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* Lautenberg Center for General and Tumor Immunology, Hebrew University-Hadassah Medical School, Jerusalem, Israel;
Department of Obstetrics and Gynecology, Hadassah University Hospital, Mount Scopus, Jerusalem, Israel; and
Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Praha, Czech Republic
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The HLA-G protein possesses some unusual characteristics, including restricted expression (mainly on EVT cells), limited polymorphism, and alternatively spliced mRNA variants (8). One of the major functions suggested for the HLA-G protein was the inhibition of NK killing. Indeed, the expression of HLA-G on the cell surface protected susceptible target cells from NK-mediated cytotoxicity (9, 10, 11, 12, 13).
Several NK receptors were suggested as being able to recognize HLA-G (14), and recently two such receptors were identified. The first is KIR2DL4 (CD158d), which is expressed either on all NK cells derived from PBLs (15) or only on decidual NK cells (16). The second is leukocyte Ig-like receptor-1 (LIR-1; also designated ILT-2 or CD85j), a member of the LIR/ILT family of proteins, which is expressed on most myelomonocytic cells, B cells, dendritic cells, and subsets of T and NK cells (17, 18).
LIR-1 binds to a wide range of HLA class I molecules, including HLA-G (19), and delivers a negative signal that inhibits killing by NK and T cells (20). It contains four extracellular Ig-like domains and four intracellular immunoreceptor tyrosine-based inhibitory motifs, which transduce the inhibitory signal by binding the Src homology domain-containing tyrosine phosphates (21, 22). In the present study we focused on understanding the molecular basis for the LIR-1 and HLA-G interactions.
Comparison between the sequence of the HLA-G molecule and other class I MHC molecules revealed two unique cysteine residues located in positions 42 and 147 in HLA-G extracellular domains 
1 and 2, respectively. The possible influence of these cysteines on NK function was investigated by mutating both cysteine residues to serines. The mutants were transfected into 721.221 cells and tested in binding, cytotoxicity, and serotonin release assays. A dramatic decrease in the binding of the LIR-1 Ig to the mutated HLA-G transfectants was observed compared with the wild-type molecule. Accordingly, the mutated HLA-G transfectants were less effective in inhibition of NK killing or of RBL/LIR-1 Fc
R-induced serotonin release.
Immunoprecipitation experiments with anti-HLA-G Abs and analysis by two-dimensional gel electrophoresis suggested a possible role for the cysteine residues in the expression of high molecular weight complexes of HLA-G protein on the cell surface. The cysteine residue located in position 42 is shown to be critical for the expression of HLA-G complexes on the cell surface. Such HLA-G complexes most likely bind with increased avidity to the LIR-1 and consequently efficiently trigger the inhibitory function of LIR-1.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The cell lines used in this work are the EBV-transformed class I MHC-negative human B cell line 721.221 (23), 721.221 transfectants, RBL (rat basophilic leukemia) cells, and RBL cells transfected with LIR-1 (24).
Point mutations in HLA-G cDNA were performed by PCR, using the following primers: 5' primer (including the KpnI site and Kozak sequence), 5'-GGGGTACCCCGCCGCCACCATGGTGGTCATGGCACCACGA-3; 3' mut S42C primer, 5'-CATCCTCGGAGACGCCGAGTC-3'; 3' mut C147S primer, 5'-CATTGGCCGCCTCAGACTTGCGCTTGGAG-3'; 3' primer (including the EcoRI site), 5'-CGGAATTCCGTCAATCTGAGCTCTTCTTTCTCCACAG-3'; 5' mut S42C primer, 5'-CGACTCGGCGTCTCCGAGGATG-3'; and 5' mut S147C primer, 5'-CTCCAAGCGCAAGTCTGAGGCGGCCAATG-3'. cDNA was cloned into the PCDNA3 vector (Invitrogen, San Diego, CA) and transfected into 721.221 cells as previously described (25).
NK cells were isolated from PBL using the human NK cell isolation kit and the autoMACS instrument (Miltenyi Biotec, Bergisch-Gladbach, Germany). NK cells were kept in culture as previously described (25).
