Modulation of HLA-G antigens expression in myelomonocytic cells

Modulation of HLA-G Antigens Expression in Myelomonocytic Cells Myriam Onno, Gae¨lle Le Friec, Ce´line Pangault, Laurence Amiot, Vale´rie Guilloux, Bernard Dre´nou, Sylvie Caulet-Maugendre, Patrice Andre´, and Rene´e Fauchet ABSTRACT: As trophoblast cells and macrophages share cellular characteristics, we investigated the expression of HLA-G antigens during the myelomonocytic differentiation. Analyses with the 87G and 16G1 monoclonal antibodies demonstrated that HLA-G was not expressed in peripheral blood monocytes, in in vitro differentiated dendritic cells and macrophages, and in resident mononuclear phagocytes infiltrating healthy tissues. Conversely, activated macrophages and dendritic cells localized in tumoral biopsies of some lung carcinomas expressed HLA-G antigens. Induction of HLA-G expression at the cell surface of the monohistiocytic cell line U 937 with different cytokines strongly suggests that cytokines secreted during inflammation may be involved in this specific upregulation. Bronchoalveolar macrophages collected from patients suffering from acute HCMV pneumonitis also expressed HLA-G molecules. In vitro, we thus demonstrated

ABBREVIATIONS CTL cytotoxic T lymphocyte HCMV human cytomegalovirus PBMC peripheral blood mononuclear cells BAL bronchoalveolar lavages bp base pairs BSA bovine serum albumin CD cluster of differentiation FITC fluorescein isothiocyanate GM-CSF granulocyte-macrophage– colony stimulating factor

From the Laboratoire Universitaire d’He´matologie et de la Biologie des Cellules Sanguines (M.O., G.L.F., C.P., L.A., V.G., B.D., R.F.), Faculte´ de Me´decine, CRI INSERM 9606, Rennes; the Laboratoire d’Anatomopathologie (S.C.M.), Rennes Cedex; and the INSERM U503 (P. A.), Ecole Normale Supe´rieure, Lyon Cedex, France. Address reprint requests to: Dr. Myriam Onno, Laboratoire Universitaire d’He´matologie et de la Biologie des Cellules Sanguines, Faculte´ de Me´decine, Universite´ de Rennes I, 2 Avenue du Pr Le´on Bernard, 35043 Rennes Cedex, France; Tel: ⫹33 (299) 33-68-85; Fax: ⫹33 (299) 28-41-52; E-Mail: [email protected]. Received August 1, 2000; accepted September 14, 2000. Human Immunology 61, 1086 –1094 (2000) © American Society for Histocompatibility and Immunogenetics, 2000 Published by Elsevier Science Inc.

that HLA-G antigens are produced during viral reactivation in the macrophages generated after allogeneic stimulation of HCMV latently infected monocytes. Our data suggest that inflammatory processes in lung tissues, like tumoral transformation and HCMV acute infection, are likely to induce HLA-G molecules in infiltrating macrophages and dendritic cells. The expression of molecules capable of downregulating both the innate and adoptive immunity could be a mechanism that helps tumoral and HCMV infected cells to escape immune response. Human Immunology 61, 1086 –1094 (2000). © American Society for Histocompatibility and Immunogenetics, 2000. Published by Elsevier Science Inc. KEYWORDS: HLA-G antigens; macrophages; tolerance; human cytomegalovirus; inflammation

IE gB IL IFN-␥ KIR mAb NK cells TRITC

immediate early glycoprotein B interleukin interferon-␥ killing inhibitory receptor monoclonal antibody natural killer cells tetramethylrhodamine isothiocyanate

INTRODUCTION The function of the classical MHC-I molecules, HLA-A, -B, -C in immune recognition is well understood both in functional and structural terms. These highly polymorphic molecules constitute transplantation antigens that may be recognized by alloreactive T cells. These molecules also play an important role in the induction of a specific immune response by presenting tumoral or viral peptide antigens to T cells. In contrast, nonclassical 0198-8859/00/$–see front matter PII S0198-8859(00)00191-9

