HLA-G Expression in Human Embryonic Stem Cells and Preimplantation An Verloes, Hilde Van de Velde, Joel LeMaoult, Ileana Mateizel, Greet Cauffman, Peter A. Horn, Edgardo D. This information is current as Carosella, Paul Devroey, Marc De Waele, Vera Rebmann of September 26, 2021. and Martine Vercammen J Immunol 2011; 186:2663-2671; Prepublished online 19 January 2011;

doi: 10.4049/jimmunol.1001081 Downloaded from http://www.jimmunol.org/content/186/4/2663

Supplementary http://www.jimmunol.org/content/suppl/2011/01/19/jimmunol.100108 Material 1.DC1 http://www.jimmunol.org/ References This article cites 72 articles, 20 of which you can access for free at: http://www.jimmunol.org/content/186/4/2663.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

HLA-G Expression in Human Embryonic Stem Cells and Preimplantation Embryos

An Verloes,* Hilde Van de Velde,†,‡ Joel LeMaoult,x,{ Ileana Mateizel,†,‡ Greet Cauffman,†,‡ Peter A. Horn,|| Edgardo D. Carosella,x,{ Paul Devroey,†,‡ Marc De Waele,* Vera Rebmann,|| and Martine Vercammen*

Human leukocyte Ag-G, a tolerogenic molecule that acts on cells of both innate and adaptive immunity, plays an important role in tumor progression, transplantation, placentation, as well as the protection of the allogeneic fetus from the maternal immune system. We investigated HLA-G mRNA and protein expression in human embryonic stem cells (hESC) derived from the inner mass (ICM) of . hESC self-renew indefinitely in culture while maintaining pluripotency, providing an unlimited source of cells for therapy. HLA-G mRNA was present in early and late passage hESC, as assessed by real time RT-PCR. Protein expression was demonstrated by flow cytometry, immunocytochemistry, and ELISA on an hESC extract. Binding of HLA-G with its ILT2 receptor Downloaded from demonstrated the functional active status. To verify this finding in a physiologically relevant setting, HLA-G protein expression was investigated during preimplantation development. We demonstrated HLA-G protein expression in oocytes, cleavage stage embryos, and blastocysts, where we find it in trophectoderms but also in ICM cells. During development, a downregulation of HLA- G in the ICM cells was present. This data might be important for cell therapy and transplantation because undifferentiated hESC can contaminate the transplant of differentiated stem cells and develop into malignant cancer cells. The Journal of Immunology, 2011, 186: 2663–2671. http://www.jimmunol.org/

uman embryonic stem cells derived from the inner cell circumvent the allogeneic nature of hESC, efforts have been un- mass (ICM) of blastocysts have the capacity to self- dertaken to make the cells less immunogenic (e.g., by knocking H renew indefinitely while maintaining their pluripotent down b2-microglobulin, a nonpolymorphic small protein essential potential (1–3). Their pluripotency is demonstrated by the capacity for the expression of HLA on the cell-surface membrane) (7, 8). to differentiate into the three germ layers (ectoderm, mesoderm, Parthenogenesis (9, 10) and somatic cell nuclear transfer (11, 12) and endoderm) both in vitro, by allowing them to form embryoid have been explored to obtain autologous hESC lines, but with

