Kinesin-1 regulates the terminal transport of the Rab27a/Slp3 granules at the IS. (A) Confocal microscopy of CTLs transfected with GFP-KIF5B, DsRed-Slp3, and Flag Rab27a and conjugated with L1210 target cells (top panel, insets represent an enlarged image). Confocal microscopy of CTLs transfected with GFP-KIF5 DN, DsRed-Slp3, and Flag Rab27a and conjugated with L1210 target cells (bottom panel, insets represent an enlarged image). All images of conjugates were representative of > 90 cells observed over at least 3 independent experiments. (B) CTLs were transfected with CFP-Slp3, CFP-KIF5B, and Flag-Rab27a or CFP-Slp3, CFP-KIF5 DN, and Flag-Rab27a. Transfected cells were incubated for 3 hours alone or with a coated anti-CD3 antibody in the presence of CD107a and CD107b antibodies. The cells were then stained with anti-CD3–PE and anti-CD8–APC. Lymphocytes were gated on CD3⁺CD8⁺. Profile shows CFP versus CD107-FITC gating on CD3⁺CD8⁺ CTLs. For each condition, we perform a histogram overlay analysis of Lamp secretion by CFP⁺ cells. Each condition is representative of 2 independent experiments performed in duplicate. (C) Representative TIRF images of mCherry-KIF5B/GFP-Rab27a/GFP-Slp3 and mCherry-KIF5DN/GFP-Rab27a/GFP-Slp3 transfectant CTLs. The “pseudocolor” scales were used to indicate the intensity staining in TIRF. A vesicle was defined by a minimum of a square of 3 pixels per 3 pixels (1 pixel = 0.16 μm) of strong intensity signal (orange to purple). Scale bars represent 5 μm. The number of vesicles per cell acquired in TIRF mode is shown on the graph. Each dot represents an individual cell; and the line, the median value. For the mCherry-KIF5B/GFP-Rab27a/GFP-Slp3 condition, 20 cells have been analyzed from cumulating 3 independent experiments. For the mCherry-KIF5DN/GFP-Rab27a/GFP-Slp3 setting, 16 cells have been analyzed from cumulating 3 independent experiments. Statistical analysis was performed using a Mann-Whitney test. ***P < .0001.