Anti cd3 pe cy7

MALC obtained at day 10 of growth, were co-cultured with NK cells at ratio E:T 0.5:1 or 1:1 and treated or not with mAbs at 10 μl/ml during 4 h in presence of brefeldin A at 10 μl/mL (except of CD69 detection). Cells were then washed, fixed with 2% PFA during 4 min, washed and permeabilized with 1% saponin during 8 min. Cells were stained with anti-CD56-PE (Miltenyi Biotec), anti-CD3-PE/Cy7 (Beckman Coulter, Roissy, France), anti-CD69-PE-Cy5 (BD Biosciences), anti-perforin (PFN)-FITC (BD Biosciences), anti-IFN-γ-Alexa fluor 647 (BD Biosciences), anti-granzyme B-Alexa647 (BD Biosciences) antibodies during 30 min at 4°C in the dark. Cells were analyzed by flow cytometry with a LSRII flow cytometer (BD Biosciences).

Mφ, untreated or treated with 5 μM ZnPPIX, were added to FBS‐coated polypropylene tubes for 14 hours at 37°C, 5% CO₂, with T cells in the presence of BD GolgiPlug Protein Transport Inhibitor (BD Biosciences) containing Brefeldin A, according to the manufacturer's instructions. Treatment with 1 mM 1‐MT was directly added in co‐culture for the entire period of incubation with T cells. Autologous T cells were thawed and activated with 1 μg/mL anti‐CD3 and 5 μg/mL anti‐CD28 mAbs for 24 hours at 37°C, 5% CO₂. After 14 hours of contact, cell cultures were fixed and permeabilized by using PerFix‐nc Kit (Beckman Coulter, California) according to the manufacturer's instructions and stained for 1 hour with anti‐CD3 PE‐Cy7 (Beckman Coulter) and anti‐IL‐10 BB700 (BD Biosciences) mAbs.

Specific cell lines against T1BT* and T1BT*-Y peptide constructs were generated from naïve CD4 T cells from a HLA-DRβ1*04:01:01 donor using as APCs autologous monocyte derived dendritic cells (DCs) as described by Moser and colleagues [47] (link). CD4 T cells were purified using a naïve CD4 T cell isolation kit as manufacturer instructions (Miltenyi Biotec, Germany). DCs were pulsed with 3.75 mM of peptides T1BT* or T1BT*-Y. DCs were matured with TNF-α (25 ng/mL). Cells were co-cultured at 37°C, 5% CO₂ for 14 days and re-stimulated at 1∶10 ratio with mature dendritic cells pulsed with 3.75 mM of the same peptide used to prime. Seven days after the cells were stained with fluorescent DR4/T*-1; DR4/QNT-5 and DR4/QNT-Y tetramers assembled by stepwise addition of streptavidin-PE (Caltag, Life technologies – California, USA) to biotinylated HLA-DR4–peptide complexes at a final molar ratio of 1∶4 as described [41] (link). T cells were incubated with tetramers for 4 hours at 37°C followed by 20 min incubation with cell surface markers: anti-CD3 PE-Cy7, anti-CD4 PE-Texas red (Beckman Coulter, Palo Alto CA. USA), anti-CD45RO FITC, anti-CD62-L PECy5 (all from BD Biosciencies. San Diego CA. USA) on ice before washing with ice-cold buffer. Tetramer and Ab binding were determined using an FACS Aria II flow cytometer (BD Biosciences).