LFA-1–dependent perforin release in human CD56⁺CD8⁺ cells. (A) Detection of intracellular perforin after ICAM ligand cross-linking on total PBMCs. Human PBMCs, preactivated with IL-2 (200 ng/mL, 12 hours) were treated with ICAM-1, ICAM-2, or ICAM-3 Fc proteins (10 μg/mL, 30 minutes), then samples were cross-linked with antihuman IgG antibodies for 4 hours. Cells were stained for intracellular perforin (perforin-Cy5 conjugate) by flow cytometry. Relative population frequency is denoted by drawn gate and values are on the right of each stimulation. Results are representative of triplicate experiments. (B) Perforin release of CD56⁺CD8⁺ cells. Human PBMCs were treated with ICAM-2 (10 μg/mL) or ICAM-2 Fc–coated beads (4 × 105 beads containing 2 μg ICAM-2) in the presence of IL-2 for 12 hours and then incubated with CMFDA-labeled target HL60 cells at a 1:50 E/T ratio for 4 hours. Intracellular perforin was assessed in the CD56⁺CD8⁺ population and quantified for release as discussed in “Materials and methods.” (C) Cytolytic activity was measured in the presence of LFA-1 blocking antibody TS1/22. Blocking mAbs were incubated at 20 μg/mL, 30 minutes prior to ICAM-2 Fc treatment (10 μg/mL, 30 minutes) and subsequent antihuman IgG (5 μg/mL) application and then incubated with CMFDA-labeled target HL60 cells at a 1:50 E/T ratio for 4 hours. (D) Intracellular perforin release was quantified in the presence of LFA-1 blocking antibodies. Blocking mAbs were incubated at 20 μg/mL, 30 minutes prior to ICAM-2 Fc treatment (10 μg/mL, 30 minutes) and subsequent antihuman IgG (5 μg/mL) application. Perforin release was quantified as described for the CD56⁺CD8⁺ human population subset. Error bars show standard deviation of 3 experiments.