The alloreactive KIR⁺ NK-cell subset of KIR-KIRL mismatched donors exhibits a superior ability for degranulation in response to pediatric BCP-ALL. (A-D) NKAES cells of KIR-KIRL mismatched (SNK14B, SNK15B, and SNK20B) or matched donors (SNK9A, SNK10P, and SNK21BC), respectively, were cocultured with NALM-16 or K562 cells. Shown is the percentage of CD107a⁺ CD56⁺ population in various alloreactive or nonalloreactive NK-cell subsets. (A) Exemplified gating strategy for the identification of NK-cell subsets in donor SNK15B. In relation to NALM-16 cells (Bw4/C1), the potentially alloreactive NK-cell population (upper left square) is represented by the cells expressing KIR2DL1/S1/S4 and/or KIR3DS1 (y-axis) but not KIR2DL2/L3/S2 or KIR3DL1 (x-axis). (Note that the combined staining with a KIR3DL1/S1 [Z27.3.7] and a KIR3DL1 [DX9] antibody enables the identification of the KIR3DL1⁻S1⁺ cell population that belongs to the alloreactive subset). (B-C) Representative original histogram data obtained from a KIR-KIRL mismatched (SNK15B) and a matched donor (SNK10P). Data in (B) shows CD107a staining in NKAES-only cultures (gray, open) and NKAES cells cocultured with NALM-16 cells (black, open). Data in (C) shows the respective stainings in coculture experiments with K562 (black, open). The upper right histogram in Figure 4C delineates the respective isotype control (light gray, filled) that allows the subsequent marker position and identification of the CD107a⁺ subset in (B) and (C). In each histogram, the subtraction result ΔCD107a is given (% CD107a⁺ cells in NKAES-tumor cocultures minus % CD107a⁺ cells in NKAES-only cultures). Note that all experiments were performed with highly activated NKAES cells that exhibited a significant baseline CD107a expression. (D) Percentage of CD107a⁺ CD56⁺ cells in the indicated NK-cell subsets of KIR-KIRL mismatched (SNK14B, SNK15B, and SNK20B) or matched donors (SNK9A, SNK10P, and SNK21BC). Shown is the increase in CD107a expression of NALM-16 cocultured KIR⁺ NK-cell subsets normalized to the CD107a expression of the corresponding KIR⁻ NK-cell subset. Filled black circle, KIR⁺ alloreactive subset in KIR-KIRL mismatched donors; open circle, KIR⁺ alloreactive subset in matched donors; light gray square, KIR⁺ nonalloreactive NK-cell subset. Data represents 1 experiment performed with the 6 indicated donors. n.s., not significant.