The Rab27a/Slp3 complex is involved in lytic granule secretion. (A) CTLs were transfected with CFP-Slp1, CFP-Slp2a, or CFP-Slp3 and then incubated for 3 hours alone or with a coated anti-CD3 antibody in the presence of CD107a and CD107b antibodies. Thereafter, cells were stained with anti-CD3–PE and anti-CD8–APC antibodies. Lymphocytes were gated on CD3⁺CD8⁺. The 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 DsRed-Slp1 or DsRed-Slp3 and conjugated with L1210 target cells. The cells were then fixed, permeabilized, and stained with anti-perforin antibody (green). The lower insets represent an enlarged image. Scale bars represent 5 μm. All images of conjugates were representative of > 90 cells observed over at least 3 independent experiments performed in triplicate. (C) CTLs were transfected with CFP-Slp1 Flag-Rab27a or CFP-Slp3 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. Thereafter, cells were stained with anti-CD3–PE and anti-CD8p–APC antibodies. Lymphocytes were gated on CD3⁺CD8⁺. The 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.