All mAbs used in this work were generated in mice, including W6/32 mAb (IgG2a), directed against class I MHC molecules, anti-HLA-G mAbs, MEM-G/09, MEM-G/13B, and MEM-G/01 (all of which are IgG1, produced and characterized in the Prague laboratory), anti-trinitrophenyl (anti-TNP) IgE (BD Biosciences, Mountain View, CA), anti-LIR-1 mAb-HPF1 (IgG1), anti-CD99 mAb-12E7 (IgG1), HC10 (IgG2a) directed against class I MHC 
2-microglobulin-free heavy chain, and anti-2-microglobulin mAb BBM1 (IgG2b). The W6/32 mAb was also used in the form of F(ab')2. Digestion and purification of the F(ab')2 were performed using the ImmunoPure F(ab')2 preparation kit (Pierce, Rockford, IL) according to the manufacturer’s instructions.
Isolation of decidual NK cells
The institutional board of The Hadassah Organization approved obtaining deciduae and placenta from elective pregnancy termination procedures, according to the principles of the Helsinki Declaration. Decidual lymphocytes were isolated as previously described (26, 27, 28). Briefly, the tissue was trimmed into 1-mm pieces and enzymatically digested for 20 min, using vigorous shaking, with 1.5 mg of type I DNase and 24 mg of type IV collagenase present in 15 ml of RPMI 1640 medium. This procedure was repeated three times. After an additional 5-min incubation at room temperature without shaking, the supernatants were collected and loaded on Ficoll-density gradient to purify the lymphocyte population. NK cells were purified using the human NK cell isolation kit and the autoMACS instrument (Miltenyi Biotec). Positive (NK and NKT cells) and negative (T cells) fractions were collected and cloned (1 cell/well) in the presence of IL-2.
Cytotoxicity assay
The cytotoxic activity of NK cells against the various target cells was assessed in 5-h 35S release assays as previously described (25). In experiments in which mAb were included, the final mAb concentration was 10 µg/ml for W6/32, F(ab')2 of W6/32, HC10 and 12E7 or 1/1000 ascities dilution for anti-LIR-1 mAb (HPF1). In all the presented cytotoxic assays, spontaneous release was <25% of maximal release.
Ig fusion proteins
The cDNA encoding for the LIR-1 Ig fusion protein was provided by Dr. Cosman (Immunex, Seattle, WA). The production of LIR-1 Ig fusion protein by COS-7 cells, its purification on protein G column, and the FACS analysis for its expression were performed as previously described (29, 30, 31). The LIR-1 Ig protein was characterized by a single protein band on nonreduced SDS-PAGE and was routinely tested for its degradation.
Immunoprecipitation and two-dimensional SDS-PAGE
Cells (106/ml) were washed four times with cold PBS containing 1 mM MgCl2 and 0.1 mM CaCl2 and then biotinylated with EZ-Link Sulfo-NHS-LC-Biotin (Pierce 21335) for 30 min at 4°C. Cells were washed four times to remove unconjugated reagent and detergent-solubilized on ice in lysis buffer (PBS containing 150 mM NaCl, 50 mM Tris (pH 7.6), 0.5% Nonidet P-40, 9 mM iodoaceteamide, 5 mM EDTA, 1 mM PMSF, 10 µM leupeptin, and pepstatin A). Cell lysates (10 x 106/sample) were precleared overnight at 4°C with protein A-Sepharose beads (Zymed Laboratories, San Francisco, CA) precoated with rabbit anti-mouse IgG. Precleared lysates were then immunoprecipitated overnight at 4°C with MEM-G/09, MEM-G/13B, or BBM1 mAbs. The immunoprecipitates were washed with lysis buffer, and biotinylated proteins were eluted in the presence of SDS under nonreducing conditions. The nonreduced samples were subjected to first-dimension SDS-PAGE on 8–10% gels in special tubes (0.5 cm in diameter). The gels, each representing an immunoprecipitate, were pulled out of the tubes and reduced with 32 mM 1,4-DTT (Roche, Mannheim, Germany) at room temperature for 2 h, then placed on top of the second dimension SDS-PAGE on 8% gels, followed by elctroblotting. The blotted biotinylated proteins were visualized by streptavidin-HRP conjugate (Amersham Pharmacia Biotech, Little Chalfont, U.K.), and HLA-G chains were specifically detected by mAb MEM-G/01, followed by goat anti-mouse Ig HRP (Sigma-Aldrich, St. Louis, MO) using ECL detection.
