Il 10 apc

A total of 5 × 10⁵ WT or TNFR2 KO-MSCs were seeded in 75 cm² flasks and were activated by the addition of 10 ng/ml of premium-grade mouse TNFα (Miltenyi) in a total volume of 10 ml. After 36 h, MSCs were treated with 1 μl/ml of Golgi-Plug (protein transport inhibitor) (BD Biosciences) and incubated for another 12 h. Cells were then stained with following Abs: CD44-PE-Vio770, CD73-PE, IFNγ-APC, IL-6-PE or FITC, TNFα-FITC, IL-10-APC (Miltenyi), and anti-TGFβ-PE (BioLegend). Events were acquired on a LSRFORTESSA flow cytometer (BD Biosciences) and analyzed using FlowJo software v10 (FlowJo, LLC).

Primary human T cells (n = 52) were isolated by digestion of fresh human skin biopsies (Ø 6 mm) in RPMI containing FCS, Collagenase type IV (Worthington), and Deoxyribonuclease I (Sigma) at 37 °C overnight followed by dissociation using the gentleMACS Dissociator (Miltenyi Biotec). Freshly isolated skin T cells were passed over a cell strainer and directly used for flow cytometric analysis. For flow cytometric analysis, T cells were stimulated with PMA/Ionomycin (10 ng/ml and 1 µg/ml, respectively) (both Sigma) for 5 h in the presence of Brefeldin A and Monensin (both BD Biosciences). Surface staining was performed at 4 °C and followed by fixation/ permeabilization using the fixation/permeabilization kit (BD Biosciences). Staining of intracellular cytokines was performed at room temperature. Antibodies used were CD3-Bv650 (#563852, clone UCHT1, dilution 1:50), CD4-BV421 (#562842, clone L200, dilution 1:20), CD8-APCCy7 (#557834, clone SK1, dilution 1:20) (BD Biosciences), IL-17A-PeCy7(#512315, clone BL468, dilution 1:20), IFN-γ-PerCPCy5.5 (#506528, clone B27, dilution 1:100), TNF-α-BV510 (#502950, clone MAB11, dilution 1:100) (BioLegend), IL-22-Pe (#12-7229-41, clone 22URTI, dilution 1:20, eBioscience), IL-10-APC (#130-108-135, clone JES3-9D, dilution 1:10, Miltenyi Biotec). Flow cytometry data was analysed and visualized using FlowJo 10.7.1.

3 × 10⁴ WT or TNFR2 KO-MSCs were co-cultured in 12-well plates with 1.5 × 10⁵ (1/5 ratio) of fresh mice WT CD3⁺CD25⁻T cells in a total volume of 2 ml. After 3 days, WT-CD3⁺CD25⁻T cells were harvested. Cells were then stimulated with 1 μg/ml PMA and 0.5 μg/ml ionomycin for 4 h and 30 min (Sigma), in the presence of 1 μl/ml GolgiPlug for the last hour (BD Biosciences). They were then immunostained with CD4-VIOBLUE, CD8α- Pe-Cy7, IFNγ-APC, TNFα-FITC, IL-10-APC, IL-17-PE, IL-2-FITC (Miltenyi), and anti-TGFβ-PE (Biolegend).