bodies (4), and in vivo, when they form teratomas after injection a low success rate. Using key factors for maintaining pluripotency, by guest on September 26, 2021 in muscles or testis of SCID mice (1, 5). The potential of pluri- somatic cells were reprogrammed into induced pluripotent stem potent human embryonic stem cells (hESC) for therapeutic use cells, which allow the production of unlimited quantities of au- has been hampered by: 1) ethical concerns because of the de- tologous cells for transplantation (13–15). However, the safety of struction of human embryos; 2) their capacity to generate prim- these induced pluripotent stem cells remains to be proven (16). itive tumors posttransplantation (6); 3) the risk of infection Also, the capacity of hESC to form teratoma should be followed transmitted by retroviruses and other animal pathogens; and 4) with caution because these benign tumors may eventually develop rejection because of HLA incompatibility. HLA molecules are into malignant cancers. Indeed, hESC were shown to generate highly polymorphic. Worldwide hESC banks would need to be primitive undifferentiated tumors in engrafted human fetal tissues installed to cover all HLA types for transplantation purposes. To in SCID mice rather than teratomas (6), indicating that undif- ferentiated hESC contaminating the transplant may develop into *Department of Hematology, University Hospital Brussels, 1090 Brussels, Belgium; aggressive tumors. Whether hESC have immune-privileged pro- †Centre for Reproductive Medicine, University Hospital Brussels, 1090 Brussels, perties to escape the immune system is under current investi- Belgium; ‡Department of Embryology and Genetics, Free University Brussels (VUB), 1090, Brussels, Belgium; xCEA, I2BM, Service de Recherches en Hemato- gation. HESC have no Ag presentation capacity because they lack Immunologie, 75475, Paris, France; {Institut Universitaire d’Hematologie, 75475, HLA class II molecules and costimulatory molecules on their || Paris, France; and Institute for Transfusion Medicine, University Hospital Essen, surface membrane (17, 18). However, they express HLA class I at 45122 Essen, Germany a very low level, which increases moderately after differentiation Received for publication April 2, 2010. Accepted for publication December 14, 2010. and reaches the level of somatic cells in presence of IFN-g (19). This work was supported by grants from the Willy Gepts Foundation and University Hospital Brussels (to M.V.) and funding from Deutsche Forschunggemeinschaft DFG HLA class I expression makes them susceptible to an immune SE 1093/2-1 (to P.A.H.). M.V. is the Senior Clinical Investigator of the Fund for Sci- response by cytotoxic T cells, but whether this response occurs is entific Research-Flanders, Belgium (Fonds Wetenschappelijk Onderzoek-Vlaanderen). under debate (17, 18, 20). hESC are weakly susceptible to lysis by Address correspondence and reprint requests to Dr. Martine Vercammen, Department NK cells, which play a major role in the innate immune system of Hematology, Universitair Ziekenhuis Brussel, Laarbeeklaan 101, 1090 Brussels, Belgium. E-mail address: [email protected] when HLA class I is missing (19), but the mechanisms hereto have The online version of this article contains supplemental material. yet to be elucidated. A potential candidate playing a key role in Abbreviations used in this article: GV, germinal vesicle; hESC, human embryonic tolerance is the nonclassical HLA-G molecule. Besides its im- ; ICM, inner cell mass; IVF, in vitro fertilization; MEF, mouse embryonic munosuppressive function in transplantation, autoimmunity, and fibroblast; mESC, murine ; MI, metaphase I; MII, metaphase II; tumor progression (21, 22), HLA-G expression is associated with SSEA, stage-specific embryonic Ag; TE, trophectoderm. placentation and protection of the allogeneic fetus from the ma- Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 ternal immune system (23). HLA-G can be found as seven www.jimmunol.org/cgi/doi/10.4049/jimmunol.1001081 2664 HLA-G IN UNDIFFERENTIATED hESC AND EMBRYOS isoforms, including four membrane-bound (HLA-G1 to -G4) and Embryos In Vitro, and the couples’ informed consent. Female patients three soluble (HLA-G5 to -G7) forms, due to the alternative splic- underwent controlled ovarian stimulation (40) and oocyte retrieval (41). ing of the HLA-G primary transcript (24). The HLA-G1 molecule Oocytes were either immature at the germinal vesicle (GV) or the meta- phase I (MI) stage or were matured in vitro until the metaphase II (MII) and its soluble counterpart G5 have been studied the most. They stage. Oocytes were denuded from surrounding cumulus and corona cells have an identical extracellular structure, which is classic HLA before injection (42). Embryos obtained after IVF or intracytoplasmic class I-like: a H chain of three globular domains noncovalently sperm injection (43) were included in the study if assessed unsuitable for bound to b -microglobulin and a nonapeptide (21). The function transfer or cryopreservation at the day of transfer (days 3 or 5 of pre- 2 implantation development). All research embryos were derived from nor- of HLA-G1 is broad; it inhibits: 1) the proliferation of T cells mally fertilized oocytes (2PN) with a normal morphology and delayed and peripheral blood NK cells (25, 26); 2) the maturation and (maximum 1 d) developmental timing. function of dendritic cells (27, 28); 3) the cytolytic function of uterine and peripheral blood NK cells (29, 30); 4) the Ag-specific Standard and real-time RT-PCR cytolytic function of CTL (31); and 5) the alloproliferative re- Total RNA was extracted from undifferentiated hESC using the RNeasy sponse of CD4+ T cells (25, 32). The soluble counterpart HLA- Mini kit (Qiagen) and a DNAse treatment (RNase-Free DNase Set; Qiagen) G5 or soluble HLA-G1, which is generated by proteasomal cleav- was performed in all the samples. First-strand cDNA Synthesis Kit (GE age from the cell membrane, has similar functions (31). HLA-G Healthcare) with the NotI-d(T) 18 primer was used to reverse-transcribe the isolated RNA. exerts these broad functions through direct binding with its re- HLA-G transcripts in hESC were quantified by real-time RT-PCR (ABI ceptors present on B, T, NK, and APCs. Three main HLA-G re- Prism 7000 SDS; Applied Biosystems) and compared with those of the ceptors have been described: ILT2/LILRB1, ILT4/LILRB2, and positive control JEG-3 by the comparative cycle threshold method, exactly

