Anti cd3ε antibody

Carboxyfluorescein succinimidyl ester-labeled effector spleen cells (0.5–1.5 × 10⁵) were cultured with or without Treg cells for 72 h as described (10 (link), 30 (link)). Labeled effector cells from TNFR2-deficient mice were used to avoid interferences by activation of the TNFR2 on effector cells in the cultures. Duplicate or triplicate cultures were stimulated with soluble anti-CD3ε antibodies (0.5 µg/ml, BD Bioscience) for 72 h. Interleukin 10 (IL-10) was quantified using the Duo Set ELISA (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. To determine the suppressive activity of myeloid suppressor cells, CFSE-labeled spleen cells (2 × 10⁵) were stimulated with anti-CD3ε (0.25 µg/ml, BD Bioscience) and anti-CD28 (0.125 µg/ml, BD Bioscience) and cultured with or without different numbers of bone marrow-derived myeloid cells. Proliferation of CD4 or CD8 T cells was determined by flow cytometry. Nitrite concentrations in the supernatants were determined using Griess reagent measuring the optical density at 540 nm.

Splenocytes from DEREG mice used to generate PBS⁺ Tregs, Aβ⁺ Tregs or KLH⁺ Tregs were obtained by mechanical disruption of spleen. The splenocytes were passed through a 40-μm cell strainer and cultured with 5 µg/mL plate-bound anti-CD3ε antibodies in combination with 2 µg/mL soluble anti-CD28 antibodies (BD Biosciences). The cells were stimulated with Aβ (10 µg/mL) or KLH (10 µg/mL) and bvPLA2 (0.4 µg/mL). After 4 days, CD4⁺CD25⁺ Tregs were isolated using magnetic-activated cell sorting (MACS) according to the manufacturer's protocol (CD4⁺CD25⁺ Regulatory T Cell Isolation Kit, Miltenyi Biotec).