HLA-G Antigens in Myelomonocytic Cells

MHC-I HLA-G molecules have been described as inhibitors of the cellular immune response. The HLA-G gene is characterized by a limited polymorphism and the alternative transcription of spliced mRNAs that encode at least seven different isoforms, namely the membranebound HLA-G1, -G2, -G3, -G4 and soluble HLA-G5, -G6, -G7 proteins [1]. HLA-G antigens are primarily expressed in fetal trophoblast cells that invade the maternal decidua. These invading trophoblast cells fail to express MHC-I HLA-A, -B or MHC-II molecules. Cell surface HLA-G1 and soluble HLA-G5 molecules can bind peptides derived from a variety of intracellular proteins. In vivo, cell surface expression of HLA-G molecules may affect cytotoxicity and antigen presenting functions through binding to LIR-1, LIR-2 and p49 inhibitory receptors [2– 4]. Furthermore, soluble HLA-G molecules impair peripheral blood NK lytic activity [5], show strong MLR suppression [6] and trigger CD95/ CD95 ligand-mediated apoptosis in activated CD8⫹ cells [7]. These different in vitro functional studies strongly suggest that cell surface and soluble HLA-G antigens may act as strong immunosuppressive molecules in vivo. The trophoblast, which forms a physical barrier between the mother and developing fetus, is a component of the host immune system during pregnancy. Of the classical immune cells, it most closely resembles the macrophage, also present in high numbers in the pregnant uterus. The macrophages and trophoblast, as cell classes, share characteristics such as phagocytosis, syncytialization, invasiness, permissiveness to HCMV, expression of the proteins CD4, CD14, IgG receptor (FcR), granulocyte-macrophage– colony stimulating factor (GM-CSF), colony stimulating factor-1 (CSF-1), IL-1, IL-6, tumor necrosis factor (TNF-␣), transforming growth factors (TGFs), platelet-␣derived growth factor (PDGF), and receptors for theses cytokines. In the uterus, both cell types appear regulated by a common element, the uterine epithelium, that secretes cytokines such as CSF-1, GM-CSF, TNF-␣, TGF␤, IL-6, and leukemia inhibitory factor (LIF). These common characteristics and regulation led us to investigate the possible expression of HLA-G antigens in myelomonocytic cells. MATERIALS AND METHODS Cell Lines Human foreskin fibroblasts (HFF), Jeg3 (choriocarcinoma), U937 (monohistiocytic leukemia), and THP-1 (monocytic leukemia) were obtained from the ATCC. These cell lines were maintained according to the recommendations of the supplier. Culture media (Life Technologies, Inc., Cergy-Pontoise, France) were supplemented with 10% FCS, 1 mM sodium pyruvate, 2 mM

