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HLA-G expression in human melanoma cells: protection from NK cytolysis
Journal of Reproductive Immunology 43 (1999) 183 – 193 www.elsevier.com/locate/jreprimm
HLA-G expression in human melanoma cells: protection from NK cytolysis Francisco Adria´n Cabestre´ a, Philippe Moreau a, Be´atrice Riteau a, El Che´rif Ibrahim a, Caroline Le Danff a, Jean Dausset b, Nathalie Rouas-Freiss a, Edgardo D. Carosella a, Pascale Paul a,* a
CEA, Ser6ice de Recherches en He´mato-immunologie, DSV/DRM, Hoˆpital Saint-Louis, Centre Hayem, 1 A6enue Claude Vellefaux, 75475 Paris Cedex 10, France b Fondation Jean Dausset, CEPH, 27, Rue Juliette-Dodu, 75010 Paris, France
Abstract Expression of the non-classical HLA-G class I antigen is physiologically restricted to a limited number of tissues including trophoblasts, and is thought to play a role in establishing tolerance of the fetus by the maternal immune system. We investigated whether ectopic expression of HLA-G could also be detected in tumor cells and confer them the ability to escape immune cytotoxic responses. High levels of all alternatively spliced HLA-G transcripts could be detected in melanoma cells by RT-PCR. Analysis of biopsies from a melanoma patient revealed a higher HLA-G transcription level in skin metastasis as compared to healthy skin, while specific amplification of the HLA-G5 transcript was only observable in the tumor. HLA-G protein expression could also be detected in two melanoma cell lines. HLA-G-positive tumors inhibit cytotoxic lysis by the NK cell line YT2C2-PR. This inhibition is not observed with B-EBV cell lines bearing matched class I specificities, and is thought to occur through interaction of HLA-G with inhibitory receptors that are distinct from known KIRs interacting with HLA-E or classical class I molecules. Together, these results confirm that HLA-G expression at the surface of tumor cells can participate in the evasion of antitumoral immune responses and favor tumor progression. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: HLA-G; Melanoma cells; NK lysis; Tumor escape
Presented at the First International Conference on HLA-G, Paris, July 1998. * Corresponding author. Tel: + 33-1-53-72-21-42; fax: + 33-1-48-03-19-60. E-mail address: [email protected] (P. Paul)
0165-0378/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 5 - 0 3 7 8 ( 9 9 ) 0 0 0 3 7 - 6
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1. Introduction Many features distinguish the non-classical class I HLA-G protein from other class I molecules encoded within the major histocompatibility complex, including its tissue restricted expression, low polymorphism and the presence of alternative transcription of the HLA-G gene which generates at least six mRNA isoforms which have the capacity to encode four membrane-bound proteins (named HLA-G1–G4) and two soluble proteins (G5 and G6) which lack the transmembrane domain anchoring the HLA class I molecule to the cell membrane (Carosella et al., 1996). The role of the HLA-G protein in immune tolerance was first suggested by its specific expression on trophoblast cells (Kovats et al., 1990) at the feto–maternal interface, which is devoid of HLA-A and B classical class I molecules. HLA-G interacts with several described killing inhibitory receptors of the immunoglobulin superfamily (KIRs) including p49 (Cantoni et al., 1998), ILT2 and ILT4 (Colonna et al., 1997) expressed at the surface of NK cells but also on T cell subsets, dendritic cells, B cells and monocyte lineages (Meyaard et al., 1997; Colonna et al., 1998). Direct interaction of HLA-G with the widely expressed CD94/NKG2A KIR receptors or the C-type lectin superfamily has been recently refuted (Braud et al., 1998; Lee et al., 1998) with the demonstration that the non-classical HLA-E molecule is the ligand for this receptor. The HLA-G peptide leader, is as efficient as other class I peptides to allow HLA-E expression at the cell surface and can thus indirectly favor such interactions. The capacity of HLA-G to function as an antigen which can inhibit cytotoxic NK effector function of the maternal immune system against its semi-allogenic fetus (Pazmany et al., 1996; Rouas-Freiss et al., 1997a) has led us to investigate how ectopic expression of HLA-G in tumors could favor escape of HLA-G expressing tumor cells from immunosurveillance.
