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Annals of Oncology Advance Access originally published online on September 9, 2007
Annals of Oncology 2007 18(11):1804-1809; doi:10.1093/annonc/mdm356
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© 2007 European Society for Medical Oncology

gynecologic tumors

HLA-G expression in human ovarian carcinoma counteracts NK cell function

A. Lin1,2, W.-H. Yan1,*, H.-H. Xu1, M.-F. Gan3, J.-F. Cai3, M. Zhu1 and M.-Y. Zhou1

1 Medical Research Center
2 Human Tissue Bank
3 Department of Pathology, Taizhou Hospital of Zhejiang Province, Wenzhou Medical College, Linhai, Zhejiang, People's Republic of China

* Correspondence to: Dr W.-H. Yan, Medical Research Center, Taizhou Hospital of Zhejiang Province, Wenzhou Medical College, Linhai, Zhejiang, 317000, People's Republic of China. Tel: +86–576–85199347; E-mail: yanwhcom{at}yahoo.com


   Abstract
Top
 Abstract
introduction
 materials and methods
 results
 discussion
 Acknowledgements
 References
 
Background: Human leukocyte antigen-G (HLA-G) is an important immunotolerant which could be a part of the strategies applied by malignant cells applied to avoid host immunosurveillance. Aberrant expression of HLA-G has been found in ovarian carcinoma. The aim of this study was to evaluate the HLA-G expression in ovarian cancer tissues and to explore its function in vitro.

Materials and methods: HLA-G expression in 33 primary ovarian carcinoma tissues was analyzed using immunohistochemistry with the anti-HLA-G monoclonal antibody (mAb) 4H84. Furthermore, the function of HLA-G in NK cell cytotoxicity was determined in vitro by cloning and expression of HLA-G on the ovarian carcinoma cell OVCAR-3.

Results: HLA-G expression was detected in 22/33 (66.7%) primary tumor tissues, but was absent in normal ovarian tissues (P<0.01). Cytotoxicity studies showed that HLA-G expression dramatically inhibits cell lyses by NK-92 cells (P<0.01), which could be restored by the anti-HLA-G conformational mAb 87G (P<0.01).

Conclusion: HLA-G was expressed in a significant number of primary ovarian carcinoma tissues, and HLA-G expression in OVCAR-3 could directly inhibit NK-92 cell lysis. Taken together, our results indicated that expression of HLA-G plays an important role in evasion of ovarian cancer cells from host immunosurveillance.

Key words: HLA-G, immune response, NK cell, ovarian carcinoma


   introduction
Top
 Abstract
 introduction
materials and methods
 results
 discussion
 Acknowledgements
 References
 
HLA-G is a non-classical HLA class I molecule from the major histocompatibility complex (MHC) which was initially reported to be restricted to extravillous cytotrophoblasts and confers protection to the fetus from maternal immune responses [1,2]. HLA-G features a low level of allelic polymorphism and encodes seven protein isoforms generated by alternative splicing of its primary mRNA [3]. Extensive studies have been carried out on its functions in fetal–maternal immune maintenance. These studies suggested that HLA-G serves as a protection factor for the fetus from maternal allorecognition by the ability of HLA-G to modulate both the functions of immune component cells, such as natural killer (NK) cells, T lymphocytes, antigen presenting cells and cytokine balance during pregnancy [4–7]. The mechanisms that underlie the functions of HLA-G involve its interaction with various receptors such as immunoglobulin-like transcripts 2 (ILT2), ILT4 and killer immunoglobulin receptor 2DL4 (KIR2DL4) expressed on these immune cells [8–10].

Other than extravillous cytotrophoblasts, distribution of HLA-G currently has been found to be broader than originally reported. An increasing number of studies have reported that HLA-G expression was observed in various malignancies such as ovarian carcinoma, breast carcinoma, hematopoietic tumors, gastric carcinoma, cutaneous melanoma, endometrial carcinoma, renal cell carcinoma, mesothelioma and trophoblastic tumors [11–15]. HLA-G expression in both primary and metastatic tumor sites but not in healthy skin and tumor regression sites suggested that HLA-G expression was also associated with malignant pathogenesis [16].