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glutamine, 10 U/ml penicillin, 100 ␮g/ml streptomycin, in a humidified 5% CO2 atmosphere. JEG3 choriocarcinoma cell line was used as a positive control in the different HLA-G expression experiments. Cells were consistently free of mycoplasma infection. Peripheral Blood Leukocytes Monocytes (n ⫽ 10) were purified from peripheral blood of healthy volunteers, using magnetic Dynabeads coated with CD14 mAbs (Dynal S.A., Compie`gne, France). Enrichment of the fractions was ⬎90% as evaluated by flow cytometry. Preparation of Mononuclear Cells Blood samples were collected from a pool of healthy volunteers at the “Etablissement Franc¸ais du Sang Bretagne.” Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation on Lymphoprep (d ⫽ 1.077) (Nycomed Pharma, Oslo, Norway), washed twice with sterile Hank’s buffer saline medium, and resuspended at 1 ⫻ 107 cells/ml in Iscove’s medium (Life Technologies) containing penicillin (100 IU/ml), streptomycin (100 ␮g/ml), L-glutamine (2 mM), and 10% human AB group serum. Cells (2 ⫻ 107/well) were plated in 6-well tissue culture Falcon plates (Becton Dickinson SA, Meylan, France) at 37°C with 5% CO2. Generation of monocyte-derived macrophages by allogeneic stimulation. Equal numbers of mononuclear cells from two unrelated blood donors were mixed at a cell concentration of 1 ⫻ 107/ml in complete Iscove’s medium before plating in 6-well plates. After 48 hours of allogeneic stimulation, nonadherent cells were removed from the culture and macrophages were maintained for 5– 6 weeks. Adherent stimulated monocytes differentiated into morphologically different phenotypes of macrophages including multinucleated giant cells. At 10-days poststimulation, monocytemacrophage differentiation was demonstrated by an uniform intracellular expression of CD68 antigen. Cultures were washed and maintained in complete 60/30 medium (60% AIMV medium [Life Technology], and 30% Iscove’s medium) composed of 50% spent media and 50% fresh media, which was replenished every 3– 4 days for up to 45 days poststimulation [8]. For analysis, macrophages were collected at different time points by trypsinising and gentle scraping. Generation of dendritic cells. Mononuclear cells were plated at a concentration of 1.107/3 ml in complete RPMI 1640 medium. After 2-h incubation at 37°C in humidified 5% CO2, nonadherent cells were removed and adherent monocytes were cultured during 7 days with 1000 U/ml of IL-4 (Peprotech Inc., London, England), and 800 U/ml of GM-CSF (Leucomax 400; Novartis-Pharma, Rueil-

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Malmaison, France). Cytokines were replenished on days 2, 4, and 6. On day 7, immature dendritic cells were differentiated into mature cells by adding TNF-␣ (10 ng/ml). Stimulation With Cytokines Monocytes purified from healthy volunteers and allogeneically stimulated monocyte-derived macrophages were treated with IL-2, IFN-␥, GM-CSF, TNF-␣, IL-4, IL-6, and IL-10 (Genzyme Diagnostics, Cambridge, MA, USA) for 24 and 48 h. As the concentrations recommended by the supplier are approximate values, different dose assays were performed to determine an optimal concentration for the expression of HLA-G antigens. Similar treatments were carried out on the cell lines U937 and THP-1. Bronchoalveolar Lavages Collected From Patients Forty bronchoalveolar samples were obtained from the “Laboratoires de Bacte´riologie-Virologie et de Parasitologie” at the Centre Hospitalier de Pontchaillou (Rennes, France) after being analyzed for bacterial, fungal, and viral infections. In particular, presence of infectious HCMV was verified by the shell vial centrifugation culture assay [9]. Cells were washed twice in phosphatebuffered saline (PBS) pH 7.5, cytocentrifuged onto glass slides, and stored wrapped in aluminum foil at ⫺20°C until used. Tissue Samples Tissue samples were obtained from 53 different patients at the time of tumor resectioning. We studied 18 lung carcinomas, 5 colon carcinomas, 5 renal cell carcinomas, 5 laryngeal carcinomas, 10 breast carcinomas, 5 ovarian carcinomas, and 5 hepatocarcinomas. Fresh tumor tissue and healthy tissue samples were taken from surgical specimens immediately after removal, then snap-frozen in an OCT compound and stored at ⫺70°C. Healthy tissues were taken from the same specimens as the original tumors, but from a place that was distant from the tumor zones and was considered morphologically normal by anatomopathologists. Serial sections from each specimen were routinely stained with hematoxylin and eosin for histological examination and tumor grading. Antibodies The 87G and 16G1 mAbs were provided by D. Geraghty (Fred Hutchinson Cancer Research, Seattle, WA, USA); 87G is a murine IgG2a mAb recognizing the membranebound HLA-G1 and the soluble isoform HLA-G5. The 87G shows no cross reactivity with other MHC-I molecules when tested on diverse MHC-I type transfected cells [10]. The 16G1 is a conformation-independent murine IgG1 mAb showing complete specificity for

