Figure 3
Enterotoxin induces IL-17 production in cocultured malignant T cells. (A) Malignant (SeAx) and nonmalignant (MF1850) T-cell lines were either mono- or cocultured with vehicle (PBS) or SEA (50 ng/mL) for 16 hours. The cocultured malignant and nonmalignant T cells were sorted by FACS, and the relative levels of IL-17A and GAPDH mRNA were determined in all samples by qPCR. In each sample, the level of IL-17A mRNA was normalized to that of GAPDH mRNA and depicted as fold change compared with monocultured malignant T cells with PBS. “Malign. Cocultured” signifies IL-17A expression in malignant T cells cocultured with nonmalignant T cells, and vice versa for “Non-malign. Cocultured.” (B-C) Malignant (SeAx) and nonmalignant (MF1850) T cells were transiently transfected with nontargeting (NT) or STAT3 specific siRNA (B) or JAK3-specific siRNA (C) and monocultured for 24 hours. Then, the transfected cells were washed and cocultured in the presence of SEA (50 ng/mL) for another 24 hours before the concentrations of IL-17A in cell culture supernatants were determined by ELISA. Data are presented as percentage of IL-17A secretion relative to cocultures of malignant and nonmalignant T cells transfected with NT siRNA. (D) Malignant (SeAx) and nonmalignant (MF1850) T-cell lines were cocultured separated by Transwells with vehicle (PBS) or SEA (50 ng/mL) for 24 hours. IL-17 concentrations in the supernatants were determined by ELISA. (E) Malignant (SeAx) and nonmalignant (MF1850) T-cell lines were either monocultured with Transwells or cocultured separated by Transwells for 24 hours. The relative levels of IL-17A and GAPDH mRNA were determined in all samples by qPCR. In each sample, the level of IL-17A mRNA was normalized to that of GAPDH mRNA and depicted as fold change compared with monocultured malignant T cells with PBS. “Malign. Transwell” signifies IL-17A expression in malignant T cells cocultured with nonmalignant T cells separated by a Transwell, and vice versa for “Non-malign. Transwell.” (F) Malignant (SeAx) and nonmalignant (MF1850) T-cell lines were mono- and cocultured with vehicle (PBS), SEA, SEA plus immunoglobulin G (IgG) isotype control, or SEA plus neutralizing IL-2 antibody (aIL-2). IL-17 concentrations in the supernatants were determined by ELISA. Error bars represent standard error of the mean of 3 independent experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; mRNA, messenger RNA.

Enterotoxin induces IL-17 production in cocultured malignant T cells. (A) Malignant (SeAx) and nonmalignant (MF1850) T-cell lines were either mono- or cocultured with vehicle (PBS) or SEA (50 ng/mL) for 16 hours. The cocultured malignant and nonmalignant T cells were sorted by FACS, and the relative levels of IL-17A and GAPDH mRNA were determined in all samples by qPCR. In each sample, the level of IL-17A mRNA was normalized to that of GAPDH mRNA and depicted as fold change compared with monocultured malignant T cells with PBS. “Malign. Cocultured” signifies IL-17A expression in malignant T cells cocultured with nonmalignant T cells, and vice versa for “Non-malign. Cocultured.” (B-C) Malignant (SeAx) and nonmalignant (MF1850) T cells were transiently transfected with nontargeting (NT) or STAT3 specific siRNA (B) or JAK3-specific siRNA (C) and monocultured for 24 hours. Then, the transfected cells were washed and cocultured in the presence of SEA (50 ng/mL) for another 24 hours before the concentrations of IL-17A in cell culture supernatants were determined by ELISA. Data are presented as percentage of IL-17A secretion relative to cocultures of malignant and nonmalignant T cells transfected with NT siRNA. (D) Malignant (SeAx) and nonmalignant (MF1850) T-cell lines were cocultured separated by Transwells with vehicle (PBS) or SEA (50 ng/mL) for 24 hours. IL-17 concentrations in the supernatants were determined by ELISA. (E) Malignant (SeAx) and nonmalignant (MF1850) T-cell lines were either monocultured with Transwells or cocultured separated by Transwells for 24 hours. The relative levels of IL-17A and GAPDH mRNA were determined in all samples by qPCR. In each sample, the level of IL-17A mRNA was normalized to that of GAPDH mRNA and depicted as fold change compared with monocultured malignant T cells with PBS. “Malign. Transwell” signifies IL-17A expression in malignant T cells cocultured with nonmalignant T cells separated by a Transwell, and vice versa for “Non-malign. Transwell.” (F) Malignant (SeAx) and nonmalignant (MF1850) T-cell lines were mono- and cocultured with vehicle (PBS), SEA, SEA plus immunoglobulin G (IgG) isotype control, or SEA plus neutralizing IL-2 antibody (aIL-2). IL-17 concentrations in the supernatants were determined by ELISA. Error bars represent standard error of the mean of 3 independent experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; mRNA, messenger RNA.

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