Eur. J. Immunol. 2007. 37: 1924–1937 direct demonstration that LILRB1 interacts preferen- tially with HLA-G dimers on trophoblast cells was shown by the LILRB1-Fc fusion protein predominantly im- munoprecipitating dimeric HLA-G from normal tropho- blast cells. These results indicate that LILRB1 would mainly bind dimeric HLA-G on the trophoblast in vivo, which could represent a mechanism allowing LILRB1+ decidual APC to discriminate between the fetal tropho- blast and maternal cells expressing conventional HLA-I molecules. We have shown that soluble HLA-G5 immunopreci- pitated from the culture supernatant of transfected cells can also form a b2m-associated dimer. However, we were not able to immunoprecipitate any form of soluble HLA-G from culture supernatants of primary trophoblast cells or pre-implantation embryos, consistent with previous reports describing only very low levels of HLA-G5 transcripts [3, 4] and shed HLA-G1 protein in vivo [4]. We next investigated the possible function of cell surface HLA-G dimers stimulating LILRB1 on myelo- monocytic cells in the decidua. As shown previously, in the presence of HLA-G, MDDC still responded to various activation stimuli to mature to an antigen-presenting phenotype [41]. Another study did show a slight reduction in DC maturation markers in the presence of HLA-G tetramers [42], although IL-10 was used during MDDC differentiation in this study and is known to inhibit MDDC maturation [43, 44]. In addition to immature DC, antigen presentation by activated DC can also result in suppression of adaptive immune responses, an effect likely to be mediated by concomitant cytokine production [45, 46]. Therefore, we measured cytokine production and found that induction of IL-6 and IL-10 was increased by MDDC activated in co-culture with HLA-G+ compared to HLA-I transfectants, whereas inflammatory cytokines such as TNF-a were unaffected. IL-10 has a well characterised role as an immunosup- pressive cytokine and has been implicated in the function of regulatory T cells [47]. IL-6 is pleiotropic, but has also been shown to have immunosuppressive effects with DC [48]. Indeed, non-inflammatory gut mucosal DC co-cultured with intestinal epithelial cells produce IL-6 and IL-10 even in the presence of a pathogen [49]. This situation has clear resonance with the local environment in the uterine mucosa where DC are exposed to infiltrating HLA-G+ trophoblast cells. These results are also consistent with observations from the myelomonocytic cell line U937, which increased IL-10 and TGF-b1 production after treatment with HLA-G [50]. The most likely functional effect of HLA-G dimers on trophoblast binding to decidual HLA-DR+ cells would be down-regulation of damaging maternal T cell responses to allogeneic placental cells. Indeed, in keeping with previous reports, we observed a diminished proliferative f 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Immunomodulation 1933 response of allogeneic lymphocytes when HLA-G, compared to classical HLA-I molecules, were present on stimulator cells [34–36]. Our findings indicate that HLA-G is mediating the inhibitory effect on lymphocyte proliferation by binding to LILRB1 and B2 on responding APC. By removal of HLA-DR+ cells from the responder population, we found that HLA-G had no effect on the direct T cell response. This observation is highly relevant to the situation in vivo because trophoblast cells do not express allogeneic HLA-A, HLA-B or HLA-II molecules that could be perceived directly by maternal T cells [24]. Thus, in vivo, T cell recognition of any allogeneic trophoblast- or placenta-specific molecules is likely to be via indirect presentation by decidual HLA-DR+ cells. There is increasing evidence for the role of LILRB in inducing tolerogenic DC [42, 51–53], and our study demonstrates how this interaction has particular relevance to the placental bed, the only site where HLA-G dimers are physiologically abundant. There are potential parallels with mechanisms of other immuno- priviledged sites, where tissue-specific molecules such as thymic stromal lymphopoietin and vasoactive intestinal peptide modulate DC function [49, 54, 55]. We have only studied the functional effect of DC exposed to HLA-G on allogeneic lymphocyte proliferation. How- ever, the altered cytokine production of these DC may also modulate uterine NK cell function [56]. HLA-G has now been shown to interact with both NK cells [12] and HLA-DR+ cells, together accounting for *90% of the leukocytes at the placental implantation site [11]. Thus, as placental trophoblast cells infiltrate the uterine mucosa, they can deliver a pregnancy-specific signal to most of the local maternal leukocytes and modify their function to accommodate the feto-placental unit [57]. Materials and methods Cell lines and primary tissue The cell lines used were: human B lymphoblastoid 721.221 line transfected with HLA-C [58], HLA-G5 [28] or HLA-G1 mutated in the leader sequence to abrogate HLA-E expression [14]; murine Ltk cells transfected either with human b2m alone or with b2m and HLA-G together [59]; and the choriocarcinoma cell line JEG-3 (ATCC, Rockville, MD). Decidual and placental tissues were obtained from elective terminations of normal first-trimester pregnancies. Ethical approval for the use of these tissues was obtained from the Cambridge Local Research Ethics Committee. Decidual leukocytes and trophoblast cells were isolated as described [25]. Briefly, decidual tissue was disaggregated with collagenase and mononuclear cells were purified on Lymphoprep. The trophoblast was released from the chorionic villi by trypsin digestion and macrophages were depleted by adherence to plastic. After culture overnight on fibronectin, 50–80% of trophoblast cells label HLA-G+ by flow cytometry with the mAb G233 [2]. www.eji-journal.eu