cPLA₂ activation is necessary for NKG2D cytolytic co-stimulatory functions. Inhibition of cPLA₂ blocks NKG2D augmentation of cytolytic T cell functions and AA reverses this blockage. Co-stimulation of cytolytic T cell functions by NKG2D was studied using three different PB-CTL clones (#411, #414, and #348) that were generated and cultured with low IL-2 (5 U/ml), in which NKG2D does not mediate direct cytolysis. Representative experiments of clone 411(top) and 414 (middle) are shown. PB-CTLs were pretreated with vehicle or CF3. Redirected cytolysis was performed against ⁵¹Cr-labeled FcgR⁺ P815 targets (E/T ratio of 50:1) in the presence of anti-CD3 mAbs at the indicated concentration in combination with either anti-NKG2D mAbs or isotype control IgG1 at fixed concentration (1 µg/ml). (bottom) A rescue experiment was performed by adding AA to CF3-pretreated cells 1 h after co-culture with target cells. A summary of three independent experiments for an anti-CD3 mAb concentration of 12.5 ng/ml with means ± SD is shown. P values are from a Tukey-adjusted pairwise comparison.