SEs stimulate malignant T cells to express IL-10 in the presence of benign T cells. (A) SA was isolated from the skin of CTCL patients. Shown are representative pictures of the involved skin lesions. (B-D) Malignant (SeAx) and benign (MF1850) T-cell lines were mono- and cocultured in the absence (−) or presence of SA isolated from involved and uninvolved skin of (B) an SS patient and (C-D) MF patients. After 24 hours, the concentrations of IL-10 in the cell culture supernatants were determined by ELISA. (E) Malignant (SeAx) and benign (MF1850) T-cell lines were mono- and cocultured with media (−), supernatant from SA from involved skin (SA sup.) or SA sup. plus a pool of antibodies against HLA-DP, HLA-DQ, and HLA-DR (anti-MHC-II, 10 μg/mL) for 24 hours. Subsequently, the concentrations of IL-10 in the culture supernatants were determined by ELISA. Error bars represent standard error of the mean (SEM) of 3 replicate cultures. (F) Representative flow cytometric analysis of MHC-II expression on the malignant (SeAx) and benign (MF1850) T-cell lines. Dashed lines represent isotype control staining and solid lines with fill MHC-II staining. (G) Malignant (SeAx) and benign (MF1850) T-cell lines were mono- and cocultured with vehicle (phosphate-buffered saline [PBS]) or soluble egg antigens [SEA] (50 ng/mL) for 24 hours and the relative expression of IL-10 and GAPDH mRNA measured by qPCR. In each sample, the level of IL-10 mRNA was normalized to that of GAPDH mRNA and depicted as fold change when compared with malignant T cells cultured with PBS. Error bars represent SEM of 3 independent experiments. (H) Malignant (SeAx) and benign (MF1850) T-cell lines were mono- and cocultured with vehicle (PBS) or SEA (50 ng/mL) for 24 hours and the concentration of IL-10 in the cell culture supernatants analyzed by ELISA. Error bars represent SEM of 3 independent experiments. (I) Malignant (SeAx) and benign (MF1850) T-cell lines were mono- and cocultured with vehicle (PBS) or SEA (50 ng/mL) for 16 hours. Subsequently, the cocultured malignant and benign T cells were sorted by FACS and the relative level of IL-10 and GAPDH mRNA in all samples determined by qPCR. In each sample, the level of IL-10 mRNA was normalized to that of GAPDH mRNA and depicted as fold change when compared with malignant T cells cultured with PBS. Malign. (Benign) indicates IL-10 expression in malignant T cells that had been cocultured with benign T cells, and vice versa for Benign (Malign.). Error bars represent SEM of 3 independent experiments.