Serotonin release, cell haptenization, and conjugation
RBL cells (106/ml) were pulsed for 3 h at 37°C in RPMI 1640/10% FCS containing 2 µCi/ml [3H]serotonin (DuPont-NEN, Boston, MA). Cells were washed, incubated at 37°C for an additional hour, then washed again and resuspended at 4 x 106 cells/ml. For cell haptenization, 721.221 cells (5 x 106 cells/ml) were incubated for 15 min at 37°C in PBS containing 0.5 mM trinitrobenzene sulfonic acid (Sigma-Aldrich), then washed twice in 50 ml of PBS and resuspended in RPMI 1640/10% FCS medium at 8 x 106 cells/ml. To measure stimulation-dependent serotonin release, 4 x 105 haptenized cells were cocultured with 2 x 105 [3H]serotonin-loaded RBL cells in the presence of 1 µg/well anti-TNP IgE (BD Biosciences) for 1 h in round-bottom, 96-well plates in a final volume of 150 µl. After 1 h at 37°C, 50 µl of supernatant was collected from each well, and radioactivity was measured. Serotonin release was calculated as % serotonin release = [(cpm sample - cpm spontaneous release)/(cpm total - cpm spontaneous release)] x 100. Total serotonin release was measured after incubation of the cells with 0.1 M NaOH (22).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The expression of HLA-G is mainly restricted to EVT cells. This is in marked contrast to other classical class I MHC proteins that are expressed on the surface of almost every nucleated cell. Comparison of the sequence of HLA-G to other class I MHC molecules revealed two unique cysteine residues in positions 42 and 147 that are localized in extracellular domains 1 and 2, respectively (Fig. 1).
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The results shown above demonstrate that the cysteine mutations did not affect mAb binding. One of the receptors recognizing HLA-G is LIR-1 (ILT-2) (17, 18). We next tested whether the generated cysteine mutations would affect LIR-1 Ig binding. Efficient LIR-1 Ig binding was observed to the wild-type HLA-G transfectants. In contrast, the LIR-1 Ig binding to both of the mutated HLA-G proteins was significantly decreased (Fig. 3). Importantly, the level of HLA-G protein expression of all HLA-G transfectants was similar, as detected by the W6/32 mAb or the anti-HLA-G mAbs (see Figs. 2 and 3 legend and data not shown). Similar results were obtained when the LB33 Mel B1 cells expressing the various HLA-G proteins were used (data not shown). These results indicate that the cysteine residues are important for efficient binding of LIR-1 to the HLA-G molecule.
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3 domain of the molecule (32). In contrast, the unique cysteine residues of the HLA-G protein are located in the 1 and the 2 domains. Nevertheless, we tested whether the mutations in the cysteine residues altered the HLA-G conformation by using the conformationally dependent W6/32 mAb. W6/32 mAb was reported to bind to a combination of epitopes composed of residues in the 2-microglobulin, 2, and 3 domains of the class I MHC molecule (33, 34). Preincubation of the targets with W6/32 mAb abolished the binding of LIR-1 Ig to all the HLA-G transfectants (Fig. 4). As described above, the level of HLA-G protein expression detected by W6/32 mAb or the anti-HLA-G mAbs on all the HLA-G transfectants was similar (data not shown). The anti-HLA-G mAbs MEM-G/09 and MEM-G/13B could not block LIR-1 Ig binding (data not shown). The binding site for both the anti-HLA-G mAbs is as yet unknown. These results suggest that the epitopes recognized by the W6/32 mAb were not affected by the cysteine mutations.
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The functional relevance of the above observation, suggesting the involvement of the cysteine residues in efficient HLA-G recognition by the LIR-1 receptor, was tested using NK killing assays. NK clones were generated as described in Materials and Methods. One hundred and twenty independent NK clones were tested in killing experiments against HLA-G-transfected target cells. About 20% of the NK clones tested were inhibited by HLA-G, and the expression of the LIR-1 receptor was observed on the surface of all of them. One representative clone expressing the LIR-1 receptor is shown in Fig. 5A.