Ovarian cancer is the leading cause of death from gynecologic cancer in women [17]. Previous reports indicated that HLA-G was expressed in solid ovarian cancer and soluble HLA-G (sHLA-G) was significantly higher in malignant effusions compared to their benign counterparts, indicating that HLA-G was a potential tumor marker for malignant ovarian cancer. Furthermore, HLA-G expression in effusions from advanced-stage ovarian carcinoma cells had been proposed as a possible marker of tumor susceptibility to chemotherapy [18,19].

The objective of the current study was to analyze the expression of HLA-G in primary ovarian carcinoma and its roles in NK cell function modulation in vitro. Results revealed that HLA-G was expressed in a significant number of ovarian carcinoma tissues, and NK cell cytolysis inhibition by HLA-G was observed in vitro.


   materials and methods
Top
 Abstract
 introduction
 materials and methods
results
 discussion
 Acknowledgements
 References
 
tissue samples
The acquisition of paraffin tissues was approved by the Medical Ethical Review Committee of the Wenzhou Medical College affiliated Taizhou Hospital of Zhejiang Province. A total of 33 formalin fixed, paraffin-embedded ovarian serous carcinomas tissue samples and 13 normal ovaries were retrieved from the clinical pathology files.

immunohistochemistry
Four-micrometer thick sections of the paraffin-embedded tissue blocks were cut and mounted on polylysine coated slides. They were dewaxed in xylene and rehydrated through a graded series of ethanol. After deparaffinization, antigen retrieval treatment was performed at 120°C (autoclave) for 5 min in a 10 nmol/l sodium citrate buffer (pH 6.0). Endogenous peroxidase activity was blocked by using a 0.03% hydrogen peroxide solution at room temperature for 30 min. Then, anti-HLA-G mAb 4H84 (1:500) was applied and incubated overnight at 4°C. After that, a thorough washing in a 0.01 M phosphate-buffered saline (PBS) solution was performed. Subsequently, binding sites of the primary antibody were visualized using a Dako EnVison kit (Dako, Denmark) in accordance with the manufacturer's instructions. Finally, sections were counterstained with hematoxylin and mounted with glycerol gelatin. HLA-G staining in ovarian carcinoma tissues was determined by three pathologists. Membrane or combined membrane and cytoplasmic expression of HLA-G were interpreted as positive. The pathologists were blinded to any clinical details related to the patients.

cells and antibodies
The human ovarian carcinoma cell line OVCAR-3 and the choriocacinoma cell lines JEG-3 and JAR (ATCC, MD, USA) were grown in complete RPMI 1640 media supplemented with 10% heat-inactivated fetal bovine serum (FBS). The NK cell lines NK-92 (ATCC, MD, USA) were grown in alpha minimum essential media ({alpha}-MEM) without ribonucleosides and deoxyribonucleosides (Gibco BRL, USA) supplemented with 12.5% heat-inactivated FBS (Gibco BRL, USA), 12.5% horse serum (Gibco BRL, USA), 0.2 mM inositol, 0.1 mM 2-mercaptoethnol, 0.02 mM folic acid, and 200 U/ml of rIL-2 (Bioscience, NY).

Anti-HLA-G conformational mAb 87G (IgG1) was a kind gift from Dr Geraghty (Fred Hutchinson Cancer Research Center, Seattle, WA, USA), and the mAb 4H84, IgG1 antidenatured HLA-G heavy chain was kindly provided by Dr McMaster (Department of Stomatology, University of California, San Francisco, CA, USA).

