Soluble anti cd28 mab

Spleens were removed from C57BL/6 mice and minced. Splenic red blood cells were removed with ammonium-chloride-potassium lysis buffer (0.15M NH₄Cl, 1mM KHCO₃, 0.1mM Na₂ EDTA, pH 7.2∼7.4). The cells were resuspended in complete media containing RPMI 1640 supplemented with 5% fetal bovine serum and 1% antibiotics (all from Gibco, Grand Island, NY, USA). CD4+ T cells were isolated using a CD4+ T cell isolation kit (Miltenyi Biotec, San Diego, CA, USA), according to the manufacturer's protocol. The purity of isolated CD4+ T cells was assessed as > 95%. Negatively selected non-CD4+ cells were regarded as antigen-presenting cells (APCs) and irradiated at 3,000 rad before coculture. Isolated CD4+ T cells were stimulated with plate-bound anti-CD3 (0.5 mg/mL) and soluble anti-CD28 (1 mg/mL) (both from BD Biosciences) in the presence or absence of CsA (30 ng/mL) and KRGE (3 μg/mL or 10 μg/mL) for 72 h. For Th17 cell-polarizing condition, isolated CD4+ T cells were stimulated with plate-bound anti-CD3 mAb (0.5 mg/mL), soluble anti-CD28 mAb (1 mg/mL), anti-interferon (IFN) γ (2 mg/mL), anti-IL-4 (2 mg/mL), anti-IL-2 (2 mg/mL), IL-6 (20 ng/mL) (all from R&D Systems, Minneapolis, MN, USA), and transforming growth factor-beta (2 ng/mL, PeproTech, London, UK) for 72 h [16] (link), [17] (link).

PBMCs of HLA-A²⁺ were isolated from the blood of healthy donors (Table I), following the provision of informed consent, using Ficoll gradient centrifugation at 600 × g for 20 min at room temperature, followed by washing in PBS, re-suspension at a concentration of 1×10⁶ cells/ml and activated by soluble anti-CD3ε mAb (OKT3, 30 ng/ml, R&D Systems, Inc., Minneapolis, MN, USA) and soluble anti-CD28 mAb (1 ng/ml, R&D Systems, Inc.) and 300 IU/ml recombinant human IL-2 (R&D Systems, Inc.) at 37°C for 48 h. Human PBMCs and Jurkat/E6-1 cells (cat. no. TIB-152; American Type Culture Collection, Manassas, VA, USA) were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 100 U/ml penicillin-streptomycin at 37°C and 5% CO₂.
The HEK293 human embryonic kidney cell line, and the HepG2 (HLA-A²⁺), Huh-1 (HLA-A²⁺) and BEL-7402 (HLA-A²⁻) human hepatocellular carcinoma cell lines (Guangdong Province Key Laboratory for Biotechnology Drug Candidates, Guangzhou, China) were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 100 U/ml penicillin-streptomycin at 37°C and 5% CO₂.