Pre coated anti cd3 antibody

Naive CD4⁺ T cells from the spleens of 6-8 weeks old male GREAT mice were prepared using magnetic bead cell sorting (Miltenyi Biotec, Bergisch-Gladbach, Germany). The purity of CD4⁺CD44^lowCD62L^high T cell subset was validated by flow cytometry. These naïve CD4⁺ T cells were stimulated with 5 μg/ml pre-coated anti-CD3ϵ antibody (Biolegend) and 1 μg/ml soluble anti-CD28 antibody (Biolegend) in culture medium containing 10 ng/ml IL-12 (Peprotech, Cranbury, NJ, USA), 10 ng/ml IL-2 (Peprotech) and 10 μg/ml anti–IL-4 antibody (BD Biosciences) for 3 days. The culture medium was RPMI 1640 medium (plus 50 μM β-mercaptoethanol) supplemented with 10% FBS, 1% GlutaMax, and 1% Pen/Strep (Gibco, Shanghai, China). 1 × 10⁵ naive CD4⁺ T cells were cultured in 96-well plates with 100 μl culture medium per well.

Healthy volunteers were recruited from the medical students at the Second Xiangya Hospital and written informed consent was obtained. Human study was approved by the Ethics Committee of the Second Xiangya Hospital of Central South University. Peripheral blood mononuclear cells (PBMCs) from the peripheral blood of healthy volunteers were separated by Histopaque (Sigma-Aldrich, St. Louis, MO). Naive CD4⁺ T cells from PBMC were isolated by negative selection using Miltenyi beads according to the manufacturer’s instructions. The purity of CD4⁺CD45RA⁺CD45RO^- T cell subset was validated by flow cytometry. The purified naïve CD4⁺ T cells were stimulated with 5 μg/ml pre-coated anti-CD3ϵ antibody (Biolegend) and 2 μg/ml soluble anti-CD28 antibody (Biolegend) in culture medium containing 10 ng/ml IL-12 (Peprotech, Cranbury, NJ, USA), 10 ng/ml IL-2 (Peprotech) and 10 μg/ml anti–IL-4 antibody (eBioscience) for 5 days. The culture medium was RPMI 1640 medium supplemented with 10% FBS, 1% GlutaMax, and 1% Pen/Strep (Gibco, Shanghai, China). 1 × 10⁵ naive CD4⁺ T cells were cultured in 96-well plates with 100 μl culture medium per well. The culture medium was refreshed on day 3.