Fig. 3.

DCs pulsed with gp96-peptide complexes derived from SMMC-7721 cells induced antitumor CTLs specific for SMMC-7721. 2 × 10⁵ DCs, which had been pulsed with gp96-peptide complexes derived from SMMC-7721 or E. coli for 4 h at 37°C, were cocultured with 1 × 10⁶ autologous T cells for 7–10 days in the presence of 20 units/ml human IL-2 in each well of 24-well culture plate. The stimulated T cells were harvested and the cytotoxicity was determined by testing LDH release. a DCs were incubated with 40 μg/ml gp96-peptide complexes and then used as stimulators for autologous T cells, and SMMC-7721 cells were used as target cells. DCs pulsed with human gp96-peptide complexes were indicated as gp96 stimulation, unpulsed DCs indicated as DC control, and T cells cultured by themselves indicated as T control. b DCs were incubated with different concentrations of human gp96-peptide complexes and then used as stimulators for autologous T cells, and SMMC-7721 cells were used as target cells; c DCs were incubated with 30 μg/ml human gp96-peptide complexes and then used as stimulators for autologous T cells, and SMMC-7721 cells or NK sensitive cell line K562 cells were used as target cells; d DCs were incubated with 30 μg/ml human gp96-peptide complexes and then used as stimulators for autologous T cells, and SMMC-7721 cells or another HCC cell line BEL-7402 cells were used as target cells. The target cells were also preincubated with an anti-MHC class I antibody (W6/32; 1:50 dilution) and assayed for lysis, indicated as SMMC-7721/blocked or BEL-7402/blocked, and lysis for the target cells without preincubation with anti-MHC class I antibody were indicated as SMMC-7721/nonblocked or BEL-7402/nonblocked. e DCs were incubated with 30 μg/ml human gp96-peptide complexes and then used as stimulators for autologous T cells, and autologous DCs pulsed with human gp96-peptide complexes or unpulsed DCs were used as target cells. The ratio of effector: target was 10. f DCs were incubated with 40 μg/ml gp96-peptide complexes derived from SMMC-7721 cells or recombinant human gp96 isolated from E. coli and then used as stimulators for autologous T cells, and SMMC-7721 cells were used as target cells. The results are expressed as mean ± SD and each value represents the mean of three replicates