Anti cd4 apc h7

Cells collected from patients and normal controls were cultured at a concentration of 1^∗10⁶ cells/mL in 2 mL tubes containing 1 mL of RPMI 1640 + FCS 10% (Lonza, Basel, CH). Cells were stimulated overnight with Dynabeads Human T-Activator CD3/CD28 (Life Technologies, Carlsbad, CA, USA). The detection of IL-17 production was analysed using the IL-17 Secretion Assay (Miltenyi Biotec, Bergisch Gladbach, D) following the manufacturer's instruction. Briefly, cells were washed with 2 mL of cold buffer at 300 ×g for 5 minutes at 4°C, and the pellet was resuspended in 90 μL of cold medium. Cells were then incubated with 10 μL of IL-17 Catch Reagent for 5 minutes in ice and cultured in 1 mL of warm medium at 37°C for 45 minutes under slow continuous rotation. Cells were then washed with cold buffer and resuspended in 75 μL of cold buffer; 10 μL of IL-17 Detection Antibody APC, 10 μL of anti-CD3 PerCP (Becton Dickinson, Franklin Lakes, NJ, USA), and 5 μL of anti-CD4 APC-H7 (Becton Dickinson) monoclonal antibodies were added. Incubation was carried out in ice for 10 minutes. Finally, cells were washed and resuspended in an appropriate volume of PBS and acquired on a FACSCanto II cytometer (Becton Dickinson). Analysis was performed with FlowJo 9.3.3 software (Tree Star, Ashland, OR, USA).

Splenocytes were isolated and stimulated overnight with 10 μg/ml of Traspain or 10 μM of TEWETGQI peptide in the presence of anti-CD154 PE and anti-CD107 PE-Cy7. Brefeldin A plus monensin were added to cultures during the last 12 h of incubation. Dead cells were stained with LIVE/DEAD^TM Fixable Blue Dead Cell Stain Kit (Life Technologies). Surface staining was performed with anti-CD3e V500, anti-CD4-APC-H7 (BD), and anti-CD8α-Brilliant Violet 650 (BioLegend). Cells were fixed at RT with PFA 2%, permeabilized in 0.5% saponin and stained using anti-IFN-γ Brilliant Violet 711 (BioLegend) and anti-TNF-α eFluor450 (eBioscience) in accordance with the manufacturer's instructions.

To detect antigen-specific T cells, spleen or blood cells were first labeled with the H2Kk-TEWETGQI dextramer-APC (Immudex) and then with anti-CD3e V500, anti-CD4-APC-H7 (BD), and anti-CD8α-Brilliant Violet V650 (BioLegend) according to the manufacturer's instructions.

Cells harvested from overnight stimulation cultures were incubated with live/dead aqua V510 for 15 min on ice. The cells were then surface stained for 30 min on ice using anti-CD3-FITC (BioLegend, clone UCHT1, 1:200, Cat# 300406), anti-CD4-APC-H7 (BD Pharmingen™, clone RPA-T4, 1:200, Cat# 560158), and anti-CD8-PerCPCy5.5 (BD Biosciences, clone RPA-T8, 1:200, Cat# 560662) antibodies. After fixation and permeabilization with Cytofix and Perm (BD Biosciences, Cat# 554714) on ice for 15 min, intracellular staining was performed on ice for 30 min using anti-TFNα-PE-Cy7 (BD, clone MAb11, 1:200, Cat # 557647) and anti-IFNγ-APC (BD Pharmingen™, clone B27, 1:200, Cat# 554702) antibodies. After the final wash step, the cells were resuspended in 200 µL of FACS buffer. Samples were acquired using an FACSAria III instrument (BD Biosciences, San Diego, CA, USA) and analysed using the FlowJo software (Treestar, San Carlos, CA, USA).