Report of the Wet Workshop for Quantification of Soluble HLA-G in Essen, 2004

Report of the Wet Workshop for Quantification of Soluble HLA-G in Essen, 2004 Vera Rebmann, Joël LeMaoult, Nathalie Rouas-Freiss, Edgardo D. Carosella, and Hans Grosse-Wilde ABSTRACT: Membrane-anchored and soluble human leukocyte antigen HLA-G (sHLA-G) molecules exert strong inhibiting signals after interaction with their cognate receptors ILT2 (CD85j), ILT4 (CD85d), and KIR2DL4 (CD158d) that are differentially expressed by natural killer cells, T cells, and antigen-presenting cells. These inhibitory functions can become operative in conditions in which such immune cells try to attack viral infected or tumor cells. Recently, clinical studies showed that sHLA-G molecules are also relevant in the prediction of allograft acceptance after heart transplantation, liver-kidney cotransplantation, and the successful implantation and development of embryos after in vitro fertilization. In view of this diagnostic potential, reliable methods for the measurement of sHLA-G molecules in various body fluids are of interest. Thus, the aims of the Wet Workshop for measurement of sHLA-G held in Essen, Germany (at the Institute of Immunology October 18 –20, 2004) were to select and validate HLA-G– specific enzyme-linked immunosorbent assay (ELISA) formats and purified standard HLA-G proteins, which can be easily generated and used as consensual references. To this end, the antibody combinations monoclonal antibody (mAb) MEM-G/9 (capture) ⫹ anti-␤2m (detection) and the mAb 5A6G7 (capture) ⫹ mAb W6/32 (detection) were chosen in an ELISA format for the simultaneous determination of shed HLA-G1 ⫹ soluble HLA-G5 (sHLA-G1 ⫹ ABBREVIATIONS ELISA enzyme-linked immunosorbent assay HLA human leukocyte antigen HRP horseradish peroxidase Ig immunoglobulin

INTRODUCTION Human leukocyte antigen (HLA)-G is a nonclassical class I HLA. Originally, it was thought that in nonpathologic

From the Institute of Immunology, University Hospital of Essen, Essen, Germany (V.R., H.G.-W.) and Service de Recherches en Hémato-Immunologie, CEA-DSV-DRM, Hoˇpital Saint Louis, IUH, Paris, France (J.L., N.R.-F., E.D.C.). Address reprint requests to: Dr. Hans Grosse-Wilde, Institut für Immunologie, Universitätsklinikum Essen, Virchowstr. 171, D-45122 Essen, Germany; Tel: ⫹49-201-723-4200; Fax: ⫹49-201-723-5906; E-mail: [email protected]. Human Immunology 66, 853– 863 (2005) © American Society for Histocompatibility and Immunogenetics, 2005 Published by Elsevier Inc.

HLA-G5) and for the exclusive detection of HLA-G5 molecules, respectively. As standard, protein HLA-G5 molecules were purified from insect SF9 cells coinfected by HLA-G5 ⫹ human ␤2m and characterized for their antigenic determinants. A total of 24 members in 13 teams participated in the 3-day sHLA-G Wet Workshop. All workshop materials, protocols, standard reagents, and samples were provided to each team by the organizers. The Wet-Workshop results clearly demonstrated that (1) the HLA-G5 standard reagent was equally detected by both ELISA formats; (2) sHLA-G1 ⫹ G5 and HLA-G5 molecules, respectively, were specifically detected by the two ELISA formats; and (3) both ELISA formats measure reproducibly the amounts of sHLA-G. The comparison of the two ELISA results obtained evidenced that in healthy donors sHLA-G1 molecules can exist in body fluids besides HLA-G5. Moreover, a novel soluble HLA-G structure can be predicted that is recognized by the mAb 5A6G7 ⫹ mAb W6/32 antibody combination, but not by the one of mAb MEM-G/9 ⫹ anti-␤2m. Human Immunology 66, 853– 863 (2005). © American Society for Histocompatibility and Immunogenetics, 2005. Published by Elsevier Inc. KEYWORDS: International workshop; sHLA-G specific ELISA; HLA-G5 standard

mAb PBS rHLA

monoclonal antibody phosphate-buffered saline recombinant HLA

situations, HLA-G expression was restricted to extravillous cytotrophoblast, thymic epithelial cells, and cornea [1–3]. Recent studies, however, showed that HLA-G is expressed (1) by allografts [4 – 6] and infiltrating mononuclear cells within the transplanted tissues [6], (2) during inflammatory diseases and by lesion-infiltrating The Wet Workshop for Quantification of soluble HLA-G in Essen, 2004 was partly supported by Serotec Ltd (UK, Oxford). Received April 13, 2005; accepted May 4, 2005. 0198-8859/05/$–see front matter doi:10.1016/j.humimm.2005.05.003

