Figure 2.

Anti-CD2 and anti-NKG2D monoclonal antibodies (mAbs) blocked natural killer (NK) cytotoxicity against MPN3. (a) A standard ⁵¹Cr-release assay was performed with interleukin-2 (IL-2) -activated human primary NK cells as effector cells and MPN3 as target cells. NK cells were pre-incubated with 10 μg/ml of designated blocking mAbs or with their isotype control. (b) Reverse transcription–polymerase chain reaction for porcine CD58 and ULBP1 genes was performed with RNAs isolated from MPN3 and primary porcine aortic endothelial cells (PAEC). The expression of messenger RNA of porcine GAPDH genes was shown as an internal control for house-keeping genes. Reaction mixture without complementary DNAs was used as negative control. (c) CD107a assay was performed to assess degranulation events, as described in the Materials and methods. IL-2-activated human primary NK cells were pre-incubated with designated mAbs before incubation with MPN3 cells. Six hours later, CD107a expression was detected using anti-CD107a mAb by fluorescence-activated cell sorting. Degranulation was measured by expression of CD107a on CD56⁺ NK cells. The values represent the percentages of CD56⁺ CD107a⁺ cells, which are representatives of a minimum of three independent experiments.