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HLA-G soluble molecules [10]. The W6/32 mAb is a murine IgG2a mAb, which binds to MHC-I heavy chains associated with ␤2-m. Macrophages and dendritic cells were phenotyped with an anti-CD68 mAb (Kp1 clone, IgG1 subclass; Dako SA, Trappes, France) and an anti-CD1a mAb (clone BL-THY-1, IgG1 subclass; Monosan, Uden, The Netherlands), respectively. Three antibodies against HCMV genes products were used: two polyclonal rabbit serums against the IE-pp86 and IEpp72 antigens (gift of J. Nelson, Department of Molecular Microbiology and Immunology, Oregon Health Sciences University, Portland, OR, USA) and a mAb against the glycoprotein B (gB) (clone HCMV37; Chemicon International Inc., Temecula, CA, USA). Detection of Viral HCMV DNA in Monocytes Because not all HCMV-seropositive individuals carry HCMV in their monocytes and a substantial proportion of seronegative individuals are indeed HCMV carriers [11], latent infection was tested by detection of HCMV DNA. DNA was prepared from adherent PBMCs using the DNA Qiagen blood kit (Qiagen, Courtaboeuf, France). HCMV-specific primers from exons 1 and 2 of the major immediate early UL123 gene were used in nested PCR reaction [11]. DNAs prepared from uninfected and infected HFF were used as negative and positive controls, respectively. Final amplification products (332 bp) were run on a 1.7% agarose gel containing ethidium bromide and were visualized with UV light. Flow Cytometry Flow cytometry was performed on 3 ⫻ 105 cells. Macrophage Fc receptors were blocked by pre-incubating cells for 60 min in a 5% human AB-group serum. Cells were incubated with primary antibodies for 45 min at 4°C. In each experiment, an equivalent concentration of a mouse isotype-matched control antibody was substituted for the specific antibody. After washing, cells were incubated for 45 min with an antimouse goat IgG F(ab⬘)2 fraction conjugated with phycoerythrin (Immunotech, Marseille, France). Fluorescence analysis was performed with a Facscalibur flow cytometer (Becton Dickinson). Immunostaining Purified and cultured cells were cytocentrifuged on glass slides and fixed for 10 min in acetone at 4°C. Frozen tissues were cut into 5-␮m thick sections, allowed to dry at room temperature for 6 –12 hours, and then fixed for 10 min in cold acetone. Samples were stored wrapped in aluminum foil at ⫺20°C until used. Immunoperoxydase labeling. Staining was carried out at room temperature using the LSAB 2 kit peroxidase

HLA-G Antigens in Myelomonocytic Cells

(Dako). Samples were incubated for 20 min in 3% BSA, 40% human AB-group serum in TBS to eliminate nonspecific bindings. The following primary antibodies were used: anti-HLA class I mAb, W6/32 (2 ␮g/ml); antiHLA-G mAbs, 87G (5 ␮g/ml), and 16G1 (4␮g/ml); anti-CD68 mAb, KP1 (4 ␮g/ml); anti-CD1a mAb, BLTHY-1 (5 ␮g/ml); anti-CD3 mAb (5 ␮g/ml); antiHCMV IE-pp86 (1:100); anti-HCMV IE-pp72 (1:100); and anti-HCMV gB (10 ␮g/ml). After incubation with the antimouse or antirabbit biotinylated antibodies, endogenous peroxidase activity was blocked in 0.3% hydrogen peroxide in methanol for 30 min. Samples were then incubated for 10 min with peroxidase-labeled streptavidin and staining completed with a freshly prepared substrate chromogen, 3% 3-amino-9-ethylcarbazole in N,N-dimethylformamide. Finally, specimens were counterstained with Harris’ hematoxylin and mounted in aqueous Aquatex mounting medium (Merck, Darmstadt, Germany). Immunofluorescence Staining Endogenous fluorescence was quenched through incubation of fixed cells in 50 mM NH4Cl for 10 min. Samples were incubated for 20 min in 3% BSA, 40% human AB-group serum in PBS so as to eliminate nonspecific bindings. Identification of HLA-G-positive cells infiltrating lung tumoral tissues. The 87G mAb (5 ␮g/ml) was applied for 30 min on sections and detected by sequential incubations with biotinylated goat antimouse IgG2a mAb and FITC-labeled streptavidin. To identify the HLA-G positive cells, the same preparations were saturated once again by 3% BSA, 40% human AB-group serum in PBS. The sections were then incubated for 30 min with the anti-CD1a, BL-THY1 or anti-CD68, KP1 mAbs. A goat antimouse IgG1 TRITC labeled was then applied on sections for 30 min. Colocalization of HLA-G molecules and HCMV antigens in allogeneically stimulated monocyte-derived macrophages. The 87G (5 ␮g/ml) or 16G1 (4 ␮g/ml) mAbs were applied for 30 min onto slides and detected by sequential incubations with biotinylated goat antimouse mAb and FITC-labeled streptavidin. To search for HCMV expression in HLA-G positive cells, the same preparations were saturated once again with 3% BSA, 40% human ABgroup serums in PBS. Samples were then incubated for 30 min with the anti-HCMV IE polyclonal sera or the anti-gB monoclonal mAb. After washing, goat antimouse or antirabbit TRITC-labeled antibodies were applied for 30 min. Samples were then mounted in a fade retardant me-