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FIGURE 1 Biochemical characterization of human leukocyte antigen (HLA)-G5 standard reagent. (A) Silver stain of HLA-G5 (lanes 1, 2) purified from HLA-G5 and ␤2-microglobulin transfected SF9 cell lysates revealed typical HLA-G5–specific double bands with molecular weights (MW) of 35 and 34 kDa and a protein having a similar MW as albumin (66 kDa). (B) Western blot analysis showed purified HLA-G5 after (⫹) and before (-) deglycosylation using the HLA-G specific monoclonal antibody (mAb) MEM/G1 as detection antibody. (C–E) Antigenic determinants of HLA-G5 were analyzed after immunoprecipitation of the standard reagent at concentrations of 100, 50, and 25 ng/ml (lanes 1, 2, and 3) using the HLA class I–specific mAb W6/32 and the sHLA-G1⫹G5–specific mAbs MEM-G/9, and MEM-G/11 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis using the polyclonal antibody RaHC specific for HLA class I heavy chain (C), the mAb MEM-G/1 specific for HLA-G (D), and the polyclonal rabbit antibody RaHLA-G/I-4 specific for intron-4 sequence of HLA-G (E). Arrows on the right-hand side indicate immunoglobulin G heavy chain and HLA-G5 specific bands. On the left-hand side the sizes of the molecular weight markers (MWM) are shown.

HLA-G–positive antigen-presenting cells and T cells [7–9], (3) by tumor tissues (reviewed in [10 –13] and tumor infiltrating antigen-presenting cells and T cells [14, 15], and (4) on monocytes and T cells from HIV patients [16]. Whereas the gene structure of HLA-G is highly homologous to that of the other HLA class I genes [1], analysis of its transcription has led to the identification of specific alternative mRNA splicing products. The HLA-G primary transcript has been shown to generate seven alternative mRNAs able to encode four membranebound (HLA-G1, -G2, -G3, and -G4) and three secreted (HLA-G5, -G6, and -G7) protein isoforms (Figure 1) [17–20]. HLA-G1, which derives from the translation of the complete HLA-G transcript, has a structure similar to that of classical HLA class I molecules: a heavy chain

constituted of three globular extracellular alpha1, alpha2, alpha3 domains noncovalently associated with ␤2microglobulin and a nonapeptide. Although it is expressed as a membrane-bound molecule, HLA-G1 can be shed or proteolytically cleaved from the cell surface, as already described for the classical HLA class I molecules [21]. The other membrane-bound HLA-G isoforms lack one or two globular domains, but all contain the alpha1 domain. HLA-G truncated isoforms are thought not to associate with ␤2-microglobulin or peptides. Secreted isoforms of HLA-G i.e., HLA-G5, -G6, -G7 are the soluble counterparts of HLA-G1, -G2, and -G3, respectively. Membrane-bound and secreted pairs share the same extracellular structure and they differ only at their C-terminus: whereas the membrane-bound isoforms have a transmembrane region (encoded by exon 5) and an

sHLA-G Workshop Report

intracytoplasmic tail (encoded by exons 6 – 8), these are replaced in secreted isoforms by a short hydrophilic tail that is encoded by the 5= sequences of intron 4 (HLA-G5 and HLA-G6) or intron 2 (HLA-G7) [17–20]. The presence of intron-encoded amino acids allows for the discrimination between shed or proteolytic cleaved HLA-G molecules and secreted HLA-G isoforms. Functional assays demonstrated that both soluble and membrane-bound HLA-G isoforms inhibit allogeneic proliferation of T cells [22–24], natural killer cell cytotoxicity [25–29], and antigen-specific T-cell cytotoxicity [30, 31]. HLA-G exerts its inhibitory functions via high-affinity interaction with three inhibitory receptors: ILT2 (LILRB1/ CD85j), ILT4 (LILRB2/CD85d), and KIR2DL4 (CD158d) [32–35]. The physiologic importance of HLA-G is clear in the context of pregnancy: HLA-G is expressed as early as the blastocyst stage and is crucial for fetal-maternal tolerance [25, 36 –39]. In pathologic conditions, HLA-G expression is associated with acceptation of allogeneic transplants [4, 5, 40] and immune escape of tumors [13, 41]. The clinical potential of HLA-G is now beginning to unveil. Indeed, (1) preeclampsia is associated with decreased sHLA-G protein levels in both maternal serum and fetal placental tissues [42] and (2) studies performed in embryos after in vitro fertilization demonstrated that implantation and clinical pregnancy outcome are correlated with soluble HLA-G production by the growing embryos [43– 46]. Moreover, (3) malignancy of ovarian cancers is associated with high soluble HLA-G titers in tumor ascites [47], (4) the role of HLA-G in immune escape has been emphasized by its involvement in the resistance to IFN-␣ therapies observed in some melanoma patients [48, 49], and (5) HLA-G expression is significantly associated with an unfavorable outcome and immunodeficiency in chronic lymphocytic leukemia and is a better independent prognostic factor than the currently used ZAP-70 or CD38 status [50]. Finally (6) HLA-G in situ expression by heart and liver allografts and high serum levels of HLA-G5 and sHLA-G1 were significantly associated with no acute and chronic graft rejection [4, 5, 40]. Thus, HLA-G has become a relevant marker in various pathologies, and a methodology to precisely measure soluble HLA-G levels is increasingly in demand. The titration of soluble HLA-G levels in body fluids has been achieved by several laboratories using nine different enzyme-linked immunosorbent assay (ELISA) formats that were set up with various capture and detection antibody pairs (Table 1). These ELISA formats detect either sHLA-G1 ⫹ G5, HLA-G5 specifically, or multiple HLA-G isoforms including truncated ones. Of note, capture and detection antibody pairs are routinely used according to locally adapted ELISA procedures.

