Anti mouse il 4

CD4 T cells were isolated from spleens of C57BL/6 mice using the Mojosort™ CD4 T cell isolation kit (BioLegend) and resuspended in iTreg differentiation media supplemented with 10ng/ml IL-2 (BioLegend), 10ng/ml TGFβ (BioLegend), 80 ng/ml all-trans retinoic acid (Millipore Sigma, St. Louis, MO) and 2.5 μg/mL soluble anti-CD28 (BD Biosciences). Cells were seeded into wells of a 12-well tissue culture plate pre-coated with anti-hamster IgG (Sigma-Aldrich, St. Louis, MO) plus 1 mg/ml anti-CD3 (clone 145–2C11, BioLegend) and stimulated for 7 days at 37°C. Th1-like iTregs were generated by adding 10 ng/ml IL-12 (BioLegend) on day 3 only, or on days 3 and 5 of differentiation. The cells were collected on day 7 of differentiation. Th1 cells were generated by culturing CD4 T cells with plate-bound anti-CD3ϵ (5 μg/mL) plus anti-CD28 (2.5 μg/mL) in Th1 cell differentiation media supplemented with IL-2 (10ng/ml), IL-12 (10ng/ml), and anti-mouse IL-4 (1 μg/ml; BioXcell, West Lebanon, NH). Th1 cells were collected for experiments on day 4 of differentiation. Media with a 1:1 mixture of RPMI 1640 and Dulbecco’s modified Eagle medium (GE Life Sciences, Pittsburgh, PA) supplemented with 10% FBS (Peak Serum, Wellington, CO), 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/mL penicillin, and 100 mg/mL streptomycin (GE Life Sciences) were used for cell culture.