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dium and viewed using a fluorescent microscope equipped with relevant excitation and detection filters. Enzyme-Linked Immunoabsorbent Assay (ELISA) Microtiter plates (Nunc maxisorp F; Polylabo) were coated overnight with the anti-HLA-G mAb 87G at the concentration of 5 ␮g/ml in carbonate buffer (0.05M, pH 9.6). After three washes in PBS containing 0.05% Tween 20, plates were saturated with 5% BSA in PBS for 2 h at 37°C. Cell culture supernatants (100 ␮l) were added to each well and incubated for 3 hours at 37°C. Supernatants were removed and plates were then washed three times with PBS. Then biotinylated W6/32 mAb was added at the concentration of 2.5 ␮g/ml in PBS containing 2.5% BSA. After 1-h incubation at 37°C, plates were washed three times with PBS and peroxidase-conjugated streptavidin (Gibco) was added at 1/1000 dilution. After 30-min incubation at 37°C and five washes with PBS, plates were incubated with 100 ␮l of p-nitrophenyl phosphate (Gibco). Relative concentrations were estimated from optical densities at 405 nm SDS-PAGE and Western Blotting Proteins were extracted using a lysis buffer containing 1% NP-40 and 0.5% sodium deoxycholate and sonicated for 30 seconds to shear off chromosomal DNA. Solubilized proteins were loaded on a 12% mini-SDS-PAGE (75 ␮g per lane) and transferred onto a PVDF membrane using an electrophoretic transfer apparatus. Quality of protein samples was checked by incubating the membrane in the Ponceau S reagent. Membranes were saturated with 5% nonfat dry milk in PBS and incubated with the 16G1 mAb (4 ␮g/ml). Alkaline phosphatase linked antimouse antibody is added and then reacted with CDP-Star chemiluminescent substrate (New England Biolabs GmbH, Schwalbach, Germany). RESULTS Absence of HLA-G Antigen Expression in Unstimulated Myelomonocytic Cells HLA-G antigen expression was searched in monocytes isolated from different normal individuals, in vitro produced dendritic cells and allogeneically differentiated monocyte-derived macrophages at around 10 days of culture. Flow cytometry analyses using the anti-HLA-G mAb, 87G showed that HLA-G antigens were absent at the cell surface of monocytes, dendritic cells, and macrophages. In the same way, no soluble HLA-G antigens were detected by Western blotting and ELISA. Conversely, both cells expressed membrane class Ia molecules: dendritic cells and macrophages expressed twoand fourfold more class Ia molecules than parental monocytes.

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FIGURE 1 Induction of HLA-G antigens in stimulated U937 cells. Detection of HLA-G antigens at the cell surface of U937 cells treated with IFN-␥ (100 U/ml), IL-2 (220 U/ml); GM-CSF (100 U/ml); IL-2 (100 U/ml) and IFN-␥ (500 U/ml) and IFN-␥ (100 U/ml) and GMCSF (100 U/ml) by flow cytometry.

