International Immunology

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International Immunology, Vol. 11, No. 6, 889-898, June 1999
© 1999 Japanese Society for Immunology

HLA-G in the human thymus: a subpopulation of medullary epithelial but not CD83⁺ dendritic cells expresses HLA-G as a membrane-bound and soluble protein

Valérie Mallet, Astrid Blaschitz¹, Laura Crisa², Christian Schmitt³, Sylvie Fournel, Ashley King⁴, Yung Wai Loke⁴, Gottfried Dohr¹ and Philippe Le Bouteiller

INSERM U395, CHU Purpan, BP 3028, 31024 Toulouse Cedex 3, France
¹ Institute of Histology and Embryology, Karl-Franzens-University, 8010 Graz, Austria
² Department of Molecular and Experimental Medicine, The Scripps Research Institute, SBR5, La Jolla, CA 92037, USA
³ CNRS UMR 7627, CHU Pitié Salpétrière, 75013 Paris, France
⁴ Department of Pathology, University of Cambridge, Cambridge CB2 1QP, UK

Correspondence to: P. Le Bouteiller

	Abstract

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The human MHC class Ib gene HLA-G is transcribed and translated in different placental cell subpopulations during pregnancy. In addition to this restricted tissue distribution, HLA-G proteins were also recently detected in the thymus of HLA-G transgenic mice, as well as in some human thymic epithelial cells (TEC). There was a need to further define the phenotype of the HLA-G-expressing cells in the human thymus as well as the type of translated forms that they produce. Using several HLA-G-specific mAb and immunohistochemistry performed on cryosections of human thymi at different ages, we found that the HLA-G-expressing cells are present on medullary cells exhibiting the epithelial morphological type 6. Co-localization experiments performed by double or triple immunofluorescence staining demonstrate that these HLA-G-expressing cells express various cytokeratins, epithelial cell markers but not the CD83 dendritic cell marker. We further show by ELISA measurements that a subset of primary cultured human TEC also expresses soluble HLA-G. Therefore, HLA-G protein tissue distribution is not restricted solely to placental cells. A subpopulation of medullary TEC also expresses HLA-G both at their cell surface and in secreted form, raising the question of the functional significance of such MHC class Ib molecules. Whether thymic soluble and/or membrane-bound HLA-G contribute to inhibit NK cells or to a negative selection of autoreactive T cells which could be harmful in case of pregnancy and/or to a positive selection of viral peptides/HLA-G-restricted CD8⁺ T cells remains to be demonstrated.

Keywords: ELISA, immunohistochemistry, thymic epithelial cells

	Introduction

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HLA class Ia molecules are cell surface glycoproteins that are expressed on most human somatic tissues (1). They are extremely polymorphic and play a crucial role in the immunosurveillance against virally infected or neoplastic cells, by presenting foreign or altered-self peptides derived from cytosolic proteins to CD8⁺ T cells (2). They are also the ligands of NK (and other cell) inhibitory or stimulatory receptors and are able to modulate the cytotoxic potential of these immune cells (3). Except for HLA-C, these MHC class Ia molecules are absent in the trophoblast cell subpopulations that constitute the materno-fetal interface during pregnancy (4–6). This lack of expression is likely to prevent the initiation of a maternal immune response to paternally derived antigens (4).

In contrast, class Ib molecules such as HLA-G exhibit a much lower polymorphism (7) and are more restricted in their tissue distribution (8). HLA-G proteins have been detected in different placental cell subpopulations during pregnancy, and in particular in invading extravillous cytotrophoblast, endothelial cells of fetal blood vessels in the chorionic villi, amnion cells and possibly chorionic villous macrophages (4,5,9–12). These placental localizations suggest that HLA-G may exert immunosuppressive functions during pregnancy to prevent potentially harmful maternal alloimmune reactions. This could be achieved through the ligation to particular NK-, or other cell type-, inhibitory receptors, together or independently of HLA-E, another MHC class Ib molecule recently brought to light (13,14). In the case of uterine infection, HLA-G may also be an efficient viral peptide presenter (15,16) to limit the spread of infection to the fetus. In addition to its expression during fetal life in these specific locations, HLA-G has also been shown to be induced by IFN- on macrophages or blood monocytes, suggesting they may also play a role during inflammatory processes (17,18). HLA-G could also exert non-immunological functions such as influencing the rate of embryonic cleavage (19) in a fashion similar of the preimplantation embryo development (Ped) locus in mouse which was shown to be the Qa2 antigen encoded specifically by the Q9 gene (20).