2. Materials and methods
2.1. Cell lines The IGR (HLA-A2, -A3, -B58), M8 (HLA-A1, -A2, -B12, -B40) melanoma cell lines, kindly provided by Dr F. Jotereau (INSERM U211, Nantes, France), and J.G. Guillet (INSERM U445) DRAN (HLA-A2, -A3, -B7, -B35, -CW5, -CW7) melanoma primary cells culture, kindly provided by Franc¸oise Farace (Institut Gustave Roussy, Villejuif, France), and NK-mediating YT2C2-PR subclone were maintained in
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RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 10 mg/ml gentamicin, and fungizone 0.5 mg/ml (Sigma) and cultured in a 37°C, 5% CO2-humidified incubator. The human HLAG-positive JEG-3 choriocarcinoma cell line (American Type Culture Collection) was cultured in DMEM (Sigma) supplemented with 10% heat-inactivated fetal calf serum, 10 mg/ml gentamicin, and 2mM L-glutamine. The B-EBV (HLA-G-negative and HLA-E-positive) cell lines BM (HLA-A29, -B61, -Cw2), HOM (HLA-A3, -B27, -Cw1), SPO (HLA-A3, -B7, -Cw7) and SWEIG (HLA-A2, -B44, -Cw5) cell lines were obtained during the Tenth International Histocompatibility Workshop and were maintained in RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 10 mg/ml gentamicin, and fungizone 0.5 mg/ml (Sigma).
2.2. Tumor samples Tissue samples from a melanoma patient were obtained from the Institut Gustave Roussy (Villejuif, France), after obtaining the prior informed consent of the patient, and included healthy skin resected at the border of the surgical wound and a skin metastasis. Immediately after surgical excision, samples were removed under aseptic conditions, frozen in liquid nitrogen, and stored until RNA extraction could be carried out. A diagnosis of melanoma metastasis was confirmed by cytological examination on formalin-fixed, hematoxylin/eosin-stained tissue sections.
2.3. Immunofluorescence analysis of melanoma cells Standard methods were used to carry out immunocytochemistry on cytospun melanoma cells which had been fixed in acetone, then rinsed in PBS and blocked in normal rabbit serum (DAKO) in PBS. Samples were incubated with primary antibody for 1 h at room temperature, followed by incubation with a secondary FITC-conjugated anti-mouse immunoglobulin antibody (DAKO). Sections were counterstained with DAPI (Sigma) nuclear dye and mounted with anti-fading mounting medium (DAKO). Fluorescence was analyzed with an MRC 1024 confocal microscope (Bio-Rad, Hempstead, UK). Following mAbs were used: W6/32, IgG2a anti-HLA class I heavy chains associated with b2m (Sigma), and 87G, IgG2b anti-HLA-G, which detects the HLA-G1 isoform, kindly provided by D. Geraghty (Seattle, WA, USA).
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2.4. RT-PCR analysis of HLA-G transcripts Total RNA was extracted using the RNA NOW reagent (Biogentex) according to the manufacturer’s recommendations and checked by electrophoresis in a 1.5% agarose denaturing gel. cDNA was prepared from 10 mg of total RNA, by using oligo-(dT)12 – 18 primer and M-MLV reverse transcriptase (Gibco-BRL). HLA-G-specific amplifications were carried out as previously described (Paul et al., 1998) using G.257 (exon 2) and G3.U (3%-UT) primers to detect all alternatively spliced HLA-G mRNA. Specific amplification of the soluble HLA-G5 transcript was conducted with G.526 (exon 3) and G.i4b (intron 4) primers. Co-amplification of b-actin cDNA was carried out in each experiment with b-actin amplimer sets (Clontech) for 16 cycles in order to evaluate comparative amounts of RNA in samples. The specificity of PCR products was confirmed by Southern blotting of the fragments onto nylon membranes (Hybond N+, Amersham, France) and hybridization with HLA-G-specific oligonucleotide probe G.R (exon 2) and HLA-G5-specific probe I4F located in intron 4. The same membranes were then probed with an b-actin-specific probe. The filters were exposed to Kodak (Biomax) films between two amplifying screens at −80°C.
2.5. Cytotoxicity assays The cytolytic activity of the YT2C2-PR subclone effector cells against melanoma cells and B-EBV cell lines was assessed in 4 h 51Cr-release assays in which effector cells were mixed with 5×103 51Cr-labeled targets (100 mCi of [51Cr]sodium chromate, Amersham, UK) in U-bottomed microtiter plates. After 4 h at 37°C in a humidified 5% CO2 incubator, 50 ml of the supernatant was collected for liquid scintillation counting (Wallac 1410, Pharmacia, France). The percentage of specific lysis was calculated as follows: percent specific lysis=[(cpm experimental release−cpm spontaneous release)/(cpm maximum release−cpm spontaneous release)]×100. Spontaneous release was determined by incubation of labeled target cells with medium. Maximum release was determined by solubilizing target cells in 0.1 M HCl. Results are presented as a mean of five experiments.
3. Results
3.1. High le6els of HLA-G transcripts can be detected in human melanoma cells Semi-quantitative RT-PCR analysis of the described HLA-G mRNA
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alternative transcripts were performed on melanoma cells using pan-HLA-G primers. IGR and DRAN melanoma cells exhibited high levels of HLA-G mRNA (Fig. 1). All HLA-G isoforms are enhanced in these cells. In contrast, only very low levels of HLA-G transcription was detected in the M8 melanoma cell line.