Induction of HLA-G Antigens in the Monohistiocytic Cell Line U937 We investigated the possibility of HLA-G protein synthesis in myelomonocytic cells under inflammatory conditions. HLA-G antigen expression was thus searched in monocytes and allogeneically stimulated monocyte-derived macrophages after stimulation with different cytokines. Whatever the analysis techniques we used, no HLA-G protein expression was induced in these myelomonocytic cells. On the other hand, we were able to induce HLA-G molecules at the cell surface of the cell line U937 treated with IFN-␥ (100 U/ml), IL-2 (220 U/ml), and GM-CSF (100 U/ml) for 48 h. Each stimulation was performed six times: the HLA-G gene was induced with variable percentages of positive cells. In one assay, the level of HLA-G expression was 25% with IFN-␥, 15% with IL-2, 51% with IL-2 and IFN-␥ and 85% with GM-CSF and IFN-␥ (Fig. 1). This protein expression by U937 cells appears transient because no antigens were recovered after 48 h of treatment. No induction was observed in the U937 cells treated with GM-CSF, IL-4, IL-6, and IL-10 alone. We were also

unable to induce HLA-G antigens expression in the THP-1 cell line. HLA-G-Positive Tumor-Infiltrating Macrophages and Dendritic Cells in Lung Carcinoma Biopsies The induction of HLA-G proteins in the monohistiocytic cell line U937 by different cytokines suggested that HLA-G molecules would be expressed in activated macrophages or dendritic cells under specific inflammatory conditions. We first tested this hypothesis by analyzing antigen-presenting cells associated with tumoral tissues. Antigen-presenting cells infiltrating liver, colon, breast, larynx, ovary, or renal cell carcinomas did not display HLA-G staining with 87G mAb. Conversely, the presence of HLA-G antigens was revealed in some small interstitial macrophagelike cells infiltrating well-differentiated squamous lung carcinomas and lung adenocarcinomas (Fig. 2A). Some positive cells also displayed a characteristic dendritic shape and long cytoplasmic processes extending between malignant cells (Fig. 2A). To identify these positive cells, we performed double immunofluorescence labeling by sequentially incubating

HLA-G Antigens in Myelomonocytic Cells

FIGURE 2 HLA-G antigen expression in macrophages and dendritic cells infiltrating pathological lung tissues. (A) Immunoperoxydase labeling of a welldifferentiated squamous cell lung carcinoma. Staining were processed using the LSAB 2 kit Peroxydase (Dako SA, Trappes, France): (1) 87G mAb strongly labeled cells with dendritic shape; (2) large macrophages located in the interalveolar walls of peritumoral stroma were also positive for HLA-G expression (87G mAb); and (3) negative isotypic IgG2a control. (B) Expression of HLA-G molecules in alveolar macrophages collected from patients with HCMV pulmonary infection: (1) immunocytochemical staining was monitored using the 87G mAb, alveolar macrophages displayed a strong cytoplasmic HLA-G expression (red-brown staining); and (2) negative isotypic IgG2a control.

FIGURE 3 HLA-G expression in one allogen-stimulated primary macrophage culture generated from two HCMV latently infected donors. Macrophage differentiation was carried out by allogeneic stimulation of PBMCs from two unrelated healthy blood donors. (A) Flow cytometry analysis demonstrated a cell surface expression of HLA-G molecules in 27% of macrophages (87G mAb). The profile shows a simultaneous decrease of MHC-I cell surface expression (W6/32 mAb). (B) Double-label immunofluorescence was performed with the anti-HCMV IE-pp86 rabbit serum (left) or the antiHCMV gB mAb (right) (rhodamine) and the antiHLA-G mAb 87G (fluorescein). Fluorescence staining is visualized with a confocal microscope. Left: cellular colocalization of IE-86 (red nucleus) and HLA-G (green cytoplasm) was observed in few macrophages at 20 days poststimulation. Right: the photograph illustrates the colocalization of HCMV glycoprotein and HLA-G antigens in the cytoplasm of macrophages displaying HCMV replication (orange fluorescent staining).

