HLA-G–mediated suppression is reversed after neutralization of HLA-G and independent of cell-cell contact. (A) Suppression assay at a ratio of 1:1 HLA-G^pos to CFDA-SE–labeled HLA-G^neg CD4 T was performed in the absence of antibody (second panel), in the presence of an IgG isotype control (third panel), and in the presence of anti–HLA-G1/5 blocking antibody (87G; fourth panel). Proliferation was measured by flow cytometry at day 4. (B) Suppression assay at a ratio of 1:1 HLA-G^pos to CFDA-SE–labeled HLA-G^neg CD4 T was performed either conventionally or in a transwell chamber: CFDA-SE–labeled CD4 HLA-G^neg T cells were placed in the lower chamber while CD4 HLA-G^pos were added to the upper chamber with no cell-cell contact to CD4 HLA-G^neg responder cells (ratio 1:1). Proliferation of CFDA-SE–labeled cells was measured by FACS. Graphs in panels A-B show representative experiments reproduced at least 3 times. (C) Supernatants of suppression assays with a CD4 HLA-G^neg/CD4 HLA-G^pos ratio of 1:1 (coculture) and 1:0 (control) were transferred to new proliferation assays of CFDA-SE–labeled CD4 HLA-G^neg T cells. Suppressive activity of supernatant was measured after 4 days by FACS analysis. Graph shows summary of 6 independent experiments. Error bars indicate standard deviation.