Deficient patient NK cell numbers and cytolytic functions. Evaluation of the NK cell cytotoxicity by 4-hour ⁵¹Cr-release assay against (A) K562, (B) 721.221, or (C) Raji target cells using PBMCs. For the Raji cells, lysis was evaluated with or without the addition of rituximab to opsonize the cells as indicated in the legend. For K562 and 721.221 target cells, 1000 U/mL IL-2 was added to the assay for its duration as indicated in the legends. Statistical significance was calculated using the Mann-Whitney U test. (D) Phenotypic evaluation of NK cells demonstrating forward versus side scatter gating strategy (left), CD56 versus CD3 with the square gate denoting classic NK cells (middle-left), the presence of CD16 on the gated CD56⁺/CD3⁻ NK cells using 2 different clones of anti-CD16, B73.1, and 3G8 (middle right), and the expression of CD56 as a percentage of maximal expression on the gated NK cells (right). Results for the patient (top) and a control (bottom) are shown. Evaluation of NK cell subsets and functional markers are for gated CD56⁺/CD3⁻ classic NK cells only. Fluorescence intensity for an IgG isotype control (black) is shown compared with that for the antigen-specific antibody for a control (blue) and the patient (red). The control IgG for the patient is shown but was similar to that for the control donor. All results are representative of at least 3 independent repeats. NKp44, NKG2C, and IL-15RB were performed on a sample separate from the others.