In the absence of 2B4-CD48 interactions, activated NK cells undergo perforin-dependent fratricide. LAK cells activated with IL-2 in vitro were used as effectors and targets in cytotoxicity assays. (A) WT and 2B4 KO LAK cells were used as effectors against WT LAK cells. (B) WT LAK cells were used as effectors against WT and CD48 KO LAK cells. (C) WT LAK cells were used as effectors against untreated, or anti-CD48 antibody–coated WT, or β2m KO LAK cells. To coat LAK targets, cells were incubated for 15 minutes in 10 μg/mL anti-CD48 antibody at room temperature. Coated cells were then washed and used in killing assays. (D) WT and perforin KO LAK cells were loaded with chromium and incubated in the presence of anti-CD16/CD32 blocking antibody plus anti-2B4, anti-CD48, anti-NK1.1, or anti-H-2K^b antibody. Chromium release, which indicates lysis due to fratricide, was measured after 6 hours of incubation. (E) WT and perforin KO LAK cells were incubated in the presence of anti-CD16/CD32 blocking antibody plus anti-2B4, anti-CD48, or anti-NK1.1 antibody. Annexin V and PI staining was measured after 6 to 7 hours of incubation with blocking antibody. The percents of Annexin V⁺, PI⁺ apoptotic cells are noted in the upper right quadrants of the FACS dot plots. Results are representative of 4 independent experiments. Error bars are standard deviation (SD).