The presence of both membrane-bound and soluble HLA-G translated products during fetal and adult life strongly suggests that there is a tolerance status against this MHC class Ib protein, preventing unwanted autoreactive T cell destruction of HLA-G-expressing cells. The analysis of HLA-G expression in the thymic cell types, such as epithelial and/or dendritic cells, regulating selection processes was therefore an important goal. First indications showing that HLA-G was present in the human thymus came from early studies showing the presence of HLA-G transcripts in this central lymphoid organ (21). Subsequently, several studies have demonstrated the presence of HLA-G mRNA in the thymus of HLA-G transgenic mice (22,23). Finally, a more recent study, using a mAb specific for a peptide sequence present in the 1 domain of HLA-G, demonstrated that HLA-G is expressed in the human thymus on a subset of medullary and subcapsular epithelial cells (24). Nevertheless, that study also showed that HLA-G immunoreactivity in the thymic medulla extended to other cellular elements which appeared not to be epithelial.

In the present study, by using several anti-HLA-G-specific mAb, we further investigated the cell compartment expressing this non-classical MHC class I in the thymus. We showed that HLA-G is mainly expressed in a subset of thymic medullary epithelial cells, but not in CD83⁺ dendritic cells. We further demonstrate that HLA-G⁺ medullary epithelial cells exhibit the phenotypic and morphological characteristics of a type 6 thymic epithelium in situ, and express both membrane-bound and soluble HLA-G proteins in culture.

	Methods

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Human tissues and cell lines
Normal human thymi were obtained as discarded tissue through the Department of Thoracic Surgery, Laënnec Hospital (Paris) and the Children's Hospital of San Diego (San Diego, CA) of otherwise healthy children (newborn, and 2, 4, 7 and 14 years old) undergoing surgery for cardiovascular procedures. Tissues were immediately snap frozen and stored in liquid nitrogen. Tonsils from a 7-year-old child and lymph nodes from a newborn baby were obtained by surgery from the Department of Forensic Medicine, University of Graz, Austria. The human gestational choriocarcinoma cell lines JAR and JEG-3 were obtained from the ATCC (Rockville, MD). JAR transfected with HLA-G1s (JAR-G1s) was obtained as follows: cDNA from JEG-3, prepared as described below, was used. The HLA-G1s construct was derived from modifications of the megaprimer method of PCR (25); two different specific amplifications were attempted with two sets of primers: Gs.4 (26) and Gc2.2 (27), modified at the end by insertion of a KpnI site; Gs.4 and G5'XbaI (3'-AGCCCCGCGGTACTGGTGGTAGGAACAGATCTCCC-5'); a third amplification was performed using these two PCR products and the G 5'XbaI and Gc2.2 (KpnI) primers. Amplified HLA-G1s cDNA was excised from a 1% agarose gel using the QIAquick gel extraction kit (Qiagen, Courtaboeuf, France), cloned into PCR-Script (Stratagene-Ozyme, Montigny le Brettoneux, France) and sequenced manually with the Sequenase version 2.0 DNA sequencing kit (Amersham, Les Ulis, France). Finally, HLA-G1s cDNA was digested with XbaI and KpnI, and subcloned into pcDNA3.

Human thymic epithelial cells (TEC) cultures
Enriched TEC populations were prepared as previously described (28). Thymic fragments were obtained from children (<4 years old) undergoing cardiac surgery (Department of Thoracic Surgery, Laënnec Hospital, Paris). Tissue was cut into 0.02 cm² pieces and washed 3 times in PBS. Explants were anchored into tissue culture flasks for 3 h at 37°C and 5% CO₂ in air. Tissue culture medium was then added, consisting of a 2:3 mixture (v/v) of Dulbecco's modified Eagle's medium (DMEM) and Ham's F-12 (HAM) medium (Gibco/BRL, Gaithersburg, MD), and supplemented with 5% FCS, 100 IU/ml penicillin, 100 µg/ml streptomycin, 20 ng/ml epidermal growth factor, 0.4 µg/ml hydrocortisone, 8 ng/ml choleratoxin, 5 µg/ml insulin and 1 ng/ml triiodotyronine (all from Sigma, St Louis, MO). Culture medium was changed every 3–4 days and, at day 14, explants were removed, adherent cells were washed with PBS/0.02% EDTA to eliminate fibroblasts and trypsinized (Trypsin–EDTA; Sigma) and used immediately or re-expanded in DMEM/HAM. By immunofluorescence, thymic stromal cell populations contained at least 80% of cytokeratin (CK)⁺/vimentin^– (epithelial) cells. The 24–48 h cell culture supernatants from 5x10⁶ cells were used for ELISA test.

