T Cell Activation Assay with Allogenic DCs

1×10⁵ T cells were co-cultured with either 2×10⁴ allogeneic DCs or glutaraldehyde fixed CHO cells in 96-well plates for 5 days (unless otherwise stated) in RPMI 1640 supplemented with 10% FBS, 50U/mL penicillin and streptomycin and 200μM L-glutamine. Stimulations were treated with anti-CD3 (OKT3; 500ng/mL, unless otherwise stated), anti-CD28 (9.3; 500ng/mL) or toxic shock syndrome toxin (TSST)-1 at indicated concentrations (Toxin Technology, Sarasota, FL, USA). Alternatively, T cells were stimulated for 5 days with Dynabeads® Human T-activator CD3/CD28 beads (Life Technologies). Where indicated stimulations were treated with abatacept (20μg/mL; Bristol Myers Squibb), 1,25(OH)₂D₃ (10nM; Sigma-Aldrich) or Cyclosporin A (CsA; Sigma-Aldrich). The concentration of 1,25(OH)₂D₃ used was determined from previous experience (19 (link)) and similar to those used by others (20 (link), 21 (link)). The vehicle for 1,25(OH)₂D₃ was ethanol, which was diluted to a final concentration of 0.01% (v/v) during experiments; vehicle controls showed no effect upon experimental outcomes. PBS vehicle was used for all other reagents.