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. 2020 May 14;10(5):57. doi: 10.1038/s41408-020-0324-3

Fig. 4. FOXP3 upregulation depends on MHC class II and IL-2.

Fig. 4

a Malignant cells (SeAx) and nonmalignant (MF1850) T cells were cultured alone or together and in the presence of either vehicle (PBS), S. aureus enterotoxin A (SEA) wild type (SEAwt), or MHC-II binding-deficient SEA mutants (SEAD227A or SEAF47A/D227A). FOXP3 expression was determined by qPCR. In each sample, the level of FOXP3 mRNA was normalized to that of GAPDH mRNA and it is depicted as fold change compared to malignant cells with PBS. b Malignant cells (SeAx) and nonmalignant (MF1850) T-cell lines were cultured alone, co-cultured separated by transwells, and cultured in the presence of vehicle (PBS) or SEA for 24h. The co-cultured malignant and nonmalignant cells were sorted by FACS, and the relative level of FOXP3 and GAPDH mRNA were determined in all samples by qPCR. In each sample, the level of FOXP3 mRNA was normalized to that of GAPDH mRNA and is depicted as fold change compared to mono-cultured malignant cells with PBS. “Malign. transwell” signifies FOXP3 expression in malignant cells in transwell with nonmalignant cells and vice versa for “nonmalign. transwell”. “Malign. co-cultured” signifies FOXP3 expression in malignant cells co-cultured with nonmalignant cells and vice versa for “nonmalign. co-cultured”; (n=3). c FACS sorted primary Sézary syndrome (SS) cells were cultured in the presence of vehicle (PBS), SE, IL-2, or PMA+Ionomycine for 24h. FOXP3 expression was determined by qPCR. In each sample, the level of FOXP3 mRNA was normalized to that of GAPDH mRNA and it is depicted as fold change compared to malignant cells with PBS. d Primary SS cells and nonmalignant (MF1850) T-cell lines were either cultured alone, co-cultured separated by transwells, or cultured together with vehicle (PBS) or a pool of SE (SEA, SEB, SEC2, SED, and SEI) for 24h. The co-cultured malignant cells and nonmalignant cells were sorted by FACS, and the relative level of FOXP3 and GAPDH mRNA were determined in all samples by qPCR. In each sample, the level of FOXP3 mRNA was normalized to that of GAPDH mRNA and it is depicted as fold change compared to mono-cultured malignant cells with PBS. “Malign. transwell” signifies FOXP3 expression in malignant cells in transwell with nonmalignant cells and vice versa for “nonmalign. transwell”. “Malign. co-cultured” signifies FOXP3 expression in malignant cells co-cultured with nonmalignant cells and vice versa for “nonmalign. co-cultured”. e Malignant cells (SeAx) and nonmalignant (MF1850) T cells were co-cultured for 3 days in the presence of vehicle (PBS) or SEA, and then spun down and washed three times and resuspended in fresh media. FOXP3 expression in malignant cells was analyzed by flow cytometry after 0, 24, 48, and 72h. f Malignant cells (SeAx) and nonmalignant (MF1850) T cells were cultured together, and in the presence of SEA and either anti-human IL-2, anti-human IL-15, a combination thereof, or isotype control. FOXP3 expression was determined by western blotting.