Figure 4

Induction of functional T cells in vitro by GPC-3 peptide-loaded DC. a. PBMC (1 × 10⁵/well), depleted of HLA class II positive cells, from 3 healthy HLA-A2 positive subjects were stimulated twice with autologous, monocyte-derived DC (1 × 10⁴/well), which had been pulsed with 1 μM peptides for 3 hours, matured with LPS and γ-irradiated, in serum-free X-Vivo medium supplemented with IL-2 (20 U/ml) and IL-7 (10 ng/ml). T cell proliferation was measured by ³H-thymidine incorporation, Stimulation Index is ratio of T cell proliferation due to peptide-pulsed DC ÷ control DC. b. CD8+ enriched T cells were stimulated twice by autologous, γ-irradiated, peptide-pulsed, matured DC. The ability of these CD8+ T cells to lyse HepG2 cells was assessed by chromium release assay. Target cells (HepG2) were labelled with 200 μCi Na₂⁵¹CrO₄ and plated (5 × 10³ cells/well) in round-bottomed 96 well plates. Effector cells were added at graded effector/target ratios and after 4 hours incubation at 37°C, 50 μl of the culture supernatant was harvested and radioactivity counted in a scintillation counter. Spontaneous release was <15% in all assays. Error bars reflect standard error of mean of 3 experiments.