Anti il 2

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).

Recombinant human RANKL (rhRANKL) and M-CSF were purchased from R&D Systems (Minneapolis, MN, USA). NAC was obtained from Sigma Chemical Co. (St. Louis, MO, USA). Anti-human IL-17, anti-interferon γ (anti-IFN-γ), anti-IL-10, anti-IL-2, anti-IL-1β, anti-TNF-α, anti-IL-6, and anti-RANKL antibodies were purchased from R&D Systems.

Human PBMCs were co-cultured with anti-HER2/CD3-TDB (50 ng/mL) and KPL-4 for 7 days in the presence or absence of pomalidomide (1 μmol/L) and anti-IL2 (2 μg/mL; R&D Systems), followed by CD8⁺ T-cell purification as described above. Five thousand conditioned CD8⁺ T cells were re-cocultured with freshly plated SKBR3 cells (1:2 ratio) and single-agent anti-HER2/CD3-TDB (8 ng/mL). Caspase 3/7 green dye for apoptosis (Sartorius) was added in the culture. Caspase 3/7 activity was detected by IncuCyte Zoom. Images were analyzed for the number of apoptotic green objects/well.