Antibodies
Characteristics and origins of the mAb used in this study are listed in Table 1. The following HLA-G recognizing mAb were used: G233 (29), BFL.1 (30), 87G (15) and 16G1, directed against a 20mer peptide derived from the C-terminal sequence of soluble HLA-G (31).

View this table: [in this window] [in a new window]   Table 1. mAb used in this study

 
Flow cytometry analysis and cell sorting
Indirect immunofluorescence staining was performed a described (32). TEC were incubated either with G233, anti-HLA-G (29) or 10.3.6, anti-murine MHC class II I-A^k, used as negative control (33) followed by F(ab')₂ goat anti-mouse IgG secondary antibody conjugated with phycoerythrin (PE) (Coulter-Immunotech, Marseille, France). Samples were analyzed on a Coulter Epics Elite flow cytometer (Margency, France) gated to exclude non-viable cells.

Immunohistochemistry
Immunohistochemical stainings were performed on cryo-sections (5 µm) of the tissues described above. Frozen sections were air-dried for 2 h, fixed in acetone for 5 min at room temperature and rehydrated in PBS for 10 min. Single stainings were performed as follows, using a HRP-LSAB kit (Dako, Glostrup, Denmark), as previously described (10). After an endogenous peroxidase blocking step, primary antibody incubations were performed at appropriate dilutions using an antibody diluent (Dako) for 30 min. Slides were washed with PBS 3 times, followed by incubation with biotinylated goat anti-mouse Ig for 10 min. After three washes in PBS, they were further incubated with streptavidin–peroxidase for 10 min. After three washes in PBS, the peroxidase labeling was developed with 3-amino-9-ethylcarbazole (Sigma, Vienna, Austria) giving a red–brown reaction product. Negative controls were incubated with a mixture of mouse IgG1, IgG2a and IgG2b isotypic control mAb (Dako). Some single stainings also used an alkaline phosphatase system, as previously described (24). After extensive washes in distilled water, slides were counterstained with Mayer's hemalum and mounted in Kaiser's glycerol–gelatin. Photographs were taken with an Axiophot microscope (Zeiss, Oberkochen, Germany).

Confocal laser scanning microscopy and immunofluorescence analysis
Cryosections of human thymus were fixed in acetone for 5 min, rehydrated in PBS and incubated in protein blocking solution (LSAB kit). For double immunostainings, the slides were incubated for 30 min in unconjugated primary G233 mAb diluted in antibody diluent (Dako). After three washes in PBS, the slides were incubated with the biotinylated second anti-mouse antibody for 10 min, followed by incubation with streptavidin–RPE–Cy5 (Dako) for 15 min. The slides were then washed again in PBS and incubated in a blocking solution made of mouse isotype irrelevant antibodies (same isotype as the second directly conjugated antibody, i.e. IgG1 for MNF116 and IgG2b for CD83). Sections were then incubated with FITC-conjugated MNF-116 (Dako) or FITC-conjugated CD83 (Immunotech) mAb for 30 min in the dark. For triple immunofluorescence staining, G233–RPE–Cy5 and MNF116–FITC incubations were performed as described above, followed by a final incubation in PE-conjugated CD83 mAb for 30 min. After washes in PBS, the slides were mounted in Moviol and examined, using a laser scanning confocal microscope. Controls were done by incubation with isotypic control mouse Ig (mixture of IgG1, IgG2a and IgG2b), followed by biotinylated anti-mouse IgG and either by streptavidin–FITC, streptavidin–RPE–Cy5 or streptavidin–PE.

RT-PCR analysis
RNA extraction was performed on JEG-3 cell line, and on 20,000 G233(+) and G233(–) sorted TEC. Total RNA was extracted using the Bioprobe-RNAzol kit (Montreuil-sous-Bois, France), according to the manufacturer's instructions. RNA from TEC cells was precipitated using glycogen as a carrier (34). cDNA was prepared from 5 µg of total RNA from JEG-3, and from total RNA extracted from TEC, by using oligo(dT)_12–18 primer and 200 U of reverse transcriptase from Superscript II kit (Life Technologies, Cergy Pontoise, France). One-fifth of this reaction was used as template for PCR amplification of HLA-G transcripts expressed in TEC. Specific amplifications of the different HLA-G transcriptional isoforms were performed with the following primer sets: Gc1.2 and Gc2.2 for the three membrane-bound isoforms (27); Gs.4 (26) and Gc2.2 for both membrane-bound and soluble forms. PCR products were analyzed by electrophoresis into 1% agarose gel and visualized by ethidium bromide staining. ß-Actin was amplified as a control of RNA quality. 3'ß (5'-TCCCTGGAGAAGAGCTACGAG-3') and 5'ß (5'-CATCTGCTGGAAGGTGGACA-3') ß-actin primers (35) amplified a 350 bp fragment.

