S. FournelX. HueM. Aguerre-GirrC. SolierM. LegrosC. Praud-BrethenouM. MoussaG. ChaouatA. BerrebiA. BensussanF. LenfantP. Le Bouteiller

Comparative reactivity of different HLA-Gmonoclonai antibodies to soiubie HLA-Gmoiecuies

Key words:

Soluble HLA-G: HU-G isoforms; HLA-Gmonoclonal antibodies; ELiSA

Acknowledgments:This work was supported by Institut National deia Sante et de la Recherche Medicaie, byAssociation pour ia Recherche sur le Cancer(9489), by Universite Paui Sabatier and byEtablissement Frangais des Greffes. We wish tothank Dr. F. C. Grunnet for providing us with theSHLA-B7 and mHLA-B7 transfectants, Dr. D.Geraghty, Dr. H. Waldmann and Pr. J.R Revillardfor their gifts of mAbs, Pr. E. Weiss. Dr. S. Maier.Dr. H, Grosse-Wilde and Dr. V. Rebmann forproviding us a caiibrated, standard sHLAG. A.Blascliitz is acknowledged for her helpfui advicein the estabiisment of ELiSA procedure and G.Cassar for the flow cytometry analysis.

Received 37 October 1999, revised,accepted for puOllcation 20 Match 2O00

' Munkseaard 2000TTssue Antigens . ISSN 0001-2815

nssue Antigens 2000; S6; 510-518Printed in Denmark . All rights reserved

MQstract: Different HLA-G monoclonal antibodies (inAbs) were first evalu-

ated for their capability to identify soluble HLA-G (sHLA-G) in ELISA. Three

of them, namely 87G, BFL.1 and MEM-G/9, when used as coating mAbs

together with W6/32 capture mAb, identified p2-mia'oglobulin (|32m)-associ-

ated-sHLA-G but not soluble HLA-B7 (sHLA-B7) in cell culture super-

natants from transfected cells. By comparison, the anti-HLA class 1 mAb 90

did recognize both sHLA-G and sHLA-B7. By using these HLA-G mAbs,

sHLA-G was identified in amniotic fluids as well as in aiiture supernatantsof first trimester and term placental explants but not in cord blond. Intron

4-retaining sHLA-G isoforms were identified in some amniotic fluids by the

use of an intnin 4-spedfic mAb {16G1). Reactivity of these different HLA-

G mAbs was then compared to determine their respective binding sites on

soiubie and membrane-bound HLA-G. Using both ELISA and flow cytome-

try analysis, we showed that they did not compete with each other, which

suggested that they did not recoipiize the same determinants. Finally, we

report that two mAbs directed against the a l domain of HLA class I heavy

chain (mAb 90 and YTH 862) did compete with 87G. therefore demonstrat-ing that this latter mAb recognized an epitope localized on this external

domain of HLA-G.

HLA-G class Ib gene is characterized by a low polymorphism,

unique structural featui-es and a limited tissue distribution (1, 2).

Major structural characteristics of HLA-G are as follows. First, the

promoter region of HLA-G differs in many ways from the other

major histocompatibitity compiex (MHC) class I genes (3,4). Second,

the cytoplasmic domain of HLA-G is shorter than in class Ia mol-

ecules (5). Third, the primary HLA-G transcripts are differentially

spliced, giving rise to six different isoforms (6-9). Four of them

encode membrane-bound products lacking either one or two exter-

nal domains, two others generate soluble proteins. These soluble

forms of HLA-G (sHLA-G). a full-length sHLA-Gl and a shorter

sHLA-G2 form lacking the a2 domain, are encoded by alternatively

spliced transcripts that retain intron 4 which contains a stop codon,

Authors'

S.X. Hue',M.C. Soller'.M. Legros'.C. Praud-Brethenou',M. Mousao'.G. CfiaouBl'.A. BerrebP.A. Bensussan',F. Lenfant'.R Le Boutatl>9r>

'Insmj! Nsilonai de laSante et lie la RecdercheM^icale U395, Hdpital dePurpan, Toulouse. France.