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In agreement with the binding experiments, the inhibitory effect of HLA-G was significantly reduced when both the 42 and 147 cysteine mutants were assayed against the NK clones (Fig. 5, B and C). The still observed minimal inhibition was the result of LIR-1 interaction with the mutated HLA-G proteins, as the anti-class I MHC mAb W6/32, the F(ab')2 of W6/32 mAb, or the anti LIR-1 mAb abolished this modest inhibition (Fig. 5, B and C).
HLA-G mainly functions in the maternal-fetal interface where the EVT cells are in direct contact with maternal lymphocytes, of which 70% are decidual NK cells. These cells are characterized by the CD16- CD56bright phenotype (35, 36). We therefore subsequently tested the decidual NK clones in killing against the various HLA-G transfectants. One representative decidual NK clone expressing the LIR-1 receptor is shown in Fig. 5D. In general, and in agreement with previous results (16), decidual NK cells have a decreased killing activity against class I MHC-negative target cells. In accordance with the killing assays performed with peripheral NK clones, efficient inhibition of lysis mediated by the .221/HLA-G transfectants was observed, while the mutations in both 42 and 147 cysteine residues resulted in an impaired inhibitory effect (Fig. 5, E and F). Blocking the target cells with W6/32 mAb or the effector cells with anti LIR-1 mAb had little or no effect and did not restore the NK-mediated lysis (Fig. 5, E and F). This is probably because virtually all decidual NK cells express one or more inhibitory receptors capable of interacting with the HLA-G molecules, including LIR-1 and KIR2DL4 (16), or with the HLA-E molecule, including CD94/NKG2A (24, 37). Among these three receptors, KIR2DL4 was demonstrated to play a predominant role in preventing the NK-mediated lysis of HLA-G by decidual NK cells (16). The W6/32 mAb, which blocks the interaction between LIR-1 and HLA-G, probably does not interact with the binding site of the KIR2DL4 receptor on HLA-G, and therefore restoration of killing by decidual NK clones could not be observed.
To directly test the interaction between LIR-1 and the various HLA-G transfectants, we used the RBL cells transfected with LIR-1 (18, 22, 24). RBL/LIR-1 transfectants were labeled with [3H]serotonin, incubated with anti-TNP mAb of the IgE isotype, and cocultured with the various .221/HLA-G transfectants, previously haptenized with TNP. As shown in Fig. 6, FcR-induced serotonin release was inhibited when RBL/LIR-1 cells were incubated with .221/HLA-G cells. The inhibition was less pronounced when HLA-G C147S cells were used, and only a slight inhibition was observed when HLA-G C42S cells were assayed (Fig. 6). Incubation of all HLA-G transfectants with parental RBL cells showed no significant change in serotonin release (Fig. 6).
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Several explanations might account for the decreased LIR-1 recognition of the mutated HLA-G protein. It is unlikely that both cysteine residues are in contact with LIR-1, as the LIR-1 binding site on HLA-G is located in the 3 domain (32). It is also unlikely that the protein conformation was grossly altered due to the mutations, since conformationally dependent mAb recognized all HLA-G transfectants in a similar manner (Fig. 2). It is still possible, however, that the cysteine residues are involved in the formation of HLA-G covalent oligomers present on the cell surface that may exhibit increased avidity for LIR-1.