KIR2DL4 (21), among which ILT2 and ILT4 are clearly inhibitory as described by Moreau et al. (44). For real-time RT-PCR, the primers used Downloaded from amplified mRNA coding for the various HLA-G isoforms indistinctively. receptors. ILT2 is expressed on B, some NK and T cells, and all To gain insight into which HLA-G isoform mRNAs were present, HLA- monocytes/dendritic cells, whereas ILT4 is mainly present on cells G–specific PCR amplifications were performed exactly as in Moreau et al. of the myeloid lineage, such as monocytes/dendritic cells (33, 34). (41) using pan–HLA-G primers (G.257-F/G.1004-R) and HLA-G5/6– In contrast, KIR2DL4, an HLA-G–specific receptor that is mainly specific primers (G.526-F/G.i4b-R), allowing the detection of all alterna- restricted to CD56bright NK cells, has inhibitory as well as acti- tively spliced HLA-G mRNAs and HLA-G5 and -G6 mRNA, respectively. The discrimination of the HLA-G products was conducted by ethidium vating functions (35–38). bromide labeling after electrophoresis. http://www.jimmunol.org/ To gain insight in the tolerogenic nature and developmental origin of hESC, we have analyzed the expression of HLA-G in Flow cytometry human oocytes, embryos, and hESC. We showed that HLA-G is hESC were washed with PBS and harvested as a single-cell suspension present in hESC, human oocytes, and preimplantation embryos using Cell Dissociation Solution (Sigma-Aldrich). The cells were incubated throughout different developmental stages with a downregulation for 15 min at room temperature with the HLA-G mAbs MEM-G/9 (mouse in ICM cells during blastocyst growth. The ILT2 binding assay IgG1; sc-51678, Santa Cruz Biotechnology; 2 mg/ml) and G233 (mouse IgG2a; 11-494-C100, Exbio; 1 mg/ml), a mouse TP25.99 mAb (IgG1; 1 mg/ gives strong evidence that HLA-G molecules on hESC have the ml), and a mouse anti–stage-specific embryonic Ag (SSEA)-4 Ab (IgG; capacity to be functionally active. MAB4304, Millipore; 10 mg/ml). After washing, cells were subsequently by guest on September 26, 2021 stained with the secondary Abs Alexa Fluor 488-conjugated F(ab9)2 frag- ments of goat anti-mouse IgG (A11017; Molecular Probes), FITC-conju- Materials and Methods gated F(ab9)2 fragments of goat anti-mouse IgG1, and PE-conjugated hESCs F(ab9)2 fragments of goat anti-mouse IgG2a (1072-02 and 1082-09; South- ern Biotechnology Associates) for 15 min at room temperature. Isotype con- Three fully characterized VUB hESC lines were used (39) (http://www. trols mouse IgG (Caltag Laboratories), IgG1 (BD Biosciences), and IgG2a hESCreg.com): two of them (VUB01 and VUB02) were derived from (Southern Biotechnology Associates) were performed at equal concentra- surplus in vitro fertilization (IVF) embryos presumed to be genetically tion as the primary Abs. Fluorescence was detected on propidium iodide- normal, whereas the third line (VUB04_CF) was derived from an IVF negative cells by using the Coulter Cytomics FC 500 (Beckman Coulter). shown by preimplantation genetic diagnosis to be heterozygous for the pF508del mutation in the CFTR (cystic fibrosis) gene. Briefly, the cells Indirect immunocytochemistry were cultured in 10% CO2 at 37˚C in hESC medium (KODMEM [Invi- trogen, Auckland, NZ] containing 20% KO-SR [KNOCKOUT SR, Serum hESC colonies