Annexin V stainings and DNA analysis of NK cells bearing activating receptor incubated with the corresponding specific sHLA ligand.

Dose-response and kinetics of activating receptor–mediated apoptosis. (A) The NK cell clone 45 bearing the activating form of C-type lectin inhibiting receptor CD94 was incubated for 48 hours with medium alone (top left dot plot) or sHLA non-A, -B, -C, and -G (that is, putative sHLA-E and F) alone (top right dot plot) or after preincubation with anti-CD94 mAb (bottom left dot plot) or with anti-CD94 mAb (1 μg/mL) followed by GAM-coated beads to obtained optimal cross-linking (bottom right dot plot). Cells were then stained with FITC-conjugated annexin V and PI and analyzed on a FACSort. Results are expressed as log green fluorescence intensity versus log red fluorescence intensity (au), and shown in the lower right portion of each dot plot is the percentage of annexin V⁺PI⁻ cells; that is, apoptotic cells. (B-C) DNA analysis with PI labeling or laddering of the NK cell clone 45 upon incubation for 72 hours with medium alone (Bi; C, lane 1) or sHLA non-A, -B, -C, and -G alone (Bii; C, lane 2) or after covering of CD94/NKG2 complex with anti-CD94–specific mAb (Biii; C, lane 3). In panel A, NK cells were labeled with PI and analyzed on a FACSort. Results are expressed as log red fluorescence intensity (arbitrary units [au]) (x-axis) versus cell number (y-axis). Numbers in each subpanel indicate the percentage of DNA content less than 2n; that is, apoptotic DNA. In panel C, DNA isolated from NK cells was subjected to agarose gel electrophoresis. DNA markers are shown on the left in base pairs (bp). (D) Titration of sHLA-I–induced apoptosis. The NK cell clone 255 bearing the activating receptor of CD158a was incubated with increasing amounts of sHLA-Cw4 for 48 hours, and apoptosis was evaluated after labeling with FITC–annexin V. Results are expressed as the percentage maximal apoptosis. Maximal apoptosis corresponds to 45% of apoptotic cells. (E) Kinetics of activating receptor–mediated apoptosis. The NK cell clone 255 activating receptor⁺CD158a⁺ (▪) or the NK cell clone 10 (activating receptor⁺ CD94⁺) (▴) was incubated with either sHLA-Cw4 or sHLA non-A, -B, -C, and -G (that is, putative HLA-E) for the indicated periods of time, and apoptosis was evaluated by staining cells with FITC–annexin V. ● indicates apoptosis of the NK cell clone 255 incubated with medium alone. Results are expressed as the percentage of apoptotic cells that are annexin V⁺ but PI⁻.