Fig 6.
Defective TCR signaling in LATKO CTLs. (A and B) TCR-mediated protein phosphorylation. CTLs were restimulated with anti-CD3 (5 μg/ml) and anti-CD8 (1 μg/ml) for the indicated time points before lysis. Cell lysates were analyzed by Western immunoblotting with anti-pTyr, phospho-specific, or pan antibodies against LAT, Erk, PLC-γ1, PKCθ, and Akt. The data shown are from one representative of three independent experiments. (C) Calcium mobilization. CTLs were loaded with the calcium indicator, indo-1. Calcium flux was induced by anti-CD3 and anti-CD8 cross-linking and analyzed by FACS. The data are representative of three independent experiments. (D) Time-lapse montages of both ratiometric Fura-2 images and the differential interference contrast of CTLs. CTLs were preloaded with Fura-2, mixed with Ova peptide-loaded L1210/Kb cells, and imaged at intervals of 30 s. The first time point (0′00″) marked the initial contact of a T cell with a target cell. Fura images are pseudo-colored, with warmer colors indicating higher intracellular calcium concentrations. Upper rows, WT CTLs; bottom rows, LATKO CTLs. (E) Quantitative analysis of calcium flux by live cell imaging. CTLs were pretreated with or without CTLA4-Ig and anti-ICAM-1 antibody for 30 min before incubation with target cells. The integrated value of calcium mobilized into the cytosol for each cell was generated by summing the changes in the relative calcium ratio (340/380 nm) at each time point within the first 10 min. Each dot represents one CTL. Horizontal bars indicate the mean. n.s., not significant; *, P < 0.05, **, P < 0.005. The data shown are from two independent experiments. (F) Calcium mobilization in the presence of U73122 (0.5 μM). (G) Effect of PLC inhibitor on CTL-mediated killing. CTLs were pretreated with U73122 (0.5 μM) or dimethyl sulfoxide (DMSO) for 30 min and then incubated with target cells in the presence of these inhibitors for 45 min before FACS analysis. The data shown are from one representative of three independent experiments.