reverse transcription-polymerase chain reaction (RT-PCR)
The choriocarcinoma cell lines JEG-3 and JAR were used as an HLA-G positive and negative control, respectively. Total RNA was extracted from JEG-3, JAR and OVCAR-3 cells with TRIZOL reagent (Invitrogen, NY, USA) according to the manufacturer's instructions. Two micrograms of total RNA was then reverse transcribed into first-strand cDNA with Moloney murine leukemia virus reverse transcriptase (Invitrogen, NY, USA) and oligo(dT)12–18 primers (Invitrogen, NY, USA) in a 20 µl reaction. Subsequently, 1 µl of synthesized cDNA was subjected to PCR in a 50 µl reaction using 2 U of Taq DNA polymerase (Roche, Indianapolis, USA) with corresponding HLA-G primers. For HLA-G1 cloning, the forward primer 5'-TCGGATCCCTCATTCTC CCCAGACGCCA-3' containing a BamH1 site, and the reverse primer 5'-CCC GATCGATTGAGACAGAGACGGAGACAT-3' containing a Cla1 site were used. PCR conditions for the full length HLA-G1 cloning were as follows: 94°C, 1 min; 58°C, 1 min; 72°C, 2 min for 35 cycles followed by a final extension of 10 min at 72°C. The PCR product was ligated to the pGEM®-Teasy vector (Promega, WI, USA) and confirmed by sequencing. Primers used for HLA-G isoform transcripts expression in OVCAR-3 cells were according to the report by Yao et al. [20] which were designed to distinguish each HLA-G mRNA isoform individually, namely HLA-G1~G6. An additional set of primers was designed to amplify all isoforms of HLA-G together (pan HLA-G). Primers are listed in Table 1. PCR was conducted for one cycle of 3 min at 94°C and then 40 cycles of 30 s at 94°C, 30 s at 58°C, and 1 min at 68°C. All PCR products were separated on 1.5% agarose gels and visualized with ethidium bromide (Sigma, St. Louis, MO, USA) on top of a UV light illuminator.


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  		Table 1. RT-PCR primers for amplification of HLA-G mRNA isoforms [20]

 
HLA-G transfection of OVCAR-3 cell line
The OVCAR-3 cells were transfected with the recombinant pVITRO2-mcs vector (Invivogen, NY, USA) containing HLA-G1 using LipofectamineTM 2000 reagent (Invitrogen, NY, USA) according to the manual instructions. The transfectants were screened with Hygromycin B (Amresco, OH, USA) and HLA-G expression was monitored by flow cytometry (BD FACSCalibur, MI, USA) with mAb 87G.

cytotoxicity assay and HLA-G specific mAb blocking assay
Cytotoxicity was performed using CytoTox96® Non-Radioactive Cytotoxicity Assay Kit (Promega, MD, USA). Effector NK-92 cells were cultured with rIL-2-containing medium on the day before assay. Effector/target ratio was optimized. During the cytolysis assay, effector cells were mixed with 1x104 target cells at a 10:1 E:T ratio in V-bottom 96-well plates (Costar, Cambridge, MA) as the protocol instructed. For the restoration of cytotoxicity by HLA-G specific mAb 87G blockade, targets were preincubated with 5 µg/ml and 10 µg/ml 87G respectively for 30 min before the effector NK-92 cells were added. Isotype IgG was added as a control. Target cell spontaneous release and maximal release of LDH and the effector cell spontaneous release of LDH were determined by incubating these cells in medium alone. Each assay was performed in quadruplicate and the results are expressed as percentages of lysis ± SD. The percentage of specific lysis was determined as follows:

Formula

statistical analysis
All statistical analyses were carried out with the SPSS software program. Correlations between the staining of HLA-G and ovarian cancers or normal ovaries were calculated using the {chi}2 test. Cytotoxicity differences between groups were analyzed for significance by a two-sided Student's t-test. A probability of <0.05 was considered statistically significant.


   results
Top
 Abstract
 introduction
 materials and methods
 results
discussion
 Acknowledgements
 References
 
HLA-G expression in ovarian carcinoma tissues
Immunohistochemical analysis of ovarian carcinomas revealed that HLA-G was expressed in 22 (66.7%) of 33 ovarian serous carcinomas. The positive tumor cells showed with both membranous and cytoplasmic staining pattern, ranging from single cells to large clusters of positive tumor cells (Figure 1). HLA-G was not detected in normal ovarian tissues (data not shown).


Figure 1
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  		Figure 1. Ovarian carcinoma cells express HLA-G. (A) HLA-G-negative primary serous ovarian carcinoma. (B) and (C) two HLA-G-positive primary serous ovarian carcinoma with combined membrane and cytoplasmic expression.

 
transfection and expression of HLA-G in OVCAR-3 cells
Exogenous HLA-G1 was cloned, sequence confirmed and transfected into the OVCAR-3 cells. HLA-G1 transcript and cell surface HLA-G expression were measured by RT-PCR and flow cytometry, respectively. The HLA-G1 isoform transcript was highly synthesized in HLA-G transfected OVCAR-3 cells (OVCAR-3-G) (Figure 2A), and cell surface stable HLA-G1 expression was detected by flow cytometry with a geometric mean (GM) of 53.78 ± 5.4 (Figure 2B). Note, the HLA-G3 isoform transcript was observed both in OVCAR-3 and in OVCAR-3-G cells.


