Eur. J. Immunol. 2007. 37: 1924–1937 been shown to result in reduced responder T cell proliferation [34–36]. Most reports infer that HLA-G is directly inhibiting T cells, although a molecular mechanism has not been defined as HLA-G-restricted TCR have not been described in humans [34, 35]. Given the strong binding of dimeric HLA-G to LILRB1 and the observed increased IL-6 and IL-10 production by MDDC exposed to HLA-G, we sought to determine whether it is the interaction between trophoblast HLA-G and LILRB on antigen-presenting cells (APC) that results in down- regulation of allogeneic T cell proliferation. Immunomodulation 1931 We therefore established a one-way MLR using PBMC as responders and 721.221 cells as stimulators. The stimulators were either HLA-I (parent), HLA-G1 transfectants expressing HLA-G dimers as well as monomers, or HLA-C transfectants to control for the effect of a conventional HLA-I molecule. At a 1 : 1 stimulator/responder ratio, the presence of HLA-G reduced the alloproliferative response compared to the parent cell line, by a magnitude consistent with previous reports (p = 0.03) (Fig. 7A) [34–36]. In contrast, a marginally increased response was seen Figure 7. Allogeneic lymphocyte proliferation is inhibited by HLA-G interacting with LILRB receptors on HLA-DR+ cells. (A) Irradiated 721.221 stimulator cells transfected with various HLA-I molecules were cultured with PBMC responders from a healthy donor. Proliferation measured by [3H]thymidine incorporation after 5 days was reduced in the presence of HLA-G but not a classical HLA-I molecule. Reduced proliferation to HLA-G transfectants was abrogated by addition of anti-LILRB and anti-HLA-I mAb. (B) When HLA-DR+ cells were depleted from the responder population, a reduced proliferative response with no additional inhibitory effect by HLA-G was observed. One result is shown, SD of triplicates. Three to ten independent experiments under each condition were performed and these were analysed together for statistical significance. (C) Flow cytometry analysis of the responder population confirming depletion of HLA-DR+ cells. f 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu