Slp2a-hem is recruited at the CTL-target cell contact zone and a dominant-negative form of Slp2a decreases cytotoxic granule exocytosis. (A) Microscopic analysis of wild-type LAK-T cells transfected with GFP-Slp2a-hem and DsRed-Rab27a and conjugated with L1210 target cells. (B) Microscopic analysis of wild-type LAK-T cells transfected with GFP-Slp2a C2 and DsRed-Rab27a and conjugated with L1210 target cells. (C) Confocal microscopy of wild-type LAK cells transfected with GFP-Slp2a-hem and unlabeled Rab27a and conjugated with L1210 target cells. Cells were then fixed, permeabilized, and stained with an antiperforin. Two different conjugates are shown. Scale bars represent 5 μm. Data are representative of 5 (A,B) or 4 (C) independent experiments. (D) The release of cytotoxic granule BLT esterase activity from transfected LAK-T cells is shown. Untransfected LAK-T cells (NT) or those transfected with GFP alone (GFP), or SHD of Slp2a tagged with GFP (GFP-SHD) were incubated on anti-CD3–coated plates for 4 hours. Cell supernatants were collected and assayed by enzyme-linked immunosorbent assay (ELISA) for serine esterase. Data are the mean of 3 independent experiments. Error bars show standard deviation among experiments. Transfection efficiency for each construct was between 40% and 50% as determined by FACScan analysis of GFP production (data not shown). **P < .01 (bracketed comparison).