Figures & data
Figure 1. Influence of MOI on GFP or tyrosinase expression by TC71, A375 and RD-ES cells. (A) Cells were infected using different MOI of MVA-GFP. Viable cells were evaluated 16 h after infection for mean fluorescence intensity of GFP-positive cells. Data are given as mean ± SEM of three independent experiments. (B) Infection with MVA-hTyr and assessment of tyrosinase protein expression by western blot analysis 16 h after infection. Control represents cells that were not infected.
Figure 2. Kinetics of GFP or tyrosinase expression of TC71, A375 and RD-ES cells after MVA infection. A375, TC71 and RD-ES cells, infected with MVA-GFP at an MOI of 10, were analyzed 0, 4, 8, 16, 24, 48 and 72 h post infection by flow cytometry and western blot analysis, respectively. (A) The percentage of GFP-positive cells. (B) Mean fluorescence intensity of MVA-GFP-infected cells and not infected control cells over time. Results of A and B are given as mean ± SEM of three independent experiments. (C) Tyrosinase expression of TC71 and RD-ES cells evaluated by western blot analysis. Control cells were not infected (lane 1), or infected with MVA wild-type not containing the cDNA for tyrosinase (lane 3).
Figure 3. IFN-γ secretion of TryF8 T cells induced by cell lines infected with MVA-hTyr. (A) MVA-hTyr-infected A375, TC71 and RD-ES cells and melanoma cell line SK23-Mel, which expresses tyrosinase endogenously, were co-cultured with CTL clone TyrF8 at an E:T ratio of 1:7 for 24 h. Background IFN-γ production by T cells was below 10 pg/mL. IFN-γ content in supernatants was quantified by ELISA. Control cells were either not infected (bar 1) or infected with MVA-wild type not containing the tyrosinase cDNA (bar 3). There was no significant difference in IFN-γ production comparing hTyr-transfected A375 and TC71 cells (p = 0.087). (B) MVA-hTyr-infected A375 and TC71 cells were co-cultured with TyrF8 T cells at an E:T ratio of 1:5 and ELISPOT analysis was performed. Control cells were infected with MVA wild-type. TyrF8 alone produced less than 10 spots (data not shown). Results are given as mean ± SEM of three independent experiments.
Figure 4. IFN-γ secretion of TyrF8 T cells achieved by primary sarcoma cells infected with MVA-hTyr. Primary HLA-A2+ sarcoma cells (n = 4 patients) were infected with MVA wild-type (bar 1) or MVA-hTyr (bar 2) as indicated and co-incubated with TyrF8 CTLs at an E:T ratio of 1:5 for 24 h. Control cell line SK23-Mel (HLA-A2 positive with endogenous tyrosinase expression) was used at the same E:T ratio. IFN-γ ELISPOT was performed. Results are shown as mean ± SEM of four independent experiments. P values indicate statistical significance between MVA wild-type control (1) and MVA-hTyr infected (2) cells.
Figure 5. IFN-γ secretion of TyrF8 T cells by infected primary sarcoma cells after heat treatment. Primary sarcoma cells were either exposed to 37°C or 41.8°C for 2 h. After 6 h of recovery at 37°C, cells were infected with MVA wild-type (bar 1) or MVA-hTyr (bar 2). Thereafter TyrF8 cells were added at an E:T ratio of 1:5 for 24 h. HLA-A2- and tyrosinase-positive SK23-Mel cells served as control. Results of the IFN-γ ELISPOT of three independent experiments are given. P values indicate statistical significance between MVA wild-type control (1) and MVA-hTyr infected (2) cells in the 37°C and heated samples.
Figure 6. IFN-γ secretion of TyrF8 T cells by infected TC71 sarcoma cell line and A375 melanoma cell line after exposure to 4-OH-ifosfamide. TC71 sarcoma cells (A), or A375 melanoma cells (B), were treated with ifosfamide and infected with MVA-hTyr (2). Cells treated with ifosfamide (100 or 200 µM) but either not infected (0) or infected with MVA wild-type (1) served as controls for background IFN-γ. IFN-γ production of TyrF8 after stimulation with SK23-Mel cells is depicted as reference value for T cell activity. IFN-γ content in 4-h co-culture supernatants was quantified by ELISA. Results are shown as mean ± SEM of three independent experiments.
Figure 7. IFN-γ secretion of TyrF8 T cells by infected TC71 sarcoma cell line and A375 melanoma cell line after exposure to 4-OH-ifosfamide and heat. TC71 sarcoma cells (A), or A375 melanoma cells (B), were simultaneously treated with ifosfamide and heat shock (41.8°C, 2 h) and thereafter infected with MVA-hTyr (2). As controls, similarly treated cells but not infected (0) or cells infected with the MVA wild-type (1) were used. IFN-γ production of TyrF8 after stimulation with tyrosinase-expressing SK23-Mel cells is depicted as reference value for T cell activity. IFN-γ content in 4-h co-culture supernatants was quantified by ELISA. Results are shown as mean ± SEM of three independent experiments. Only the results of cells with concomitant heat shock treatment are shown, because cells without heat shock showed comparable values. For both cell lines p values indicate statistical significance as to whether ifosfamide- and heat-treated cells were MVA-hTyr-infected or not.
