Anti mouse cd3

Mouse and human cytokines used for in vitro polarizations as well as anti-human CD3 and anti-human CD28 were purchased from Biolegend. Anti-mouse CD3, anti-mouse CD28 and mouse and human neutralizing antibodies to IL-4 and IFN-γ were purchased from BioXCell. Anti-CK2α and anti-CK2β antibodies for detection by flow cytometry were purchased from AbCam and Calbiochem, respectively. Secondary antibodies for flow cytometry were purchased from Jackson ImmunoResearch. Flow cytometry antibodies against mouse CD4, IL-17A, IFN-γ, CD25 and CD45.1 and human CD3, CD4 and Foxp3 were purchased from Biolegend. Flow cytometry antibodies against mouse CD44, Foxp3 and GM-CSF and human IL-17A were purchased from eBioscience. Aqua Live/Dead Viability dye, CFSE proliferation dye and 2-NBDG were purchased from ThermoFisher Scientific. Flow cytometry antibodies and isotype controls for phosphorylated S6, Akt, STAT3, STAT5 and SMAD2/3 were purchased from Cell Signaling. Immunoblotting antibodies against phosphorylated T389, S371 and total S6 Kinase p70 and phosphorylated S129, S473 and total Akt were purchased from Cell Signaling, and antibody against mouse β-actin was purchased from abcam.

Naïve CD4⁺ T cells (CD25⁻Foxp3-GFP⁻CD4⁺CD8⁻TCRβ⁺CD62L⁺CD44⁻) cells were sorted from pooled spleens and lymph nodes after CD4⁺ cell enrichment using the Dynabeads™ FlowComp™ Mouse CD4 Kit according to the manufacturer’s instructions. Tissue culture treated 96-well flat-bottom plates (USA Scientific) were coated with 1 μg/mL anti-mouse CD3 and 1 μg/mL anti-mouse CD28 antibodies (BioXCell) in 200 μL 1x PBS at 37°C for 2 hours and then washed once with 1x PBS. Cells were cultured on these plates for 2–4 days with 1 ng/ml recombinant human TGF-β and varying concentrations of recombinant human IL-2 before staining and analysis. To assay the stability of Foxp3 expression, naïve CD4⁺ T cells were cultured in Treg cell induction conditions with or without 0.25 mM ascorbic acid-2-phosphate (Sigma) for 4 days. Foxp3-GFP⁺ cells were then sorted and cultured on new plates coated with or without 1 μg/mL anti-CD3 and 1 μg/mL anti-CD28 antibodies (BioXCell) in the presence of 100 U/mL recombinant IL-2 for 1–4 days. At the end of culture, cells were harvested and stained with a viability dye (BioLegend) followed by cell fixation and Foxp3 staining.