“Single-KIR” NK-cell lines specifically detect HLA class I allotypes. (A) FACS analysis of KIR (CD158a, 158b, and CD158e) expression by healthy donor-derived NK cells stained with a single anti-CD158 mAb (x-axis) versus a mixture of 2 anti-CD158 mAb (y-axis), as indicated. Single KIR-expressing CD158a⁺, CD158b⁺, and CD158e⁺ NK-cell subpopulations of 14%, 5%, and 2%, respectively, are indicated. (B) FACS analysis of CD158a⁺, CD158b⁺, and CD158e⁺ cells after FACS sorting and expansion in culture for up to 21 days revealed a purity of single KIR-expressing NK-cell lines of more than 97%. Numbers on plots are percentages of total CD56⁺CD3⁻ cells. (C) Cytotoxicity of CD158a⁺, CD158b⁺, and CD158e⁺ NK-cell lines against 721.221 control cells (open symbols) and 221 transfectants with HLA class I allotypes belonging to group C1, C2, and Bw4 (closed symbols) at the indicated effector-to-target (E/T) ratios. (D) Cytotoxicity of CD158a⁺ and CD158e⁺ NK-cell lines against K562 cells (filled and open circles, respectively) and against leukemic blasts from a patient with AML M2 (filled and open squares, respectively). The HLA class I allotype of the patient is indicated.