'Biologle de la RelationMatemo Foetale. IMSERM U131, HOpltai AntoineBeclSre. Cfamart, Frarice.

^Service de GyrtecclogleObst^rique. HOpltal UGisue. Toulouse. Fiance.

*Facun6 de M^Oecine deCr#tell, INSERM U4it8,CrSteil, France

Corraapondance to;pn 11 tope Le BnutelllerInsitiul National de la Sant^

et de la Rechetctie

Centm HospKalo.Univefsiiaire Oe Purpan,BP 3026

31024 Toulouse Cedax 03FranceFa»: +33 562 748 386Tel: +33 562 748374

510

Fournel et a l : HLA-6 mAb reactivity

thus precluding the translation of transmembrane and cytoplasmic

domains {7, 10, 11).

Constitutive expression of HLA-G translated products has been

reprodudbly detected in placenta and thymus. Within the plaeenta,

the following cellular subtypes were found to bind HLA-G mAbs

by immunohistochemistry: invading extravillous cytotrophoblast

(12, 13-15) endothelial cells lining fetal vessels in the chorionic villi

(13), mesenchymal Hofbauer cells (16) and amnion epithelial cells

(17, 18). Soluble forms of HLA-G have been detected in anmiotic

fluids and plasma (18-20) and, in one report, in some syncytiotro-

phoblast (16). HLA-G was also found in some medullary thymic

epithelial cells (21, 22).

Functions of HLA-G are now emerging (1). HLA-G binds nonam-

er peptides (23, 24) and is capable of presenting them to CD8+ T

cells (25, 26). HLA-G is the ligand of few killer cell immunoglobuiin-

like receptors (2, 27) and modulates HLA-E cell surface expression

upon binding to a nonamer derived from the HLA-G leader se-

quence (28).

A number of groups have reported the use of HLA-G monoclonal

antibodies (mAbs) (14,16,18,23,29). Only partial characterization of

these reagents has been made and very few comparative studies have

been carried out to examine their respective reactivities (1). Investi-

gations on their potential use in ELISA for the detection of soluble

forms of HLA-G are also limited (16, 19), and little information is

available about which antigenic determinant they can recognize. By

comparing the reactivity of three HLA-G mAbs in ELISA, we show in

this report that they al! identified p2-microglobulin (p2m)-associated

sHLA-G but not sHLA-B7, that they did not compete with each other

and therefore did not recognize the same epitopes. Additional compe-

tition experiments performed by flow cytometry with mAbs directed

against the a l domain of HI.A class I molecules demonstrate that the

antigenic determinant recognized by the 87G anti-HLA-G mAb is

located on this external domain of HLA-G.

(29), 87G agG2b; gift of Dr. D. Geraghty, Seattle, WA. USA) (23) and

MEM-G/9 agGl. Exbio, Prague, Czech Republic) are HLA-G-specific

mAbs. MEM-G/9 mAb was raised against native, properly folded,

p2m-associated HI.A G. It reacts with HLA-G on the cell surface but

not with denatured molecules, under the conditions of Western blot-

ting. MEM-G/1 (igGl, Exbio, Prague, Czech Republic) reacts with the

denatured HLA-G HC. 16G1 (IgG2a. gift of Dr. D. Geraghty, Seattle,

WA, USA) is specific for a synthetic peptide corresponding to the

amino acid sequence of sHLA-G intron 4 (23). 10.3.6 anti-murine MHC

class n I-A"* (IgG2a) (34) and control isotypic mAbs (Dako, Trappes,

France) were used as negative controls.