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TABLE 1 ELISA formats published for the quantification of sHLA-G molecules Capture mAb W6/32 MEM-G/9 MEM-G/9 87G 16G1 16G1/16A1 4H84 G233 5A6G7 a

Detection mAb

Specificity

Anti-␤2m sHLA-G1 ⫹ G5a Anti-␤2m sHLA-G1 ⫹ G5 W6/32 sHLA-G1 ⫹ G5 Anti-␤2m sHLA-G1 ⫹ G5 Anti-␤2m sHLA-G1 ⫹ G5 Rabbit anti-Intron 4 HLA-G5 3C/G4 56B W6/32 HLA-G5

Reference 51 41,52 43 51,53 53 54 42 55 57

After depletion of sHLA-A, -B, -C, and -E by mAb TP25.99.

Furthermore, when setting up a soluble HLA-G ELISA test, the thorny issue of the standard protein arises. Here again, purified soluble HLA-G proteins to be used as standard in ELISA testing range from biologic sample– derived protein [42] to crude or purified supernatants or lysates of various cells transfected with HLA-G1 or HLA-G5 [43, 51, 52, 54 –56], none of these standard proteins claiming to be perfect. The bottom line is that even though these methodologies are all valid and yield semiquantitative results that accurately reflect soluble HLA-G concentration differences, the diversity of methodologies prevents result comparisons between teams. The aims of the 2nd International Workshop on HLA-G (Paris, France, 2003) and of the Wet-Workshop for quantification of soluble HLA-G (Essen, Germany, 2004) were to select and validate HLA-G ELISA formats and purified HLA-G proteins that can be easily set up and used as consensual references. We chose, for the soluble HLA-G ELISA workshop (Essen, Germany, 2004), to validate two ELISA formats, one for the titration of sHLA-G1 ⫹ G5 and one for the specific titration of HLA-G5. For the titration of sHLA-G1 ⫹ G5, we selected the MEM-G/9 ⫹ anti-␤2m format because (1) it is the one that has been used the most, (2) MEM-G/9 is widely used and considered a reliable monoclonal antibody, and (3) both MEM-G/9 and anti-␤2-microglobulin (␤2m) are commercially available. For the titration of HLA-G5 specifically, we selected the 5A6G7 ⫹ W6/32 format because (1) monoclonal antibody (mAb) 5A6G7 has been validated during the 2nd International Workshop on HLA-G (Paris, France, 2003) and is considered a very specific antibody toward the intron-4 sequence of HLA-G5 and (2) both mAb W6/32 and mAb 5A6G7 are commercially available. As standard protein, we selected HLA-G5 purified from HLA-G5 ⫹ human ␤2m cotransfected insect SF9 cells, because (1) HLA-G5 should be recognized indiscriminately by both ELISA formats and (2) the protein produced by HLA-G5 ⫹ human ␤2m coinfected insect SF9 cells is correctly folded (see Results)

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in an environment devoid of any other human molecules in general, and HLA molecules in particular. The design of the 3-day sHLA-G Wet Workshop was as follows: 13 teams with a total of 24 participants were provided with all materials and protocols necessary to perform both ELISA formats and were asked to measure soluble HLA-G concentrations in the provided samples using the standard protein for calibration. The samples were chosen to address the issues of (1) the quality and reliability of the standard protein in both assays (i.e., the quality of the standard curve); (2) the specificity of both assays for sHLA-G1 ⫹ G5 and HLA-G5, respectively; and (3) the reproducibility of the results obtained among the 13 participants teams. We found that these two soluble HLA-G ELISA formats are specific, reliable, and allow sHLA-G1 ⫹ G5 or HLA-G5 titration up to 80 ng/ml in cell culture supernatants and biologic specimens such as plasma/serum, ascites, and amniotic fluid. We propose that these two ELISA formats, as well as the standard protein used during the workshop (kept at the Service de Recherches en Hémato-Immunologie, Commissariat à l’Energie Atomique, Paris, France, and at the Institute of Immunology, University Hospital of Essen, Germany) be used as references for other soluble HLA-G titration methodologies. MATERIALS AND METHODS Workshop Participants P. Contini ([email protected]), F. Puppo ([email protected]), DIMI University of Genoa, Italy; R. Rizzo ([email protected]), Dip. Exp. and Diagnostic Medicine, Section of Medical Genetics, University of Ferrara, Italy; T. Barona ([email protected]), J. Machado Caetano ([email protected]), Faculdade de Ciencias Medicas, Department Immunologia, Lisboa, Portugal; L. Amiot ([email protected]), Y. Sebti ([email protected]), Laboratoire d’Hematologie, Immunologie, Rennes, France; J. Marin Sanchez ([email protected]), Servicio de Immunologia, Hospital Universitario Reina Sofia, Cordoba, Spain; C. Brown ([email protected]), Histocompatibility and Immunogenetics Department, National Blood Service, London; B. Kumpel (belinda.kumpel@nbs. nhs.uk), Bristol Institute of Transfusion Sciences, National Blood Service, Bristol, UK; C. Schieferstein ([email protected]), K. Bleymehl (k-bleymehl@ gmx.de), Medizinische Klinik I, Universitätsklinikum Frankfurt, Germany; A. Steinborn ([email protected]), Frauenklinik, Universitätsklinikum Frankfurt, Germany; H. Juch ([email protected]), Histologie und Embryologie, Medizinische Universität Graz, Austria; R. Roussev ([email protected]), Millenova Immunology Labs and CARI Reproductive Insti-

