FasL-Fas interaction is responsible for activating receptor–mediated NK cell apoptosis.

(A) The NK cell clone 10 (CD94⁺ activating) was incubated with sHLA non-A, -B, -C, and -G (that is, putative HLA-E and HLA-F) for 48 hours alone or after preincubation with anti-Fas mAb (5 μg/mL) or in the presence of anti-FasL mAb (5 μg/mL) or anti-Fas plus anti-FasL mAbs (5 μg/mL plus 5 μg/mL) in combination. Apoptosis was evaluated by labeling NK cells with FITC–annexin V. The effect of the covering of CD94 (achieved by preincubating NK cells with anti-CD94–specific mAb) is shown for comparison. Results are expressed as the percentage of apoptotic cells (annexin V⁺ but PI⁻). “nil” indicates apoptosis in medium alone. (B-D) ELISA for the presence of FasL in supernatant recovered from the NK cell clone 262 (CD158a⁺ activating) (B) or A1.25 (CD158b⁺ activating) (C) or 1 (CD94⁺activating) (D) incubated for 48 hours with the corresponding sHLA-I allele ([B] sHLA-Cw4; [C] sHLA-Cw3; [D] sHLA non-A, -B, -C, and -G) alone or after covering of activating receptor with specific mAb ([B] anti-CD158a plus sHLA-Cw4; [C] anti-CD158b plus sHLA-Cw3; [D] anti-CD94 plus sHLA non-A, -B, -C, and -G). Some experiments were performed upon optimal cross-linking of the indicated activating receptor with specific mAb (1 μg/mL) followed by GAM-coated beads (4 per cell) ([B] CD158a-XL; [C] CD158b-XL; [D] CD94-XL) or with unrelated sHLA-A2 allele (C) or sHLA-Cw3 after covering of CD8 (C) with anti-CD8 mAb (OKT8, 1 μg/mL). (E) Analysis of mRNA coding for FasL in the NK cell clone 262 bearing activating form of CD158a upon incubation for 6 hours with sHLA-Cw4 alone or after covering of CD158a⁺ with specific anti-CD158a mAb. “nil” represents mRNA coding for FasL in NK cells incubated with medium alone, and results obtained after optimal cross-linking of CD158a achieved using anti-CD158a mAb followed by GAM-coated beads (CD158a-XL) are shown for comparison.