Values are given as 2−delta
CT. RORγt primer (Metabion, Planegg-Martinsried, Germany) and probes were obtained from Eurogentec (Cologne, Germany) using the previously described sequences . t-bet and PNOC panel were purchased from Applied Biosystems (Foster City, CA, USA) with the numbers Mm00450960_m1 and Mm00803087_m1. To analyse cytokine release during aTreg restimulation, this website supernatants were collected and stored at –80°C. Cytokine content was quantified using the CBA kit (FlowCytomix) from Bender MedSystems® (Vienna, Austria). The supernatants were prepared according to the manufacture’s protocol. Samples were analysed on a FACSCalibur (Becton Dickinson, San Jose, USA). To determine the frequency of Treg cells, cells were stained for CD3ε-PerCP (clone
145–2C11, Biolegend Fell, Germany), CD25-PE (clone 3C7, Miltenyi® Biotec) and Foxp3-FITC Stem Cell Compound Library molecular weight (clone FJK-16, eBioscience). The cells were first stained for the surface expression of CD3ε and CD25 for 15 min at 4°C. Cells were then washed, fixed and permeabilised (30 min; 4°C) using the buffer from the Foxp3 staining kit (eBioscience) followed by an intracellular staining for Foxp3 and/or Helios-AlexaFluor 647 (clone 22F6, Biolegend Fell, Germany) for 30 min at 4°C. The percentage of CD4+CD25+Foxp3+ Treg cells was determined on a FACSCalibur (BD). The maturation of B cells was measured using CD19-FITC (clone 6D5, Miltenyi® Biotec), IAb–PE (clone M5/ 114.15.2, eBioscience) and CD86-Biotin (clone GL-1)/Streptavidin-PerCP (both Biolegend, Fell, Germany). Data were analysed with CellQuest software. For intracellular cytokine staining, cells were harvested after 7 days of primary culture washed once and restimulated with 1 μg/mL ionomycin and 10 ng/mL phorbol myristate acetate (both Biotrend Chemikalien GmbH, Cologne, Germany) for 4 h at 37°C. After 2 h, 2 μg/mL Brefeldin A (Sigma-Aldrich Chemie
GmbH, Steinheim, Germany) was added to imbed the cytokines inside the cells. Subsequently, cells were labelled with the live/dead stain (Fixable Viability Dye eFluor 506, eBioscience), their surface expression of CD3ε and CD25 (15 min; 4°C), and additionally fixed and BCKDHA permeabilised with the Foxp3 staining kit. Intracellular staining for IFN-γ allophycocyanin (clone XMG1.2, Biolegend), IL-17-FITC (clone ebio17B7, eBioscience) and Foxp3– Alexa Fluor 488 (FJK-16s, eBioscience) was done for 30 min. Samples were measured by LSR II (BD) and analysed with FlowJo software (Treestare, Ashland, OR, USA). Neuropilin-1 was stained on the surface of the cells using Neuropiln-1-PerCP (R&D Systems) CD40L staining was done as described by Kirchhoff et al. . aTreg cells were isolated from primary culture and restimulated with allogeneic B cells. To prevent exportation and degradation of CD40L, we added 5 μg/mL Brefeldin A after 2 h of stimulation. The next day CD40L (PE, R&D Systems) was stained intracellularly using the Foxp3 staining kit.