p44/42 MAPK and Syk activities map to distinct junctures in the LFA-1–dependent adhesion of CD56⁺CD8⁺ NK cells. (A) Src inhibition decreases lysis of target cells. CMFDA-labeled K562 cells were incubated at 1:25 E/T ratio of targets that had been treated with either IL-2 of IL-12 (100 ng/mL, 12 hours) in the presence of increasing concentrations of the src inhibitor PP2. Specific lysis is plotted. (B) Conjugate formation of CMFDA-labeled HL60 cells and CD56⁺CD8⁺ cells. Conjugate flow cytometric assay was performed on PBMCs treated with indicated chemicals (10 μM, 30 minutes) prior to treatment with ICAM-2 (10 μg/mL, 30 minutes). CMFDA-labeled HL60 cells were incubated at 1:25 E/T ratio for 5 minutes and fixed with 2% paraformaldehyde directly. Cells were then immunolabeled with CD8 and CD56 antibodies and gated for CD8⁺CD56⁺ cell populations, and percent HL60 fluorescence was calculated relative to total HL60 cells. (C) Intracellular perforin levels (perforin-PE conjugate) after E/T cell mixing in the presence or absence of 10 μM PD98059 (preincubated for 30 minutes before cell mixing). Low and medium perforin-containing subsets are gated and displayed for CD56 and CD8 markers as denoted by arrows. Perforin medium subsets (CD56⁺CD8^med, CD56⁺CD8^high) were further gated and displayed for granzyme A levels, as denoted by color-coded gates. Similar analysis was performed for perforin low subsets. Numbers at the top of each gate represent the population frequency in each section. (D) Perforin content in CD56CD8 subsets following E/T cell mixing of target HL60 cells in the presence of 20 μM PD98059. Some cells were stimulated with ICAM-2 (10 μg/mL) prior to incubation with target cells. Median fluorescent values are displayed for CD56CD8 subsets at various E/T ratios. (E) Phospho-p44/42 detection after LFA-1 and CD16 cross-linking. PBMCs were incubated with 1 μg/1 × 106 cells of β₂ integrin mAb (clone CTB104) and CD16 (clone 3G8), followed by goat antimouse IgG (Fab′)₂. Cells were stimulated at 37° C for 15 minutes before being processed for intracellular flow cytometry. Error bars in panels B, C, and D show standard deviation of 3 experiments.