Anti cd28 mab clone 37.51

The normal CD4⁺CD25⁻T cells were labeled with carboxyfluorescein-succinimidyl-ester (CFSE, Invitrogen, Carlsbad, CA) according to the manufacturer's instructions and then mixed with CD4⁺CD25⁺Tregs from rmGas6-treated or normal mice in a ratio of 1 : 1. Anti-CD3 monoclonal antibody (clone 145-2C11) (mAb) combined with anti-CD28 mAb (clone 37.51) (eBioscience, San Diego, CA) at a final concentration of 1 μg/ml was added to the culture for stimulation. The proliferation of cells was measured by flow cytometry using a FACScan (BD Biosciences, Mountain View, CA). The percentage of suppression was calculated as [1 − (% Teff proliferation with Treg cells/% Teff proliferation without Treg cells)] × 100.

Proliferation Assays. CD4⁺CD25^high (all Foxp3⁺) cells isolated from the secondary lymphoid organs were magnetically sorted from the spleen of Foxp3-GFP-KI C57BL/6J mice. They were loaded with 5 μM carboxyfluorescein succinimidyl ester (CFSE) (Life Technologies) and cultured (5× 10⁴ cells per well) in RPMI medium 1640 supplemented with 5% (vol/vol) FCS (Life Technologies), 1% antibiotics, and 5 × 10⁻⁵ M β-mercaptoethanol. Cells were plated in 96-well round-bottomed culture plates, either alone or with sorted MPP at 1:1 T:MPP cell ratios, and stimulated with 2.5 μg/ml of anti-CD3 mAb (clone 145–2C11) and 5 μg/ml of anti-CD28 mAb (clone 37.51, eBioscience) for 4 days. Inhibitors were added at 5 to 20 µg/ml: anti-CD137L (clone TKS-1, Biolegend), anti-CD80 (clone 16-10A1, Biolegend), anti-GITRL (clone 5F1, Biolegend), anti-CD86 (clone GL-1, BD Biosciences), anti-OX40 (polyclonal goat IgG, R&D Systems), anti-TGFβ (clone 2G7, grown in our laboratory), anti-IL10 (clone JES052A5, R&D).