3.2. A tumor biopsy exhibits higher le6el of HLA-G transcription than healthy skin RT-PCR analysis of RNA extracted from both tumor skin metastasis and healthy skin tissue of the same melanoma patient revealed that HLA-G transcription is higher in the tumor site than in the healthy skin biopsy. Specific amplification of the soluble HLA-G5 transcript corresponding to the full-length soluble HLA-G protein is also found in the tumor site while undetectable in healthy skin (Fig. 2).
3.3. HLA-G protein expression correlates with inhibition of NK-mediated cytolysis by IGR and DRAN melanoma cells Immunofluorescence analysis of the DRAN melanoma cells using the anti HLA-G 87G antibody revealed the presence of HLA-G proteins (Fig. 3A). Although the HLA-G1 isoform was only faintly detected in IGR cells by
Fig. 1. Analysis of HLA-G mRNA in melanoma cells. Pan-HLA-G primers G.257 and G3.U were used for PCR amplification of HLA-G transcripts corresponding to all known HLA-G isoforms. cDNA from choriocarcinoma JEG-3, was used as control for high HLA-G transcription. IGR, M8 and DRAN correspond to cDNA amplification of melanoma cells. HLA-G specific bands were revealed by hybridization with the GR-specific probe located in exon 2. Bands corresponding to HLA-G1, -G2, -G3, -G4, and -G5 transcripts are indicated by arrows. PCR products co-amplified in the same reaction by b-actin primers were detected on the same membrane by a b-actin probe.
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Fig. 2. Detection of HLA-G transcripts in human melanoma and healthy skin biopsies. The pan-HLA-G primers G.257 and G3.U were used for RT-PCR amplification of HLA-G transcripts from an ex vivo skin metastasis (SM) and from healthy skin biopsy (HS) obtained from the same patient. JEG-3 was used as control of high HLA-G transcription. HLA-Gspecific bands were revealed by hybridization with the GR-specific probe located in exon 2. HLA-G5 amplification was conducted with G.526 and G.i4b primers HLA-G5 specific band was detected by hybridization with the I4F specific probe located in intron 4. Bands corresponding to HLA-G1, -G2, -G3, -G4, and -G5 transcripts are indicated by arrows. PCR products co-amplified in the same reaction by b-actin primers were detected on the same membrane by a b-actin probe.
immunoprecipitation with a high amount of cells, we have previously shown that other HLA-G protein isoforms, corresponding to HLA-G2 and -G3 may be expressed in this cell line (Paul et al., 1998). We then investigated the capacity of HLA-G-positive melanoma cells to inhibit NK-mediated cell lysis. Classical class I expression was detectable in these cells and could participate in the inhibition of NK cell activity. Cytotoxicity assays were thus performed using the YT2C2-PR NK subclone which (i) exhibits NK cytotoxic activity against class I-negative K562 cells, (ii) is inhibited by HLA-G1 or HLA-G2 K562 transfectants, and (iii) does not bear any of the known KIR receptors for classical class I (Rouas-Freiss et al., 1997b). DRAN melanoma cells which express HLA-G1 clearly inhibit NK cell lysis by the YT2C2-PR NK subclone (Fig. 3B) while the HLA-G-negative M8 melanoma cell line is efficiently lysed. As previously described (Paul et al., 1998), inhibition of NK lysis was also observed using the IGR melanoma cell line. In order to further confirm that this inhibition was not due to
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Fig. 3. Inhibition of NK lysis by HLA-G-positive melanoma cells. (A) DRAN melanoma cells are stained with the mAb 87G by using the standard immunohistochemical methods. (B) Effect of HLA-G expression by IGR and DRAN melanoma cells on sensitivity to lysis by the YT2C2-PR subclone. Several HLA-G-negative B-EBV cell lines [HOM (A3, B27, Cw1), BM (A29, B61, Cw2), SPO (A3, B7, Cw7), SWEIG (A2, B44, Cw5)], were lysed by the YT2C2-PR subclone. This subclone was used as an effector at 50:1 effector/targer ratio. Results are expressed as the percentage of specific lysis recorded in a 4 h 51Cr release assay. Spontaneous release never exceeded 10% of the maximum release.
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other classical class I antigens or to HLA-E expression, we tested the ability of EBV-transformed cell lines, which specificities were matched to that of the IGR melanoma cell line, to inhibit NK lysis by the YT2C2-PR subclone. As shown in Fig. 3B, the four matched B-EBV cell lines bearing HLA class I and E molecules where efficiently lysed by YT2C2-PR suggest-ing that these molecules do not participate in the inhibition of NK lysis by IGR or DRAN melanoma cells.