ELISA for soluble HLA-G
Soluble HLA-G class I levels were measured with a sandwich ELISA. Microtiter plates (Becton Dickinson) were coated with 87G (1 µg/ml) or BFL.1 mAb (2 µg/ml) in 0.1 M carbonate buffer, pH 9.5. After three washes in PBS containing 0.05% Tween 20, plates were saturated with 250 µl of PBS containing 4% BSA for 2 h at 37°C. Culture supernatants (100 µl) were added to each well. After incubation for 2 h at 37°C, plates were washed 3 times in PBS and biotinylated W6/32 mAb (100 µl, 5 µg/ml) was added to each well and incubated for 1 h at 37°C. Plates were washed 5 times and further incubated with a peroxidase-conjugated streptavidin (Dako) for 30 min at 37°C. After five washes, 100 µl of o-phenyldiamine peroxidase substrate (Sigma) was added to each well and incubated for 15 min at room temperature. The reaction was stopped by addition of 100 µl of 2 N H₂SO₄. The relative concentrations of soluble HLA-G was estimated from absorbancy of the yellow product at 492 nm. All samples were assayed in duplicates. Statistical significance was assessed by using the Wilcoxon test on paired values.

	Results

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Human thymic HLA-G-expressing cells are present in the medulla and mainly exhibit the epithelial cell type 6 morphology
Cryosections of human thymus were immunostained using four different HLA-G-specific mAb, i.e. G233 (29), 87G, 16G1 (15) and BFL.1 (30). These mAb were previously shown to label mainly extravillous cytotrophoblast (G233 and 87G) or chorionic fetal endothelial cells (16G1 and BFL.1) on sections of first trimester human placenta (5,8,10,29). On cryosections of human newborn thymus, we found that the G233 mAb clearly stained cells within the medulla, very few reaching the cortico-medullary junction (Fig. 1a and b). Similar results were obtained on three other newborn thymus samples (data not shown) as well as on human thymi of 4- and 14-year-old donors (Fig. 1c and d). Most stained cells were observed in the central part of the medulla, either located in the Hassall's corpuscules or scattered around them. Staining intensities of the labeled cells were heterogenous. Few branched cells were strongly stained in the whole cytoplasm including their protusions, whereas others, located around the Hassalls' corpuscules, were slightly stained (Fig. 1b) and could sometimes be more easily detected without counterstaining (data not shown). Cortical and subcapsular thymic cells did not show any specific immunoreactivity with the G233 mAb (Fig. 1a, c and d). Isotypic control antibodies gave no staining (Fig. 1e). Some of the G233⁺ cells were clearly identified as epithelial cells of type 6, according to previously established morphological criteria such as the presence of protusions and bright nuclei (36). These cells comprised the outer cellular layer of Hassall's bodies or were located in their immediate surroundings (Fig. 1b and c). Other HLA-G⁺ cells could also belong to epithelial cells of type 5 which are normally found in the medulla and cortico-medullary region, and are often arranged in small groups (36). Staining of thymic sections with the 87G mAb gave similar results with regard to the location and epithelial cell types detected, although the intensity of labeling was always lower than the one obtained with the G233 mAb and fewer cells seemed to be stained (data not shown). Neither BFL.1 nor 16G1 mAb gave positive stainings (data not shown). Using the same immunostaining procedure, none of the four anti-HLA-G mAb tested, including G233 and 87G, labeled any cell in cryosections of human tonsil or lymph node similarly processed (data not shown).

View larger version (118K): [in this window] [in a new window]   Fig. 1. Immunohistochemical localization of HLA-G-expressing cells in human thymus of a newborn baby (a and b), and 4-year-old (c) and 14-year-old (d and e) donors. Cryosections were incubated with the HLA-G-specific G233 mAb (a–d) or a control mouse IgG antibody (e), followed by a biotin–streptavidin peroxidase detection system developed with 3-amino-9-ethylcarbazole, yielding a red–brown reaction (a and b) or with an alkaline phosphatase detection method (c, d and e), as previously described (24). Sections (a) and (b) were counterstained with Mayer's hemalum. (a, c and d) HLA-G-expressing cells are essentially present in the medulla (m) and absent in the cortex (c). (b) High magnification of medullary cells stained with G233 mAb, showing positive stained cells arranged around Hassall's corpuscles (Hc), either as an outer wall exhibiting the morphological type 6 TEC or scattered in the surroundings, sometimes as branched shaped cells. (a) White bar = 90 µm; (b) white bar = 22 µm; (c, d and e) black bars = 20 µm.

