SEA induces IL-10 expression via a mechanism that is dependent on cell-cell contacts between malignant and benign T cells as well as secretion of IL-2 by benign T cells. (A) Malignant (SeAx) and benign (MF1850) T cells were mono- and cocultured with vehicle (PBS) or SEA (50 ng/mL). In cocultures, the malignant and benign T cells were either cultured in transwells where the 2 subsets are separated by a cell-impermeable filter (indicated by a line) or together without a cell-impermeable filter (no line). The supernatants were harvested after 24 hours and the concentrations of IL-10 determined by ELISA. Error bars represent SEM of 3 independent experiments. (B) Malignant (SeAx) and benign (MF1850) T cells were cultured alone and together with SEA for different periods of time. At each given time point, the cells were harvested and the cocultured cells sorted into pure populations of malignant and benign T cells by FACS. Finally, the relative levels of IL-2, IL-10, and GAPDH mRNA in each sample were analyzed by qPCR. The levels of IL-2 and IL-10 mRNA were normalized to that of GAPDH and depicted as fold change when compared with benign and malignant T cells respectively at time point zero. Malign. (Benign) indicates cytokine expression in malignant T cells that had been cocultured with benign T cells, and vice versa for Benign (Malign.). Data are representative of 2 independent experiments. (C) Malignant (SeAx) and benign (MF1850) T cells were mono- and cocultured in the presence of SEA (100 ng/mL) together with an IL-2 neutralizing antibody (Anti-IL-2, 1 μg/mL), an isotype control antibody (1 μg/mL), or vehicle (PBS) for 24 hours before the concentrations of IL-10 in the cell culture supernatants were determined by ELISA. Error bars represent SEM of 3 independent experiments.