4. Discussion Analysis of HLA-G mRNA levels in tumor cells confirms that HLA-G can be activated at the transcriptional level in some melanoma cells. This enhancement of HLA-G transcripts does not seem to be the reflect of in vitro culture conditions, as amplification of HLA-G mRNA levels are also observed in tumor biopsies of a melanoma patient, while only basal levels of HLA-G transcription are found in healthy skin of the same patient. The transcript corresponding to the soluble full-length isoform is only detected in the tumor site while not observable in healthy skin. We characterized two HLA-G mRNA-positive (DRAN, IGR) and one negative (M8) melanoma cell lines which exhibit differential HLA-G transcription and protein isoforms expression, and use these cells as models to study the impact of tumoral HLA-G expression on NK responses. As previously shown, IGR cells inhibit NK lysis. In contrast, the M8 melanoma cell line in which no HLA-G protein expression could be detected was used as a control and was efficiently lysed by YT2C2-PR NK cells (Paul et al., 1998). DRAN melanoma primary cultured also express HLA-G proteins and inhibit NK lysis by the YT2C2-PR clone. We further showed that HLA-G-negative B-EBV-transformed cell lines, which exhibit classical class I allelic specificities matching those of IGR, are efficiently lysed by the YT2C2-PR NK subclone. We thus conclude that sole HLA-E and classical class I molecules expression, if not associated with HLA-G, cannot account for inhibition of NK lysis by IGR and DRAN melanoma cells. Transfection of HLA-G-negative M8 melanoma cells with HLA-G1 also resulted in inhibition of NK activity (data not shown) thus confirming the implication of HLA-G in the inhibition of NK-mediated cell lysis by HLA-G expressing melanoma cells. Although other mechanisms that could be implicated in resistance to NK lysis (Pardoll, 1998), such as Fas ligand expression (Hahne et al., 1996), cannot be excluded, all together these results suggest that, even in cells that express other HLA-class I molecules, HLA-G can inhibit NK lytic activity of the YT2C2-PR NK clone. YT2C2-PR does not bear any of the known
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KIR receptors known to interact with HLA-E and classical class I molecules and has been shown to bear a new receptor for the HLA-G ligand on these cells that remains to be defined (Rouas-Freiss et al., 1997b). This expression could also have other implications in vivo as HLA-G interacts with various KIRs expressed on subsets of NK, T cells, B cells, dendritic cells and mononuclear blood cells (Meyaard et al., 1997; Moretta et al., 1997; Colonna et al., 1998). HLA-G expression at the surface of tumor cells could thus also modulate the immune functions of other antitumoral response effector cells, such as antigen-presenting cells and cytotoxic T lymphocytes. It was recently demonstrated that HLA-E is the ligand for the widely CD94/NKG2A C-type lectin inhibitory receptor (Borrego et al., 1998; Braud et al., 1998; Lee et al., 1998). Since the indirect immunomodulatory effect on HLA-E expression is also a property of various classical HLA class I alleles, the role of HLA-G expression would appear even more decisive in situations where classical class I molecules are repressed. HLA-G would then be the only source of leader peptides able to stabilize HLA-E expression and favor inhibition mediated by CD94/NKG2A. In this regard, in HLA class I loss tumor cell variants and trophoblast cells which are devoid of MHC class I, HLA-G would appear as a major immunotolerance antigen able to inhibit cytotoxic functions. The mechanisms that participate in ectopic activation of HLA-G gene transcription and protein expression in subsets of melanoma cells remain to be defined and could implicate various factors provided by the tumor microenvironment. Several cytokines including IL-10, TGFb and IL-15 have also been shown to upregulate KIRs (Carayol et al., 1998; Mingari et al., 1998a,b). The investigation of the relevance of HLA-G expression in tumors could provide a new understanding of factors favoring tumor progression and be taken into account in future design of cancer therapies.
Acknowledgements We are grateful to Marie Franc¸oise Avril, Jean Gerard Guillet, Frederique Anne Le Gal, Franc¸oise Farace, Sylvaine Mercier and Pierre Duvillard for providing us melanoma biopsies and cell lines. We thank Daniel Geraghty for providing us HLA-G antibodies. We thank Noah Hardy for reading and correcting the manuscript. This study was supported by grants from the MENRT (F.A.C.), the French Commissariat a` l’Energie Atomique, the Association pour la Recherche sur le Cancer and Etablissement Franc¸ais des Greffes.
