditioned medium derived from IDO-positive CNE2 cells impairs cytolytic activity of PBLs
We also investigated if PBL cytotoxicity could be restored when IDO was removed. Activated PBLs were incubated with CNE2-CM derived from IFNγ-treated or untreated CNE2 cells containing 100 U/mL IL-2 for 96 h, and restored groups were then transferred to fresh medium containing 100 U/mL IL-2 and incubated for 24 h. The cytolytic activity of each group was analyzed by LDH release assay. PBLs cultured in CNE2-CM from IFNγ-treated CNE2 cells exhibited decreased cytolytic activity against target cells, compared with those in CNE2-CM from untreated CNE2 cells (Fig. 5D). However, the cytolytic activity of PBLs cultured in CNE2-CM derived from IFNγ-treated CNE2 cells was fully restored when the CNE2-CM was replaced by fresh medium for 24 h. In the presence of 100 μM 1 MT, both un-restored and restored groups maintained a similar level of cytolytic activity. These results suggest that the tumor milieu created by IDO-positive NPC cells could significantly impair the cytolytic function of activated lymphocytes, providing a potential mechanism for immune evasion in patients with NPC.
Expression of IDO in tumor tissues from patients with NPC
Unlike most carcinomas in which TILs are infrequently observed, NPC has intense lymphocyte infiltration in the tumor tissue [25, 28], though these infiltrated lymphocytes apparently have limited ability to eliminate the tumor. The present study demonstrated, for the first time, that expression of IDO occurred in the NPC-derived cell line CNE2 and in tumor tissues from patients with NPC. Furthermore, we have shown that the expression of IDO in CNE2 cells was inducible by activated PBLs and that the cytolytic function of PBLs was impaired in the tumor milieu created by enzyme activity of IDO, providing a novel insight into the role of IDO in tumor immune evasion in NPC.
The infiltration of lymphocytes to tumor sites suggests that an immune response has been initiated, and Tang et al found that TILs in undifferentiated NPC are in an activated state . However, NPC still progresses in most patients, and the prognosis of advanced stage NPC is frustratingly poor , indicating that tumor immune evasion occurs in these patients. Several mechanisms have been proposed to explain this e.g., the abnormal expression of IL-10 and Bcl-2 in NPC cells [31, 32], as well as the impairment of IFNγ secretion and perforin expression in CD8+ T cells . In this study, we found that the immunosuppressive enzyme IDO was highly expressed in tumor tissues from NPC patients, suggesting that IDO-mediated immunosuppression may be involved in immune evasion, and so contribute to NPC progression.
Previous studies have demonstrated posttranslational control of IDO, implying that IDO expression and activity may not be correlated [34, 35]. However, in the present study, analysis of Kyn production proved that the IDO expressed in CNE2 cells possessed functional enzymatic activity, and suggesting that active IDO was induced in NPC cells by IFNγ. We also showed that IFNγ induced IDO expression in CNE2 cells at concentrations as low as 5 U/ml, implying that IDO expression in NPC cells could be easily induced by low levels of IFNγ. The time course of IDO expression in CNE2 cells treated with IFNγ indicated that, once IDO protein was synthesized, it degraded slowly (>72 h), which could result in a long-lasting effect of IDO in NPC patients, resulting in severe tryptophan deprivation and Kyn accumulation at tumor sites. Hence, dense infiltration of lymphocytes or systemic administration of IFNγ could induce IDO expression and cause persistent, enhanced IDO activity in NPC patients.
