Anti cd28 clone 9

Naive and memory human CD4⁺ T cells were cultured on anti-CD3 (clone OKT3, 10 μg/mL; BioXCell) and anti-CD28 (clone 9.3, 2 μg/mL; BioXCell) coated plates with 20 U/mL IL-2 in RPMI 1640 (Lonza), supplemented with 10% heat-inactivated FCS (BioSera), penicillin/streptomycin and 2 mM L-glutamine (both Lonza), 2-mercapto-ethanol (Gibco), HEPES buffer (Lonza), 100 mM sodium pyruvate (Gibco), and and 1% nonessential amino acids (Sigma). Cells were split and media replenished as necessary.
Th1, Th2, and Th17 cells were cultured with autologous irradiated APCs (CD4⁻ cells) at a 1:5 ratio, with 100 U/mL IL-2 and 1 μg/mL anti-CD3 (OKT3) in X-VIVO 15 media (Lonza), supplemented with 5% human AB serum (Sigma), penicillin/streptomycin, and L-glutamine. GSK-3 inhibitors were added as above and cells were split as necessary. To determine cytokine production, on day 7 of T-cell culture, cells were stimulated with PMA (50 ng/mL) and ionomycin (1 μg/mL) for 4 h, in the presence of Golgi Stop. Cells were stained with fixable viability dye ef780 (eBioscience) and surface-stained with anti-CD4 Alexa700. Following fixation and permeabilization, intracellular staining for anti-IL-10 ef660, anti-IFNγ PerCPCy5.5, anti-IL-17A PE, and anti-IL-4 FITC (all eBioscience) was carried out.

15 kD poly-glutamic acid (from Alamanda Polymers) was dissolved in water to form 20 mg/ml and sonicated for 10 min. An equal volume of 4 mg/ml 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (Thermo Fisher) in water was added, and the solution was mixed for 5 min at RT. The resulting activated PGA was then combined with antibodies at a 4:1 molar ratio in phosphate buffered saline (PBS) and mixed for 6 h at RT. To remove unlinked PGA, the solution was exchanged 3 times against PBS across a 50,000 NMWCO membrane (Millipore). Antibody concentrations were determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific). The antibodies we used for T cell experiments were anti-CD3 (clone OKT3, BioXCell; Cat# BE0001-2), anti-CD4 (clone OKT4, BioXCell; Cat# BE0003-2), anti-CD8 (clone OKT8, BioXCell; Cat# BE0004-2), and anti-CD28 (clone 9.3, BioXCell; Cat# BE0248). Clone C1.18.4 was used as a control antibody (BioXCell; Cat# BE0085). For HSC transduction, we used polyclonal goat anti-human CD105 (R&D Systems; Cat# AF1097) and non-specific polyclonal goat IgG antibodies (Biolegend; Cat# 400102). There were no dilutions for all above antibodies. We coupled them to polyglutamic acid at a 4:1 molar ratio.