Cells

The human gestational choriocarcinoma cell lines JAR and JEG-3 and

the lymphoblastoid cell tines .221 and CIR were obtained from the

American Type Culture Collection (Rockville, MD, USA). JAR-Gl

cells, transfected with the full-length membrane-bound HLA-Gl en-

coding cDNA, and JAR-sGl, transfected with the full-length, intron 4

retaining soluble HLA-Gl (sHLA-Gl) cDNA, have been already de-

scribed (13, 35). 221-sGl cells were similarly transfected with sHLA-

Gl encoding cDNA. ClR-sB7 cell line transfected with sHLA-B7 en-

coding cDNA, and ClR-mB7, transfected with membrane HLA-B7 en-

coding cDNA, were kindly provided by Dr. F. C. Grumet (Stanford,

CA, USA) and have been previously described (36). Cell lines were cul-

tured in RPMI 1640 with Glutamax (Life Technologies. Cergy-Ponto-

ise, France) supplemented with 10% FCS, 1 mM sodium pyruvate, 100

rU/ml penicillin and 100 ng/ml streptomycin (Sigma, St Quentin Fal-

lavier, France). Cultures were maintained in a humid atmosphere at

37°C containing 5% CO2. Cell culture supernatants were collected,

centrifuged at 1200 rpm for 5 min at 4°C, and stored at -20°C until

use. In some cases they were concentrated five times using Macrosep

lOK (10 kDa cutoff) centrifugal concentrator (Paul Filtron Corpor-

ation, Northborough, MA, USA).

Material and methods

Monoclonal antibodies

W6/32 mAb (IgG2a) recognizes a framework determinant expressed

on p2m-assodated HLA class I heavy chain (HC) (30); both human

mAb 90 (IgG 1, gift of Pr. j . P Revillard, Lyon, France) and rat YTH862

(IgG2b, gift of Dr. H. Waldmann, Oxford, UK) recognize a framework

determinant located on the a l domain of HLA class I HC (31); HCA2

mAb (IgGl, gift of Pr. H. Ploegh, Cambridge, MA, USA), recognizes

free HC of HLA-A, -E, -F and -G as well as some selected -B and -C

alleles (32,33); BFL.l agG2a, Coulter Immunotech, Marseille, France)

Amniotic fluids and cord blood sera

Amniotic fluids were obtained by amniotie puncture of healthy

pregnant women (16 to 24 weeks of pregnancy). Cord blood sera

were obtained from term placentas. Samples were centrifuged at

2000 rpm for 5 min, concentrated five times as described above, or

not concentrated, and stored at - 2 0 ^ .

Placental supernatants

Supernatants were prepared as previously desaibed (37). Briefly,

first trimeter and term placentas were obtained from healthy preg-

Tissue Anti^ns 2000: 65: 510-518 511

Foume! et a l : HLA-G mAb reactivity

tiant women, and 1.5 g of tissue was washed in HBSS and incubated

in 10 ml of RPMl-10% FCS. At various times, supernatants were

harvested, and stored at -20°C.

Specific soluble HLA-G (sHLA-G) ELISA

96-well microtiter plates {Corning Costar Corporation, Cambridge,

MA. USA) were coated with different mAbs at the indicated concen-

trations in O.IM carbonate buffer pH 9.5 for 1 h at 37''C then over-

night at 4°C. After three wash^ with phosphate-buffered saline

(PBS) containing 0.05% Tween 20 (PBS-Tween), plates were satu-

rated with 100 til of PBS containing 4% BSA overnight at 4T. Cell

culture supernatants (50 |il) were added to each well. After incuba-

tion for 2 h at 37°C, plates were washed three times with PBS-

Tween. Biotinylated mAb (50 \i\) was added to each well and incu-

bated for 1 h at 37X. Plates were washed five times with PBS-

Tween and further incubated with a peroxidase-conjugated avidin

(Dako) for 15 min at 3rC. After five washes with PBS-Tween. 50

nl of ortho-phenyl-diamine peroxidase substrate (Sigma) was added

to each well and incubated for 15 min at room temperature. The

reaction was stopped by addition of 50 nl of 2N H2SO4. The relative

1.5

I

e.5

8

•C1i)-sB70221-561

.AFig. 1. Comparative reactivity of different HLA-G mAbs in ELI-SA. Microwells were coated either with 87G (2 jiK/ml), BFL.l (2 ng/ml).

