Dendritic cells pulsed with hsp70-peptide complexes derived from human hepatocellular carcinoma induce specific anti-tumor immune responses

Figure 3 Generation of specific CTLs against SMMC-7721 by DCs pulsed with hsp70-peptide complexes derived from SMMC-7721 cells. 1 × 10⁵ DCs were incubated with 20 μg/mL hsp70- peptide complexes derived from SMMC-7721 for 4 h at 37°C and then cocultured with 1 × 10⁶ autologous T cells in each well of 24-well culture plate for 7-10 d in the presence of 20 U/mL human IL-2. The stimulated T cells were harvested by density gradient centrifugation and the cytotoxicity was determined by testing LDH release. As a control, T cells cocultured with unpulsed DCs or by themselves were also used as effectors. (A ) T cells were cocultured with autologous DCs pulsed with hsp70-peptide complexes (indicated as hsp70 stimulation) or unpulsed (indicated as DC control), or by themselves (indicated as T control), and SMMC -7721 cells were used as target cells; ( B) T cells were cocultured with autologous DCs pulsed with hsp70-peptide complexes, and SMMC-7721 cells (indicated as SMMC-7721 as target) or NK-sensitive cell line K562 cells (indicated as K562 as target) were used as target cells; (C) T cells were cocultured with autologous DCs pulsed with hsp70-peptide complexes and SMMC- 7721 cells (indicated as SMMC-7721 as target) or Hela cells (indicated as Hela as target) were used as target cells; (D) T cells cocultured with autologous DCs pulsed with hsp70-peptide complexes were used as effector cells and SMMC-7721 cells or Hela cells were used as target cells. The ratio of effector cells:target cells was 10:1. The target cells were preincubated with an anti-MHC class I antibody (W6/32; 1:50 dilution) or not, indicated as blocked and nonblocked separately. (E) T cells cocultured with autologous DCs pulsed with hsp70- peptide complexes were used as effector cells and autologous DCs pulsed with hsp70-peptide complexes or unpulsed were used as target cells (indicated as DC pulsed or DC unpulsed separately). The ratio of effector cells:target cells was 10:1. The results are expressed as mean±SD and each value represents the mean of three replicates.