To test this hypothesis, wild-type and mutated .221/HLA-G transfected cells were cell surface biotinylated, lysed, and then immunoprecipitated as described in Materials and Methods. The lysates were immunoprecipitated using conformational anti-HLA-G mAbs, MEM-G/09 or MEM-G/13B (Fig. 7), and then were analyzed by two-dimensional (nonreduced/reduced) SDS-PAGE. To prevent post-lysis disulfide bond formation of HLA-G molecules, iodoacetamide was included in the cell lysis buffer in all immunoprecipitation experiments. As shown in Fig. 7A, the wild-type HLA-G is expressed on the cell surface as homotrimers, homodimers, or monomers. Their molecular masses were, as expected, 
120, 80, and 40 kDa, and the ratios of their expression (calculated by densitometric analysis) were 0.1, 0.5, and 1 for the homotrimers, homodimers, and monomers, respectively. The HLA-G oligomers can be found on the cell surface in different ratios, suggesting that the formation of HLA-G oligomers is a dynamic process. For example, when the amount of the monomers on the cell surface decreased, the amount of the dimers increased respectively, and the various HLA-G oligomers can be found on the cell surface in ratios of 0.1, 1, and 0.6 for the homotrimers, homodimers, and monomers, respectively. The HLA-G mutants were also tested in the immunoprecipitation experiments. Mutating the cysteine located in position 147 completely abolished HLA-G trimer formation and expression on the cell surface, leaving dimers and monomers only in ratios of 0.86 and 1, respectively (Fig. 7B). Importantly, mutating the cysteine residue located in position 42 abolished both the formation of HLA-G trimers and dimers, leaving the expression of monomers only (Fig. 7B). The control immunoprecipitation of .221/HLA-Cw3 proteins showed only the monomeric pattern of expression of HLA-Cw3 on the cell surface (Fig. 7B). No protein bands were observed when the control .221 cells were used in the immunoprecipitation experiments (data not shown).
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These results demonstrate that the cysteine-dependent complexes of HLA-G are expressed on the cell surface and that these complexes may play a crucial role in recognition by the LIR-1 receptor.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study we show that the two unique cysteine residues located in positions 42 and 147 are involved in the formation and expression of HLA-G homodimers and homotrimers on the cell surface. Mutating these cysteine residues to serines dramatically impaired recognition of the HLA-G protein by the LIR-1 receptor. This resulted in impaired binding of the LIR-1 to HLA-G, decreased inhibition of NK lysis by HLA-G, and decreased inhibition of FcR-induced serotonin release from RBL/LIR-1 cells. Importantly, the cysteine mutations probably did not alter the conformation of the HLA-G protein (Figs. 2 and 4). The cysteine residues probably also play an important role in the recognition of HLA-G by other receptors, such as KIR2DL4, since LIR-1-independent inhibition was observed when decidual NK clones were assayed against HLA-G, while virtually no inhibition was observed when both cysteine mutants were used (Fig. 5).
The expression of HLA-G oligomers on the cell surface can be schematically diagrammed, as shown in Fig. 8. The Cys residue located in position 42 is critical for the formation of HLA-G oligomers. Mutating this residue abolished the formation of dimers and trimers, whereas mutating residue 147 only partially affected the formation of HLA-G dimers (Figs. 7B and 8B). The structure of the HLA-G protein is as yet unsolved; however, the critical role of residue 42 is supported by the superimposition of the HLA-G molecule onto the HLA-A2 crystal structure. Such superimposition reveals that the Cys42 residue is located on a loop projecting out from the MHC class I molecule (39). The Cys147 residue, on the other hand, is predicted to be located on a position pointing into the groove. The concealed position of Cys147 residue probably contributes to its limited ability to interact with another Cys147; however, interactions with Cys42 can occur (Fig. 7). The trimer formation might occur either because the superimposition does not accurately reflect the HLA-G structure or because the HLA-G on the cell surface might be found in a slightly different conformation, enabling the interaction of Cys147 with Cys42. In addition, the disulfide bond that is generated between Cys147, which points into the groove, and Cys42 might affect either peptide composition or peptide stability of the HLA-G proteins, resulting in limited amounts of HLA-G trimers on the cell surface. These HLA-G trimers might be stabilized by the two other HLA-G proteins that are connected via Cys42.
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The proposed model does not rule out the existence of smaller amounts of higher HLA-G multimers on the cell surface or exclude the presence of intrachain disulfide bridges formed by these cysteine residues. Although the observed complexes are likely to be HLA-G oligomers (Fig. 7), our results and the proposed model do not preclude the existence of another as yet unknown protein(s) that might bind the HLA-G molecule via disulfide bonds and interact, either directly or indirectly, with the LIR-1 receptor.
