T cells generated by DC transfected with GPC-3 mRNA are functional in vitro GPC-3 mRNA transfected DC but not mock transfected DC induced
proliferation of autologous T cells (Figure 2a), indicating that T cells reacting to GPC-3 epitopes are represented in the peripheral T cell repertoire. ELISPOT assay for interferon-gamma production found that DC expressing GPC-3 generated significantly more T cells producing interferon-gamma than mock transfected DC (53 ± 15 versus 4 ± 3 spots per well, respectively; p < 0.01) (Figure 2b). P505-15 These data demonstrate that monocyte-derived DC transfected with GPC-3 mRNA and matured with LPS were able to process and present GPC-3 derived epitopes, resulting in the proliferation of autologous T cells, which were functional as assessed by interferon-gamma production. Figure 2 T cells generated by DC transfected with GPC-3 mRNA are functional in vitro. PBMC were depleted of HLA class II positive cells and co-cultured with autologous, γ-irradiated, LPS matured DC in serum-free X-Vivo medium supplemented on days 1, 3 and 7 of culture with IL-2 (20 U/ml) and IL-7 (10 ng/ml). After 7 days, T cells were re-stimulated with the same DC for a further 5 days. a. T cell proliferation (1 × 105/well) was measured by
3H-thymidine incorporation, T cells this website were cultured alone, with DC (1 × 104/well) transfected with 20 μg GPC-3 mRNA, or mock transfected DC. b. ELISPOT assay for interferon-γ production was performed on T cells (1 × 105/well) stimulated by DC transfected with 20 μg GPC-3 mRNA or mock transfected DC. Assessment of binding affinity of GPC-3 peptides to HLA-A2 Among the 6 GPC-3
peptides tested, peptides 1, 2, 4 and 5 (GPC-3 229-237, 522-530, 186-194 and 222-230, respectively) showed significant binding affinities, whereas peptides 3 and 6 (GPC-3 299-307 and 169-177, respectively) did not show significant binding under the conditions used in these experiments (Figure 3). However, none of the GPC-3 peptides exhibited very strong binding to HLA-A2, as all demonstrated weaker binding than the “”immunodominant”" AFP peptide (GVALQTMKQ). Figure 3 Binding affinity of GPC-3 peptides MYO10 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 MEK162 in vivo 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.