 
Medullary thymic HLA-G⁺ cells co-express several epithelial cell markers but not the CD83 dendritic marker
In order to define the phenotype of HLA-G⁺ thymic medullary cells, we first performed single immunostainings of thymic sections with various mAb specific for a panel of CK epithelial cell markers. Some of these mAb, including MNF116 and NCL5D3, show a broad pattern of reactivity against several CK, whereas the others recognized a single CK protein (Table 1). This study allowed us to define which of the different CK markers had the same compartmental localization as the HLA-G-expressing cells. Results are summarized in Table 2 and illustrated in Fig. 2. We subdivided the different thymic compartments in which epithelial cells were present into subcapsular, cortical and medullary, including Hassall's corpuscles. HLA-G-expressing cells are located in the medullary compartment (Fig. 2a). The MNF116 mAb, directed against CK5, 6, 8, 17 and 19, stained epithelial cells present in the three different compartments (Fig. 2b). The CK7⁺ cells were present in the medulla and in the subcapsular part, but not in the cortex (Fig. 2c). Only the CK13 had the same compartmental distribution as G233 (cf. Fig. 2a and d) and 87G⁺ cells (data not shown): it stained cells in the medullary region, including Hassall's corpuscles, and not in the subcapsular and cortical compartments (Fig. 2d), although the staining intensity is much higher for CK13 than for G233. We next extended this study to investigate markers that distinguish two other cell types present in the thymic microenvironment, i.e. macrophages (CD163⁺) and dendritic cells (CD83⁺). We used the Ber-Mac3 mAb which recognizes the CD163 macrophage marker (37) and the HB15A mAb that reacts against CD83 dendritic marker (38). CD83⁺ cells were found in the cortico-medullary junction and the medulla, but there were very few cells in the cortex (Fig. 2e). CD163⁺ cells were located in the trabeculae, cortex and cortico-medullary junction but less within the medulla (Fig. 2f). None of CD83⁺ or CD163⁺ cells were present in the Hassall's corpuscules (Fig. 2e and f respectively).

View this table: [in this window] [in a new window]   Table 2. Immunohistochemical localization of various CK among the different compartmental TEC: comparison with HLA-Ga

 

View larger version (195K): [in this window] [in a new window]   Fig. 2. Immunohistochemical localization of HLA-G, dendritic, epithelial and macrophage cell markers within thymic compartments: comparative study. Serial cryosections of the same human newborn thymus were incubated either with HLA-G-specific G233 mAb (a); CK5-, 6-, 8-, 17- and 19-specific clone MNF116 mAb (b); CK7-specific clone OV-TL12/30 (c); CK13-specific clone KS13.1 (d); CD83 dendritic-specific HB15A mAb (e); CD163 macrophage-specific Ber-Mac3 mAb (f). A peroxidase labeled streptavidin–biotin detection system was used, developed with 3-amino-9-ethylcarbazole, giving a red–brown reaction. Sections were counterstained with Mayer's hemalum. Both cortex (c) and medulla (m) with Hassall's corpuscles (Hc) are seen. White bar = 40 µm.

 
Two- or three-color laser scanning confocal microscopy was then used to further assess the epithelial cell type of HLA-G⁺ cells in the thymic medulla and to investigate whether some of these HLA-G-expressing cells could also be of dendritic cell type (Figs 3 and 4). Among the various cell surface markers that have been used to identify dendritic cells, we have chosen CD83, which was recently identified as a specific selective marker of dendritic lineage that has aided in their characterization (38). Dual labeling revealed that epithelial marker revealed by MNF116 mAb (Fig. 3a) and HLA-G revealed by G233 mAb (Fig. 3b) were co-localized in the same cells (Fig. 3c). In contrast, the use of CD83 dendritic marker (Fig. 3d) and of HLA-G mAb (Fig. 3e) demonstrated that HLA-G⁺ cells did not belong to the CD83⁺ dendritic lineage since when double stained with G233 and CD83 mAb, there was no co-localization of these markers (Fig. 3f). Triple staining with MNF116–FITC (green), CD83–PE (red) and G233–Cy5 (blue) further confirmed that a subpopulation of epithelial cells (MNF116⁺) expressed HLA-G (G233⁺) in the medulla, whereas CD83⁺ cells in this compartment were always negative for HLA-G (Fig. 4). A summary of all of these immunohistochemical and laser scanning microscopy studies is illustrated in Fig. 5.

