Anti cd3 cd28 human t activator dynabeads

Thawed CD8⁺ T-effector cell lines were cultured for 6 h in AIM-V containing 2% human AB serum in the presence of one of the selected best-binding peptides (1 μM; synthesized by JPT Peptide Technologies Inc). In addition, CD8⁺ T-effector cell lines cultured with medium only (negative control) or in the presence of anti-CD3/CD28 Human T-Activator Dynabeads (11131D, Thermo Fisher Scientific) (positive control) were included. During the last 4 h, Brefeldin (555,029, BD) A and Monensin (554,724, BD) were added. Stimulated cells were stained with fluorochrome-conjugated antibodies, anti-CD3-BUV395 (563,546, BD), anti-CD4-BV711, anti-CD8-BV786, and Fixable Viability Stain 780 (565,388, BD). For intracellular staining, cells were fixed, permeabilized and stained with anti-IFNγ-APC (554,702, BD) and anti-TNF-FITC (2,204,971, Thermo Fisher Scientific), using the Foxp3/transcription factor staining buffer set (00–5523-00, Thermo Fisher Scientific). Cells were acquired on an LSRFortessaX20 (BD) and analyzed using FlowJo (V10/Treestar).

Conventional CD25⁻ skin T cells were sorted by flow cytometry, labeled with CellTrace Violet (Thermo Fisher Scientific) for 10 min at room temperature, washed, and cultured for 5 d with autologous skin dendritic cells (DCs) at a ratio of 1:5 (DC/T cell) in the presence of either 50 ng/ml IL-15 or 50 ng/ml IL-7 + 50 ng/ml IL-15. As a positive control, cells were stimulated with anti-CD3/CD28 Human T-Activator Dynabeads (Thermo Fisher Scientific) at a bead/cell ratio of 1:2 in the presence of 30 U/ml IL-2.