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Pardoll, D.M., 1998. Cancer vaccines. Nature Med. 4, 525 – 531. Paul, P., Rouass-Freiss, N., Khalil-Daher, I., Moreau, P., Riteau, B., Le Gal, F.A., Avril, M.F., Dausset, J., Guillet, J.G., Carosella, E.D., 1998. HLA-G expression in melanoma: a way for tumor cells to escape from immunosurveillance. Proc. Natl. Acad. Sci. USA 95, 4510–4515. Pazmany, L., Mandelboim, O., Vales-Gomez, M., Davis, D.M., Reyburn, H.T., Strominger, J.L., 1996. Protection from natural killer cell-mediated lysis by HLA-G expression on target cells. Science 274, 792–795. Rouas-Freiss, N., Marchal-Bras Goncalves, R., Menier, C., Dausset, J., Carosella, E.D., 1997a. Direct evidence to support the role of HLA-G in protecting the fetus from maternal uterine natural killer cytolysis. Proc. Natl. Acad. Sci. USA 94, 11520 – 11525. Rouas-Freiss, N., Marchal, R.E., Kirszenbaum, M., Dausset, J., Carosella, E.D., 1997b. The alpha1 domain of HLA-G1 and HLA-G2 inhibits cytotoxicity induced by natural killer cells: is HLA-G the public ligand for natural killer cell inhibitory receptors? Proc. Natl. Acad. Sci. USA 94, 5249–5254.
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HLA-G expression in human melanoma cells: protection from NK cytolysis Francisco Adria´n Cabestre´ a, Philippe Moreau a, Be´atrice Riteau a, El Che´rif Ibrahim a, Caroline Le Danff a, Jean Dausset b, Nathalie Rouas-Freiss a, Edgardo D. Carosella a, Pascale Paul a,* a
CEA, Ser6ice de Recherches en He´mato-immunologie, DSV/DRM, Hoˆpital Saint-Louis, Centre Hayem, 1 A6enue Claude Vellefaux, 75475 Paris Cedex 10, France b Fondation Jean Dausset, CEPH, 27, Rue Juliette-Dodu, 75010 Paris, France
Abstract Expression of the non-classical HLA-G class I antigen is physiologically restricted to a limited number of tissues including trophoblasts, and is thought to play a role in establishing tolerance of the fetus by the maternal immune system. We investigated whether ectopic expression of HLA-G could also be detected in tumor cells and confer them the ability to escape immune cytotoxic responses. High levels of all alternatively spliced HLA-G transcripts could be detected in melanoma cells by RT-PCR. Analysis of biopsies from a melanoma patient revealed a higher HLA-G transcription level in skin metastasis as compared to healthy skin, while specific amplification of the HLA-G5 transcript was only observable in the tumor. HLA-G protein expression could also be detected in two melanoma cell lines. HLA-G-positive tumors inhibit cytotoxic lysis by the NK cell line YT2C2-PR. This inhibition is not observed with B-EBV cell lines bearing matched class I specificities, and is thought to occur through interaction of HLA-G with inhibitory receptors that are distinct from known KIRs interacting with HLA-E or classical class I molecules. Together, these results confirm that HLA-G expression at the surface of tumor cells can participate in the evasion of antitumoral immune responses and favor tumor progression. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: HLA-G; Melanoma cells; NK lysis; Tumor escape
Presented at the First International Conference on HLA-G, Paris, July 1998. * Corresponding author. Tel: + 33-1-53-72-21-42; fax: + 33-1-48-03-19-60. E-mail address: [email protected] (P. Paul)
0165-0378/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 5 - 0 3 7 8 ( 9 9 ) 0 0 0 3 7 - 6
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1. Introduction Many features distinguish the non-classical class I HLA-G protein from other class I molecules encoded within the major histocompatibility complex, including its tissue restricted expression, low polymorphism and the presence of alternative transcription of the HLA-G gene which generates at least six mRNA isoforms which have the capacity to encode four membrane-bound proteins (named HLA-G1–G4) and two soluble proteins (G5 and G6) which lack the transmembrane domain anchoring the HLA class I molecule to the cell membrane (Carosella et al., 1996). The role of the HLA-G protein in immune tolerance was first suggested by its specific expression on trophoblast cells (Kovats et al., 1990) at the feto–maternal interface, which is devoid of HLA-A and B classical class I molecules. HLA-G interacts with several described killing inhibitory receptors of the immunoglobulin superfamily (KIRs) including p49 (Cantoni et al., 1998), ILT2 and ILT4 (Colonna et al., 1997) expressed at the surface of NK cells but also on T cell subsets, dendritic cells, B cells and monocyte lineages (Meyaard et al., 1997; Colonna et al., 1998). Direct interaction of HLA-G with the widely expressed CD94/NKG2A KIR receptors or the C-type lectin superfamily has been recently refuted (Braud et al., 1998; Lee et al., 1998) with the demonstration that the non-classical HLA-E molecule is the ligand for this receptor. The HLA-G peptide leader, is as efficient as other class I peptides to allow HLA-E expression at the cell surface and can thus indirectly favor such interactions. The capacity of HLA-G to function as an antigen which can inhibit cytotoxic NK effector function of the maternal immune system against its semi-allogenic fetus (Pazmany et al., 1996; Rouas-Freiss et al., 1997a) has led us to investigate how ectopic expression of HLA-G in tumors could favor escape of HLA-G expressing tumor cells from immunosurveillance.
