Naive CD4 T cells (0.4 × 106) in 24 well plates (Costar) pre-coated with anti-CD3 (154-2C11, 5 μg/ml, BioXcell) and anti-CD28 (37.51, 2 μg/ml, BioXcell) were cultured in RPMI containing 10% FCS and polarizing cytokines. The cytokines were: Th1, mIL-12 (10 ng/ml, Biolegend) and anti-mIL-4 (11B11, 2 μg/ml, BioXcell); Treg, hTGF-β1 (3 ng/ml, Biolegend), mIL-2 (20 ng/ml, Biolegend), anti-mIFN-γ (XMG1.2, 2 μg/ml, BioXcell) and anti-mIL-4; Th17, mIL-6 (10 ng/ml, Biolegend), hTGF-β1 (2 ng/ml), anti-mIFN-γ, and anti-mIL-4. Cells stimulated in ‘neutral’ conditions (anti-mIL-4 plus anti-mIFN-γ without added cytokines) were considered Th0 cells.
Where studied, protease inhibitors, AEBSF (Pefabloc) and E64 (Sigma-Aldrich), E64D (Santa Cruz), z-Phe-Ala-fmk (Enzyme Systems), CA074-OMe (EMD Millipore), Ns-Ile-Trp-CHO (IW-CHO, Enzo Life Sciences), CLIK195 (provided by Guo-Ping Shi), and the AEP inhibitor LI-1 [15 (link)], were added at the start of culture. Unless otherwise indicated, differentiated cells were harvested after 3 days for Western blot, peptidase assay or active site labeling or were restimulated for 4h with PMA (50 ng/ml) and ionomycin (750 ng/ml) (Sigma-Aldrich) in the presence or absence of Brefeldin A for flow cytometry or ELISA, respectively.
Where studied, protease inhibitors, AEBSF (Pefabloc) and E64 (Sigma-Aldrich), E64D (Santa Cruz), z-Phe-Ala-fmk (Enzyme Systems), CA074-OMe (EMD Millipore), Ns-Ile-Trp-CHO (IW-CHO, Enzo Life Sciences), CLIK195 (provided by Guo-Ping Shi), and the AEP inhibitor LI-1 [15 (link)], were added at the start of culture. Unless otherwise indicated, differentiated cells were harvested after 3 days for Western blot, peptidase assay or active site labeling or were restimulated for 4h with PMA (50 ng/ml) and ionomycin (750 ng/ml) (Sigma-Aldrich) in the presence or absence of Brefeldin A for flow cytometry or ELISA, respectively.