Kinetics and dose-response of NK cell apoptosis induced by different sHLA-I alleles.

The NK cell population NK-1 (90% CD8⁺, A), the NK cell clones Cl. C8.6 (CD8^intermediate, B), and Cl. H2-50 (CD8^bright, C) were incubated with medium alone (nil), or 4 μg/mL sHLA, or anti-CD8 mAb (OKT8, 1 μg/mL; OKT8-XL), or anti-CD54 mAb (14D12D2, 1 μg/mL; CD54-XL) followed by 4-per-cell GAM-coated magnetic beads to achieve cross-linking of the corresponding molecule or anti-Fas mAb (CH-11, 1 μg/mL; Fas-XL) and analyzed for their reactivity with FITC–annexin V at different time points (1, 2, and 3 days). Results are expressed as the percentage of annexin V⁺ PI⁻ cells. The NK-1 cell population and the NK cell clones analyzed were CD94⁺ and KIR2D⁻. (D) The NK cell clone Cl.S2 (CD8^bright) was incubated with increasing amounts of either sHLA-I or with sHLA-G1 (0.5, 1, 2, 3, 4, and 8 μg/mL). After 48 hours, apoptosis was assessed by staining with FITC–annexin V. Nil: apoptosis in the absence of any addition. (E) The NK cell clone Cl.S2 (CD8^bright) was incubated with 4 μg/mL sHLA-I isolated from serum of healthy donors or with sHLA-A2, sHLA-Cw4, sHLA-B46, or sHLA-G1 (from 721.221 cells transfected with the corresponding HLA-I allele) alone or after pretreatment of NK cells with anti-CD8 mAb (OKT8, 1 μg/mL), to avoid interaction of sHLA-I with CD8 or with anti-CD54 mAb as control mAb, and apoptosis was evaluated after 48 hours. Results are expressed as the percentage of annexin V⁺ PI⁻ cells and are representative of 4 independent experiments by using 4 different NK cell clones from 4 healthy donors.