2. Materials and methods
2.1. Cell lines The IGR (HLA-A2, -A3, -B58), M8 (HLA-A1, -A2, -B12, -B40) melanoma cell lines, kindly provided by Dr F. Jotereau (INSERM U211, Nantes, France), and J.G. Guillet (INSERM U445) DRAN (HLA-A2, -A3, -B7, -B35, -CW5, -CW7) melanoma primary cells culture, kindly provided by Franc¸oise Farace (Institut Gustave Roussy, Villejuif, France), and NK-mediating YT2C2-PR subclone were maintained in
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RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 10 mg/ml gentamicin, and fungizone 0.5 mg/ml (Sigma) and cultured in a 37°C, 5% CO2-humidified incubator. The human HLAG-positive JEG-3 choriocarcinoma cell line (American Type Culture Collection) was cultured in DMEM (Sigma) supplemented with 10% heat-inactivated fetal calf serum, 10 mg/ml gentamicin, and 2mM L-glutamine. The B-EBV (HLA-G-negative and HLA-E-positive) cell lines BM (HLA-A29, -B61, -Cw2), HOM (HLA-A3, -B27, -Cw1), SPO (HLA-A3, -B7, -Cw7) and SWEIG (HLA-A2, -B44, -Cw5) cell lines were obtained during the Tenth International Histocompatibility Workshop and were maintained in RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 10 mg/ml gentamicin, and fungizone 0.5 mg/ml (Sigma).
2.2. Tumor samples Tissue samples from a melanoma patient were obtained from the Institut Gustave Roussy (Villejuif, France), after obtaining the prior informed consent of the patient, and included healthy skin resected at the border of the surgical wound and a skin metastasis. Immediately after surgical excision, samples were removed under aseptic conditions, frozen in liquid nitrogen, and stored until RNA extraction could be carried out. A diagnosis of melanoma metastasis was confirmed by cytological examination on formalin-fixed, hematoxylin/eosin-stained tissue sections.
2.3. Immunofluorescence analysis of melanoma cells Standard methods were used to carry out immunocytochemistry on cytospun melanoma cells which had been fixed in acetone, then rinsed in PBS and blocked in normal rabbit serum (DAKO) in PBS. Samples were incubated with primary antibody for 1 h at room temperature, followed by incubation with a secondary FITC-conjugated anti-mouse immunoglobulin antibody (DAKO). Sections were counterstained with DAPI (Sigma) nuclear dye and mounted with anti-fading mounting medium (DAKO). Fluorescence was analyzed with an MRC 1024 confocal microscope (Bio-Rad, Hempstead, UK). Following mAbs were used: W6/32, IgG2a anti-HLA class I heavy chains associated with b2m (Sigma), and 87G, IgG2b anti-HLA-G, which detects the HLA-G1 isoform, kindly provided by D. Geraghty (Seattle, WA, USA).
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2.4. RT-PCR analysis of HLA-G transcripts Total RNA was extracted using the RNA NOW reagent (Biogentex) according to the manufacturer’s recommendations and checked by electrophoresis in a 1.5% agarose denaturing gel. cDNA was prepared from 10 mg of total RNA, by using oligo-(dT)12 – 18 primer and M-MLV reverse transcriptase (Gibco-BRL). HLA-G-specific amplifications were carried out as previously described (Paul et al., 1998) using G.257 (exon 2) and G3.U (3%-UT) primers to detect all alternatively spliced HLA-G mRNA. Specific amplification of the soluble HLA-G5 transcript was conducted with G.526 (exon 3) and G.i4b (intron 4) primers. Co-amplification of b-actin cDNA was carried out in each experiment with b-actin amplimer sets (Clontech) for 16 cycles in order to evaluate comparative amounts of RNA in samples. The specificity of PCR products was confirmed by Southern blotting of the fragments onto nylon membranes (Hybond N+, Amersham, France) and hybridization with HLA-G-specific oligonucleotide probe G.R (exon 2) and HLA-G5-specific probe I4F located in intron 4. The same membranes were then probed with an b-actin-specific probe. The filters were exposed to Kodak (Biomax) films between two amplifying screens at −80°C.
2.5. Cytotoxicity assays The cytolytic activity of the YT2C2-PR subclone effector cells against melanoma cells and B-EBV cell lines was assessed in 4 h 51Cr-release assays in which effector cells were mixed with 5×103 51Cr-labeled targets (100 mCi of [51Cr]sodium chromate, Amersham, UK) in U-bottomed microtiter plates. After 4 h at 37°C in a humidified 5% CO2 incubator, 50 ml of the supernatant was collected for liquid scintillation counting (Wallac 1410, Pharmacia, France). The percentage of specific lysis was calculated as follows: percent specific lysis=[(cpm experimental release−cpm spontaneous release)/(cpm maximum release−cpm spontaneous release)]×100. Spontaneous release was determined by incubation of labeled target cells with medium. Maximum release was determined by solubilizing target cells in 0.1 M HCl. Results are presented as a mean of five experiments.