Western blot analysis demonstrated that CM from activated PBLs was able to induce IDO expression in CNE2 cells. PBLs are known to functionally express multiple inflammatory cytokines, including IFNγ and TNFα, which are believed to be potent inducers of enzyme expression . The ability of PBLs to induce IDO expression was also observed after culture in CNE2-CM derived from IDO-expressing CNE2 cells, suggesting that the IDO-generating condition did not directly impair cytokine release. This is in agreement with the results of previous studies, which found that the T cell activation markers, including IL-2, IFNγ, CD69, CD25, and CD71, were still expressed under conditions depleted of tryptophan and/or enriched in Kyn [13, 37]. It has also been reported that merely activating the immune system, such as by adoptive T cell transfer using a conventional lymphoblastoid cell line-reactivated preparation [38, 39], or by vaccination with dendritic cells [40, 41], might be inadequate for immunotherapy, and may result in up-regulation of IFNγ and/or TNFα production, leading to IDO expression in tumor cells . Further studies are needed to determine if IDO expression in CNE2 cells is directly induced by these cytokines, if these cytokines act synergistically to increase the expression of IDO, or if there is an independent pathway of IDO expression that exists in the CM from activated PBLs.
In the present study, we found that IFNγ-induced IDO expression in CNE2 cells did not promote apoptotic death of PBLs. IDO-expressing cells have been reported to promote apoptosis of blood T cells due to the cytotoxicity of tryptophan metabolites, such as Kyn, but not through tryptophan deficiency. Terness et al. and Frumento et al. showed that Kyn concentrations > 100 μM suppressed T cell proliferation by inducing cell death, whereas concentrations < 30 μM did not cause T cell apoptosis [26, 42]. In the current study, IDO-expressing CNE2 cells generated Kyn in an IFNγ dose-dependent manner, and Kyn concentration in the cell culture system did not exceed 10 μM after treatment with 50 U/ml of IFNγ, which was too low to promote T cell death. Although it is difficult to extrapolate from in vitro to in vivo situations, the lack of a cytotoxic effect due to the presence of Kyn at a low concentration in the tumor microenvironment could explain previous observations of high densities of lymphocytes in NPC tumor tissue.
Once expressed in the tumor cells, IDO creates a local environment depleted of tryptophan and enriched in Kyn, thus rendering TILs susceptible to proliferation arrest and/or dysfunction. In our in vitro study, we found that exposure to CNE2-CM derived from IDO-expressing CNE2 cells dramatically weakened the cytolytic function of PBLs against target cells, but this attenuation could be reversed by addition of the IDO inhibitor, 1 MT. In this case, the down-regulation of the cytolytic function of PBLs did not appear to be due to the production of viral IL-10 (vIL-10), which was frequently observed in NPC tumor tissue , because vIL-10 was not detectable in CNE2 cells by reverse transcription polymerase chain reaction analysis (data not shown). These results suggest that PBLs are susceptible to the IDO-generating environment and that blocking IDO activity could restore their anti-tumor activity. We demonstrated that the cytolytic activity of PBLs could be fully restored by the replacement of CM from IDO-expressing CNE2 cells with fresh medium. Previous studies also found that IDO-mediated T cell proliferation arrest was overcome by the addition of tryptophan and removal of metabolites from the medium [9, 26, 42]. These observations suggest that the IDO-mediated suppression of PBL cytotoxicity is a reversible event, and that blocking IDO activity in tumor cells could help to re-establish the anti-tumor immune response, especially in tumors with high densities of infiltrating lymphocytes, such as NPC.
In conclusion, we identified IDO expression in NPC and found that the enzyme could be efficiently induced by low dose IFNγ in the NPC cell line CNE2. PBLs exposed to the milieu generated by IDO-expressing CNE2 lost their cytolytic function, but did not undergo promoted cell death. We suggest that the negative effect of IDO expression in NPC cells on the cytolytic function of PBLs could contribute to the lack of efficacy of current immunotherapeutic strategies. Blocking both two isoforms of IDO, IDO1 and IDO2, by using their inhibitors L- and/or D-1-methyl-tryptophan  may provide a potential means of improving the outcomes of immunotherapy for the treatment of NPC.
human nasopharyngeal carcinoma cell line
Peripheral blood mononuclear cells
conditioned medium from CNE2 cells
high-performance liquid chromatography
- 1 MT:
This work was supported by National Science Foundation of China (No. 30873032 and No. 30672510) and the Bureau of Guangdong Provincial Science and Technology (No. 2008A030201007 and No. 2007B030704007).
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