Supporting our results, a recent report suggested the presence of disulfide-linked dimers of HLA-G on the cell surface that are formed via the Cys42 residue (39). The role of residue 147 or the functional relevance of these dimers was not investigated (39). Importantly, in this study we show that both dimers and trimers of HLA-G are expressed on the cell surface, that both Cys42 and Cys147 residues are involved in the formation of these high molecular HLA-G complexes on the cell surface, and that these complexes play a crucial role in recognition by the LIR-1 receptor.
Although a relatively small amount of these trimers is found on the cell surface (Fig. 7, A and B, and Fig. 8A), they probably play a critical role in the LIR-1 recognition of HLA-G. This is indicated by the fact that both cysteine mutations give a similar pattern of decreased recognition of the LIR-1 receptor, and that the common phenomenon for both the mutations was the absence of expression of HLA-G homotrimers on the cell surface (Figs. 7 and 8).
HLA-G is the only class I MHC protein reported to date that might be expressed on the cell surface as trimers. A recent report showed that aberrant HLA-B27 disulfide-linked dimers may be present on the cell surface under some circumstances (40); in this case, the disulfide bond was formed between two extracellular 1 domains (Cys67).
Our findings may reconcile the discrepancies previously reported regarding recognition of HLA-G by NK cells. While it is well established that inhibition of NK lysis can be observed when using intact cells expressing the HLA-G protein on the cell surface (10, 14, 15, 37) (Fig. 5), no recognition of NK cells by HLA-G was observed when HLA-G tetramers were used (41). We suggest that this absence of recognition by HLA-G tetramers might be due to the special organization of the HLA-G proteins on the cell surface (Figs. 7 and 8). As we demonstrate in this study, an efficient recognition of HLA-G by NK cells might depend on the presence of high molecular complexes of HLA-G on the cell surface. It is possible that when the HLA-G proteins are expressed as tetramers, they are not found in the most suitable arrangement to be optimally recognized by the NK cell receptors.
The HLA-G protein is expressed on the placenta when HLA-A and -B class Ia molecules are largely absent (3, 4). This may indicate its important role in the immunological tolerance of the fetal semiallograft facing the maternal immune system. It was therefore surprising to learn that the binding affinities of the inhibitory receptor LIR-1 to HLA-G are low (kilodaltons in the micromolar range) (32). It had also been previously suggested that high local concentrations of class I molecules would have to be presented to trigger the inhibitory function of LIR-1 (32). In this study we show that HLA-G has probably developed a unique mechanism to enhance LIR-1 binding by the expression of disulfide-linked oligomeric structures on the cell surface. This pattern of expression might increase LIR-1 avidity to the HLA-G protein and enable efficient inhibition of NK cells.
Similar effects of increased avidity were observed when including a free cysteine in the KIR2DL1 receptor. This disulfide-linked KIR2DL1 bound its ligand, the HLA-Cw4, at a molar ratio of one dimer to one HLA-Cw4 molecule. In addition, the dimer bound more tightly to HLA-Cw4, suggesting the occurrence of a second binding event that increases the overall avidity of the KIR2DL1 dimer for HLA-C (42).
The HLA-G protein has unique characteristics that distinguish it from other HLA class I molecules, such as tissue-restricted distribution, limited polymorphism, and impaired spontaneous endocytosis (43). Here we present another unique characteristic of HLA-G, which is the presence of high molecular complexes of HLA-G on the cell surface, that is absent in any other class I MHC molecules. This might be especially important in the maternal-fetal interface, where there must be strict control of the interaction between HLA-G and the inhibitory receptors that bind it.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Ofer Mandelboim, Lautenberg Center for General and Tumor Immunology, Hadassah Medical School, 91120 Jerusalem, Israel. E-mail address: oferman{at}md2.huji.ac.il
3 Abbreviations used in this paper: EVT, extravillous cytotrophoblast; LIR-1, leukocyte Ig-like receptor-1; TNP, trinitrophenyl.
Received for publication December 10, 2002. Accepted for publication May 29, 2003.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2-microglobulin affects binding of class I MHC molecules by the W6/32 antibody. Immunogenetics 49:312.[Medline]
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