Anti cd3 anti cd28 antibody

PBMCs were isolated from venous blood of healthy donors using lymphocyte separation medium (Lonza Biologics, Portsmouth, NH) and density centrifugation. PBMCs were then cultured at 1×10⁶ cells mL of RPMI 1640, 10% heat-inactivated fetal bovine serum (FBS), 200 mM L-glutamine, and 1% penicillin/streptomycin (Lonza Biologics, Portsmouth, NH). Cells were activated with 1 μg/mL anti-CD3/anti-CD28 antibodies (BD Biosciences, San Jose, CA) and 100 units of recombinant human IL-2 (AIDS reagent program, Germantown, MD) for three days. In some cases CD8+ T cells were further isolated from PBMCs. CD8+ T cells were isolated using an untouched CD8+ T cell Isolation Kit (Miltenyi Biotec, Germany) on an AutoMACS Separator (Miltenyi Biotec, Germany). CD8+ T cells were cultured for three days with 1 μg/mL anti-CD3/anti-CD28 antibodies (BD Biosciences, San Jose, CA) and 100 units of recombinant human IL-2 (AIDS reagent program) at 1×106 cells/mL of RPMI 1640. In some experiments, cells were stained with carboxyfluoresceinsuccinimidyl ester (CFSE, Invitrogen Life Technologies) prior to culture and treatment with 5 μM IWP-2. Three days later, cells were run on a BD FACSVerse flow cytometer (BD Biosciences) to determine percentage of proliferating cells.

Naive CD4⁺T cells were isolated from spleens of MRL/lpr mice by using the Dynabeads Untouched Mouse CD4 Cell Kit (Life Technologies AS, Norway). For Tfh cell differentiation, naive CD4⁺T cells were cultured for 5 days with 10 ng/ml IL-21 (PeproTech, USA), 10 ng/ml IL-6 (PeproTech), and 25 µl of anti-CD3/anti-CD28 antibody (BD Biosciences Pharmingen, USA) with various doses of Baicalin (dissolved in DMSO and diluted with PBS) or a DMSO-PBS vehicle as a control. For Foxp3⁺ regulatory T cell differentiation, sorted naive CD4⁺T cells were cultured for 5 days with 5 ng/ml TGF-β (PeproTech), 30 U/ml IL-2 (PeproTech, USA), and 50 ng/ml anti-CD3/anti-CD28 antibody (BD Biosciences Pharmingen) with or without Baicalin as described above. For some differentiation experiments, 10 μM mammalian target of rapamycin (mTOR) agonist (MHY1485; MedChem Express, USA),or 200 ng/ml mTOR antagonist rapamycin (LC Laboratories) was added with 40 μM Baicalin for 5 days.
For some experiment, sorted naive CD4⁺T cells were cultured for 5 days with 5 ng/ml TGF-β, 30 U/ml IL-2, and 50 ng/ml anti-CD3/anti-CD28 antibody with or without 40 μM Baicalin for 5 days, then these 1 × 10⁶ Baicalin-induced Foxp3⁺ regulatory T cells or 1 × 10⁶ vehicle-induced Foxp3⁺ regulatory T cells were injected intravenously into MRL/lpr mice weekly for 4 weeks.