Methodological platform and relative degranulating capacity of lymphocyte subsets. (A-D) Freshly isolated, resting PBLs alone or mixed with target cells and mAbs as indicated were incubated for 2 hours at 37°C. (A,B) Thereafter, cells were stained with fluorochrome-conjugated anti-CD3, -CD56, and -CD107a mAbs and analyzed by flow cytometry. (A) Lymphocytes were gated on forward scatter/side scatter plots, followed by gating of CD3⁺, CD3⁻CD56⁺, and CD3⁻CD56⁻ cell subsets. (B) Induced CD107a surface expression (ΔCD107a⁺) was calculated as the percentage of CD107a⁺ cells after indicated stimulation subtracted from the percentage of CD107a⁺ cells after incubation of PBLs alone. For gating on CD3⁺ cells, a fluorochrome-conjugated anti-CD3 mAb was used. Values represent the mean (± SD) of 6 donors. (C,D) After indicated stimulation, cells were stained with fluorochrome-conjugated anti-CD3, anti-CD4, anti-CD8, anti-CD14, anti-CD16, anti-CD62L, anti-CD107a, and anti-CCR7 mAbs. (C) T cells were gated on forward scatter/side scatter plots for lymphocytes (R1 gate), followed by forward scatter/forward area plots (R2 gate), and gated on CD3⁺CD14⁻ cells (R3 gate). Contour plots show CD3⁺CD14⁻ T-cell populations (R3 gate) overlayed with dots representing CD3⁺CD14⁻CD107a⁺ T cells (R4 gate). The frequencies of degranulating T cells are indicated. Numbers indicate the percentage of degranulating T cells within each quadrant. (D) Induced CD107a surface expression was determined for indicated CD3⁺CD14⁻ T-cell subsets. Values represent the mean (± SD) of 5 donors. Numbers indicate mean frequencies of CD3⁺CD14⁻ T-cell subsets and their SD. (E) Resting PBLs were stained with anti-CD3, anti-CD4, anti-CD8, anti-CD14, anti-CD16-, anti-CD56, and anti-CD62L mAbs, followed by fixation, permeabilization, and intracellular staining with anti-CD107a, antiperforin, antigranzyme A, or anti-CD63 mAbs. Values represent relative mean fluorescence intensity (R-MFI), where MFI values for indicated staining have been subtracted for MFI values for isotype control mAbs. Values represent mean (± SD) of 5 donors.