Tumour infiltrating T cells isolated three weeks after transplantation are more inhibited Groups of four mice were subcutaneously inoculated with 2 × 105 of TC-1 tumour cells in the flank. Mice were terminated at 2 or 3 weeks after TC-1 tumour inoculation. Tumours were dissociated and digested with collagenous D and DNase I. Mononuclear cells were harvested by Ficoll separation and incubated with cell stimulation cocktail in the presence of monensin overnight at 37 °C/5% CO2. Cells were surface stained for CD3, CD4 or CD8, and PD-1 followed by intracellular staining for IFN-γ, granzyme B or perforin. (a) Percentage PD-1 + IFN-γ + or PD-1-IFN-γ + of CD3 + CD4+ cells; (b) Percentage of PD-1 + IFN-γ + or PD-1-IFN-γ + of CD3 + CD8+ cells; (c) Percentage of PD-1+ granzyme B+ or PD-1- granzyme B+ of CD3 + CD8+ cells; (d) Percentage of PD-1+ granzyme B+ or PD-1- granzyme B+ of CD3 + CD4+ cells; (e) Percentage of PD-1+ perforin+ or PD-1- perforin+ of CD3 + CD8+ cells. The results represent one of two independent experiments.