MDSCs induced by HLA-G express PIR-B and are immunosuppressive. (A-B) Mice injected with M8-pcDNA cells, M8-HLA-G1 cells, or M8-HLA-G1 cells pretreated with the 87G mAb (M8-HLA-G1 + anti–HLA-G) were killed at day 21 after injection. Mononuclear cells were purified from blood (A) or spleens (B) and stained with the indicated antibodies. Analyses were performed by flow cytometry. Data are from 1 representative mouse of 3. (C) Immunostaining of purified HLA-G protein bound to MDSCs. Gr1⁺ cells were purified from spleens of mice injected with M8-HLA-G1 cells. The purity (> 95%) was attested by flow cytometry (inset). Cells were incubated with HLA-G⁻ medium (control), a purified form of HLA-G at 180 ng/mL (HLA-G), or HLA-G + anti–PIR-B (clone 259.2). HLA-G binding to cells was detected by anti–HLA-G and goat anti–mouse conjugated to FITC through confocal microscopy analysis. (D) NK cells were purified from spleens of M8-HLA-G1–injected mice 3 days after injection. Gr1⁺ cells were purified from spleens of mice injected with M8-pcDNA or M8-HLA-G1 cells 21 days after injection. The Gr1⁻ counterpart was also tested. A classic 4-hour ⁵¹Cr-release cytotoxic assay was conducted with NK cells as effectors (E), M8-HLA-G1 cells as targets (T), and Gr1⁺ or Gr1⁻ cells as a third party (I). Results are expressed as the mean percentage lysis from triplicates. Data are from 1 representative mouse of 3. (E) Mice were injected subcutaneously with M8-pcDNA or M8-HLA-G1 cells alone at day 0 or with an anti–PIR-B Ab (clone 259.2) at days −1, 0, and 1, and M8-HLA-G1 cells at day 0 (M8-HLA-G1 + anti-PIR-B). Tumor growth was monitored at the indicated time points. Data represent the means ± SD from 1 representative experiment with 3 mice in each group. Inset shows cell-surface expression of PIR-B on PBMCs of M8-HLA-G1–injected mice detected with the clone 259.2.