DNAM-1 and NKG2D are both involved in the NK-cell–mediated killing of allogeneic activated T cells. (A) A classic ⁵¹Cr-based cytotoxicity assay was performed using IL-2–activated NK cells as effectors and 3-day SEB-activated allogeneic PBMCs as targets at the indicated E:T ratios. The assay was performed in the presence of neutralizing mAbs against NKG2D and/or DNAM-1 or against CD56 used as an isotype control. The data shown are representative of 1 of 6 donors tested. (B) Mean ± SE of lytic units (LU) calculated on 6 different donors. (C) NK cells, used as effectors, were incubated at 37°C with allogeneic, CFSE-labeled, 3-day SEB-stimulated PBMCs used as targets at an E:T ratio of 6.25:1, 12.5:1, 25:1, and 50:1. After 4 hours, cells were stained with 7-AAD, as described in “Immunofluorence and FACS analysis.” Analysis of target cells was performed by gating CFSE⁺ cells. An E:T ratio of 12.5:1 is shown. Numbers represent the percentage of cells in each quadrant. The experiment shown is representative of the 8 performed. (D) The percentage of lysed cells was analyzed by calculating the percentage of 7-AAD⁺ target cells in the CFSE^low (◇) or CFSE^high (□) populations. The donor analyzed is the same as that in panel C.