Analysis of cytokine production and cytotoxicity in different NK-cell subsets after activating receptor stimulation. (A) Analysis of intracellular IFN-γ and TNF expression in CD56^bright, CD56^dim CD62L⁺, and CD56^dim CD62L⁻ NK cells after stimulation with a combination of plate-bound mAbs against NKp30, NKp46, NKG2D, 2B4, and CD2 or isotype control mAb; 1 representative experiment of 6 is shown. (B) Analysis of NK-cell subset cytotoxicity after K562 stimulation; mean ± SEM of 9 independent experiments is shown. (C) Ex vivo staining of granzymeA (GRA), granzymeB (GRB), perforin (Prf; open histograms), and of corresponding isotype control (gray-filled histograms) in CD56^bright, CD56^dim CD62L⁺, and CD56^dim CD62L⁻ NK cells; 1 representative donor of 3 is shown. MFI and percentage of positive cells is depicted. (D) CD56^dim NK cells derived from HLA-Bw4 donors were sorted for NKG2A⁻ CD62L⁺ and NKG2A⁻ CD62L⁻ cells and stimulated for 6 hours with K562. CD107a expression was analyzed within competent KIR3DL1⁺ 2D KIR⁻ NKG2A⁻ and hyporesponsive KIR3DL1⁻ 2D KIR⁻ NKG2A⁻ NK-cell subsets after staining for KIR3DL1 (indicated as 3DL1) and 2D KIR and gating on the indicated subsets. Mean ± SEM of 8 independent experiments is shown. *P < .05; **P < .01; ***P < .0001 as calculated by Wilcoxon test.