Anti rorγt pe

Splenocytes were collected from each mouse and the proportion of each T helper cell subset was analyzed: Th1 cells (CD4+ CD25+ T-bet+), Th2 cells (CD4+ CD25+ GATA-3+), Th17 cells (CD4+ CD25+ ROR-γt+), and Treg cells (CD4+ CD25+ Foxp3+). For analysis of the proportion of the T helper subsets, splenocytes were stained with antibodies to CD4 and CD25 (FITC rat anti-mouse CD4 and APC rat anti-mouse CD25; BD Biosciences, San Jose, CA, USA). Cells were washed, fixed, and intracellular staining was performed according to the manufacturer’s protocol for the Foxp3/transcription factor staining buffer set (eBioscience, San Diego, CA, USA) and intracellular antibodies (PE rat anti-mouse Foxp3; BD Biosciences, PE anti-RORγt, PE anti-T-bet, and PE anti-GATA-3; eBioscience). Fluorescence signals from 10,000 cells were counted, and flow cytometry analysis was carried out using a FACSAria flow cytometer (BD Biosciences). The proportion of CD138-positive cells was also determined (PE rat anti-mouse CD138, BD Biosciences).

DCs were harvest after 24 h treatment and stained with different antibodies raised against specific surface markers: Anti-CD11C- PerCP-Cy5.5, Anti-CD86-APC, Anti-CD40-FITC, Anti-CD80-PE-Cy7, Anti-MHCII-PE, Anti-CD274 (PD-L1)-PE, Anti-CD273(PD-L2)-FITC, Anti-OX40-L-APC for 30 mins. Co-cultured cells were harvest and then mixed with different antibodies raised against specific surface markers: Anti-CD4-PerCP-Cy5.5 (eBioscience, The Netherlands), anti-CD25-FITC (eBioscience, The Netherlands), anti-CD69-PE (eBioscience, The Netherlands), anti-CCR-6-PE (eBioscience, The Netherlands), for 30 minutes. Staining for intracellular markers were performed according manufacturer’s protocol, (eBioscience, Foxp3 staining set, Bio connect, The Netherlands). Antibodies used for intracellular markers where anti-Foxp3-PE-Cy7 (eBioscience, the Netherlands), anti-RORγt-PE (eBioscience, The Netherlands). Matching Isotype controls were used to minimize the influence of nonspecific binding and proper gate setting. All incubations were performed on ice and protected from light. In total a minimum of 50,000 cells were counted and analyzed using FACS Canto II and FACS Diva software (BD Biosciences, The Netherlands).

Th17-differentiated CD4⁺ T cells were stained with anti-CD4-APC (eBioscience, CA, USA). Cells were then permeabilized in flow cytometry fixation and permeabilization buffer (eBioscience) and stained with an anti-RORγT-PE (eBioscience). Flow cytometry analysis was performed using a BD Fluorescence Activated Cell Sorter Canto I (BD). Data were analyzed using FlowJo (BD).

The following antibodies were used: For cell sorting: anti-CD4-PerCPCy5.5, anti-CD62L-APC, anti-CD44-PE and anti-NRP1-APC (all BD, NJ). For intracellular staining: anti-IL-13-PE and anti-IL-17A-PE are from BD (NJ). Anti-IFN-γ-FITC, anti-T-bet-PECy.7, anti-GATA3-PE, anti-RORγt-PE and anti-FOXP3-APC were purchased from eBioscience (CA). In brief, cells were stimulated for 2 hours with PMA and ionomycin with the addition of brefeldin A (GolgiPlug; BD, NJ). Afterwards, cells were fixed with 4% formyl saline, permeabilized with 0.1% saponin buffer and stained with fluorescent antibodies before analyzing on a FACS Verse (BD, NJ). Events were collected and analyzed with Flow Jo software (Tree Star, Ashland, OR).

CD4⁺ T cells were resuspended at 2 million cells/ml and stimulated with anti-CD3/anti-CD28 (1 μl/ml) under neutral or Th1- (50 ng/ml IL-12, 125 ng/ml anti-IL-4), Th2- (40 ng/ml IL-4, 50 ng/ml anti-IFN-γ), Th17- (50 ng/ml IL-6, 2 ng/ml TGF-β1) polarizing conditions. At 72 h, T cells were stimulated by phorbol myristate acetate (PMA) and brefeldin A (BFA) (Sigma-Aldrich) for 6 h. Subsequently, cells were stained with anti-CD4-FITC (CAT#85-11-0041-82), anti-IFN-γ-PE (CAT#85-12-7311-82), anti-IL-4-APC (CAT#85-17-7041-82), anti-IL-17A-PE (CAT#85-12-7177-81), anti-T-bet-PE (CAT#85-12-5825-80), anti-GATA-3-PE (CAT#85-12-9966-41) and anti-ROR-γt-PE (CAT#85-12-6981-80) (all eBioscience) at 4°C for 30 min and analyzed by flow cytometry. For activation assays, CD4⁺ T cells were treated with anti-CD3/anti-CD28 antibodies for 48 h. Cells were harvested and stained with anti-CD69-PE (CAT#85-12-0691-81, eBioscience) at 4°C for 30 min, and then analyzed by flow cytometry.