MEM-G/9 (ascite 1/5000). 16G1 (2 Mg/mi), mAb 90 (ascite 1/100), MEM-G/1(ascite 1/1000). HCA2 (liybridoma supernatant 1/5) or isotypic control Ab

(mixture of IgG2a. IgGl and IgM at 2 jig/ml). Then cell culture super-nalants (non concentrated for 87G, MEM-G/9 and 16G1 mAbs, 5X a>ncen-trated for the other mAbs) were added and bound molecules identified by

using biotinylated W6/32 mAb (10 Mg/nil), as described in Materials andMethods. To assess ELISA specificity, cell culture supernatants of 221 sGl

cells (white bar) were compared to culture supernatants of ClR-sB7 cells(black bar). Results are expressed as means±SD of absorbancy units (492

trni) from four (S7G, MEM-G/9), five (BFL.1, mAb90. MEM-G/1, HCA2. con-trol Ab) or two (16G1) separate experiments. Mean OD obtained with 87Gwas 2.84 ±0^6.

concentrations of sHLA-G or sHLA-B7 were estimated from absorb-

ance measui-ed at 492 nm (duplicate wells) on a MRX-ELISA

microplate reader (Dynatech. Guyancourt, France). A standard

supernatant of sHLA-G/.221 transfectant, produced by Prof. E.

Weiss and Dr. S. Maier (Mtmich, Germany), and calibrated by Drs.

H. Grosse-Wilde and V. Rebmann (Essen, Germany), as previously

described (38), served as reference in standard calibration curves.

Row cytometry competition studies

Cells (0.5x10^) were incubated with blocking mAb diluted in PBS

supplemented with 1% FCS and 0.2% NaN3 for 30 min at AX. After

two washes in the same meditun, 87G mAb (5 [lg/ml in PBS/FCS/

Azide) was added to the cells for an additional 30 min at 4°C. The

cells were then washed and incubated with phycoerythrin-conju-

gated streptavidin (Coulter Immunotech) for 30 min at 4X. After

subsequent washes, cells were analyzed on a Coulter Epics Elite

fiow cytometer (Margency, France) gated to excluded non-viable

cells.

Results

Several HLA-G mAbs identify sHLA-G In cell culture

supernatants of sHLA-G transfectants

To investigate the ability of several HLA-G mAbs to detect sHLA-

G present in cell culture supernatants, different HLA-G-recognizing

mAbs were coated to the microtiter plates and the biotinyIated-W6/

32 anti-HLA class I mAb was used to reveal the bound molecules.

As shown in Fig. 1. four HLA-G mAbs, namely 87G, BFL.l. MEM-

G/9 and 16G1, although with different affinities, specifically recog-

nized sHLA-G in cell culture supernatant from a lymphoblastoid

cell line transfected with sHLA-G-encoding cDNA (.221-sGl). By

comparison, no HLA-G mAb recognized sHLA-B7 in cell culture

supernatant from a lymphoblastoid cell line transfected with sHLA-

HLA-B7-encoding cDNA (ClR-sB7). The different signal intensities

obtained in ELISA with these three HLA-G mAbs are likely to re-

flect different levels of binding affity. mAb 90 which binds to a

common epitope of HLA class I HC (31), including HLA-G (P. Le

Bouteiller, tmpublished data), did recognize both sHLA-G and

SHLA-B7. In contrast, neither MEM-G/1 nor HCA2 mAbs, which

were raised against denatured HLA class I HC. nor isotypic control

Abs, reacted against sHLA-G or sHLA-B7.

We further examined the sHLA-G specificity of 87G, MEM-G/9

and BFL.l mAbs. Whereas these mAbs identified sHLA-G in .221-

sGl culture supernatants, none of them detected any soluble protein

512 nssue Antf ens 2000: Sft;

Foumel et al : HLA-G mAb reactivity

in cell culture supernatants from 2l\, CIR or ClR-sB7 cells (Fig. 2).