were plated in a four-well Multidish Nunclon (Nunc) Replacement for ES cells; Invitrogen], 2 mM glutamine [Invitrogen], 1% previously coated with inactivated MEF. Oocytes and human embryos were nonessential amino acids [Invitrogen], 1 mM b-ME [Sigma-Aldrich, St. individually stained in 50-ml droplets in a 96-well plate (Cellstar; Greiner Louis, MO], 4 ng/ml basic fibroblast growth factor [Invitrogen], and 1% Bio One). Fixation was performed with 3.7% formaldehyde (Merck; VWR penicillin/streptomycin [Invitrogen]) on mytomycin-inactivated CF1 mouse International) for 10 min at room temperature. The samples were sub- embryonic fibroblasts (MEF) and were passaged by mechanical dissocia- sequently washed and permeabilized with 0.1% Triton X-100 (Sigma- tion each 5 to 6 d. HESCs were used at early and late passages for all three Aldrich) for 20 min at room temperature. Samples were incubated over- lines, respectively. night at 4˚C with a mouse IgG1 anti–HLA-G mAb MEM-G/9 (10 mg/ml, Exbio; or 2 mg/ml, Santa Cruz Biotechnology), a mouse IgG1 anti-KRT18 Cell lines mAb (1 mg/ml; ab15830, Abcam), or a rabbit polyclonal IgG anti-NANOG Ab (3 mg/ml; ab21624, Abcam). Control reactions for nonspecific binding The human HLA-G–expressing choriocarcinoma cell line, JEG-3 (Amer- were included in each experiment and carried out by replacing the mouse ican Type Culture Collection), was used as a positive control. JEG-3 cells Abs with a mouse IgG1 mAb (BD Biosciences) and the rabbit Ab with were cultured in DMEM (Sigma-Aldrich) supplemented with 10% FCS, 2 rabbit serum (R9133; Sigma-Aldrich) at equal concentration as the primary mM L-glutamine, 0.1 mM nonessential amino acids, and 1 mM sodium Abs. Alexa Fluor 488- and 633-conjugated goat anti-mouse F(ab9) frag- pyruvate. The M8 melanoma cell line transfected with a pcDNA3.1 hy- 2 ments (A11017 and A21053; Molecular Probes) and Alexa Fluor 488- gromycin vector was used as a negative control, whereas the HLA-G1– conjugated goat anti-rabbit F(ab9) fragments (A11070; Molecular Probes) transfected line was used as a positive control. Cells were grown in RPMI 2 were used as secondary Abs at a concentration of 10 mg/ml for 2 h at 4˚C 1640 medium (Sigma-Aldrich) supplemented with 10% FCS, 200 mM L- in the dark. All primary and secondary Ab solutions were prepared in PBS m glutamine, 10 mg/ml gentamicin, 250 g/ml fungizone, and 50 mg/ml supplemented with 2% BSA (Sigma-Aldrich). Extensive washing with hygromycin B. PBS supplemented with 2% BSA was performed between all steps. After Oocytes and human preimplantation embryos staining, oocytes and embryos were mounted between two glass coverslips (24 3 50 mm) in SlowFade Gold antifade reagent (Molecular Probes). To Human preimplantation embryos and oocytes were obtained for research at prevent squeezing, round glass coverslips (Ø, 10 mm) were put between the Brussels Center for Reproductive Medicine with approval of the In- the coverslips. Wells containing hESC were covered with SlowFade Gold stitutional Ethical Committee, the Committee for Scientific Research on antifade reagent (Molecular Probes). Confocal scanning microscopy with The Journal of Immunology 2665 an Ar-HeNe laser (488/633) (IX71 Fluoview 300; Olympus) was per- formed to record the fluorescent images.

