Figure 4

HGF restrains cytotoxicity of antigen-specific CD8 ⁺ cells in a perforin-dependent manner. (a) The cytolytic capacity of day 5-cultured CD8⁺ T cells was tested using EL-4 cells as targets. CD8⁺ T cells isolated from splenocytes cultured with HGF demonstrated dramatically lower specific cytotoxicity compared to CD8⁺ T cells isolated from splenocytes cultured alone (right panel). gp100_25-33-stimulated CD8⁺ T cells did not lyse non-pulsed EL-4 target cells (left panel). Results are the mean ± SEM of three independent experiments carried out in triplicates. (b) The 4 h cytotoxicity assays (effector : target ratio, 20 : 1) were performed with CTLs pretreated with (+) or without (-) 1000 nM of concanamycin A (ConA) for 2 h. Data are represented as percent of specific lysis above the specific lysis of the EL-4 targets without adding exogenous peptide. A representative experiment of two is shown. *P <0.05 by Student’s t-test. (c) Flow cytometry analysis of day 5-cultured CD8⁺ T cells showed that addition of HGF did not decrease the level of expression of CD3ζ on a per cell basis, as indicated by comparative geometric mean of fluorescence (GMEAN). Final GMEAN values are the result of the ratio between the GMEAN obtained with the experimental antibody and the isotype control. Representative histograms of day 0 and 5 of three independent experiments with similar results are shown.