Using 87G as coating mAb, sHLA-G was also detected in culttire

supernatants of JAR transfected with a cDNA encoding sHLA-Gl

(JAR-sGl), whereas no soluble protein was identified in the culture

medium either from tintransfected JAR cells or from JAR cells frans-

fected with a cDNA encoding membrane HLA-Gl QAR-Gl) (Fig. 2).

Using serial concentrations of a calibrated sHLA-G reference (20),

we established standard curves for 87G and MEM-G/9 HLA-G

mAbs (Fig. 3). Both curves revealed linearity for sHLA-G concen-

trations in the range of 30 to 260 ng/ml.

We then used the ELISA described above (coating mAb : 87G)

to investigate the presence of sHLA-G in few biological fiuids and

culttu'e media from placental explants. As shown in Fig. 4A, sHLA-

G was identified in amniotic fitiids of healthy pregnant women (19-

24 weeks of pregnancy) but not in cord blood sera. Similar results

were obtained with MEM-G/9 coating mAb (data not shown). It

should be noted that cord blood sera always gave a quite high

background in ELISA after coating the plates with isotypic IgG

control, which was never the case for amniotic fluids or the different

cell culture supernatants (these latter control values have been sub-

sfracted from the values shown in Fig. 4). In contrast, by using

mAb 90 (anti HLA-A, -B, -C, -G) or B1.23. 2 (anti-HLA-B and -C but

not HLA-G) coating mAbs (29) and W6/32 detection mAb, we have

observed that the cord blood sera did contain sHLA class Ia mol-

ecules (data not shown). sHLA-(; was also detected in culture super-

natants from first trimester or term placenta explants (Fig. 4B).

Based on ELISA standard curves (see Fig. 3), we estimated that the

concenfrations of sHLA-G in amniotic fluids were in the range of

35 to 130 ng/ml, depending on the samples, and in the range of 30

to 60 ng/ml for placental supernatants. Tbe use of 87G and MEM-

G/9 mAbs did not allow to disaiminate between sHLA-G isoforms

possibly derived from shedding or proteolytic cleavage of mem-

brane molecules and those retaining intron 4. Therefore we used a

conformation independent mAb 16G1 which reacts uniquely with

Fig. 2. Specific detection of sHLA-G with 87G, IVIEMG/9 andBFL.l coating inAbs. (A), 87G (1 ^g/ml), (B), MEM-G/9 (1/5000) and (C),

BFL.1 (2 |ig/ml) were coated on microtiter plates and biotinylated W6/32mAb (10 |ig/nil) was added to revealed the bound sHLA-G molecules (see

Materials and methods). To assess the specificity of the EUSA, cell culturesupernatants from 221-sGl and JAR-sGl cells were used as positive control

and those from JAR. JAR-Gl (which express membrane HLA-Gl), 221, CIRand C1R-SB7 cells were used as negative control. All culture supernatants

were used non concentrated, except JAR-sGl which was concentrated 5times. Results are expressed as means±SD of absorbancy units (492 nm) on

ten separate experiments for 87G. seven for BFL.1 and five for MEM-G/9mAb.

nssue Anti^ns 2000: 55: 510-518 513

Fournel et al : HLA-G mAb reactivity

87G r = 0.9738

UIU

492

oO

0.5

0

1

0.8

0.6

0.4

0.2

MEM-G /9 = 0.999

0 50 100 150 200 250 300

Concentration of sHLA-G1 (ng/ml )

Fig. 3. ELISA standard curves for sHLA-G using 87G or MEM-G/9 mAbs. Varying concentrations of calibrated standard supernatant of

sHLA-Gl/,221 transfectant (see Material and methods) were coated into

miooplates. Binding of either 87G (2 ng/ml) or MEM-G/9 (1/5000) mAbs

followed by biotinylated W6/32 was measured by ELISA as described in

Material and nfiethods. Results are expressed as means of duplicates.

