CTLs and NK cells expressing the STXBP2^R65Q mutation exhibit impaired functions. (A) Cytotoxicity assay to measure CTL-mediated cell killing. PBMCs from control (black) and P1 cells (red) were cultured with CD3/CD28 beads for 5 days and then incubated for 48 hours with (solid line) or without (dashed line) 200 U/mL of IL-2. Equivalent numbers of CD8⁺ T-cells (Effector) were purified from control and P1 PBMCs and then incubated with anti-CD3 antibody in the presence or absence of P815 target cells (Target) at the indicated cell ratios. The killing assay was run for 4 hours at 37°C, and the amount of lactate dehydrogenase released into the supernatant was quantified using a CytoTox 96 assay. (B) NK cytotoxicity was assessed using control and P1 PBMCs incubated with K562 target cells, as described in panel A. Target-cell lysis was normalized to the number of NK cells in the PBMCs to obtain the percent NK-specific lysis. (C) CD107a assay to measure degranulation. PBMCs from control and P1 were incubated in the presence (stim; blue) or absence (unstim; red) of K562 cells for 4 hours at 37°C. Cells were stained using anti-CD107a-PE, anti-CD56-APC, anti-CD8-FITC, and anti-CD3-PerCP antibodies and analyzed by flow cytometry. CD3^–CD8^–CD56⁺ cells were gated and analyzed for the appearance of CD107a on the surface after incubation with target cells. Plots are representative of 3 independent experiments. (D) Graph showing the percentage of cells that increased CD107a staining on stimulation. The term “δ CD107a” reflects the difference between the percentage of NK cells expressing CD107a after K562 stimulation and the percentage of cells expressing surface CD107a after incubation with medium. (E) Mean fluorescence intensity (MFI) values in the CD107a-PE channel of unstimulated (unstim) vs stimulated (stim) cells. Results are the mean ± SD of 3 independent measurements for P1 and 10 different normal controls. *P < .01.