Expression of Munc18-2^R65Q in control CTLs reduces cytolytic activity. (A) CD8⁺ T cells from normal control donors were transduced with lentiviral particles encoding for ECFP-Munc18-2^R65Q, ECFP-Munc18-2^WT, or ECFP alone. Seven days posttransduction, the cytotoxic activity against anti-CD3-coated P815 cells was tested at the indicated effector to target-cell ratios. (B) Expression levels of ECFP-Munc18-2^R65Q and ECFP-Munc18-2^wt were compared to endogenous Munc18-2 by western blot analysis using anti-Munc18-2 antibody. (C) CD107a degranulation assay for ECFP-expressing cells. ECFP⁺ cells were purified by cell sorting and were incubated in the presence (stim; blue) or absence (unstim; red) of CD3-coated P815 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 cell surface following incubation with target cells. Plots are representative of 2 independent experiments. (D) Graph showing the percentage of cells that increased CD107a staining after stimulation and the mean fluorescence intensity (MFI) values in the CD107a-PE channel of unstimulated (u) and stimulated (s) cells. Results are the mean ± standard deviation of 2 independent measurements. *P < .01. (E-F) Schematic representation depicting the mode of action of Munc18-2 during lytic granule secretion in a normal control (E) or patient cell carrying the STXBP2^R65Q mutation (F). Step 1: soluble Munc18-2^WT and Munc18-2^R65Q can bind monomeric STX11 on the acceptor membrane (probably the plasma membrane). Step 2: lytic granules approach the plasma membrane on cell activation. Step 3: both Munc18-2^WT and Munc18-2^R65Q facilitate SNARE-complex assembly between specific v-SNAREs (such as Vamp8) and target-SNAREs (such as STX11 and SNAP23). Step 4: Munc18-2^R65Q, but not Munc18-2^WT, may arrest some SNARE complexes in a partially zippered state and thus uncouple their coordinated action, which is required for membrane fusion. Step 5: top view of a lytic granule docked at the plasma membrane. The dominant-negative effect of Munc18-2^R65Q might result from the fact that it inactivates some of the SNARE complexes involved in the membrane fusion reaction, thereby reducing the energy generated and inhibiting lytic granule fusion with the plasma membrane.