the intron 4 retaining soluble form of HLA-G (23). We compared its

reactivity with 87G and MEM-G/9 mAbs by testing eleven other

amniotic fiuid samples in ELISA. Results are shown in Fig. 5. Very

similar results were obtained with 87G and MEM-G/9 mAb: both

mAbs detected the same variations of the sHLA-G level of express-

ion, according to amniotic fluid samples. BFL.1 HLA-G mAb also

gave very similar results (data not shown). When 16G1 mAb was

used, the most positive amniotic fluids (samples 8 and 13) were the

same as those detected by the other HLA-G mAbs. Sample 17 ap-

peared more positive with 16G1 mAb than with the other HLA-G

mAbs whereas a slight reactivity was detected in samples 16 and

18. These data demonstrated that at least part of sHI A-G present

in amniotic fluids was intron 4 retaining sHl.A-G is<}form. 16G1

mAb also reacted with placental cell supernatants (Solier et al.,

manuscript in preparation),

Different HLA-G mAbs do not bind to the same epitopes on

sHLA-GI

As demonstrated above, 87G, BFL-1, MEM-G/9, mAb 90 and W6/

32 mAbs recognize sHLA-G in culture media from different sHLA-

G transfectants. To compare the reactivities of these various HLA-

G mAbs in ELISA, we used different combinations of coating and

detection mAbs. Microwells were coated either with 87G, BFL.1,

1 2 3 4 5 6 7 1 2 3 4 5 6 7 8 9

AmnJotic fluids Cord blood

24h 48h 12h 24h 48h

•' trim, placentas Term placentas

Fig. 4. Detection of sHLA-G in amnioticfluid, cord blood and cell culture super-natants of first trimester and term placen-ta! explants. A) Seven samples of amni-otic fluids (numbers 1-7). concentrated 5times, and 9 samples ol c<ird blood sera(numbers 1-9), non-concentrated, wereevaluated usii^ sHLA-G-specific ELISA(coating mAb: 87G; detection mAb: bi-otinylated W6/32 mAb). Values are expressedas mean absorbancy units (492 nm) of three sep-

arate EL1SA±SD after substraction of thevalues obtained with the isotypic a)ntrol coatedAbs. Numbers 4 and 9 of cord blocid sjiniples:

OD=0. B) Culture supernatants of first tri-mester or term placental explants wereharvested at indicated times and thenevaluated non-concenlrated using sHLA-G-specific ELISA. Values are expressed as meanabsorbancy units (492 nm) ±SD of eight first tri-mester and fifteen term placental explants. 87G

mAb was coated at 1 pg/ml and biotinylated W6/32 added at 10 Mg'nil. 221-sGl and ClR-sB7 cul-

ture supernatants were used as positive andnegative controls, respectively.

514 TIssw Antigens 2000: BS: 510-51B

Fournel et al : HLA-G mAb reactivity

Fig. 5. Detection of sHLA-G in amnioticfluid: comparative study using three differ-ent HLA-G mAbs. Eleven Tion-concentratedsamples of amniotic fluid (numbers 8-18) were

evaluated using sHLA-G-spedfic ELISA. CoatingmAbs were as follows: 87G (2 g/mI), MEM-G/9 (ascite 1/5(XK)), or 16G1 (2 ig/ml). Detection

mAb: biotinylated W6/32 mAb. Values are ex-pressed as mean absorbancy units (492 nm) of

duplicates ±SD. Values obtained with the iso-typic control coated Abs were all negative. 221-

sGl and C1R-SB7 culture supernatants wereused as positive and negative controls, respec-

tively. OD obtained with 87G and MEM-G/9 on221-sGl were 2.66 and 1.14, respectively.

