1932 Richard Apps et al. with HLA-C transfectants (Fig. 7A) [34]. Consistent with previous results [36], mAb specific to HLA-I or LILRB1/2 molecules that are known to block HLA-G:LILRB binding (Fig. 4) [33, 37] reversed the inhibition of proliferation observed in the presence of HLA-G (Fig. 7A). To test whether HLA-DR+ cells expressing LILRB were responsible for the HLA-G-mediated inhibition of proliferation, we next depleted these cells from responder populations. In an MLR, responder T cell proliferation results both from direct stimulation by the allogeneic HLA of stimulator cells as well as from indirect stimulation of allogeneic stimulator cell anti- gens presented by responder APC. We found that if HLA- DR+ cells are depleted from the responding PBMC population (Fig. 7C), the proliferative response to the parent cell line is reduced as expected, since only directly reacting T cells can respond (Fig. 7B). However, there is no additional inhibitory effect in the presence of HLA-G after removal of HLA-DR+ cells from the responding PBMC. Therefore, we conclude that HLA-G only inhibits the indirect APC-dependant stimulation of proliferation. Taken together, our findings indicate that the interaction of HLA-G dimers with LILR expressed by HLA-DR+ cells is directing myelomonocytic cells to down-regulate T cell proliferative responses. Discussion We have shown that a significant proportion of HLA-G molecules present at the surface of normal human trophoblast cells in vivo exist as a dimer. Previously, there were concerns that these dimers were merely an artefact of overexpression in transfectants, particularly as we and others have found that they are difficult to detect on JEG-3 choriocarcinoma cells [17, 19]. This homodimer is of the conventional, b2m-associated HLA-I complex. Crystal structure [20] and mutational studies [18] have demonstrated that the dimeric form of HLA-G is formed by disulphide bonding between cysteine 42 residues of the heavy chain a1 domain. This b2m-associated dimeric complex is likely to be unique to HLA-G, as among HLA-I molecules a cysteine at position 42 is specific to HLA-G. HLA-B27, for example, can also form disulphide-linked multimers in vitro, but these are mediated by bonding between cysteines at position 67 and require heavy chain dissociated from b2m [22]. The unique conformation of HLA-G as a homodimer of conventional, b2m- associated class I complexes crucially maintains recog- nition by the LILRB1 receptor because co-crystal structures confirm that a significant proportion of this receptor's interface with HLA-I molecules is with the b2m molecule [15]. In healthy individuals, HLA-G expression is restricted to fetal trophoblast cells that f 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Eur. J. Immunol. 2007. 37: 1924–1937 invade the uterine decidua and myometrium in placentation [11]. It is possible that HLA-G dimers could act as a pregnancy-specific signal modulating the local uterine immune response. The HLA-G dimers we have demonstrated at the surface of EVT will come into contact with virtually all the maternal leukocytes of the decidua [11]. The dominant population of NK cells may bind endocytosed HLA-G via KIR2DL4 [12], although it is not known if HLA-G dimerisation affects KIR2DL4 binding. In con- trast, preferential binding of the HLA-G dimers to LILR molecules has been well described. The inhibitory receptors, LILRB1 and LILRB2, both bind all HLA-I molecules, with a higher affinity for HLA-G attributed to sequence differences in the a3 domain [16]. HLA-G free heavy chains observed at the surface of transfectants reduce LILRB1 binding to b2m-associated HLA-G complexes and potentially modulate LILRB1 stimulation [19]. Recently, the increased avidity of HLA-G dimer- isation has been shown to substantially increase binding to LILRB. A LILRB1-Fc fusion protein bound more strongly to wild-type HLA-G than to HLA-G where the cysteine is mutated to a serine at position 42, abrogating dimerisation [18, 19]. This was supported by surface plasmon resonance measurements of recombinant monomeric and dimeric HLA-G complexes binding LILRB molecules [20]. The increased binding avidity of HLA-G dimers translates into augmented signalling through LILRB1. This was shown by LILRB1-mediated inhibition of serotonin release triggered by IgeR, inhibition of NK killing [18] and a LILRB1 chimera NFAT-GFP reporter cell assay [20]. We therefore investigated LILR expression in the decidua and binding to HLA-G dimers on trophoblast cells. Previous studies in humans have only demon- strated binding of HLA-G tetramers to peripheral myelomonocytic cells [38]. We detected LILRB1 and B2 expression by all decidual HLA-DR+ cells, consistent with results detecting expression on decidual macro- phages [39]. In contrast to peripheral myelomonocytic cells, LILRB1 and not LILRB2 appears to be predomi- nantly expressed in the decidua. LILRB1 was also detected on 20% of NK and 10% of T cells in the decidua. This is similar to the expression on peripheral PBMC [13, 40], although the function of LILRB1 on these other leukocytes remains unclear. Having detected LILRB expression by decidual myelomonocytic cells, we demonstrated binding of LILRB1-Fc fusion proteins to HLA-G at the surface of normal trophoblast cells. The specificity of this interac- tion was confirmed by blocking mAb to both LILRB1 and HLA-I molecules. In keeping with the previous findings that LILRB1 preferentially binds to HLA-G dimers, no binding was demonstrable with JEG-3 cells that mainly displayed conventional monomeric HLA-G. A more www.eji-journal.eu