Soluble anti human cd28

Human PBMCs were stimulated with immobile purified anti-human-CD3 (OKT3), soluble anti-human-CD28 (2 µg/mL; CD28.2; both from BioLegend, San Diego, CA, USA) or 0.35 µM sodium pyrithione (Sigma-Aldrich). For fast zinc signals, anti-CD3 was coated to beads, whereas, for activation for 24–72 h, anti-CD3 was coated to a 24-well culture plate. Anti-CD3 was coated to Dynabeads™ Pan Mouse IgG (Invitrogen by Thermo Fisher Scientific, Eugene, OR, USA) according to the manufacturer’s protocol. The beads were washed in isolation buffer (PBS + 0.1% BSA + 2 mM EDTA) and incubated for 30 min with 1 µg anti-CD3 per 1 × 10⁷ beads at 2–8 °C. Afterwards, beads were washed and stored in isolation buffer. Before usage, coated beads were resuspended in a culture medium. When anti-CD3 was coated to a 24-well culture plate, the plate was first coated with 2.6 µg/mL goat anti-mouse IgG (Jackson ImmunoResearch, Ely, UK) over night at 4 °C. Then, wells were washed with balanced salt solution (BSS) and PBS, then incubated with 2 µg anti-CD3 in 0.5 mL PBS + 0.1% BSA at 37 °C for 2 h. After washing with PBS, 1 mL with 1 × 10⁶ PBMCs were added and further stimulated with 2 µg/mL soluble anti-CD28. Cells were incubated for 24–72 h at 37 °C.

Human PBMC
(2 × 10⁵ cells/200 μL/well in
a 96-well flat bottom plate) labeled by CFSE (4 μM, Molecular
Probe) were treated with plate-bound anti-human CD3 (1.25 μg/mL,
#317302, BioLegend) and soluble anti-human CD28 (2 μg/mL, #302902,
BioLegend) antibodies with or without 2D216 (5 μM)
for 3 days. Supernatants were subjected to IL-2 and IFN-γ ELISA.
T cell division was analyzed by the MACSQuant Analyzer 10 (Miltenyi
Biotec) using APC-conjugated anti-human CD3 antibodies (#17–0038-42,
eBioscience). The % divided, the percent of the live CFSE-labeled
CD4⁺ T cells that entered division, was calculated using
FlowJo (version 10.6.1, Becton Dickinson).