IB I I 12 13 14 15 16 17 18

Hmniotic fluids

MEM-G/9. W6/32 mAbs or with mAb 90, then the bound sHLA-G

molecules were revealed either by biotinylated-W6/32, -87G or

-WL.] mAbs. As shown in Fig. 6. all the combinations using HLA-

G mAbs or W6/32, albeit with different affinities, allow the detection

of sHLA-G in culture supernatants of 221-sHLA-G, suggesting that

these various mAbs recognize different epitopes on sHLA-G mol-

ecules. However, combinations using 87G, MEM-G/9 or, to a lesser

extent, BFL.l coating mAbs, followed by biotinylated W6/32 detec-

tion mAb, gave the strongest specific signals. In all these combi-

nations, SHLA-B7 is never recognized (Fig. 6, left panel). Interest-

1.5

0.5

Coating mAb

i

C1R-SB7 n221-sG1

JDetection mAb Biotinylated W6/32 BiotinylatedB7G BlotinyiatedBFL.1

Fig. 6. Comparative ELISA using different combinations of coat- the bound sHLA-G molecules (see Materials and methods). Non-concen-ing and detection mAbs. 87G (1 ng/ml), BFL.1 (2 ng/m!), MEM-G/9 (asdte

1/5O(X)), mAb 90 (ascite 1/100), W6/32 (2 ng/ml) and isotype control Ab(IgG2a-(-IgGl -HigM; 2ng/nil) were coated on microwells and biotinylated W6/

), 87G (5 ng/ml) or BFL.l (5 Mg/ml) mAbs were added to revealed

trated cell culture supernatants from 221-sG] cells (white bar) were used as

positive control and those from ClR-sB7 cells (black bar) were used asnegativa control. Results are expressed as means±SD of absorbancy units(492 nm) on three separate experiments.

T7ssue Anti^ns 2000; 65: 510-518 515

Fournel et al : HLA-G mAb reactivity

ingly, when mAb 90 was used as coating mAb and biotinylated 87G

as detection mAb, no protein was detected (Fig. 6, medium panel),

suggesting that 87G mAb recognized the same or a near epitope on

the a l domain of sHLA-G.

87G mAb recognizes a determinant iocaiized on the a l

domain of HLA-G

We next compared the epitope distribution on membrane-bound

HLA-G by flow cytometry analysis. Trophoblast-derived JEG-3

cells, which are known to constitutively express HLA-G (7, 33),

have been incubated firstly, with various mAbs which recognize

HLA-G (blocking mAbs) and, secondly, with biotinylated 87G

mAb. As shown in Fig. 7, pre-incubation of JEG-3 with 87G

mAb almost abolished the binding of biotinylated 87G. as com-

pared to a preincubation with 10.3.6 control mAb or with HCA2

mAb (this latter mAb having been previously shown not to bind

to the cell surface of JEG-3: data not shown). BFL.l, MEM-G/9

and W6/32 mAbs did not prevent the binding of biotinylated

87G, demonstrating that these mAbs did not recognize the same

epitope as 87G mAb on membrane-bound HLA-G. In contrast,

mAb 90 and YTH862, which both recognize a framework deter-

minant on the a l domain of HLA class I HC (31), inhibited the

binding of 87G mAb. Altogether, these results and those men-

tioned above (ELISA) strongly suggest that 87G mAb did recog-

nize an epitope mapped on the a l domain of membrane-bound

and sHLA-G.

4,00

3.00

2.00

1.00

BiocklngmAb :

Detection mAb : Biotinylated87G

Fig. 7. Competition assays of various HLA-G mAbs : flow cytome-try analysis. JEG-3 celts were pre-incubated either with 87G (5 ng/ml),

10.3.6 (negative control, undiluted hybridoma supernatant), HCA2 (non-

diluted hybridoma supernatant), BFL.1 (5 ng/ml), MEM-G/9 (a.scite, 1/1000),W6/32 (5 ng/ml), mAb 90 (ascite, 1/100) or YTH862 (non-diluted hybridomasupernatant). Then, after intensive washes, cells were incubated with bi-

otinylated 87G mAb (2.5 fig/ml) followed by phycoerythrin-conjugatedstreptavidin. Binding of S7G mAb was evaluated by flow cytometry, as

described in Material and methods. Results are expressed as mean fluor-escence intensity (MFO ±SD on three separated experiments.