View larger version (105K): [in this window] [in a new window]   Fig. 3. HLA-G molecules co-localize with CK MNF116 epithelial cell marker (green) but not with the CD83 dendritic marker (red): images obtained by two-color immunofluorescence confocal laser scanning microscopy. Thymus cryosections were fixed in acetone and labeled with the following antibodies: FITC-conjugated MNF116 (a), unconjugated G233 followed by a biotinylated anti-mouse IgG and a streptavidin–RPE–Cy5 (b) or both (c); FITC-conjugated CD83 (d), unconjugated G233, followed by a biotinylated anti-mouse IgG and a streptavidin–RPE–Cy5 (e) or both (f). An overlay diagram is shown in which co-localized HLA-G and MNF116 epithelial marker appear yellow/orange (c). Cells positive for HLA-G are negative for the CD83 dendritic marker (f). One result representative of three independent experiments is shown. Scanning field: 10 µm2.

 

View larger version (49K): [in this window] [in a new window]   Fig. 4. Triple staining further confirms that most HLA-G+ cells (blue) are MNF116+ (green) and do not express CD83 (red). Thymus cryosections were fixed in acetone and incubated with the following antibodies: in green (a): FITC-labeled MNF116; in red (b): PE-labeled CD83; in blue (c): unconjugated G233, followed by a biotinylated anti-mouse IgG and a streptavidin–RPE–Cy5. Co-localization was obtained by the following sequential labelings: first, unconjugated G233, followed by a biotinylated anti-mouse IgG and a streptavidin–RPE–Cy5; second, FITC-labeled MNF116 and third, PE-labeled CD83 (d), HLA-G molecules co-localize with the epithelial marker, giving a turquoise-blue staining (white arrows), but not with the CD83 dendritic marker. One result representative of three independent experiments is shown. Scanning field: 20 µm2.

 

View larger version (97K): [in this window] [in a new window]   Fig. 5. Diagram showing some features of the HLA-G-expressing cells in the human thymus, including thymic compartmental localization, cell morphology and some phenotypic markers. Such a compilation of the immunohistochemical and immunofluorescence studies shows that HLA-G-expressing cells are located in the medulla and express different epithelial cell markers. In contrast, HLA-G-expressing cells do not express dendritic nor macrophage markers.

 
A subpopulation of purified human TEC expresses HLA-G, both at the transcriptional and protein (including soluble) levels
Presence of cell surface HLA-G molecules on a subpopulation of human TEC was demonstrated by a flow cytometry analysis performed on a primary culture of purified TEC (26), using HLA-G-specific mAb. The G233 mAb stained ~15% of the cells (Fig. 6a), as did the 87G mAb (data not shown). Similar results were obtained on several TEC samples (data not shown). In order to further define which of the HLA-G isoforms were present in these HLA-G-expressing TEC, we performed RT-PCR analysis on G233⁺ sorted TEC, using different sets of HLA-G-specific primers, capable of discriminating between membrane-bound and soluble HLA-G isoforms (Fig. 6b). Amplified cDNA products from G233⁺ and G233^– sorted TEC, as well as from the control JEG-3 cell line, were analyzed by agarose gel electrophoresis, followed by ethidium bromide staining. Using membrane-bound HLA-G-specific primers, three predominant bands of ~1200, 900 and 600 bp were detected in JEG-3 (Fig. 6b, upper panel). These bands correspond to the expected sizes of the HLA-G1, -G2 and/or -G4, and -G3 isoforms respectively (27,31,39). In the G233⁺ sorted TEC, the two upper bands were clearly seen. In contrast, no HLA-G-specific cDNA could be amplified in the G233^– sorted cells TEC. By using another set of primers specific for the soluble forms of HLA-G, soluble HLA-G1 isoforms were also detected in G233⁺ TEC as well as in JEG-3 cell line, but not in G233^– TEC (Fig. 6b, middle panel). ß-Actin cDNA was amplified as a control of RNA quality, a single band being obtained for each cell type (Fig. 6b, lower panel). Therefore, it can be concluded that both membrane-bound and soluble HLA-G transcriptional isoforms are produced in G233⁺ TEC.

