Purified CD4+ T cells were prepared using immunomagnetic negative selection (Miltenyi Biotec). Briefly, lymphocytes were allowed to react with CD4+ T cell biotin-antibody cocktail (biotin-conjugated monoclonal antibodies against CD8a, CD11b, CD11c, CD19, CD45R (B220), CD49b (DX5), CD105, anti-MHC class II, Ter-119, and TCRγ/δ) and then incubated with antibiotin microbeads, followed by magnetic separation. The purity of the resulting CD4+ T cell populations was examined using flow cytometry, and was consistently above 95%.
T-helper cell differentiation was conducted as previously described [26 (link)]. Briefly, naive CD4 T cells (0.4 × 106) in 24-well plates (Costar) precoated with 5 μg/ml of anti-CD3 (154-2C11, BioXcell) and 2 μg/ml of anti-CD28 (37.51, BioXcell) were cultured in RPMI containing 10% FCS and polarizing cytokines at the various concentrations of ECF. The cytokines used were Th1, mIL-12 (10 ng/ml, Biolegend), and anti-mIL-4 (11B11, 2 μg/ml, BioXcell). Cells stimulated in “neutral” conditions (anti-mIL-4 and anti-mIFN-γ without cytokines) were considered Th0 cells. The differentiated cells were harvested after 3 days and restimulated for 4 h with 50 ng/ml of PMA (Sigma-Aldrich) and 750 ng/ml of ionomycin (Sigma-Aldrich) in the presence or absence of Brefeldin A for flow cytometry.
T-helper cell differentiation was conducted as previously described [26 (link)]. Briefly, naive CD4 T cells (0.4 × 106) in 24-well plates (Costar) precoated with 5 μg/ml of anti-CD3 (154-2C11, BioXcell) and 2 μg/ml of anti-CD28 (37.51, BioXcell) were cultured in RPMI containing 10% FCS and polarizing cytokines at the various concentrations of ECF. The cytokines used were Th1, mIL-12 (10 ng/ml, Biolegend), and anti-mIL-4 (11B11, 2 μg/ml, BioXcell). Cells stimulated in “neutral” conditions (anti-mIL-4 and anti-mIFN-γ without cytokines) were considered Th0 cells. The differentiated cells were harvested after 3 days and restimulated for 4 h with 50 ng/ml of PMA (Sigma-Aldrich) and 750 ng/ml of ionomycin (Sigma-Aldrich) in the presence or absence of Brefeldin A for flow cytometry.
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