Figure 3

Binding affinity of GPC-3 peptides to HLA-A2. T2 cells were plated into 96-well U-bottomed plates at 1 × 10⁵ cells per well in 200 μL X-Vivo (Biowhittaker) and cultured overnight at 18°C to increase cell surface HLA-A2 expression. a. 3 hours after pulsing with increasing concentration of GPC-3 peptides, positive control (AFP) peptide or negative control (random) peptide plus 5 nM β₂ microglobulin and incubation at 37°C, T2 cells were stained with a FITC-conjugated HLA-A2 specific antibody and examined by flow cytometry; b. T2 cells were stained with a FITC-conjugated HLA-A2 specific antibody and examined by flow cytometry at time points after the cells had been incubated for 3 hours at 37°C with 100 μM peptide, 5 nM β₂ microglobulin and 5 μg/ml Brefeldin A. The data shown are representative of three independent experiments.