Discussion

In this report, we have shown that three HLA-G mAbs. namely,

87G, BFL.1 and MEM-G/9 did recognize sHLA-G molecule, in it;

p2-m-assodated form. They could not recognize HLA-G free HC for

the following reasons: firstly, the detection mAb W6/32 used in ELI-

SA only recognizes a conformational monomorphic determinant ex-

pressed on p2m-associated HLA class I HC (30); secondly, none of

the MEM-G/1 and HCA2 mAbs, which both react with denatured

free HLA-G HC, did boimd proteins from sHLA-G containing super-

natants, showing that such free HC were not present in the cell

culture supernatants used; finally, we have previously shown that

both 87G and BFL.l were able to recognize affinity purified p2m-

assodated-HLA-G in ELISA (35). These results confirm prervious

studies performed by other groups: Fujii et al. (7) mentioned that

87G detected sHLA-G in ELISA and Puppo et al. (19) recently iden-

tified sHLA-G in JEG-3 culture supernatant, as well as in some

biological fluids, by using W6/32 and biotinylated BFL.1 as captui¥

and detection mAbs respectively, after immunodepletion of HLA

class Ia and H1.A-E molecules with mAb TP25.99. To our knowl-

edge, no data has been reported yet with MEM-G/9 mAb.

None of these HLA-G mAbs did recognize sHLA-B7 present in

cultxire supernatant of transfected cells, although it carmot be tot-

ally excluded that they also reacted with some unfrequent sHLA

class Ia alleies. However, this is unlikely for the following reasons:

firstly, they did not detect any soluble protein in all cord bkxxl

samples tested, although we have shown by using mAb 90 or

Bl.23.2 coating mAbs that these cord sera did contain some sHLA-

class Ia molecules, as already reported by other groups (19); second-

ly, both 87G (23) and BFL.1 (29) were described not to bind to pe-

ripheral blood mononuclear cells of various donors expressing vari-

ous HLA class la allelea The three HLA-G mAbs used in this study

clearly recognized intron 4 retaining sHLA-Gl isoform, since only

this species was produced by JAR and 221 transfectants. In con-

trast, they were unlikely to detect the intron 4 retaining sHLA-G2

shorter form lacking the o2 domain, since W6/32 (used as coating

or detection mAb) was shown to recognize an epitope in the a2

domain (31), absent in sHLA-G2 (7).

sHLA-Gl has been detected in amniotic fluid, which is in accord-

ance with several previous reports (18, 20). It was shown that at

least part of the sHLA-G in amniotic fluid derived from HLA-G

cleaved from the membrane (18). The reactivity of 16G1 mAb

specifically raised against intron 4 amino acid that we observed in

some amniotic fiuid samples demonstrated that, in addition to shed-

ded or cleaved sHLA-G, intron 4 retaining sHLA-G isoforms were

also present.

516 nssue Antigens 2000: 66: 510-518

Fournel et al : HLA-G mAb reactivity

We did not find any sHLA-G in cord blood, which is in contrast

with other findings (19). However, knowing that human serum does

contain p2m-free class I HC (39) and that the HLA-G mAbs used in

this study could not detect such a form, it cannot be excluded that

p2m-free sHLA-G HC were present in cord blood.

Both types of analysis, performed in ELISA and fiow cytometry,

came to the same conclusion that 87G mAb recognizes an epitope

localized on the a l domain of HLA-G HC. A similar conclusion was

drawn from previous investigations (6,40). On the contrary, absence

of competition between 87G, BFL.1 and MEM-G/9 suggest that the

antigenic determinants recognized by these HLA G mAbs are dif-

ferent.

Such HLA-G mAbs should be useful tools to investigate the

possible immunoregulatory functions sHLA-G might exert in nor-

mal and pathological conditions (41. 42). Further epitope mapping

study of these HLA G reagents together with cristallography of

HLA-G molecule may help in more critical interpretation of such

future functional studies.

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