Soluble HLA-I alleles trigger NK cells to produce IFN-γ upon ligation of activating receptor.

(A) The NK cell clone 262 (CD158a⁺ activating) was incubated for 24 hours with either sHLA-Cw4 or unrelated sHLA-A2 allele alone or with sHLA-Cw4 after covering of CD158a or CD8 with either anti-CD158a– or anti-CD8–specific mAb (1 μg/mL), respectively. (B) Production of IFN-γ upon incubation of the NK cell clone 262 with the indicated increasing amounts of the specific ligand sHLA-Cw4. (C) IFN-γ production by the NK cell clone 45 (CD94⁺activating) in the presence of sHLA non-A, -B, -C, and -G alone (that is, putative HLA-E) or after covering of CD94 with anti-CD94 mAb (1 μg/mL). The amount of IFN-γ produced upon incubation of the NK cell clone 45 (CD94⁺activating) with anti-CD94 or anti-CD16 or anti-CD54 mAb followed by GAM-coated beads (4 per cell) (to induce optimal cross-linking of the indicated molecules, CD94-XL, CD16-XL, CD54-XL) is shown for comparison. (D) The NK cell clone 262 (CD158a⁺activating) was incubated with sHLA-Cw4 (▪) or with anti-CD158a mAb followed by optimal cross-linking with GAM-coated beads (4 per cell) (■) for 24 hours alone or in the presence of increasing doses of CsA (5, 50, 500 ng/mL). ▴ indicates IFN-γ produced by the same NK cell clone in medium alone. Culture supernatant was recovered and analyzed for the presence of IFN-γ by ELISA. Results are expressed as nanograms per milliliter and are representative of 3 independent experiments.