Figure 3

Induction of HCC cells-specific CTL by DCs/allo-HCC. A, Nonadherent PBMCs from healthy donors stimulated by unirradiated DCs/allo-HCC (upper panel) or DCs mixed with allo-HCC cells (lower panel) were cocultured with the HCC cells at a ratio of 10:1. After 4 hr culture, cells were examined under a microscope. After 12 hr culture, floating cells were harvested and adherent cells were examined (magnification, ×20) (magnification, × 100 in upper corner panel). B, T cells stimulated by unirradiated DCs/allo-HCC were cocultured with PKH26-labeled allo-HCC cells at the indicated ratios. Target cells were preincubated with control IgG (solid circles) or anti-MHC class I mAb (W6/32; 1:100 dilution, open circles).C, T cells were stimulated with 2 types of fusion cell preparation from three healthy donors: 1) unirradiated DCs/allo-HCC in the absence of HCCsp (■) and 2) unirradiated DCs/allo-HCC/sp in the presence of HCCsp (□). T-cell proliferation assay was performed by Cell Titer 96 Nonradioactive Cell Proliferation Assay Kit according to the protocol. D, After stimulation with the 2 types of fusion cell preparation from four healthy donors, percentage of IFN-γ-positive CD4⁺ or CD8⁺ T cells was assessed. E, After stimulation with the 2 types of fusion cell preparation from three healthy donors, T cells were incubated with PKH-26 labeled allo-HCC cells at a ratio of 60:1. Percentage of cytotoxicity (mean ± SD of 3 replicates) was determined by flow cytometry-CTL assay. F, After stimulation with the 2 types of fusion cell preparation, CD8⁺ T cells (HLA-A2+) from three healthy donors were analyzed by HLA-A2/WT1 pentameric assay. CD8+ T cell reactivity to WT1 was shown as the percentage of double-positive population (CD8+ pentamer+) among all CD8+ T cells. For each group, the mean ± SD of three experiments is shown. *, Significant differences.