Figure 3

Functions of TAA-specific CTL are impaired when target melanoma cells are cultured in MCTS. (A) Interferon-gamma secretion by CTL clones upon stimulation with HBL, D10, or NA8 cells cultured in 2D or in MCTS. Cytotoxic T-lymphocyte clones specific for HLA-A^*0201-restricted gp100_280–288 or Melan-A/MART-1_27–35 epitopes and displaying corresponding tetramer binding profiles (right panels) were co-incubated for 24 h at 2.5 : 1 E:T ratio in the presence of similar numbers of HBL or D10 melanoma cells (HLA-A^*0201+,gp100+,Melan-A/MART-1+), cultured in 2D (□) or in 3D (▪). Interferon-gamma secretion was measured by ELISA in culture supernatants. Data are reported as average of triplicate measurements. Error bars represent the standard deviation of the mean concentration of triplicate cultures. (B) The FasL, perforin, and granzyme B gene expression in CTL stimulated by 2D or 3D cultured HBL. Cells from a CTL clone specific for HLA-A^*0201-restricted Melan-A/MART-1_27–35 epitope were co-cultured for 24 h at 2.5 : 1 E:T ratio in the presence of HBL melanoma cells cultured in 2D (□) or in 3D (▪). Total cellular RNA was extracted and reverse transcribed. FasL, perforin, and granzyme B gene expression were analysed by quantitative real-time PCR. Data were expressed as ratio to cells incubated in the presence of melanoma cells cultured in 2D. (C) Interferon-gamma secretion by CTL cultured with HBL cells from intact or disrupted MCTS. Cells from a CTL clone specific for HLA-A^*0201-restricted Melan-A/MART-1_27–35 epitope were stimulated for 24 h at 1 : 1 E:T ratio in the presence of HBL melanoma cells cultured in 2D (□), in 3D (▪) or following MCTS disruption (). Interferon-gamma secretion was measured by ELISA in culture supernatants. Data are reported as average of triplicate measurements. Error bars represent the standard deviation of the mean concentration of triplicate cultures.