3. Results
3.1. High le6els of HLA-G transcripts can be detected in human melanoma cells Semi-quantitative RT-PCR analysis of the described HLA-G mRNA
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alternative transcripts were performed on melanoma cells using pan-HLA-G primers. IGR and DRAN melanoma cells exhibited high levels of HLA-G mRNA (Fig. 1). All HLA-G isoforms are enhanced in these cells. In contrast, only very low levels of HLA-G transcription was detected in the M8 melanoma cell line.
3.2. A tumor biopsy exhibits higher le6el of HLA-G transcription than healthy skin RT-PCR analysis of RNA extracted from both tumor skin metastasis and healthy skin tissue of the same melanoma patient revealed that HLA-G transcription is higher in the tumor site than in the healthy skin biopsy. Specific amplification of the soluble HLA-G5 transcript corresponding to the full-length soluble HLA-G protein is also found in the tumor site while undetectable in healthy skin (Fig. 2).
3.3. HLA-G protein expression correlates with inhibition of NK-mediated cytolysis by IGR and DRAN melanoma cells Immunofluorescence analysis of the DRAN melanoma cells using the anti HLA-G 87G antibody revealed the presence of HLA-G proteins (Fig. 3A). Although the HLA-G1 isoform was only faintly detected in IGR cells by
Fig. 1. Analysis of HLA-G mRNA in melanoma cells. Pan-HLA-G primers G.257 and G3.U were used for PCR amplification of HLA-G transcripts corresponding to all known HLA-G isoforms. cDNA from choriocarcinoma JEG-3, was used as control for high HLA-G transcription. IGR, M8 and DRAN correspond to cDNA amplification of melanoma cells. HLA-G specific bands were revealed by hybridization with the GR-specific probe located in exon 2. Bands corresponding to HLA-G1, -G2, -G3, -G4, and -G5 transcripts are indicated by arrows. PCR products co-amplified in the same reaction by b-actin primers were detected on the same membrane by a b-actin probe.
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Fig. 2. Detection of HLA-G transcripts in human melanoma and healthy skin biopsies. The pan-HLA-G primers G.257 and G3.U were used for RT-PCR amplification of HLA-G transcripts from an ex vivo skin metastasis (SM) and from healthy skin biopsy (HS) obtained from the same patient. JEG-3 was used as control of high HLA-G transcription. HLA-Gspecific bands were revealed by hybridization with the GR-specific probe located in exon 2. HLA-G5 amplification was conducted with G.526 and G.i4b primers HLA-G5 specific band was detected by hybridization with the I4F specific probe located in intron 4. Bands corresponding to HLA-G1, -G2, -G3, -G4, and -G5 transcripts are indicated by arrows. PCR products co-amplified in the same reaction by b-actin primers were detected on the same membrane by a b-actin probe.
immunoprecipitation with a high amount of cells, we have previously shown that other HLA-G protein isoforms, corresponding to HLA-G2 and -G3 may be expressed in this cell line (Paul et al., 1998). We then investigated the capacity of HLA-G-positive melanoma cells to inhibit NK-mediated cell lysis. Classical class I expression was detectable in these cells and could participate in the inhibition of NK cell activity. Cytotoxicity assays were thus performed using the YT2C2-PR NK subclone which (i) exhibits NK cytotoxic activity against class I-negative K562 cells, (ii) is inhibited by HLA-G1 or HLA-G2 K562 transfectants, and (iii) does not bear any of the known KIR receptors for classical class I (Rouas-Freiss et al., 1997b). DRAN melanoma cells which express HLA-G1 clearly inhibit NK cell lysis by the YT2C2-PR NK subclone (Fig. 3B) while the HLA-G-negative M8 melanoma cell line is efficiently lysed. As previously described (Paul et al., 1998), inhibition of NK lysis was also observed using the IGR melanoma cell line. In order to further confirm that this inhibition was not due to
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Fig. 3. Inhibition of NK lysis by HLA-G-positive melanoma cells. (A) DRAN melanoma cells are stained with the mAb 87G by using the standard immunohistochemical methods. (B) Effect of HLA-G expression by IGR and DRAN melanoma cells on sensitivity to lysis by the YT2C2-PR subclone. Several HLA-G-negative B-EBV cell lines [HOM (A3, B27, Cw1), BM (A29, B61, Cw2), SPO (A3, B7, Cw7), SWEIG (A2, B44, Cw5)], were lysed by the YT2C2-PR subclone. This subclone was used as an effector at 50:1 effector/targer ratio. Results are expressed as the percentage of specific lysis recorded in a 4 h 51Cr release assay. Spontaneous release never exceeded 10% of the maximum release.
