Phenotypic and functional features of NK cells. (A) Number of lymphocytes (black line) and CD3⁻CD56⁺ NK cells (red line) over the examination period. (B) Flow cytometric analysis of CD3⁻CD56⁺ NK cells among lymphocytes and coexpression of NKG2A and NKG2C on NK cells. (C) Expression of inhibitory KIR (KIR2DL1, KIR2DL2/3, and KIR3DL1) on CD3⁻CD56⁺ NK cells. (D) Expression of CD27, CD62L, and CD57 cell-differentiation markers on CD3⁻CD56⁺ NK cells. Numbers correspond to the proportion of positive cells. (E) Polyfunctionality assays of resting CD3⁻CD56⁺ NK cells tested against various targets including K562, 721.221, .221-AEH, and Raji cells in the presence of 1 μg/mL rituximab (E/T ratio of 1/1). Cells were stained with mAbs for CD107a degranulation, and intracellular production of IFN-γ and TNF-α. All data were analyzed with the Boolean gate algorithm of Flow Jo Version 8.8 (TreeStar). Pestle software Version 1.6 was used to remove the background, and pie charts, generated using the Spice software Version 5.2 (NIAI freeware), present the frequency of NK cells positive for 0, 1, 2, or 3 responses (to CD107a, IFN-γ, and TNF-α). Arcs depict the frequency of cells positive for CD107a, IFN-γ, and/or TNF-α, as described.¹⁰  (F) Redirect killing assays of CD3⁻CD56⁺ NK cells against the P815 cell line in the presence of 5 μg/mL mAbs specific for NKG2A and NKG2C, or the matched isotype control (Ig) (E/T ratio of 1/1). Numbers correspond to the proportion of positive cells. (G) Proliferation of NK cells after stimulation for 7 days with IL-2 and/or irradiated 721.221 or .221 AEH cells measured by cell dilution of CFSE. (H) Proliferation of NK cell after stimulation for 7 days with IL-2 and/or irradiated P815 cells plus anti-NKG2A, anti-NKG2C, or both mAbs, measured by cell dilution of CFSE.