CD56⁻CD16⁺ NK cells express low levels of activating receptors and display compromised effectors functions. (A) Freshly isolated NK cells were stained with anti-CD56 and anti-CD16 mAbs to distinguish 3 different subsets as in Figure 4. Each subset was assessed for the surface expression of the main activating receptors and for intracellular expression of perforin and granzyme B. (B) The same NK cells were cultured overnight in the presence or in the absence of rhIL-15 and then incubated with either medium alone (not shown) or with K562 for 3 hours. After incubation, different NK-cell subsets were analyzed for CD107a expression. In parallel the different NK-cell subsets were evaluated for intracellular IFN-γ in response to K562 or phorbol 12-myristate 13-acetate plus ionomycin. The percentage of positive cells and relative mean fluorescence intensity are indicated in each histogram. A representative patient from group 1 at 6 months after transplantation and, for comparison, a representative CB and a healthy donor are shown. (C) CD107a expression and (D) intracellular IFN-γ production, after incubation with K562, are shown for CD56⁻, CD56^dim, and CD56^bright NK-cell subsets from group 1 patients (n = 4; left) at 6 months after transplantation and from healthy adult controls (n = 30; right). (E) Freshly isolated NK cells were cultured overnight in medium alone and then incubated with the FcγR⁺ p815 murine cell line for 3 hours in the presence or absence of anti-CD16, anti-NKp46, or anti-NKG2C mAbs. CD107a expression is shown for CD56⁻ and CD56^dim NK-cell subsets in group 1 patients (n = 4; left) at 6 months after transplantation and in healthy adult controls (n = 6; right). (C-E) The 95% CIs for the mean and statistical significance are indicated (*P < .05).