VAMP8-carrying cells accumulate and fuse at the immune synapse before cytotoxic granules. (A–E) Bead-stimulated human CD8⁺ T cells were transfected with VAMP8-TFP and perforin-mCherry encoding constructs and imaged 24 h after transfection. (A) Selected live-cell TIRF microscopy images of VAMP8-TFP and perforin-mCherry in a transfected CTLs in contact with an anti-CD3– and anti-CD28–coated coverslip. Bar, 2.5 µm. (B) Mean dwell time of VAMP8-TFP and perforin-mCherry vesicles in the TIRF plane per cell (n = 15, paired t test, ***, P > 0.001). (C) Mean VAMP8-TFP and perforin-mCherry vesicle accumulation over time in the TIRF plane per cell (n = 15). (D) Mean fluorescence dispersion events for VAMP8-TFP and perforin-mCherry vesicles in the TIRF plane per cell (n = 15, paired t test, ***, P > 0.001). (E) Cumulative fluorescence dispersion events for VAMP8-TFP and perforin-mCherry vesicles in the TIRF plane per cell (n = 18). Error bars show means and SDs.