Monoclonal antibodies against cd3

Freshly isolated SLN cells, expanded SLN T cells or thawed PBMCs were directly stained with antibodies labeled with either FITC, PE, PE-CY5.5, PerCP-CY5.5 or allophycocyanin and analyzed by flow cytometry at 100,000 or 200,000 events per measurement, as previously described [8 (link), 9 (link)]. Monoclonal antibodies against CD3, CD4, CD8, CD56, CD19, CD25 (BD Biosciences) and latency-associated peptide (LAP) (R&D Systems Inc. Minneapolis, MN) with matching isotype control antibodies were used. Intracellular FoxP3 staining was done using the eBioscience (San Diego, CA) PE-antihuman FoxP3 staining set following the manufacturer’s instructions. CTLA4, IFN-γ, TNF-α, IL-2, IL-4 and IL-5 were stained intracellularly using the Cytofix/Cytoperm Kit with GolgiStop (BD Biosciences) as described [19 (link)]. LAP staining was performed as described [20 (link)]; CD4- and CD25-enriched and CD4- and CD25-expanded T cells were stimulated for 48 h with anti-CD3 and anti-CD28 mAbs (both at 1 μg/ml) in the presence of 10 IU IL-2 after which they were stained according to the published protocol [20 (link)].

Intracellular and extracellular staining was applied for T-cell analysis after 10 days of expansion. 2×10⁶ PBMCs were restimulated with an EBNA-1 or CMV-pp65 peptide pool (JPT, Berlin, Germany) at (1 µg/mL) or DMSO (Sigma Aldrich, Schnelldorf, Germany) as negative control for 5 h. Brefeldin A (7.5 µg/mL) (Sigma Aldrich, Schnelldorf, Germany) was added after 1 h of stimulation. Live/dead cells were discriminated using an amine reactive dye (Invitrogen, Life Technologies, Darmstadt, Germany) and stained with fluorescence conjugated monoclonal antibodies against CD3, CD4, CD8, PD-1, IFN-y, TNF-α and IL-2 (BD Biosciences, NJ, USA). Background events in DMSO controls were subtracted from events counted in response to EBNA-1 or CMV-pp65 stimulation. Data acquisition was performed on BD LSR II (Becton Dickinson, NJ, USA) and analysis was done using FlowJo software.