View larger version (36K): [in this window] [in a new window]   Fig. 6. (a) Flow cytometry analysis of cell surface HLA-G expression on cultured human TEC. Cells were labeled by indirect immunofluorescence, as described in Methods, using either G233 (anti-HLA-G) or 10.3.6 (anti-murine MHC class II I-Ak-negative control) primary mAb. HLA-G+ cells (TEC-G+) and HLA-G– cells (TEC-G–) were sorted out and used for RT-PCR analysis. (b) RT-PCR study showing that HLA-G+ TEC express both membrane-bound and soluble HLA-G transcriptional isoforms. Upper panel: RT-PCR amplification of HLA-G cDNA from JEG-3, TEC-G– and TEC-G+, using the Gc1.2 and Gc2.2 primers (27): ethidium bromide-stained agarose gel. Line `0' corresponds to a distiller water-containing negative control. Size markers on the left hand-side are indicated in bp. The Gc1.2 and Gc2.2 primers used for cDNA amplification are indicated in the HLA-G cDNA sequence from which they are derived. L, leader sequence; 1, 2, 3, external domains; Tm, transmembrane domain; C, cytoplasmic domain containing a stop codon (*); 3'UT, 3' untranslated region. G1, G2 and/or G4 and G3 arrows, on the right hand-side, point to the HLA-G1 (1200 bp), HLA-G2 and/or G4 (900 bp) and HLA-G3 (600 bp) species. Middle panel: RT-PCR amplification of HLA-G cDNA from TEC-G–, TEC-G+ and JEG-3 using the Gc2.2 (27) and Gs.4 (26) primers: ethidium bromide-stained agarose gel. Such primers reveal both soluble (Gs) and membrane-bound (Gm) isoforms. The Gs4/Gc2.2 primers used for cDNA amplification are indicated in the HLA-G cDNA sequence from which they are derived. Size markers on the left hand-side are indicated in bp. Lines `0' correspond to a distiller water-containing negative control. Due to the presence of intron 4, PCR products of the soluble HLA-G isoform are longer than the cDNA fragment obtained from the HLA-G transmembrane isoform. Lower panel: ß-actin amplified cDNA as a control of RNA quality. (c) Detection of the relative levels of soluble HLA-G proteins secreted by cultured human TEC (1, 2, 3: three different cultures), as compared with negative (JAR) and positive (JAR-G1s) cells. The assay was performed with the ELISA described in Methods, using 87G (anti-HLA-G) as first coating mAb, cell culture supernatants from 5x106 cells incubated for 2 days in 5 ml of culture media and biotinylated W6/32 mAb, as secondary mAb. Values in absorbancy units (492 nm) represent the average (±SD) of six separate experiments for each supernatant (n = 6, P 0.05).

 
Since RT-PCR analysis revealed the presence of soluble HLA-G transcriptional isoforms in HLA-G⁺ TEC, we investigated whether the corresponding translated products were secreted in culture supernatants. In order to do so, we designed a two-step ELISA for the detection of soluble HLA-G. In these ELISA experiments, the anti-HLA-G mAb (either 87G or BFL.1) was immobilized on microtiter plates and a biotinylated pan-HLA class I mAb (W6/32) was used for detection. Results of these experiments, using three different TEC culture supernatants and 87G mAb, are shown in Fig. 6(c). Although at low levels, soluble HLA-G was reproducibly detected in all TEC supernatants tested. Soluble HLA-G was also detected from supernatants of JAR-G1s, although at lower levels than TEC. No soluble HLA-G was ever detected in supernatants of untransfected JAR. Similar results were also obtained when BFL.1 mAb was used instead of 87G (data not shown).

	Discussion

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In this paper we showed that HLA-G-expressing cells in the human thymus are predominantly found in the medulla and express CK markers of the epithelial cell type. Our study demonstrates that not all the anti-HLA-G-specific mAb tested showed positive immunoreactivity to fixed cryosections of human thymus. Two HLA-G-specific mAb (G233 and 87G) clearly stained a subpopulation of thymic medullary epithelial cells, as assessed by immunohistochemical analysis of thymus cryosections and flow cytometry analysis of primary cultured TEC. These results are consistent with a previous observation using a different anti-HLA-G mAb (24), although we never found HLA-G-expressing cells in the subcapsular compartment. Two other HLA-G recognizing mAb (BFL.1 and 16G1) did not stain cells in the thymus by immunohistochemistry. These results are in accordance with a previous study showing that both BFL.1 and 16G1 mAb had the same reactivities on placental tissue sections: they both stain endothelial cells of fetal vessels in chorionic villi (10). However, BFL.1 detected soluble HLA-G isoforms in TEC culture supernatant by ELISA. Among the HLA-G⁺ cells detected in the epithelial cells, at least two populations could be identified by immunohistochemistry, one of them expressing low amounts of HLA-G, the other expressing high levels. The significance of these various levels of expression remains to be determined. We observed that some G233⁺ cells in the medulla did not express epithelial cell markers, suggesting that some HLA-G⁺ cells in the thymus could belong to a non-epithelial type. This was already observed by Crisa et al. (24), using a different anti-HLA-G mAb. Moreover, when we used double- or triple-staining laser scanning confocal microscopy, all of the G233⁺ cells were MNF116⁺, whereas none of the G233⁺ cells were CD83⁺. However, CD83 is expressed on mature, activated dendritic cells (38). Therefore, it cannot be excluded that some G233⁺/CD83^– cells are of the dendritic, unmature type. All of the data presented here have been obtained on newborn and young children thymi. It cannot be excluded that HLA-G is also expressed on other cell types, including dendritic, during the early fetal life. Results obtained on HLA-G transgenic mice showing expression of HLA-G in dendritic cells (40) are consistent with this hypothesis.

