Quantification of perforin and other granule constituents in T-cell and NK cell subsets. (A-B) Freshly isolated, resting PBMCs from healthy adult volunteers were surface stained with fluorochrome-conjugated antibodies to CD3, CD4, CD8, CD14, CD19, CD56, and CD57, followed by fixation, permeabilization, and intracellular staining with isotype control antibodies or antibodies to perforin, CD107a, granzyme A, granzyme B, or granzyme K, and analyzed by flow cytometry. Lymphocytes were gated on forward scatter/side scatter plots. (A) Perforin-expressing cells were assessed relative to expression of the lineage markers CD3, CD4, CD8, CD56, and CD57. The pie chart depicts the relative distribution of the lymphocyte subsets, as indicated. Shading reflects the relative mean fluorescence intensity (R-MFI) of perforin staining in each subset and the frequency of each subset is indicated. Results are derived from 12 donors. Numbers indicate mean ± SD frequencies of indicated subsets relative to the total perforin-expressing lymphocyte population. (B) Values represent R-MFI, where the MFI values of indicated staining have been subtracted from MFI values of isotype control antibodies. Values represent the means ± SD of 12 donors. Numbers indicate mean ± SD frequencies of indicated subsets relative to the total lymphocyte population. Statistical analyses were performed using the Wilcoxon signed-rank matched pairs test. *P < .05; **P < .01; ***P < .001.