ELISA

HLA-G molecules from hESC were solubilized by 1% octyl-b-D-gluco- pyranoside (Sigma-Aldrich), 150 mM NaCl, 10 mM Tris (pH 7.4), 2 mM EDTA, 0.2 mM PMSF, and protease inhibitors (Sigma-Aldrich). For the determination of solubilized HLA-G molecules, the HLA-G–specific mAb MEM-G/9 was used as specific capture. Bound molecules were detected by a polyclonal antiserum rabbit anti-human b2-microglobulin (DakoCyto- mation) followed by Envision goat anti-rabbit HRP (DakoCytomation). Lysis buffer was used as negative control, purified HLA-G5 protein served as standard reagent, and 3,39,5,59-tetramethybenzidine as substrate solu- tion. After stopping the enzyme reaction with 1 M H2SO4, the OD was measured at 450 nm (Biotek Instruments, Winooski, VT). For the calcu- lation of the ELISA detection limits, a standard curve starting from a concentration of 5 ng/ml was performed in geometrical dilution steps. The results obtained were subjected to the software DINTEST (Institute for Forensic and Traffic Medicine, University Hospital, Heidelberg, Germany). According to this proceeding, the detection limit of HLA-G ELISA was 0.41 ng/ml. Downloaded from ILT2 receptor binding on hESC lysate For measurement of ILT2 receptor binding to solubilized HLA-G and classical HLA class I molecules derived from hESC, Luminex-x-MAP- technology and instruments were used (Luminex). Microspheres (Lumi- nex) with color code 36 and 77 were covalently coupled with the G233 mAb specific for HLA-G and the TP25.99 mAb recognizing all HLA class I molecules except HLA-G, respectively (45). Binding of the Abs was http://www.jimmunol.org/ performed as recently described (46). G233 and TP25.99 coupled micro- spheres were simultaneously incubated with hESC lysate. The bound HLA class I molecules were exposed to recombinant human ILT2 receptor protein fused to the Fc region of human IgG1 (R&D Systems). Thereafter, the bound ILT2 receptor was detected by an anti-human ILT2 mAb (BD FIGURE 1. HLA-G mRNA expression in hESC. A, Real-time quanti- Biosciences), PE conjugated. Measurement of the microspheres was car- tative RT-PCR analysis showing relative quantities of HLA-G transcripts in ried out by the Luminex 100 IS System (Luminex). In total, 100 micro- early and late passage hESC compared with those of JEG-3 (assigned as spheres of each color were analyzed in each sample. The results were value 1). B, RT-PCR products obtained by amplification with the pan– presented in geometrical fluorescence intensity. For the determination of HLA-G primer (G.257-F/G.1004-R) (top panel), and HLA-G5/6-specific

HLA-G molecules recognized by the ILT2 receptor, HLA-G5 was used in primers that reveal transcripts for the main soluble HLA-G isoforms by guest on September 26, 2021 a concentration ranging from 10–0.625 ng/ml. Lysis buffer was used as (bottom panel). a negative control.

revealed a clear membrane staining, although expression in the Results cytoplasm was difficult to observe because of the high nuclear/ HLA-G mRNA expression in hESC cytoplasmic ratio characteristic and colony growth of hESC. To analyze whether HLA-G mRNA transcripts are present in Therefore, cytospins were made and stained with the 5A6G7 mAb hESC, real-time RT-PCR was performed on three VUB hESC that specifically recognizes the soluble isoforms HLA-G5 and -G6. lines (VUB01, VUB02, and VUB04_CF). As shown in Fig. 1A, No staining was observed in VUB01 as well as in VUB02 and expression was found in early aswellaslatepassagesofall VUB04_CF. HLA-G expression was confirmed with the 4H84 three lines. The quantities of HLA-G transcripts represent 1 to 2% mAb that recognizes all isoforms (Supplemental Fig. 1). HLA-G1 compared with those of JEG-3 cells, which are known to express membrane expression with the MEM-G/9 mAb was reproducibly a high level of HLA-G mRNA (44). detected in different passages, early and late. Coexpression of To visualize the alternative mRNA isoforms, the same cDNA NANOG in the nuclei of the cells expressing HLA-G confirmed from the same samples were also analyzed by qualitative RT- the undifferentiated status of the cells (Fig. 2A). On the contrary, PCR. The HLA-G isoform expression patterns were different in the differentiated cells that appear spontaneously at the periphery the three lines. VUB01 only expressed HLA-G1, whereas HLA-G1, of the colony after 6 d of culture and identified by lack of NANOG -G5, and -G3/7 were present in VUB02. In contrast, VUB04_CF and morphological changes have shown a downregulation of HLA- expressed all the isoforms (Fig. 1B). G expression (Fig. 2A,VUB02diff). Within one cell line, no differences in transcripts were found JEG-3 cells were used as a positive control for the Ab. MEM-G/9 between early and late passages (data not shown). did not show any signal on M8-pcDNA cells nor did the IgG1 isotype control on hESC, JEG-3, and M8-pcDNA cells (Fig. 2B). HLA-G protein expression in hESC Immunocytochemistry results were confirmed by flow cytome- To determine whether HLA-G mRNA transcripts are translated try in two lines (VUB01 and VUB04_CF). We have shown based on into proteins, different techniques were performed. Initially, im- the SSEA-4 staining that almost all cells were undifferentiated munocytochemistry was used to investigate the cellular localization (Fig. 3). of HLA-G proteins. Because no isoform-specific Abs exist, we used ELISA analysis of ∼300,000 solubilized VUB01 cells showed the MEM-G/9 mAb that specifically recognizes HLA-G1 and a concentration of 1.3 ng/ml b2-microglobulin–associated HLA-G its soluble counterpart -G5, which have been studied the most. molecules. Importantly, solubilized HLA-G molecules from VUB01 HLA-G protein expression in undifferentiated VUB01, VUB02, cells were exclusively recognized by the ILT2 receptor, whereas and VUB04_CF was investigated by confocal microscopy and other solubilized HLA class I molecules did not bind to ILT2. The 2666 HLA-G IN UNDIFFERENTIATED hESC AND EMBRYOS Downloaded from

