Eur. J. Immunol. 2007. 37: 1924–1937 PE-conjugated F(ab')2 anti-mouse IgG (Sigma). Medium was analysed for IL-6, IL-8, IL-10 and TNF-a by ELISA (R&D Systems). Mixed lymphocyte reaction Stimulator cells were 721.221 parent (5) or HLA-G1 (105), HLA-G5 and HLA-C (80) transfectants. The HLA-I-transfected cells had similar levels of expression as monitored by BBM.1 staining; the MFI is shown in parentheses above. Stimulator cells were washed, seeded at equal densities and cultured overnight without selection. These cells were then irradiated with 5000 rad and aliquoted into wells of a 96-well plate at varying densities to give stimulator/responder ratios of between 3 : 1 and 0.1 : 1. Responder cells were PBMC from healthy donors, isolated on Lymphoprep and added at 5 104 cells/well in a total volume of 300 lL. All donors demonstrated proliferation and when typed retrospectively where shown to be mismatched to the HLA-DR1 epitope expressed by 721.221 cells [58]. Donors in the experiments shown in Fig. 7 were DRB1*04/DRB1*07 in panel (A) and DRB1*03/DRB1*13 in panel (B). HLA-DR+ cells were re- moved from responder populations with azide-free HLA-DR- FITC and EasySep FITC-selection kit (Stem Cell Technologies). After 5 days, 1 lCi/well of [3H]thymidine was added, cells were harvested after a further 18 h (TomTec 96-well plate harvester), and [3H]thymidine incorporation was measured (Wallac Trilux liquid scintillation counter). For blocking experiments, azide-free HP-F1 (3 lL ascites/well), M401, M422 or W6/32 (all 0.05 lg/well) were added immediately. F(ab')2 fragments of W6/32, necessary to avoid antibody- dependant cell-mediated cytotoxicity, were generated by pepsin digestion [68]. 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