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tute, Chicago, IL; L. Fan ([email protected]), Shanghai Institute of Immunology Shanghai Second Medical University (P.R. China; M. Daouya (MD208758@ dsvidf.cea.fr), J. LeMaoult ([email protected]), N. Rouas-Freiss ([email protected]), E. Carosella ([email protected]), CEA/SRHI, Paris; and M. Dolar, T. Schreiter, V. Rebmann, H. Grosse-Wilde ([email protected]), Institute of Immunology, University Hospital of Essen, Germany. Guest: M. Suchanek ([email protected]), Exbio, Praha, Czech Republic. Antibodies and Reagents The mAb 5A6G7 was generated toward a synthetic polypeptide corresponding to the amino-acid sequence encoded by the intron-4 part retained in the secreted HLA-G5 and HLA-G6 isoforms [55]. mAb MEM-G/9 kindly provided by Serotec Ltd (Oxford, UK) and mAb MEM-G/11 (Exbio, Prague, Czech Republic) recognize exclusively native HLA-G1 and HLA-G5 molecules, whereas mAb MEM-G/1 (Exbio) binds to all denatured HLA-G molecules at the ␣1-domain. All of these mAbs were murine immunoglobulin (Ig)G1 mAbs. The antisera RaHC and the RaHLA-G/I-4 were generated in rabbits toward denatured HLA class I heavy chains and specific peptide motifs of intron-4 of HLA-G, respectively [12, 56]. Purified IgG goat anti-mouse specific for Fc fragment and mouse anti-rabbit IgG were purchased from Dianova (Hamburg, Germany). Biotinylated mAb W6/32 (IgG2a) recognizing HLA class I molecules complexed with ␤2-microglobulin (␤2m) was purchased from Leinco Technologies (St. Louis, MO) and unconjugated mAb W6/32 from Serotec Ltd. Rabbit anti-human ␤2m antiserum and the EnVision horseradish peroxidase (HRP) rabbit enhancing reagent were obtained from Dako (Copenhagen, Denmark) and AMDEX Streptavidin HRP from Amersham (Freiburg, Germany). Samples and Controls As summarized in Table 2, each Wet-Workshop team analyzed nine EDTA plasma samples from healthy donors, one ascites fluid derived from a patient suffering from ovarian cancer [47] and one amniotic fluid [51]. Two EDTA plasma samples (no. 10 and no. 11) were depleted of soluble HLA-G molecules by mAb MEMG/9 coupled to immunomagnetic beads (Dynabeads M280, Dynal, Hamburg, Germany) before serving as negative controls for the ELISA format using mAb MEM-G/9 as capture reagent. Furthermore, culture supernatant derived from HeLa cells stably transfected with HLA-B*2705 fused with exon 5 of the soluble murine MHC class I variant Q10 (rHLA-B27) served as a soluble HLA class I antigen source and as negative control for the HLA-G–specific ELISA formats [58]. Purified recombi-

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TABLE 2 List of Wet Workshop samples Sample no.

Fluid

Remarks

1 2 3 4 5 6 7 8 9 10 11 12 13

EDTA plasma EDTA plasma EDTA plasma EDTA plasma EDTA plasma EDTA plasma EDTA plasma Ascites Amnion EDTA plasma EDTA plasma Culture supernatant Culture supernatant

Healthy donor Healthy donor Healthy donor Healthy donor Healthy donor Healthy donor Healthy donor Ovarian cancer 19th week of gestation sHLA-G1 ⫹ G5 depleted sHLA-G1 ⫹ G5 depleted rHLA-B27 rHLA-G1 (purified)

nant HLA-G1 (rHLA-G1) was kindly provided by Prof. E. Weiss (Institut für Anthropologie und Genetik, LMU München, Germany), to function as positive control for the ELISA using mAb MEM-G/9 and as negative control for the ELISA using mAb 5A6G7, respectively. All samples listed were centrifuged at 1600g, divided into the respective aliquots, and stored at ⫺80°C until use. Production and Purification of HLA-G5 Production of HLA-G5 was performed by cloning HLA-G5 heavy chain and human ␤2m into baculovirus. Briefly, cDNA coding for HLA-G5 and human ␤2m were cloned into pTen21 and pTen12 vectors (Qbiogen Inc., Illkirch, France), respectively, and sequenced. Both vectors were then transfected into SF9 cells (Gibco, Paisley, UK) in the presence of linearized BacTen viral DNA. HLA-G5 and human ␤2m recombinant baculovirus clones were selected by polymerase chain reaction screening and used to co-infect primary stocks of SF9 cells. HLA-G5 molecules were extracted from infected SF9 cell lysates using the protein extraction kit “BugBuster” (Merck Biosciences, Darmstadt, Germany). After centrifugation at 20,000g for 20 min at 4°C, the supernatant fluid obtained was used for further purification steps. Supernatant was diluted with 0.05 M phosphate buffer and loaded onto a NHS-Sepharose column (Amersham Biosciences, Freiburg, Germany) coupled with mAb W6/ 32. Eluted HLA-G5 protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) and silver stain. Besides the HLA-G5-specific protein with the typical molecular weight of 35 kDa, an additional protein with a size of 66 kDa was visualized. Fractions containing HLA-G5 were pooled and a protein concentration of 5 ␮g/ml was determined by BCA protein assay (Pierce, Rockford, IL). After SDS-PAGE and silver stain of pooled fractions (Figure 1A) the densitometrical analysis (software program WinCam 2.2 [Cybertech, Berlin, Germany]) revealed that HLA-G5 mole-

