The Slp3-linker domain expression impairs Lamp release but has no effect on lytic granule polarization. (A) CTLs were transfected with CFP-Slp1 linker, CFP-Slp2 linker, or CFP-Slp3 linker. 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 3 independent experiments performed in duplicate. (B) Confocal microscopy of CTLs transfected with GFP-Slp3 linker and stained for α-tubulin (red). The insets represent an enlarged image. Scale bars represent 5 μm. All images of single cell were representative of > 60 cells observed over at least 4 independent experiments performed in duplicate. (C) Confocal microscopy of CTLs transfected with mCherry-Slp3 linker and GFP-KLC1. The insets represent an enlarged image. Scale bars represent 5 μm. All images of single cell were representative of > 60 cells observed over at least 4 independent experiments performed in duplicate. (D) Confocal microscopy of CTLs transfected with mCherry-Slp3 linker and conjugated with L1210 target cells. Cells were then fixed, permeabilized, and stained with anti-perforin antibody (green). The insets represent an enlarged image. Scale bars represent 5 μm. All images of conjugates were representative of > 60 cells observed over at least 4 independent experiments performed in duplicate.