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serial sections of the same tumor nodules with the 87G anti-HLA-G mAb and the anti-CD1a or anti-CD68 mAbs, respectively. These immunolabeling demonstrated that HLA-G molecules accumulated in the cytoplasm of some cells from the dendritic lineage (CD1a positive) and some interstitial macrophages (CD68 positive) (data not shown). Expression of HLA-G Antigens in Alveolar Macrophages During Acute HCMV Pneumonia Because macrophages expressed HLA-G antigens in inflammatory tumoral lungs, we then searched for HLA-G protein expression in alveolar cells collected from patients with different infectious pneumonia. Only cytocentrifuged preparations containing at least 85% of alveolar macrophages were analyzed. Out of 40 bronchoalveolar lavages (BAL) performed on patients suffering from pneumonitis, four displayed a positive HLA-G immunochemical staining. Between 5% and 25% of alveolar macrophages demonstrated an expression of HLA-G antigens (Fig. 2B). Interestingly, these four BAL yielded a positive HCMV culture using the shell vial centrifugation assay. They were collected from two bone marrow transplant patients and two HIV-infected patients. Furthermore, 33 and 37 kDa HLA-G soluble isoforms were identified in the protein lysate of one sample tested by Western blot (data not shown). No HLA-G expression was found in alveolar macrophages of lungs infected by bacterial or other viral pathogens (p ⬍ 0.0001). Herpes simplex viruses were isolated in two BAL samples and these did not display HLA-G expression. Induction of HLA-G Antigens in Macrophages Generated Following Allogeneic Stimulation of HCMV Latently Infected Peripheral Blood Monocytes We tried to confirm the potential upregulation of HLA-G gene activity upon HCMV reactivation in allogeneically stimulated monocyte-derived macrophages. Primary macrophage cultures were established by mixing PBMCs from unrelated blood donor pairs. We studied ten primary macrophage cultures established from PBMCs collected from healthy donors: six cultures were established by mixing PBMCs from six unrelated HCMV DNA-positive donor pairs and four others by mixing PBMCs from four unrelated HCMV DNA-negative donor pairs. HLA-G expression was monitored by flow cytometry every 5– 6 days. Cell surface and soluble HLA-G proteins were never detectable in adherent monocytes before allogeneic stimulation. Using the 87G mAb, HLA-G expressing macrophages were first detected by flow cytometry in five primary cultures established from latently infected

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monocytes at around 3 weeks poststimulation. At day 20 poststimulation, one primary culture demonstrated HLA-G cell surface expression in 27% of macrophages (Fig. 3A). Two-color staining analyses using 87G and W6/32 mAbs showed that induction of HLA-G antigens always occurs in macrophages that still express classical MHC-I molecules. No HLA-G cell surface expression was detectable after 5 weeks poststimulation. Soluble HLA-G molecules were also detected by ELISA in the supernatants of three primary cultures generated from latently infected monocytes at around 3 weeks poststimulation. On Western blot, these soluble molecules migrated as a 37 kDa soluble HLA-G5 isoform (data not shown). HLA-G antigens were never detected in the primary cultures produced by mixing uninfected PBMCs. During the culture, these uninfected macrophages always displayed an usual high level of classical MHC-I antigens at their cell surface (data not shown). We simultaneously verified whether virus was reactivated from its latent state during the macrophage differentiation process. Macrophage samples were collected every 5– 6 days for 50 days and evaluated for HLA-G and HCMV expression by double-label immunofluorescence. The presence of HCMV IE-pp86 products was demonstrated in the nucleus of some HLA-G expressing macrophages of both infected primary cultures between 16 and 20 days poststimulation. Coexpression of HCMV gB late antigen and HLA-G molecules were also observed a few days later in macrophages of five cultures (Fig. 3B). DISCUSSION In this review, we showed that HLA-G translation is tightly regulated in mature myelomonocytic cells under stressful conditions. We never detected HLA-G proteins in peripheral blood monocytes, in cultured uninfected myelomonocytic cells, nor in resident immune cells infiltrating healthy tissues. Conversely, we report the evidence for HLA-G protein expression in tumor infiltrating macrophages and dendritic cells. These HLA-Gpositive tumor-infiltrating cells were detected in 5 out 18 different lung carcinoma biopsies. The specific HLA-G expression in immune accessory cells capable of endocytosing, processing, and presenting antigens to appropriate subsets of T lymphocytes suggests that HLA-G proteins may modulate the antitumoral immune response and, therefore, induce a local immune privilege in favor of lung tumor growth. We were then able to induce HLA-G antigen expression in the monohistiocytic leukemia U937 cells after a treatment with IFN-␥. Furthermore, we demonstrated that, in synergy with IFN-␥, IL-2 and GM-CSF selectively enhance HLA-G expression at the cell surface. This

