Eur. J. Immunol. 2007. 37: 1924–1937 Receptors for HLA-G have been described on NK and myelomonocytic cells derived from peripheral blood. The NK cell receptor is KIR2DL4, a member of the killer immunoglobulin-like receptor (KIR) family. NK cells are reported to endocytose HLA-G into KIR2DL4-containing compartments, and the subsequent interactions mod- ulate NK cell cytokine secretion [12]. LILRB1 and LILRB2 are inhibitory receptors of the leukocyte immunoglobulin-like receptor (LILR) family which are mainly expressed by DC and macrophages [13]. LILRB1 and B2 bind most HLA-I molecules [13–15], but the highest affinity is for HLA-G [16]. Recently, it was discovered that HLA-G can exist as a dimer in addition to the conventional heterotrimeric HLA-I molecule conformation. Dimers of HLA-G were first described with recombinant protein in vitro [17], then later observed on the surface of both transfected cells [17, 18] and the choriocarcinoma cell line JEG-3 [19]. The HLA-G dimer is linked by disulphide-bonding between cysteine 42 residues of the heavy chain a1 domain, an exposed extracellular cysteine not involved in intramolecular Ig superfamily domain formation [18, 20]. Comparison with other HLA-I gene sequences shows that a cysteine residue at this position is unique to HLA-G [18]. Amongst other primates, cysteine 42 is conserved in the MHC-G genes of chimpanzees and gorillas that are also non-polymorphic and presumably act as functional homologues. However, in the orang-utans, Old and New World monkeys where MHC-G orthologues function as classical MHC-I mole- cules or are pseudogenes [21], cysteine 42 is substituted to a serine which has been shown to abrogate dimerisation [9, 18]. The outstanding questions are whether these HLA-G dimers are formed in vivo on normal trophoblast cells and what their function might be at the maternal-fetal interface. We now show that a significant proportion of the HLA-G present on the surface of normal first- trimester trophoblast cells exists as a dimer. This complex is a homodimer of two conventionally b2m- associated HLA-G molecules. We demonstrate LILRB1 and B2 expression by decidual myelomonocytic cells and show that a LILRB1-Fc fusion protein preferentially binds to the dimeric form of HLA-G on normal trophoblast cells. Therefore, HLA-G dimers can provide a trophoblast-specific signal to decidual myelomonocy- tic leukocytes via LILRB1. We present evidence that this interaction leads to modulation of dendritic cell (DC) function and suppression of allogeneic lymphocyte proliferation. In this way, HLA-G potentially modifies the maternal local immune response to fetal trophoblast cells. Immunomodulation 1925 Results HLA-G forms a disulphide-linked homodimer of the conventional, b2m-associated HLA class I complex To detect HLA-G dimers, surface proteins of 721.221 cells transfected with HLA-G1 cDNA were biotinylated and immunoprecipitated with a panel of HLA-I-reactive antibodies. HLA-G-specific MEM-G/11, 87G and G233 recognise the b2m-associated class I structure. W6/32 is also conformationally dependant but binds all HLA-I molecules. The immunoprecipitated complexes were first analysed by denaturing reducing PAGE, followed by Western blotting for biotin-labelled surface proteins. As expected, both HLA-G-specific and pan-class I antibo- dies detected an HLA-G band at 39 kDa, as well as co- immunoprecipitating the 13.7-kDa b2m molecule (Fig. 1A). However, when analysed under denaturing but non-reducing conditions, an additional 80-kDa band appears, indicating that some of the HLA-G is present as a disulphide-linked homodimer (Fig. 1B). Densitometry measurements of complexes detected by G233, MEM-G/ 11, W6/32 and BBM.1 estimate that 40% of the HLA-G molecules are in the dimeric form. HLA-G dimerisation may alter or obscure the 87G epitope because this antibody preferentially binds the monomeric form of HLA-G compared to other antibodies in the panel. Under non-reducing conditions, both 39- and 80-kDa bands can be co-immunoprecipitated by the anti-b2m antibody, BBM.1, indicating that the HLA-G dimers are b2m associated (Fig. 1B). Therefore, HLA-G dimers are of the conventional class I complex and not free heavy chains as described previously for HLA-B27 [22]. All these experiments were repeated with 721.221 cells trans- fected with genomic HLA-G (as opposed to cDNA) and the rodent cell line, Ltk, transfected with human b2m and HLA-G with similar results (not shown). The characteristics of HLA-G, when expressed by a trophoblast cell line, were investigated using the choriocarcinoma cell line, JEG-3. This is similar to normal EVT in that it expresses HLA-C, HLA-G and HLA-E whilst lacking HLA-A and HLA-B [10, 23, 24]. Under reducing conditions, an HLA-G band at 39 kDa, with an additional 45-kDa molecule, was detected by W6/32 (Fig. 1C). The 45-kDa species was confirmed as a classical HLA-I molecule by immunoprecipitation with T149 and B1.23.2, antibodies specific for HLA-C on these cells (Fig. 1D, E). In contrast to the findings with HLA-G transfectants, no 80-kDa band was observed under non-reducing conditions unless the blot was overexposed to the point that significant background staining was apparent (Fig. 1D, E). Densitometry measurements suggest that less than 10% of the HLA-G molecules are dimerised. Thus, the conventional f 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu