Whole Blood Intracellular Cytokine Staining (ICS) Assay

In order to determine the frequency of NAGLU-specific effector T cells, 700 µl of whole blood were stimulated for 6 hours at 37°C with NAGLU at 0.5 µg/ml, 1µg/ml or 2 µg/ml, or NAGLU peptide at 0.5 µg/ml or 1µg/ml in the presence of 1µg/ml CD28/CD49d mAbs and 10µg/ml Brefeldine A (Sigma-Aldrich, Lyon France). SEB (1 µg/ml) was used as a positive control. Blood samples were then treated with 10 fold diluted FACS Lysing solution (Becton Dickinson) and stored at -80°C until the ICS assay. Staining was performed on unfrozen blood samples, washed with PBA and permeabilized with BD FACS™ Permeabilizing Solution 2 (Becton Dickinson). The following mAbs were used: anti-CD3-V500 (clone UCHT1, BD Horizon), anti-CD4-APC (clone RPA-T4, BD Pharmingen), anti-CD8-APC-H7 (clone SK1, BD Pharmingen), anti-IFNγ-AF488 (clone B27, BD Pharmingen), anti-CCR7-PE (clone 3D12, BD Pharmingen), anti-CD45RA-PE-Cy7(BD Pharmingen), anti-IL2-PE-CF594 (clone 5344.111, BD Horizon), anti-CD69-PerCP (clone L78, BD Biosciences), and anti-TNFα-V450 (BD Horizon). Staining was done in PBA containing 0.05% saponin (Sigma-Aldrich, Lyon France), cells were fixed in 1% PFA solution and immediately acquired on CyAn Beckman Coulter. The frequency of T cells expressing CD69 and intracellular cytokines was determined with FlowJo software.