Ethylene diamine tetraacetic acid (edta)

To determine maturation status, DCs were harvested two days after addition of peptides and maturation factor LPS. Cells were stained in FACS buffer (PBS (GIBCO) containing 0.5% BSA (Sigma) and 0.5 mM EDTA (ICN Biomedicals)) for 30 minutes at 4°C with either one of two panels that contained the following maturation markers: anti-CD80-FITC, anti-CD14-PE, anti-DC-SIGN-APC, anti-HLA-DR-Pacific Blue and Live/dead-AmCyan (Invitrogen) (panel 1) or anti-CD83-FITC, anti-CD40-PE, (BD Biosciences), anti-PD-L1-APC (eBioscience), anti-CD86-Pacific Blue (BioLegend) (panel 2). Live/dead-AmCyan (Invitrogen) was included in both panels. For analysis of the co-culture, the following markers were used: anti-CD8-FITC (Sanquin), anti-CD3-PerCP, anti-TNFα-PE-Cy7, anti-IFN-γ-APC (BD Biosciences), anti-CD4-Pacific Blue (eBioscience) and Live/dead-AmCyan (Invitrogen). Four hours prior to staining, Brefeldin A (BD Biosciences) was added to the culture; then cells were stained using the Cytofix/Cytoperm kit from BD Biosciences according to manufacturer’s recommendations. Cells were measured using a FACS Canto II (BD Biosciences) and results were analyzed using FlowJo version 9.7.5 software. First, lymphocytes were gated, followed by gating of live cells, then CD3⁺ cells and finally CD8⁺ or CD4⁺ cells were placed in a quadrant with TNF-α⁺ or IFN-γ⁺ cells.

From each cultured splenocyte sample CD4⁺ T cells were isolated by positive selection using CD4 magnetic microbeads and a magnetic cell separator (Miltenyi Biotech) according to the manufacturer’s instructions. The purity of the CD4⁺ T cells was determined using flowcytometry. Briefly, the isolated cells were stained with Pacific blue-conjugated anti-CD4 (Biolegend) in FACS buffer (PBS (pH 7.2) supplemented with 0.5% BSA (Sigma Aldrich) and 0.5 mM EDTA (ICN Biomedicals). After washing, data were acquired on a FACS Canto II (BD Biosciences) and analyzed using FlowJo software (Tree Star). The purity of the isolated CD4⁺ T cells was >95%.