Figure 2
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  		Figure 2. HLA-G expresses in ovarian cancer cells OVCAR-3 and OVCAR-3-G. (A) RT-PCR analysis of the HLA-G mRNA isoforms expressed in OVCAR-3 and OVCAR-3-G. HLA-G mRNA isoforms in the JEG-3 and JAR cells are a positive and negative control, respectively. The lanes are: M, 1-kb DNA marker; Pan, Pan HLA-G; G1, HLA-G1; G2, HLA-G2; G3, HLA-G3; G4, HLA-G4; G5, HLA-G5; G6, HLA-G6. (B) Analysis of cell surface HLA-G expression in OVCAR-3 and OVCAR-3-G cells with flow cytometry. Shaded area: isotype IgG; unshaded area: HLA-G mAb 87G.

 
protection of the HLA-G transfected OVCAR-3 cells from NK cytolysis
We then investigated whether such HLA-G protein expression in ovarian cancer cell lines could be associated with decreased susceptibility to NK lysis. For this purpose, the leukemia cell line NK-92 was used as a model of the NK effector cell. NK-92 cells bear known killer receptors such as ILT2 and KIR2DL4, which are involved in HLA-G-specific recognition. NK-92 cell line is an IL-2-dependent NK-like cell line. NK-92 serves as an excellent model system to study NK cell biology and KIR functions due to its strong target cell cytotoxicity and well-defined cell surface markers. In this study, we show that HLA-G positive ovarian cancer cells could abolish NK-92 cell lysis with a percentage of 20.4 ± 4.1% for the HLA-G transfected OVCAR-3 cells, while the OVCAR-3 cells were lysed by NK-92 with a percentage of 67.0 ± 7.6%. Significant difference was found between the OVCAR-3-G and OVCAR-3 cells (P<0.01) (Figure 3A). Blocking experiments with the HLA-G specific mAb 87G were performed to exclude the involvement of other HLA molecules present on ovarian cancer cells in the inhibition of NK-92 lysis. As shown in Figure 3B, different concentration of 87G enhanced NK-92 killing of OVCAR-3-G cells, and the restored lyses was proportional to the amount of the 87G in the experiments.


Figure 3
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  		Figure 3. NK-92 cytotoxicity inhibition induced by HLA-G expression. (A) comparison of the mean cytolytic percentage of NK-92 to different targets (OVCAR-3 and OVCAR-3-G) at a E/T ratio of 10:1. Four independent experiments were performed, and the results are expressed as percentages of specific lysis ± SD. (B) restoration of cytotoxicity by HLA-G mAb 87G antibody blockade. Target cell OVCAR-3-G were preincubated with 5 µg/ml and 10 µg/ml 87G, respectively, or isotope IgG as a control antibody at a E/T ratio of 10:1. Four independent experiments were performed, and the results are expressed as percentages of specific lysis ± SD. * P<0.05, ** P<0.01.

 

   discussion
Top
 Abstract
 introduction
 materials and methods
 results
 discussion
Acknowledgements
 References
 
Ovarian cancer is the leading cause of death from gynecologic cancer [17]. Epithelial ovarian carcinoma displays unique affinity to serosal surfaces and their lining organs. Consequently, metastases to these anatomic sites and positive effusion cytology are present in the majority of patients at diagnosis of this disease [21]. Various changes, including the expression of certain tumor markers, take place during the progression of cancer, some of which are favorable for tumor development and help escape from host immunosurveillance. One such recently discovered molecule is HLA-G, which has been found to have immunosuppressive and immunomodulatory roles in cancer development [22].

HLA-G, a non-classical MHC class I antigen, has limited polymorphism and is expressed in seven isoforms. Alternative splicing of the primary HLA-G transcript leads to the synthesis of the four membrane-bound HLA-G1, HLA-G2, HLA-G3 and HLA-G4 isoforms, as well as the three soluble HLA-G5, HLA-G6 and HLA-G7 isoforms [2,3]. HLA-G antigens play a key role in the establishment and maintenance of immune tolerance by inhibiting the function(s) of immunocompetent cells. Inhibitory effects are mediated by the direct binding of HLA-G to its receptors such as ILT2, ILT4 and KIR2DL4. Consequently, HLA-G can, through these receptors, directly interact with NK cells, T lymphocytes and antigen-presenting cells, and exert its immunotolerant functions at different stages of the immune response [23].

