Protective effect of cell-surface and sHLA-E molecules against CD94/NKG2A-dependent NK cell cytotoxicity. Cell surface expression of HLA-E mediates protection toward NK cell cytotoxicity. (A) Cytotoxicity assays were performed using target cells with no HLA-E expression at the cell surface, including the class I–deficient lymphoblastoid cell lines (C1R and K562) and primary cultures of SMCs. Target cells were preincubated with culture medium, control IgG (mouse IgG1; 10 μg/mL), or anti–HLA-E mAbs (10 μg/mL) for 20 minutes at RT. (B-C) Cytotoxicity assays were performed using ECs as target cells with a regulated HLA-E expression at the cell-surface ECs. ECs were untreated (B) or activated with 100 U/mL IFNγ for 48 hours (C). ECs were labeled with 51Cr and preincubated with culture medium, control IgG (mouse IgG1; 10 μg/mL), or anti–HLA-E mAb (10 μg/mL) for 20 minutes at RT before incubation with purified NK cells for 4 hours at 37°C. (D) Blocking experiments were performed after preincubation of NK cells with anti-NKG2A, anti-NKG2C, or anti-ILT2 receptor (10 μg/mL for each). Control was achieved using an isotype-matched control IgG. Soluble HLA-E provides protection toward NK cell cytotoxicity to cells with no or low HLA-E expression at the membrane. Resting ECs (E) and SMC (F) were preincubated with culture medium (without sHLA-E) or conditioned medium from IFNγ-treated HAECs (with sHLA-E) for 20 minutes at RT before incubation with freshly purified NK cells. (G) Resting ECs were preincubated with culture medium (without sHLA-E) or conditioned medium from IFNγ-treated HAECs (with sHLA-E) for 20 minutes at RT. ECs were then treated with culture medium, control IgG (mouse IgG1; 10μg/mL), or anti–HLA-E mAbs (10 μg/mL) for 20 minutes at RT before incubation with NK cells. For all these experiments, results, expressed as mean of specific lysis ± SD, are representative of at least 3 independent experiments. *P < .01 versus control.
