1928 Richard Apps et al. Eur. J. Immunol. 2007. 37: 1924–1937 b2m band was consistently stronger in reducing blots. This is likely to be an artefact due to residual tertiary conformation under non-reducing conditions stearically hindering access of streptavidin-HRP to biotinylated residues. Reprobing of blots with anti-b2m mAb confirmed that all light chain was at 13.7 kDa (not shown) and weaker bands from some biotinylated molecular weight markers also occurred under non- reducing conditions (Fig. 1). We did not investigate free heavy chains of HLA-G, which have been observed at the surface of transfectants [19], due to the lack of an antibody recognising free heavy chains that is specific for HLA-G in vivo [31]. LILRB1, an HLA class I molecule receptor, is expressed by decidual leukocytes HLA-G binds to members of the LILR family, LILRB1 and B2 [13, 14]. Although these receptors bind a broad spectrum of HLA-I alleles, they have a higher affinity for HLA-G [16], particularly in its dimeric form [18, 20]. Therefore, we investigated LILRB1 and B2 expression by decidual leukocytes. Two different antibodies were used for both receptors. Maternal decidual leukocytes were isolated from first-trimester pregnancies and identified as T cells (CD3), NK cells (CD56) and DC or macrophages (HLA-DR) [32], followed by staining for LILR expression. LILRB1 was detected on all DC and macrophages (Fig. 3A), as well as 20% of NK cells (Fig. 3D) and 10% of T cells (Fig. 3G). Expression of LILRB2 was restricted to DC and macrophages (Fig. 3A, E, H). LILRB2 is expressed more strongly on peripheral than decidual myelomonocytic cells, whereas LILRB1 appears to predominate in the decidua (Fig. 3A, B). LILRB1-Fc fusion protein preferentially binds the dimeric form of HLA-G in vivo Predominant expression of LILRB1 by decidual leuko- cytes suggests that this receptor may be particularly important in the recognition of trophoblast HLA-I molecules. To investigate this possibility, we studied binding of a LILRB1-Fc fusion protein to cells expressing HLA-G. As predicted, LILRB1-Fc bound to 721.221 and Ltk cells transfected with HLA-G (Fig. 4E, F). The level of binding correlated with HLA-G expression (Fig. 4A, B) and the specificity was demonstrated by blocking with mAb to LILRB1 (M401) and W6/32 which is known to block LILRB1 binding to HLA-I molecules [33] (Fig. 4J, K). HLA-G-specific mAb do not bind epitopes blocking LILRB1 binding. To detect LILRB1-Fc binding to primary trophoblast preparations, it was necessary to first label contaminating leukocytes expressing FccR with lin-FITC, leaving trophoblast and stromal cells in the FITC-negative quadrants f 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Figure 3. LILRB expression by decidual leukocytes. Decidual (A) or peripheral (B) leukocytes were identified as myelomonocytic cells by forward/side scatter and HLA-DR+ gates and stained with anti-LILRB1 (dark line), anti-LILRB2 (grey line) or isotype control antibodies (filled histogram). Decidual CD56+ (C–E) or CD3+ (F–H) lymphocytes were stained with isotype control (C, F), anti-LILRB1 (D, G) and anti-LILRB2 (E, H) antibodies. These results from decidual leukocytes are representative of seven different experiments using decidua pooled from 17 samples. Similar results for both LILRB1 and 2 were observed with two different antibodies. The apparently higher isotype control mAb staining of T cells compared to NK cells results from inclusion of more cells in plots demonstrating T cell staining, due to their low frequency in decidual leukocyte populations. (Fig. 4N, O). A gate around these cells shows that the level of HLA-G was less abundant at the surface of normal trophoblast than transfectants (Fig. 4A, B, D). Despite this, binding of the LILRB1-Fc fusion protein to trophoblast cells could still be demonstrated and was blocked by mAb to LILRB1 or HLA-I (Fig. 4H, M, N, O). Interestingly, no binding of LILRB1-Fc to JEG-3 cells was detected (Fig. 4G, L) even though these cells showed higher levels of HLA-G expression than trophoblast cells (Fig. 4C, D). The failure to detect binding to JEG-3 cells, which display HLA-C, HLA-E and mainly monomeric HLA-G, suggests that LILRB1-Fc preferentially binds the dimeric form of HLA-G and not conventional HLA-I molecules. To test this, the LILRB1-Fc fusion protein was used to immunoprecipitate surface-labelled proteins from HLA-G-transfected and primary trophoblast cells (Fig. 5). As before, the G233 mAb detects both monomeric and dimeric HLA-G, with *40% of HLA-G molecules in the dimeric form. The LILRB1-Fc fusion protein strongly bound the dimeric form of HLA-G, www.eji-journal.eu