FIGURE 2. HLA-G protein expression in hESC. A, Coexpression of HLA-G (red) and NANOG (green) in undifferentiated hESC http://www.jimmunol.org/ colonies and during spontaneous differenti- ation (VUB02 diff and arrows). B, Positive and negative controls for the HLA-G Ab. Each immunofluorescent image is accom- panied by the differential interference con- trast (DIC) image. Scale bars, 50 mm. by guest on September 26, 2021

same results were obtained for VUB04_CF. Importantly, no HLA-G protein expression in embryos ILT2-specific signal was obtained for the MEF feeder cells. As To investigate whether the HLA-G surface-Ag phenotype of hESC shown in Fig. 4, the strength of the ILT2 binding signal corre- corresponded to that of the ICM cells of human blastocysts, HLA-G sponded approximately to the amount of solubilized HLA-G de- protein expression was analyzed in blastocysts of various stages. termined by the ELISA. This indicates that HLA-G molecules In all five early blastocysts, in which trophectoderm (TE) and ICM expressed on VUB01 and VUB04_CF have the capacity to in- are morphologically not yet clearly defined, HLA-G protein was teract with their cognate receptor ILT2 and should therefore be observed on the surface and in the cytoplasm of all cells. Of the functionally active. seven full blastocysts tested, six showed a clear cytoplasmic and The Journal of Immunology 2667

FIGURE 3. Flow cytometric analysis of cell-surface marker expression in VUB01 and VUB04_CF. HLA-G expression was assayed by use of two Abs: MEM-G/9, recognizing HLA-G1, and G233, recognizing all HLA-G FIGURE 4. ILT2 receptor binding test. The dotted bars correspond to isoforms. Expression of HLA class I molecules except HLA-G, was the ILT2 signal obtained by beads coupled with Ab G233, recognizing assayed by Ab TP25.99. The undifferentiated status of the cells was ex- specifically HLA-G molecules; the white bars correspond to the ILT2 amined by the SSEA-4 Ab. The black curves represent the isotype con- signal obtained by beads coupled with Ab TP25.99, recognizing all HLA trols. class I molecules except HLA-G. Mean values of the signals 6 SEM are given for the HLA-G5 standard curve and lysis buffer. membrane expression in the TE as well as in the ICM. Of the 17 Downloaded from expanded blastocysts tested, 13 displayed a clear staining in the TE out of three four-cell stage embryos, and one out of two six-cell and ICM, three showed expression in the TE and only in the outer stage embryos. cells of the ICM, and 1 was completely negative. From this stage A cytoplasmic and membrane staining was observed in four out on, HLA-G expression was more restricted to the membrane, but of six eight-cell stage embryos. In compacting and compacted cytoplasmic staining was still observed in some of them. embryos, a brighter staining was observed. The three compacting

Out of three hatching blastocysts, two expressed HLA-G in the embryos showed a cytoplasmic staining in all of the cells, whereas http://www.jimmunol.org/ TE cells as well as in all cells of the ICM, whereas in the third in the two compacted embryos, the cytoplasm as well as the blastocyst, only the TE cells and the outer cells of the ICM were membrane stained positive in all of the cells. No expression was positive. The same result was also observed in three hatched detected in the nuclei at any stage (Fig. 7). Control reactions for blastocysts (Fig. 5). nonspecific staining gave no fluorescent signals. To demonstrate that these HLA-G–positive cells were ICM cells, coexpression of NANOG and HLA-G was examined. NANOG Discussion proteins were detected in some nuclei of the HLA-G–positive Due to their pluripotent capacity, hESC are proposed as an un- ICM cells of expanded blastocysts (Fig. 6). limited source of cells for transplantation and regenerative medi- KRT18, a -determining gene that is enriched in TE cine. Immunoprivilege has been ascribed to hESC, although con- by guest on September 26, 2021 samples of human blastocysts (47, 48), confirmed the TE locali- troversial and with unknown mechanism. In this report, we clearly zation (data not shown). show the presence of HLA-G in undifferentiated hESCs and in the Further, we confirmed the initial reports of HLA-G Ag ex- corresponding pluripotent ICM cells in the human blastocyst. pression patterns in oocytes, cleavage stage, and compacted em- Real-time RT-PCR demonstrated HLA-G mRNA in three dif- bryos. Of the seven oocytes used, three were GV, two MI, and two ferent hESC lines, in agreement with the DNA microarray analysis MII. Low levels of HLA-G could be detected in two GVs, one MI, reported by Grinnemo et al. (18). Levels are low relative to those and one MII. In the GV stage, expression of HLA-G was more found in the human choriocarcinoma JEG-3 cell line. We describe pronounced in the cytoplasm, whereas in the MI and II stage, for the first time, to our knowledge, the HLA-G isoform expres- expression was more observed on the membrane. The two tested sion pattern in hESCs, which differs among different lines, al- zygotes showed a membrane and weak cytoplasmic staining. In though HLA-G1 mRNA was found in all three lines. No differ- the 14 cleavage stage embryos, a weak cytoplasmic staining was ences were observed between early and late passage within one observed in all blastomeres of three two-cell stage embryos, two line, indicating that the expression of HLA-G transcripts in hESC

FIGURE 5. HLA-G expression during blastocyst growth. Confocal microscopy images showing the expression of HLA-G (green) in TE and all (A–C)or only outer (D, E, see arrows) ICM cells. F represents the isotype control. Each image is accompanied by his differential interference contrast image and represents a section throughout the examined material. Scale bars, 50 mm. 2668 HLA-G IN UNDIFFERENTIATED hESC AND EMBRYOS

were not further characterized, nor were undifferentiated hESC examined for HLA-G expression before BMP-4 culture (50, 51). Finally, Drukker et al. (19) also reported that the hESC lines H9 and H13 express nonclassical HLA-E molecules. HLA-G plays a key role in both adaptive and innate immunity (52). HLA-G–expressing cells seem to have immune-privileged properties in transplantation, autoimmunity, and tumor progres- sion (21), but how they circumvent the immune system is largely unknown. hESC express MHC class I molecules at a very low level, which increases moderately after differentiation but reaches the level of somatic cells in presence of IFN-g (19). They have no Ag presentation potential because they lack expression of MHC FIGURE 6. ICM localization. Coexpression of HLA-G (red) and class II and costimulatory molecules on their membrane (17–19). NANOG (green) in an expanded blastocyst (bottom panels). The upper panels represent the isotype control. Each image is accompanied by its Therefore, they should not be able to induce a robust immune differential interference contrast (DIC) image and represents a section response by direct Ag presentation. This was confirmed by Li throughout the examined material. Scale bars, 50 mm. et al. (20), who showed that hESC (undifferentiated and differ- entiated) fail to stimulate proliferation of alloreactive T cells, and transplantation of these cells into human PBMC-reconstituted is stable during long-term culture. Protein expression was found by mice gave no rejection (17). However, when indirectly presented Downloaded from immunocytochemistry in three VUB hESC lines tested and con- by professional APCs, such as dendritic cells syngeneic to the firmed by flow cytometry in two lines (VUB01 and VUB04_CF). responder T cells, hESC were shown to induce CD4+ T cell pro- Remarkably, no HLA-G5/-G6 protein expression was found in liferation as well as human fibroblasts and were acutely rejected VUB02 and VUB04_CF, although they were present at the RNA in immunologically competent mice (18). We showed expression level. This probably implies that there is a regulation at posttran- of HLA-G by a specific ELISA, detecting exclusively b2-micro- scriptional level, as reviewed by Donadi et al. (49), that needs globulin–associated HLA-G molecules. The capacity of HLA-G http://www.jimmunol.org/ further investigation. to interact with its cognate receptor ILT2 recognizing especially We report for the first time, to our knowledge, HLA-G protein b2-microglobulin–associated HLA-G molecules was proven by an expression in undifferentiated hESC cultured under conventional assay that is based on the Luminex-x-MAP-technology. This offers conditions with MEFs (1). This result is in contradiction with the the possibility to analyze simultaneously solubilized HLA-G mol- data published by Drukker et al. (19), who reported no HLA-G ecules and other HLA class I molecules derived from hESC in expression in H9 and H13 hESC lines as detected by flow one sample by the use of different color-coded microspheres cap- cytometry. Discrepancies among laboratories may be explained by turing the respective molecules. The results clearly showed that differences in hESC isolation and culture methods, enzymatic HLA-G molecules derived from VUB01 and VUB04_CF were digestion for the obtainment of single cells, as well as the HLA-G recognized and other HLA class I molecules were not recognized by guest on September 26, 2021 Abs used for detection. Regarding the expression of HLA-G in by the ILT2 receptor. This observation is in line with classical differentiated hESC, we have shown by immunocytochemistry HLA class I exhibiting a low affinity to ILT2 (53–55). In con- that spontaneously differentiated cells at the periphery of the colo- clusion, the ILT2 binding assay gives strong evidence that HLA-G nies after 6 d in culture do not express HLA-G. Drukker et al. (19), molecules on hESC have the capacity to be functionally active. using flow cytometry, have shown that differentiated cells from However, more functional assays are required to provide evidence embryoid bodies and teratomas also have no HLA-G expression. that this molecule is one of the mechanisms through which hESC Recently, HLA-G expression was described in hESC colonies circumvent the immune system. driven into trophoblast differentiation by 5 d of culture on Ma- Immunosuppressive properties have been assigned to hESC- trigel in the presence of BMP-4 (50). By using the 4H84 Ab, derived mesenchymal progenitor cells in which HLA-G was which recognizes all HLA-G isoforms, HLA-G was detected in clearly shown to inhibit NK cell-mediated cytotoxicity, as well as the peripheral cells adapting trophoblast morphology. The central allogeneic T cell proliferation. Bone marrow mesenchymal stromal core cells remained morphologically undifferentiated and expres- cells do not express HLA-G on their membrane and are less resistant sed POU5F1 but no HLA-G, except for some regions where HLA- to NK mediated lysis (56). However, Selmani et al. (57) showed that G staining penetrated the core region. Unfortunately, these cells mesenchymal stem cells do secrete the soluble isoform HLA-G5,