Paul et al. have described for the first time that expression of HLA-G was found in solid tumors [24]. To date, HLA-G expression has been seen in most tumors, including ovarian cancer, lung cancer, colon cancer, melanoma, breast cancer, renal cancer, etc. [22]. Studies showed that HLA-G was detected in around 50% of patients with ovarian cancer and soluble HLA-G plasma levels are significantly increased in patients with ovarian carcinoma. In agreement with this, soluble HLA-G levels are increased in malignant ascites when compared with that of benign ones. Interestingly, HLA-G expression in cancer cells from malignant effusions has been proposed as a possible marker of tumor susceptibility to chemotherapy in patients with advanced-stage ovarian carcinoma [12,18,19].

In the current study, our data revealed that HLA-G was detected in 22 of 33 ovarian cancer tissues; however, no positive signal was observed in normal ovarian tissues. This finding indicated that HLA-G could play a role in the pathogenesis of ovarian cancer. However, the function of the HLA-G expression in ovarian cancer cells is unknown and needs to be explored.

We then investigated the function of HLA-G in the regulation of NK cell cytotoxicity against the ovarian cancer cell line OVCAR-3 in vitro. As our results indicated, OVCAR-3 did not express the transcript of HLA-G1, but did express the HLA-G3 isoform. However, no surface HLA-G was detected with the mAb 87G which recognizes the HLA-G molecule in the {alpha}1 domain [25], indicating that the HLA-G3 isoform, which lacks the {alpha}2 and {alpha}3 domain and contains only the {alpha}1 domain, did not reach the cell surface. Our data are consistent with the previous reports that only the full length HLA-G1, but no other alternative isoforms of HLA-G, were expressed on cell surface [26,27]. With the methodology of cloning, the exogenous HLA-G molecule was stably expressed in the HLA-G-negative OVCAR-3 cells. Cytotoxicity data showed that HLA-G expression could markedly decrease the lysis of NK-92 cells against targets. To evaluate this NK cell lysis inhibition induced by HLA-G expression, a blocking assay was performed. We found that the cytolysis of NK-92 cells could be dramatically restored in a dose-dependent manner when the HLA-G molecule expressed on transfected OVCAR-3 cells was blocked, indicating that HLA-G directly inhibits the cytotoxicity of NK cells against OVCAR-3.

In conclusion, our findings showed that HLA-G could be a tumor marker expressed by ovarian carcinoma. Our results also provided functional analysis of HLA-G in ovarian cancer cell line in vitro and revealed that HLA-G could directly inhibit NK cell cytotoxicity. These data support the hypothesis that HLA-G plays an important role in human ovarian carcinomas escaping from human immune surveillance and is associated with the pathogenesis of ovarian carcinomas.


   Acknowledgements
Top
 Abstract
 introduction
 materials and methods
 results
 discussion
 Acknowledgements
References
 
This work was supported by the grants from Natural Science Foundation of Zhejiang Province, China (Y205531, Y205575). We are grateful to Dr McMaster (Department of Stomatology, University of California, San Francisco, CA, USA) and Dr Geraghty (Fred Hutchinson Cancer Research Center, Seattle, WA, USA) for generously providing antibodies.

Received for publication April 27, 2007. Accepted for publication June 11, 2007.


   References
Top
 Abstract
 introduction
 materials and methods
 results
 discussion
 Acknowledgements
 References
 
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3. Paul P, Cabestre FA, Ibrahim EC, et al. Identification of HLA-G7 as a new splice variant of the HLA-G mRNA and expression of soluble HLA-G5, -G6, and -G7 transcripts in human transfected cells. Hum Immunol (2000) 61:1138–1149.[CrossRef][Web of Science][Medline]

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17. Jemal A, Murray T, Ward E, et al. Cancer statistics, 2005. CA Cancer J Clin (2005) 55:10–30.[Abstract/Free Full Text]

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