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other classical class I antigens or to HLA-E expression, we tested the ability of EBV-transformed cell lines, which specificities were matched to that of the IGR melanoma cell line, to inhibit NK lysis by the YT2C2-PR subclone. As shown in Fig. 3B, the four matched B-EBV cell lines bearing HLA class I and E molecules where efficiently lysed by YT2C2-PR suggest-ing that these molecules do not participate in the inhibition of NK lysis by IGR or DRAN melanoma cells.
4. Discussion Analysis of HLA-G mRNA levels in tumor cells confirms that HLA-G can be activated at the transcriptional level in some melanoma cells. This enhancement of HLA-G transcripts does not seem to be the reflect of in vitro culture conditions, as amplification of HLA-G mRNA levels are also observed in tumor biopsies of a melanoma patient, while only basal levels of HLA-G transcription are found in healthy skin of the same patient. The transcript corresponding to the soluble full-length isoform is only detected in the tumor site while not observable in healthy skin. We characterized two HLA-G mRNA-positive (DRAN, IGR) and one negative (M8) melanoma cell lines which exhibit differential HLA-G transcription and protein isoforms expression, and use these cells as models to study the impact of tumoral HLA-G expression on NK responses. As previously shown, IGR cells inhibit NK lysis. In contrast, the M8 melanoma cell line in which no HLA-G protein expression could be detected was used as a control and was efficiently lysed by YT2C2-PR NK cells (Paul et al., 1998). DRAN melanoma primary cultured also express HLA-G proteins and inhibit NK lysis by the YT2C2-PR clone. We further showed that HLA-G-negative B-EBV-transformed cell lines, which exhibit classical class I allelic specificities matching those of IGR, are efficiently lysed by the YT2C2-PR NK subclone. We thus conclude that sole HLA-E and classical class I molecules expression, if not associated with HLA-G, cannot account for inhibition of NK lysis by IGR and DRAN melanoma cells. Transfection of HLA-G-negative M8 melanoma cells with HLA-G1 also resulted in inhibition of NK activity (data not shown) thus confirming the implication of HLA-G in the inhibition of NK-mediated cell lysis by HLA-G expressing melanoma cells. Although other mechanisms that could be implicated in resistance to NK lysis (Pardoll, 1998), such as Fas ligand expression (Hahne et al., 1996), cannot be excluded, all together these results suggest that, even in cells that express other HLA-class I molecules, HLA-G can inhibit NK lytic activity of the YT2C2-PR NK clone. YT2C2-PR does not bear any of the known
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KIR receptors known to interact with HLA-E and classical class I molecules and has been shown to bear a new receptor for the HLA-G ligand on these cells that remains to be defined (Rouas-Freiss et al., 1997b). This expression could also have other implications in vivo as HLA-G interacts with various KIRs expressed on subsets of NK, T cells, B cells, dendritic cells and mononuclear blood cells (Meyaard et al., 1997; Moretta et al., 1997; Colonna et al., 1998). HLA-G expression at the surface of tumor cells could thus also modulate the immune functions of other antitumoral response effector cells, such as antigen-presenting cells and cytotoxic T lymphocytes. It was recently demonstrated that HLA-E is the ligand for the widely CD94/NKG2A C-type lectin inhibitory receptor (Borrego et al., 1998; Braud et al., 1998; Lee et al., 1998). Since the indirect immunomodulatory effect on HLA-E expression is also a property of various classical HLA class I alleles, the role of HLA-G expression would appear even more decisive in situations where classical class I molecules are repressed. HLA-G would then be the only source of leader peptides able to stabilize HLA-E expression and favor inhibition mediated by CD94/NKG2A. In this regard, in HLA class I loss tumor cell variants and trophoblast cells which are devoid of MHC class I, HLA-G would appear as a major immunotolerance antigen able to inhibit cytotoxic functions. The mechanisms that participate in ectopic activation of HLA-G gene transcription and protein expression in subsets of melanoma cells remain to be defined and could implicate various factors provided by the tumor microenvironment. Several cytokines including IL-10, TGFb and IL-15 have also been shown to upregulate KIRs (Carayol et al., 1998; Mingari et al., 1998a,b). The investigation of the relevance of HLA-G expression in tumors could provide a new understanding of factors favoring tumor progression and be taken into account in future design of cancer therapies.
Acknowledgements We are grateful to Marie Franc¸oise Avril, Jean Gerard Guillet, Frederique Anne Le Gal, Franc¸oise Farace, Sylvaine Mercier and Pierre Duvillard for providing us melanoma biopsies and cell lines. We thank Daniel Geraghty for providing us HLA-G antibodies. We thank Noah Hardy for reading and correcting the manuscript. This study was supported by grants from the MENRT (F.A.C.), the French Commissariat a` l’Energie Atomique, the Association pour la Recherche sur le Cancer and Etablissement Franc¸ais des Greffes.
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