FIGURE 7. HLA-G expression throughout human preimplantation development. Confocal microscopy images showing the expression of HLA-G in human oocytes, cleavage stage, and compacted embryos. Each image is accompanied by its differential interference contrast image and represents a section throughout the examined material. c, cell stage. The Journal of Immunology 2669 which inhibits NK cell-mediated cytotoxicity and IFN-g secretion lineage as well as in endothelial cells of developing blood vessels and suppresses allogeneic T cell proliferation and expansion of (71). All of these structures originate from the hypoblast. When CD4+CD25highFOXP3+ regulatory T cells. comparing hESC with their murine counterpart murine ESC HLA-G expression is also claimed to be associated with embryo (mESC), there are remarkable differences, such as culture require- implantation, the protection of the allogenic fetus from the ma- ments, growth characteristics, expression profile, and developmental ternal immune system, and placentation (23, 58–60). Membrane, potential. In fact, hESC resemble more epiblast-derived mESC shed, or soluble HLA-G comes in contact with the maternal en- (derived from postimplantation embryos), leading to the hypothesis dometrium and its immune cells (uterine CD56bright NK, T and that hESC cells are derived from human epiblast cells (72, 73). B cells), where it binds with its inhibitory receptors KIR2DL4 and Because of major ethical objections, it is impossible to try to ob- ILT2. These interactions prevent the attack of the fetus by the tain human epiblast-derived ESC for comparative studies. How- maternal immune system. In contrast, soluble HLA-G binding to ever, based on our staining results, we suggest that hESC originate the KIR2DL4 receptor also modulates the local cytokine secretion from the early ICM cells expressing NANOG before downregula- and plays also a role in the remodeling of the spiral arteries during tion of HLA-G. The possibility that in hESC HLA-G is downreg- placentation (61). Furthermore, HLA-G was claimed to be a pre- ulated and upregulated again during in vitro derivation and culture dictor for pregnancy and a promising noninvasive tool for embryo cannot be excluded at this moment. selection (58). However, further studies combining single-embryo culture and single-embryo transfer with a highly sensitive HLA-G Acknowledgments immunodetection technique in highly standardized conditions are We thank the Centre for Reproductive Medicine for providing the samples needed to determine the true value of soluble HLA-G measure- and Medical Genetics for putting the confocal microscope at our disposal, Downloaded from ment in embryo culture medium (62). the colleagues from the Human Embryonic Stem Cell Lab, and especially Because hESCs are derived from preimplantation embryos, we the head of the laboratory, Prof. K. Sermon. We also thank W. Renmans for were interested to find out which embryonic cells express HLA-G flow cytometry help, M. Daouya for technical assistance with the cytospins, and from which developmental stage onwards. We confirmed the and B. Guns for assistance with the pictures. presence of HLA-G protein from oocyte to blastocyst as described

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