Blocking experiments and functional assays. (A) Total PB NK cells were treated with beads (1) uncoated (ctr; black bars), (2) coated with sHLA-G (beads HLA-G; white bars), (3) coated with sHLA-G and treated with 87G anti–HLA-G blocking mAb (striped bars), or (4) coated with sHLA-G and treated with isotype-matched irrelevant ctr mAb (gray bars) before being admixed with NK cells. Cytofluorimetric analysis of CXCR3 or NKG2A expression has been performed. (B) Blocking experiments performed on total PB NK cells untreated (ctr; black bar) or treated with sHLA-G (white bar) or preincubated with anti-ILT2/CD85j mAb (striped bar) or isotype-matched mAb (gray bar) and then treated with sHLA-G. Cytofluorimetric analysis of CXCR3 or NKG2A expression is shown. Data are expressed as MRFI. Means of 3 different experiments ± SD are shown. Asterisks indicate significant differences. Chemotaxis was performed on total PB NK cells (C) or PB CD56^bright NK cells (D), using as chemoattractants (1) CXCL10, CXCL11, CX₃CL1, and CCL21 or (2) CCL2, CXCL10, and CCL21, respectively. Data are expressed as migration index (no. of migrated cells/no. of total cells × 100). Means of 5 different experiments ± SD are shown. Asterisks indicate significant differences. (E) IFN-γ secretion by total PB NK cells treated with anti-NKp46 mAb alone or in combination with anti-NKG2A mAb. NK cells were either untreated (black bars) or treated with sHLA-G (white bars) before mAb-mediated cross-linking of NKp46 and/or NKG2A molecules. Data are expressed as IFN-γ fold increase (nanogram per milliliter of IFN-γ secreted by specific mAb-treated cells divided by nanogram per milliliter of IFN-γ secreted by irrelevant isotype-matched treated cells). Means of 3 different experiments ± SD are shown. Asterisks indicate significant differences.