T cells in the thymus are submitted to both negative and positive selection, both processes being important for the development of the T cell repertoire and the acquisition of self-tolerance (reviewed in 41). Animal models suggest that HLA-G could influence the T cell repertoire since it has been shown that immunization of HLA-G transgenic mice with peptides that bind to HLA-G elicited a HLA-G-restricted cytotoxic T cell response (22). It should be noted also that positive T cell selection can develop at other sites than the thymus; in particular, intestine and lungs, which are rich in epithelial cells (42). One may hypothesize that such a peripheral positive selection could also occur during pregnancy in the placenta which is rich in specialized epithelial cell types expressing HLA-G: cytotrophoblast cells (11). Therefore, HLA-G anti-viral function may be efficient to eliminate virally infected cells in the uterus and protect the fetus from uterine viral infection spread. It is likely that homeostatic regulatory mechanisms (43) maintain unresponsiveness to self-HLA-G. A central tolerance could be due to the presence of HLA-G proteins in thymic medullary epithelium. A peripheral tolerance could be maintained during pregnancy when the mother's mature lymphocytes encounter fetally-derived HLA-G-expressing cells in the placenta. Whether HLA-G expressed in some trophoblast and endothelial cells during pregnancy (10) can be considered as self-antigen (i.e. presenting the same, unaltered self peptides as the HLA-G expressed in the adult mother) is unknown. The medullary compartmentalization of HLA-G and its presence on epithelial but not dendritic cells does not exclude that surviving T cells leaving the thymus could have a repertoire of receptors that are influenced by the HLA-G molecules expressed in the thymic environment and in the medullary epithelial cells in particular. However, the absence of HLA-G-expressing cells we observed in peripheral human lymph nodes suggests that T cells which could have been positively selected in the thymus would not survive due to the absence of encounter in the periphery with the same HLA-G–self-peptide complex that led to their positive selection (44). The ability to isolate such HLA-G⁺ TEC to homogeneity will help to further identify their phenotypic markers (presence or absence of cofactors and/or adhesion molecules that are essential for thymocyte death in particular) and their role in T cell education (43). Another important question is whether the T cell repertoire may be altered in the maternal thymus during pregnancy, knowing that MHC expression may be altered, that medulla area is greatly enlarged and that medullary epithelial cells proliferate (45).

We have shown that soluble HLA-G was produced by some TEC. Some immunoregulatory properties of soluble HLA class I molecules have been described (46,47). One possible function of soluble HLA-G molecules could be the inactivation of NK cells, present in the thymus, which could destroy the thymocyte fractions that lack HLA class Ia expression (48).

These results pave the way for future functional studies to investigate the role of HLA-G in central thymic, and possibly peripheral, T cell selection.

	Acknowledgments

 
The 87G and 16G1 mAb were kindly donated by Dr D. Geraghty. We wish to thank Dr John Lamberti and the staff of the Children's Hospital of San Diego, San Diego, CA and Professor Lecas, Department of Thoracic Surgery, Laënnec Hospital, Paris for assistance in obtaining human thymus. Maryse Aguerre-Girr is gratefully acknowledged for her excellent technical assistance, and Georges Cassar for flow cytometry analysis and cell sorting. We also thank Rudi Schmied for the laser scan microscopy experiments. This work was supported by funds from INSERM, ARC, Ligue Nationale Contre le Cancer and Conseil Régional Midi Pyrénées given to P. L. B. V. M. was supported by a fellowship from Ligue Nationale Contre le Cancer, Comité de la Haute Garonne. L. C. is supported by a Career Development Award from the Juvenile Diabetes Foundation. A. K. and Y. W. L. are supported by Tommy's Campaign. We are grateful to Dr Francioise Lenfant, Professor Philippe Naquet and Professor Joost van Meerwijk for helpful discussions.

	Abbreviations

 

CK	cytokeratin
PE	phycoerythrin
TEC	thymic epithelial cells

	Notes

 
The first two authors contributed equally to this work

Transmitting editor: L. Moretta

Received 14 October 1998, accepted 17 February 1999.

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