Soluble HLA-I induces apoptosis in NK cells through its interaction with CD8.

NK cell clones expressing different levels of CD8 (CD8^dull, clones Cl.8.25 and Cl.S4, panels A,D; CD8^intermediate, clones Cl.8.6 and Cl.4.25, panels B,E; and CD8^bright, clones Cl. 77.12 and Cl.S2, panels C,F), but homogeneously expressing CD94/NKG2 complex and KIR2D⁻ were incubated with medium alone (nil), or anti-Fas mAb (CH-11, 1 μg/mL) (Fas-XL) or 4 μg/mL sHLA-I or anti-CD8 mAb (OKT8, 1 μg/mL) followed by 4-per-cell GAM-coated magnetic beads (OKT8-XL) to achieve CD8 cross-linking or 4 μg/mL sHLA-I either after preincubation with anti-CD8 mAb (OKT8, 1 μg/mL) to avoid sHLA-I interaction with CD8 or anti-CD54 mAb as control mAb. Cells were then analyzed for the reactivity with FITC–annexin V after 48 hours. Results are expressed as the percentage of annexin V⁺ PI⁻ (numbers in each panel indicate the percentage of apoptotic cells). (G-L) DNA staining with PI of fixed and permeabilized NK cells (Cl.77.12) incubated with medium alone (G), or 4 μg/mL sHLA-I (H) or anti-CD8 mAb (OKT8, 1 μg/mL) followed by 4-per-cell GAM-coated magnetic beads (I) to achieve CD8 cross-linking or 4 μg/mL sHLA-I after preincubation with anti-CD8 mAb (L) to avoid sHLA-I interaction with CD8. Numbers in each panel indicate the percentage of NK cells with a less than 2n DNA content (apoptotic cells, markers). (M) DNA laddering of NK cells (Cl.77.12) incubated with medium alone (lane 1), or 4 μg/mL sHLA-I (lane 2) or anti-CD8 mAb (OKT8, 1 μg/mL) followed by 4-per-cell GAM-coated magnetic beads (lane 3) or 4 μg/mL sHLA-I, after preincubation with anti-CD8 mAb (OKT8, 1 μg/mL, lane 4). DNA markers are shown on the left.