Eur. J. Immunol. 2007. 37: 1924–1937 monomeric HLA-G was only very weakly detected, and the 45-kDa classical HLA-C molecule expressed by trophoblast cells was not seen at all. These bands were confirmed to be specific for an interaction of LILRB1 Immunomodulation 1929 with HLA-G because they were not precipitated from untransfected 721.221 cells or by an IgG1-Fc fragment. That LILRB1-Fc predominantly binds to dimeric rather than monomeric HLA-G at the surface of HLA-G transfectants and normal trophoblast cells suggests that this is likely to be the major interaction occurring in vivo. Monocyte-derived DC activated in the presence of HLA-G demonstrate altered cytokine production Given the high-affinity interaction of HLA-G dimers with LILRB1 and the expression of this receptor by all decidual HLA-DR+ cells, we next investigated whether HLA-G appeared to have any functional effect on DC. Due to the difficulty in obtaining sufficient HLA-DR+ cells from the decidua that are not activated by the isolation procedure [32], we used monocyte-derived DC (MDDC) generated in vitro. MDDC were prepared from peripheral blood, and their response to a maturation stimulus was observed in co-culture with cells expres- sing HLA-G. We used 721.221 or Ltk cells displaying dimeric and monomeric HLA-G identical to normal Figure 4. LILRB1-Fc binds to HLA-G at the surface of transfected cells and trophoblast. HLA-G expression on the indicated cells was detected by G233 staining (green) compared to untrans- fected cells (red) or with an isotype control mAb (filled histogram) (A–D). LILRB1 binding to the indicated cells was detected by LILRB1-Fc staining (green) compared to untrans- fected cells (red) or with an IgG-Fc fragment (filled histogram) (E–H). Specificity was confirmed with blocking mAb (J–M): LILRB1-Fc (green) and IgG1-Fc fragment (filled histogram) binding are shown as before and also after pre-incubation of the fusion protein with anti-LILRB1 mAb M401 (red) or of the cells with anti-HLA-I mAb W6/32 (blue). These results are representative of four different experiments for each cell type, using trophoblast from eight samples. To see LILRB1-Fc binding to trophoblast cells, contaminating leukocytes that bound the fusion protein non-specifically via FccR were labelled with a lineage marker cocktail (CD3/14/16/19/20/56-FITC) and analy- sis was performed on FITC-negative trophoblast cells (upper left quadrant). Dot plots of LILRB1-Fc binding (N) and blocking by anti-LILRB1 mAb (O) are shown. f 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Figure 5. LILRB1-Fc preferentially binds to the dimeric form of HLA-G at the cell surface. 721.221 HLA-G+ transfectants (221G), untransfected controls (221P) and normal trophoblast cells were surface-biotinylated, immunoprecipitated with the in- dicated antibodies or fusion proteins and resolved by non- reducing PAGE before Western blotting with streptavidin-HRP. These results are representative of five different experiments for each cell type. Trophoblast cells were pooled from 15 samples. The image includes lanes from separate gels. www.eji-journal.eu