cules represented 80% of total amount of protein, which corresponds to a HLA-G5 concentration of 4 ␮g/ml. Deglycosylation of HLA-G5 (Figure 1B) was performed overnight at 37°C using 15 U/ml N-Glycosidase F (Roche, Mannheim, Germany) in 0.05 M phosphate buffer (pH 7.2). The antigenic specificity of purified HLA-G5 molecules was further proven by immunoprecipitation of pooled fraction using mAb W6/32, MEM-G/9, and MEMG/11, followed by SDS-PAGE and Western blotting using RaHC, RaHLA-G/I-4, and mAb MEM-G/1 (Figure 1C– 1E) as detection antibodies, respectively. MAb MEM-G/9 ⴙ Anti-␤2m ELISA for Determination of sHLA-G1 ⴙ G5 Levels Each well of microtiter plates (Costar Corning, Bodenheim, Germany) was coated at 4°C overnight with 100 ␮l of goat anti-mouse at final concentration of 10.4 ␮g/ml. Unless otherwise stated microtiter plates were washed four times with phosphate buffered saline (PBS) with 0.05% Tween 20 (PBS-T) after each incubation step. The wells were blocked for 1 h at 37°C with 300 ␮l/well of PBS-T with 3% fatty acid free BSA (Calbiochem, La Jolla, CA) and then incubated at 4°C overnight with 100 ␮l/well of mAb MEM-G/9 (6.6 ␮g/ml in PBS). Unbound antibodies were removed and free binding sites were blocked with 3% BSA in PBS-T for 1 h at 37°C. After discarding the blocking solution, 100 ␮l of samples (1:2 diluted in PBS supplemented with 1% mouse serum) and standard reagent were added and incubated for 1.5 h at 37°C. Standard reagent (HLA-G5) was serially diluted from 80 to 5 ng/ml in PBS. Bound sHLA-G1 ⫹ G5 were detected by 100 ␮l/well of rabbit anti-human ␤2m (1:500 in PBS) for 1 h at 37°C room temperature. To enhance detection, 100 ␮l of dextran polymer conjugated with goat anti-rabbit IgG and HRP (EnVision) was added to each well and incubated for 30 min at room temperature. This dextran polymer was diluted 1:15 in PBS supplemented with 1% mouse serum. After six washing steps, 100 ␮l of substrate solution (10 ␮g o-phenylenediamine in 10 ml 0.1 M citrate buffer at pH 5.5 and 8 ␮l of 30% H2O2) were added to each well. The reaction was stopped after 10 min by 50 ␮l/well of 3 M H2SO4 and the OD measured at 490 nm (Biotek Instruments, Winooski, VT). Determination of sHLA-G1 ⫹ G5 concentrations of the samples was done by four-parameter curve fitting. MAb 5A6G7 ⴙ mAb W6/32 ELISA for Determination of HLA-G5 Levels Each well of microtiter plates was coated at 37°C for 2 h with 100 ␮l of mAb 5A6G7 diluted in 0.1 M carbonatebicarbonate buffer (Na2CO3: 0.029 M, NaHCO3: 0.069 M, pH ⫽ 9.5) at a final concentration of 5 ␮g/ml. Microtiter plates were washed five times with PBS-T. The wells were then blocked for 2 h at 37°C with 300

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␮l/well of PBS-T with 2% fatty acid free BSA (Calbiochem), washed five times with PBS-T, and incubated at 4°C overnight with 100 ␮l/well of sample and standard reagent. Standard reagent was serially diluted from 80 to 5 ng/ml. After 5 washes in PBS-T, bound HLA-G5 molecules were detected by 100 ␮l/well of biotinylated anti-pan-HLA Class I mAb W6/32 at a final concentration of 0.4 ␮g/ml for 1 h at 37°C. After five washes in PBS-T, 100 ␮l/well of a 1:8,000 dilution of streptavidin HRP (AMDEX) were added for 1 h at 37°C. After 5 washes in PBS-T, 50 ␮l of the substrate (TMB, 3,3=,5,5= tetramethylbenzidine, Sigma) were added for up to 10 minutes, and the reaction was stopped by 50 ␮l/well of HCl 1N and the OD measured at 450 nm (Biotek Instruments). Determination of HLA-G5 concentrations of the samples was done by four-parameter curve fitting. Statistics Data are presented as mean ⫾ standard error of the mean (SEM). One-way analysis of variance for repeat measurements was used to compare the ELISA results among the 13 workshop teams. Based on this analysis, two teams were found to be significantly different from the other workshop teams and therefore excluded from further analysis data. After testing for Gaussian distribution, Student’s t-test was used to compare ELISA results using mAb MEM-G/9 with those using mAb 5A6G7 as capture reagents. A two-tailed p ⬍ 0.05 was considered significant. RESULTS Characterization of the HLA-G5 Standard Protein One central aim of the Wet Workshop was to establish a standard reagent for the different ELISA formats, which can be recognized by the two capture reagents, mAb MEM-G/9 specific for sHLA-G1 ⫹ G5 and mAb 5A6G7 specific for the intron-4 – derived polypeptide of HLAG5. To this end, SF9 cells were co-infected with HLA-G5 and human ␤2m. The purified protein procured from infected SF9 cells was immunoprecipitated by mAbs MEM-G/9, MEM-G/11, or W6/32 and analyzed by SDS-PAGE and Western blot. A double band of molecular sizes of approximately 35 and 34 kDa was identified by Western blotting using RaHC, RaHLA-G/ I-4 and mAb MEM-G/1, respectively, as detection antibodies (Figure 1C–1E). Differential glycosylation processes in insect cells seem to be responsible for the two different molecular sizes of HLA-G5 molecules, because, after deglycosylation, HLA-G5 with a molecular weight of 34 kDa was preferentially detected (Figure 1B). Thus, the molecular size and the specific staining by the antiserum toward the intron-4 – derived polypeptide of HLA-G5 clearly identified the purified proteins as HLA-

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FIGURE 2 Human leukocyte antigen-G5 standard curve of enzyme-linked immunosorbent assay formats using either monoclonal antibody (mAb) MEM-G/9 ⫹ anti-human ␤2m or mAb 5A6G7 ⫹ mAb W6/32 as antibody combinations. Each point represents the mean ⫾ SEM value obtained from the different workshop teams (n ⫽ 11).

G5. The positive immunoprecipitation results with mAb W6/32, MEM-G/9, and MEM-G/11 recognizing only native HLA-G forms (Figure 1C–1E) implicated that after purification the HLA-G5 molecules were still associated with ␤2m. Thus, these purified HLA-G5 fulfilled all requirements to serve as standard reagent in ELISA formats using as antibody combinations either mAb MEM-G/9 ⫹ anti-human ␤2m or mAb 5A6G7 ⫹ mAb W6/32. Quality of the Calibration Curves The calibration curves of HLA-G5 generated by the Wet-Workshop participants run nearly identical for both ELISA formats in a concentration range from 5 to 80 ng/ml (Figure 2). This indicates that HLA-G5 molecules were captured and detected equally well by the antibody combinations used in the two ELISA formats. Specificity of the ELISA Tests The following control specimens were tested by each Wet-Workshop participant as negative controls: (1) a cell culture supernatant containing rHLA-B27 but no HLA-G molecules (no. 12), (2) two sHLA-G1 ⫹ G5depleted plasma samples (no. 10 and no. 11), and (3) as positive control HLA-G1 molecules (no. 13). As can be seen from Table 3, the rHLA-B27– containing cell culture supernatant (no. 12) and the sHLA-G1 ⫹ G5– depleted plasma sample (no. 10) were found to be negative in both ELISA formats. This indicates that the two

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TABLE 3 Wet Workshop ELISA results

Sample no.

ELISA 1 C: MEM-G/9a D: ␤2ma

ELISA 2 C: 5A6G7 D: W6/32

ELISA 1 vs. ELISA 2 p Valueb

1 2 3 4 5 6 7 8 9 10 11 12 13

59.3 ⫾ 3.5c 35.1 ⫾ 2.5 29.2 ⫾ 2.6 27.7 ⫾ 2.5 32.7 ⫾ 2.2 95.9 ⫾ 5.7 81.9 ⫾ 6.7 3.1 ⫾ 0.6 13.9 ⫾ 1.8 0.1 ⫾ 0.1 0.1 ⫾ 0.1 0.9 ⫾ 0.2 138.0 ⫾ 23.9

59.3 ⫾ 6.7 23.9 ⫾ 2.3 85.8 ⫾ 16.6 14.6 ⫾ 1.8 38.9 ⫾ 2.1 61.5 ⫾ 4.7 53.6 ⫾ 6.9 5.3 ⫾ 0.6 0.1 ⫾ 0.1 1.3 ⫾ 0.4 5.8 ⫾ 0.7 0.7 ⫾ 0.3 0.1 ⫾ 0.1

NS 0.0021 0.007 0.0006 NS 0.0002 0.009 NS ⬍0.0001 NS ⬍0.0001 NS ⬍0.0001

Abbreviations: ELISA ⫽ enzyme-linked immunosorbent assay; NS ⫽ not significant; mAb ⫽ monoclonal antibody. a C ⫽ capture mAb, D ⫽ detection mAb. b p Value (Student’s t-test). c Mean ⫾ SEM in ng/ml, n ⫽ 11.

FIGURE 3 Workshop samples containing soluble human leukocyte antigen (sHLA)-G1 molecules. Each point represents the enzyme-linked immunosorbent assay results of one Wet Workshop team obtained by the antibody combination mAb MEM-G/9 ⫹ anti-human ␤2m or monoclonal antibody (mAb) 5A6G7 ⫹ mAb W6/32. Points located under the diagonal line indicate the presence of sHLA-G1 that is not detectable by the 5A6G7 assay.

ELISA formats did not cross-react with either classical soluble HLA class I molecules or any other antigen present in human plasma, being therefore specific for sHLA-G1 ⫹ G5 and HLA-G5. Purified rHLA-G1 molecules (no. 13) were only detected by the ELISA with mAb MEM-G/9 as capture reagent (Table 3, Figure 3). This demonstrates that the two ELISA formats indeed differ in their specificity: the ELISA based on mAb 5A6G7 only identifies sHLA-G molecules containing intron-4 – derived peptide sequences (HLA-G5), whereas the ELISA with mAb MEM-G/9 recognizes both, sHLA-G1 and HLA-G5 molecules. Reproducibility of Results and Interoperator Variations Besides the four control samples, seven EDTA plasma samples from healthy donors, one ascites fluid from ovarian cancer, and one amniotic fluid (Table 2) were included in the workshop analysis. ELISA results (Figure 3) obtained for plasma samples were consistent among all 11 teams. Mean concentrations of sHLA-G1 ⫹ G5 for these plasma samples ranged from 27.7 to 95.9 ng/ml with SEM ranging between 2.2 to 6.7 ng/ml (Table 3). Mean concentrations of HLA-G5 for the plasma samples ranged from 14.6 to 85.8 ng/ml, with SEM values from 1.8 to 16.6 ng/ml (Table 3). Ascites and amniotic fluids yielded the same results when found positive. This analysis indicates that the two ELISA protocols are not especially sensitive to interoperator differences and yield reliable results.

sHLA-G1 ⴙ G5 and HLA-G5 Concentrations in Body Fluids The concentrations of sHLA-G1 ⫹ G5 and HLA-G5 in the body fluids studied were compared. Wet-Workshop sample no. 1, 5, 8, and 10 (Table 3) yielded nearly identical results with the two ELISA formats. This implies that HLA-G5 was present in these samples, but not sHLA-G1 molecules. Sample no. 10 was deliberately depleted from sHLA-G1 ⫹ G5 molecules by mAb MEM-G/9 resulting in the lack of reactivity in both ELISA formats. In four plasma samples (no. 2, 4, 6, 7; Table 3), we found that the concentrations of sHLA-G1 ⫹ G5 (MEM-G/9 test) were significantly higher than HLA-G5 concentrations (5A6G7 ELISA), as depicted in Figure 3. This means that sHLA-G1 and HLA-G5 molecules were both present in the samples. In the amniotic fluid (no. 9), no signal was detected by 5A6G7 ELISA in contrast to the MEM-G/9 ELISA (Table 3, Figure 3). This indicates that sHLA-G1, but not HLA-G5, molecules were present in amniotic fluid at the concentration found using the MEM-G/9 test. All these results are consistent with the notion that the MEM-G/9 test detects sHLA-G1 and secreted HLA-G5 indiscriminately, and that therefore values obtained with it should be equal to or higher than those obtained with the 5A6G7 ELISA which does not quantify sHLA-G1 molecules. Surprisingly, this notion proved not to be entirely accurate. Indeed, in plasma sample no. 3, all investigators found significantly higher values using the 5A6G7 test than the MEM-G/9 test (Figure 4, Table 3). This

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FIGURE 4 Wet Workshop samples containing soluble human leukocyte antigen-G molecules with a novel protein configuration. Each point represents the enzyme-linked immunosorbent assay results of one Wet Workshop team obtained by the antibody combination mAb MEM-G/9 ⫹ anti-human ␤2m or monoclonal antibody (mAb) 5A6G7 ⫹ mAb W6/32. Points located above the diagonal indicate that soluble HLA-G molecules exist that are not or weakly detectable by the MEM-G/9 assay.

means that HLA-G5 molecules exist that can be recognized by the antibody combination mAb 5A6G7 ⫹ W6/32, but not by mAb MEM-G/9 ⫹ anti-human ␤2m. This is further substantiated by plasma sample no. 11, for which we found a definite positive, even if weak, signal with the 5A6G7 ELISA format after depletion of sHLA-G1 ⫹ G5 molecules with mAb MEM-G/9. DISCUSSION The goals of the 2nd International Workshop on HLA-G (Paris, France, 2003) and especially the Wet Workshop for quantification of soluble HLA-G (Essen, Germany, 2004) reported here were to validate an ELISA test capable of measuring sHLA-G1 and HLA-G5 molecules, and another ELISA test specific for HLA-G5 that can both be used on a variety of sample types and that can be set up easily using commercially available reagents. The reason for these efforts was that even though (1) HLA-G expression is relevant in a variety of pathologic situations; (2) amongst the various HLA-G isoforms, soluble ones are the most accessible and the easiest to quantitate; and (3) several methods have been set up to measure soluble HLA-G levels, there is no consensus on a reference method that would allow standardization and comparison of results. The 3-day Wet Workshop on sHLA-G involved 24 investigators from 13 laboratories (teams) and led to the

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validation of two ELISA procedures and a standard protein. The ELISA procedure to measure soluble HLA-G1 and secreted HLA-G5 indiscriminately involved a capture by the anti-sHLA-G1 ⫹ G5 mAb MEM-G/9 and detection by anti-␤2-microglobulin. The ELISA procedure to measure secreted HLA-G5 specifically involved a capture with the anti–intron-4 of HLA-G mAb 5A6G7 and detection by anti–pan-HLA class I mAb W6/32. The standard curves for these ELISA tests were generated using recombinant HLA-G5 protein extracted from HLA-G5 ⫹ ␤2m cotransfected insect cells. The results obtained with both ELISA formats using the recombinant HLA-G5 standard protein that they both recognize indicate that these two ELISA measure HLA-G5 levels up to 80 ng/ml with the same efficiency, because superimposable standard curves were obtained. The results obtained with biologic samples show that both ELISA formats are specific and do not cross-react with other molecules including other HLA that might be contained in biologic samples, and that these tests allow for the quantification of sHLA-G1 and HLA-G5 if used in combination. Finally, even though each investigator may have obtained different standard curves, this did not affect the absolute values obtained for the samples measured, indicating that both tests generate reproducible results. Overall, these two ELISA formats reached expectations and, in our opinion, can serve as reference assays to measure soluble HLA-G molecules in culture supernatants and biologic fluids. Tests carried out during this workshop allowed us to measure sHLA-G1 ⫹ HLA-G5 and HLA-G5 specifically in different body fluids (i.e., EDTA plasma samples, amniotic fluid, and ovarian cancer ascites). The combined use of both tests revealed that soluble HLA-G molecules were present at very variable amounts in peripheral blood of healthy controls: sHLA-G1 ⫹ G5 concentrations ranged from 27.7 to 95.9 ng/ml, and secreted HLA-G5 concentrations ranged from 14.6 to 85.8 ng/ml. Furthermore, both sHLA-G1 and HLA-G5 were present in ascites and in four plasma samples (no. 2, 4, 6, 7), whereas only HLA-G5 was present in two plasma samples (no. 1, 5, 8) and only sHLA-G1 was detected in amniotic fluid. Thus, it appears that in the peripheral blood of healthy individuals, the nature and the quantity of soluble HLA-G molecules differ from one individual to another. This might be linked to differences in the source of soluble HLA-G molecules, the immune state at which they are released, or HLA-G alleles and their promoters, and might also correspond to different HLA-G functions. It has to be noted that in plasma sample no. 3, all investigators found significantly (p ⫽ 0.007) higher concentrations of HLA-G5 (ELISA: 5A6G7 ⫹ W6/32) than concentrations of sHLA-G1 ⫹ G5 (ELISA: MEM-G/09

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⫹ anti-␤2m). This was puzzling because both tests recognized recombinant HLA-G5 with the same efficiency, meaning that values obtained for sHLA-G1 ⫹ G5 should always be equal or higher than those obtained for HLA-G5 alone. To explain this result, one has to postulate that HLA-G5 (and possibly sHLA-G1) can organize itself as a structure in which the intron-4 – encoded sequences are accessible and which sufficiently resembles an HLA class I molecule to be recognized by W6/32, but which differs from sHLA-G1 ⫹ G5 normal structure, because MEM-G/9 does not recognize it. This, which happened during this workshop, is not unheard of and has been previously noticed by several of the investigators present (Rouas-Freiss unpublished and [59]. Whether this new structure of HLA-G5 is a homodimer that can be recognized by mAb W6/32 in the same way as reported for the HLA-B27 homodimer [60] is unknown. Of note, HLA-G1 is able to form disulfidelinked dimers, which could be expressed on cell surface. In principle, this can also happen to HLA-G5, as the disulfide bridge is located at amino acid position 42 within the alpha 1 domain of HLA-G molecules [61]. Thus, the Wet Workshop for quantification of soluble HLA-G in Essen, Germany, has validated ELISA formats to measure sHLA-G1 ⫹ G5 molecules or HLA-G5 exclusively. In addition the results of the workshop gave strong evidence for at least one unreported structure for HLA-G5, and possibly for sHLA-G1 as well, that can be detected by the combined use of both ELISA formats. The precise methodology of these two ELISA formats as well as reagents and a list of suppliers can be found at our website (in preparation) and the protein used for standardization is kept in ESSEN (Institute of Immunology, University Hospital of Essen) and PARIS (Service de Recherches en Hémato-Immunologie, CEA-DSV-DRM, Hôpital Saint Louis). Small amounts of standard reagent will be distributed after inquiry for the establishment such ELISA formats. REFERENCES 1. Ellis SA, Sargent IL, Redman CW, McMichael AJ: Evidence for a novel HLA antigen found on human extravillous trophoblast and a choriocarcinoma cell line. Immunology 59:595, 1986. 2. Crisa L, McMaster MT, Ishii JK, Fisher SJ, Salomon DR: Identification of a thymic epithelial cell subset sharing expression of the class Ib HLA-G molecule with fetal trophoblasts. J Exp Med 186:289, 1997. 3. Le Discorde M, Moreau P, Sabatier P, Legeais JM, Carosella ED: Expression of HLA-G in human cornea, an immune-privileged tissue. Hum Immunol 64:1039, 2003. 4. Lila N, Carpentier A, Amrein C, Khalil-Daher I, Dausset

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