1934 Richard Apps et al. HLA-I and LILR antibodies and fusion proteins Conformationally dependant mAb specific for HLA-G were G233 [2] from our laboratory, 87G [60] from Dr. D. Geraghty, and MEM-G/11 [61] from AbCam. BBM.1 (Santa Cruz Biotechnology) specifically recognises b2m [62], W6/32 (Serotec) binds molecules encoded by all HLA-I loci [63], T149 (from Dr. B. Uchanska-Ziegler) and B1.23.2 (from Dr. P. Le Bouteiller) recognise HLA-B and HLA-C allotypes [23, 64]. MEM-G/1 (Abcam) detects denatured HLA-G [65]. Anti-LILRB1 (M401) and anti-LILRB2 (M422) mAb [66] were supplied by Amgen Inc. Different anti-LILRB1 mAb (HP-F1) [37] and anti-LILRB2 mAb (42D1) [67] were from Dr. M. Lopez-Botet and Dr. M. Colonna, respectively. Isotype control antibodies were from Oxford Biotechnology. A sequenced LILRB1-Fc fusion protein construct [66] from Amgen Inc was transiently transfected into 293T cells using Lipofectamine 2000 (Invitrogen). Fusion protein was puri- fied from supernatant with protein G-Sepharose beads (Amersham Pharmacia Biotech) and assayed by Easy-Titer (Pierce) and reducing SDS-PAGE, where it resolved as a single band at *100 kDa. Biotinylation, immunoprecipitation, PAGE and Western blotting Cells were washed with cold PBS, then biotinylated with 0.2 mg/mL EZ-Link Sulfo-NHS-LC-Biotin (Pierce) in pH 8.0 PBS for 30 min at 4 C to prevent endocytic intracellular labelling. Unconjugated reagent was quenched by adding glycine to a concentration of 10 mM. Cells were then lysed in fresh ONYX lysis buffer [20 mM Tris (pH 7.4), 140 mM NaCl, 1 mM EGTA, 1% Triton, 10% glycerol, 50 mM iodoacetamide and protease inhibitor cocktail (Roche)]. This concentration of iodoacetamide has been shown to prevent post-lysis HLA-I multimerisation [30]. Labelled lysates were stored at –80 C. Thawd lysates were incubated for 90 min at 4 C with protein G-Sepharose beads coated with isotype control mAb. Precleared lysates were then immunoprecipitated for 90 min at 4 C with protein G-Sepharose beads precoated with anti- HLA antibodies or LILRB1-Fc. Immunoprecipitates were washed with ONYX containing 3 mM SDS, bound proteins were eluted with NuPAGE LDS Sample Buffer (Invitrogen) and divided into two aliquots; one was denatured by treatment at 95 C for 10 min, the other was denatured and reduced by the same process in the presence of 200 mM 2-mercaptoethanol (Sigma). Denatured samples were stored at –20 C overnight. For immunoprecipitation of soluble HLA-G, transfected 721.221 cell cultures were inoculated at 1 106 cells/mL and incubated for 2 days. JEG-3 and primary trophoblast cells were cultured as described for 2 days [2, 25]. Human pre- implantation embryo culture supernatants were obtained from Dr. R. Gosden (Cornell University). Supernatants were concentrated by centrifugal filtration if necessary (Amicon, 30 000 MWCO) and a 700-lL aliquot was precleared as before and immunoprecipitated overnight. Samples were resolved on reducing or non-reducing NuPAGE Bis-Tris 10% gels (Invitrogen), electroblotted and visualised with a streptavidin-HRP conjugate followed by ECL detection (Amersham Biosciences). Unlabelled soluble HLA-G f 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Eur. J. Immunol. 2007. 37: 1924–1937 molecules were detected with biotinylated MEM-G/1 followed by streptavidin-HRP. 2D-PAGE was performed by running a non-reducing gel as above, excising the lane and equilibrating for 30 min in reducing buffer (0.125 M Tris pH 8, 3.5 mM SDS, 0.15 M 2-mercaptoethanol). This gel slice was then positioned at 90 along the top of a new gel and run under reducing conditions before Western blotting as above. Flow cytometry Freshly isolated leukocytes were first incubated with human IgG (Sigma) to block Fcc receptors. Anti-LILRB1 and -LILRB2 antibodies were added and detected with fluorochrome- conjugated secondary antibodies. Free secondary antibody binding sites were blocked with the appropriate species Ig, before staining with directly conjugated CD56, CD3 or HLA-DR mAb (Serotec and Becton Dickinson). Data was acquired with a FACScan flow cytometer and analysed using CellQuest soft- ware (Becton Dickinson). Primary trophoblast cells cultured overnight were removed from the plate with 0.5 mM EDTA. Fcc receptors were blocked with 40% heat-inactivated human AB serum (Sigma) for G233 mAb staining experiments or with mouse anti-CD32 antibody (Stem Cell Technologies) for LILRB1-Fc staining experiments. Contaminating fetal leukocytes were also labelled with lin- FITC, a cocktail of FITC-conjugated anti-CD3, -CD14, -CD16, -CD19, -CD20 and -CD56 antibodies (Becton Dickinson). Cells were stained with G233 mAb, LILRB1-Fc or IgG1-Fc fragment (Bethyl Laboratories). Primary antibody was detected with PE- conjugated anti-mouse IgG F(ab')2 fragment (Sigma). Fusion protein binding was detected with PE-conjugated anti-human IgG (c chain specific) secondary antibody (Sigma). In blocking experiments, fusion protein was incubated with anti-LILRB1 or isotype control antibody or cells were incubated with W6/32 or the isotype control antibody prior to staining with fusion protein. MDDC experiments PBMC from healthy donor buffy coats were isolated on Lymphoprep (Axis-Shield, Norway), washed extensively and plated in plastic culture dishes at 2 107 cells/mL. After 2 h, non-adherent cells were washed away and the adherent fraction was cultured in RPMI with GM-CSF (100 ng/mL) and IL-4 (40 ng/mL) (Biosource), refreshing the medium as necessary. After 5 days, more than 80% of the cells exhibited a DC-like phenotype (LILRB4+, DC-SIGN+, CD14–) with fewer than 10% of cells CD3+. MDDC were stimulated for 12–24 h with 10 ng/mL LPS (Sigma), 500 ng/mL sCD40L (Peprotech) or 20 ng/mL TNF-a, 50 ng/mL IL-6 and 10 ng/mL IL-1b (Biosource, R&D Systems). HLA-I 721.221 or Ltk parent and HLA-G-transfected cells were irradiated with 1000 rad and added to the MDDC either with the maturation stimulus or throughout their differentiation. Cells were harvested and stained for flow cytometry with LILRB4-PC5 (Becton Dickinson), PE-conjugated antibodies to CD14 (Becton Dickinson), CD40, CD80, CD83 and CD86 (all Serotec), FITC-conjugated HLA-DR antibody (Becton Dick- inson) and unlabelled DC-SIGN (Becton Dickinson), anti- LILRB1 (M401) and -LILRB2 (M422) antibodies followed by www.eji-journal.eu