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additional result suggests that cytokines may be involved in the modulation of HLA-G protein synthesis in mature myelomonocytic cells infiltrating inflammatory tissues. In this study, we also showed that HCMV induces the expression of HLA-G molecules. Ex vivo, alveolar macrophages collected from patients undergoing HCMV pneumonitis also displayed HLA-G molecules. Although subjects from whom HCMV was not recovered had immunologic abnormalities and other pathogens due to their underlying disease, no HLA-G antigens were detected. In allogen-stimulated macrophage cultures, membrane-bound and soluble HLA-G antigens were expressed upon differentiation of latently HCMV-infected monocytes during viral reactivation. Although HLA-G transcription persists, there was no translation of these transcripts in uninfected macrophages up to 50 days poststimulation. Thus, cellular factors that dictate macrophage differentiation are not sufficient for HLA-G up-regulation. Our results are in agreement with a report, which demonstrated that unlike their classical MHC-I counterparts, HLA-G molecules stably expressed in JEG 3 trophoblastic cells are resistant to rapid degradation imposed by the HCMV gene products US2 and US11 [12]. HLA-G may possess characteristics of structure or trafficking that allow escape from HCMV associated MHC-I degradation pathway. Because classical MHC-I molecules are partially downregulated in HCMV-infected macrophages and deliver inhibitory messages to NK cells, similar inhibitory signals mediated by HLA-G molecules seem redundant. Thus, HLA-G molecules may provide additional signals to other immune effector cells. For example, HLA-G molecules could inhibit an anti-HCMV CTL response induced by viral peptides loaded on classical MHC-I complexes. In transplant recipients, the development of disease, e.g., pneumonitis, is indeed clearly correlated with absent or diminished HCMV-specific CD8⫹ CTL responses [13, 14]. A marked consequence of HCMV infection of macrophages is the suppressive effect of these virally infected cells on lymphoproliferative responses [15, 16]. In addition, it is conceivable that HLA-G molecules affect the function of myelomonocytic cells such as antigen presentation, cytokine production [2], and downregulate the antiviral adaptative immune response. In conclusion, our observations provide evidence that HLA-G antigen expression is intimately linked to the differentiation of monocytes. Circulating blood monocytes immigrate into extravascular tissue sites and differentiate as macrophages at sites of infection, injury, allograft, or tumor rejection. Initiation of an immune response at these different sites of inflammation may result in the induction of HLA-G molecules in monocyte-derived macrophages upon inflammatory cytokine

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production or reactivation of latent HCMV virus. Then immunosuppression provided by HLA-G molecules would favor tumoral progression or virus dissemination and exacerbate the severity of associated diseases. The immunological or clinical consequences of HLA-G expression in lung carcinoma and in natural HCMV infection remain to be unraveled. ACKNOWLEDGMENTS

We thank Professors M. P. Rame´e and G. Lancien for providing tissue sections; D. E. Geraghty and J. Nelson for their gift of monoclonal antibodies; and Martine Richard for her technical assistance. This work was supported by grants from the Institut National de la Recherche Scientifique et Me´dicale (CRI 9606) and from the Ministe`re de la Recherche et de l’Enseignement Supe´rieur (UPRES-EA 22-33).

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