VAMP8 knockdown does not affect proximal T cell signaling, but regulates Stx11 vesicle trafficking and fusion at immune synapses. (A–K) Bead-stimulated human CD8⁺ T cells were transfected with siRNA, as indicated, as well as LifeAct-GFP (D–F) or Stx11-mCherry (G–K). (A) CTLs were loaded with Ca²⁺-sensitive Fluo-4 and Fura red dyes and assessed by flow cytometry and stimulated as indicated. iono., ionomycin. (B and C) Western blot of CTL lysates after transfection and stimulation as indicated. (B) Blots for phospho-ERK (pT202/pY204), VAMP8, and loading control GAPDH are shown for one representative donor. (C) Densitometry analysis of phospho-ERK signaling in five individual donors. Mean values are indicated, with bars representing SDs (ANOVA). (D) TIRF microscopy images of CTLs transfected with siRNA, as indicated, and LifeAct-GFP after contact with anti-CD3 and anti-CD28 antibody-coated coverslips for 250 s. (E) Quantification of F-actin clearance based on LifeAct-GFP fluorescence in individual cells (n = 12). (F) Quantification of cell spreading was based on LifeAct-GFP fluorescence in individual cells (n = 15). (G) Selected live-cell TIRF microscopy images of mCherry-Stx11 in representative CTLs transfected with siRNA as indicated. Arrowheads indicate fusion events; vesicle 1 is indicated by closed arrowheads, and vesicle 2 is indicated by open arrowheads. (H) Mean dwell time of mCherry-Stx11 vesicles in the TIRF plane per cell (n = 15, unpaired t test, **, P > 0.01). (I) Mean mCherry-Stx11 vesicle accumulation over time in the TIRF plane per cell (n = 20). (J) Mean overall mCherry-Stx11 accumulation over time in the TIRF plane per cell (n = 20). (K) Mean fluorescence dispersion events for mCherry-Stx11 vesicles in the TIRF plane per cell (n = 15, unpaired t test, **, P > 0.01). (L) Representative TIRF image depicting concentric circle region of interest used for radial analysis and dispersion of VAMP8-TFP and mCherry-Stx11. (M) Graph depicts the distribution of cumulative VAMP8-TFP fluorescence dispersion events on a per cell basis. (N) Graph depicts the distribution of cumulative mCherry-Stx11 fluorescence dispersion events on a per cell basis. (O) Quantification of mCherry-Stx11 radial distribution in TIRF plane of CTLs cotransfected with siRNA, as indicated. siCTRL, control siRNA. Bars: (D and G) 5 µm; (L) 2.5 µm.