Soluble anti human cd28 28.2

Peripheral blood mononuclear cells (PBMCs) were isolated from donors by Ficoll-Paque PLUS (GE Healthcare) or Lymphoprep (Stemcell) gradient centrifugation. Total CD4⁺ T cells were isolated by negative magnetic selection using a CD4 T cell isolation kit (Stemcell) and CD4⁺CD25^hiCD127^lo-negCD45RO⁺ T_reg cells were sorted on a FACS Aria (BD Biosciences). T_reg cells were cultured in RPMI 1640 medium supplemented with 5% Human serum, 2 nM L-glutamine, 5 mM HEPES, and 100 U/ml penicillin, 100 μg/ml streptomycin, 0.5 mM sodium pyruvate, 0.05 mM nonessential amino acids, and 5% human AB serum (Gemini Bio-Products). 96-well round bottom plates (Corning) were pre-coated with anti-human CD3 (UCHT1) (1–2 μg/ml) and used for T_reg in vitro culture with soluble anti-human CD28 (28.2) (1–5 μg/ml) (BD Bioscience) and human IL-2 (50 U/ml). Human IL-2 was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH). T_H1-T_reg cells were induced with human recombinant IL-12 (20 ng/ml) (R&D). The Wnt/β-catenin inhibitor PKF115–584 (Tocris) was used at 200 nM and IWR-1 (Tocris) was used at 20 μM. SGK1 inhibitor GSK650394 (Tocris) was used at 10 μM. AKT inhibitor MK2206 (Tocris) was used at 5 μM. Frizzled 8 FC chimera protein (R&D) was used at 500 ng/ml.