Functional assays display a correlation of phenotypical characteristics with improved functions regarding adhesion, cytokine production and cytotoxicity of IL-12-modulated NK cells.

<p>(A) Comparison of adhesion capacity to endothelial cells. <i>Ex vivo</i> generated NK cells from day 28 of cultures including or without IL-12 were purified and subsequently used in adhesion assays on LecTERT lymphatic endothelial cells (LEC) or resting human umbilical vein endothelial cells (HUVEC). Mean values ± SEM calculated from 3 independent experiments each performed in duplicates are shown. (B) Comparison of IFN-γ production capacities. <i>Ex vivo</i> generated NK cells from day 28 were cocultured with target cells at 1∶1 ratio. IFN-γ content was measured in the supernatants by ELISA. Mean values ± SEM calculated from 4 independent experiments are shown. (C) Comparison of NK cytotoxicity. After day 28 of culture <i>ex vivo</i> NK cells differentiated in the presence or absence of IL-12 were tested for their capacity to lyse the MHC class I-deficient target cell line K562 (n = 5) and the MHC class I-positive target cell line KG1a (n = 3). Cytotoxicity was determined on the basis of remaining intact CFSE-labeled target cells and is shown as mean values ± SEM. (D) Comparison of ADCC capacity. <i>Ex vivo</i> NK cells generated with or without 0.2 ng/ml IL-12 resulting in 36% or 21% CD56⁺CD16⁺ cells, respectively, were purified at day 28 of culture and subsequently analyzed in Europium-release killing assays. The human acute lymphoblastic leukemia cell lines 721.221 and Nalm-6 were used at an effector to target ratio of 6∶1 in the presence or absence of rituximab as indicated. Mean values ± SD calculated from triplicate wells are shown for one experiment representative of two performed.