Cd25 bv421

Regulatory T cells (Treg) were defined as CD4⁺CD25⁺CD127^low/- cells. Other CD4⁺T subsets were identified by differential expression of CCR4, CXCR3, and CCR6 as previously reported (7 (link), 9 (link)): CXCR3⁺CCR4⁻CCR6⁻ (Th1), CXCR3⁻CCR4⁺CCR6⁻ (Th2), CXCR3⁻CCR4⁺CCR6⁺(Th17), and CXCR3⁺CCR4⁻CCR6⁺ (Th1Th17) (Figure 1). In addition, Th17 cells were defined as the summation of CXCR3⁻CCR4⁺CCR6⁺ (Th17) and CXCR3⁺CCR4⁻CCR6⁺(Th1Th17).
The fluorochrome-conjugated antibodies used for polychromatic flow cytometry analysis were CD3-BV510 (Biolegend, USA, catalog no. 317332), CD4-APC-Cy7 (Biolegend, USA, catalog no. 317418), CCR4-APC (Biolegend, USA, catalog no. 359404), CXCR3-PE (Biolegend, USA, catalog no. 353706), CCR6-PE-Cy7 (Biolegend, USA, catalog no. 353418), CD25-BV421 (Biolegend, USA, catalog no. 302630), CD127-FITC (Biolegend, USA, catalog no. 351312), PE-Cy7-CCR7 (Biolegend, USA, catalog no. 353226) and CD62L-FITC (Biolegend, USA, catalog no. 304804). A viability dye, 7-AAD (Becton Dickinson, USA, catalog no. 559925), was used to exclude dead cells. Isotype antibodies were also used as negative controls for every detection to set proper gating for the receptor expression. Cells were analyzed by fluorescence-activated cell sorting (FACS), using the BD LSRII cytometer and FlowJo software.

Cells used for in vitro or in vivo cytokine analysis were stimulated with PMA (20 ng/ml; Sigma-Aldrich), Ionomycin (1 µg/ml; Sigma-Aldrich), and Brefeldin A (2 µg/ml; Sigma-Aldrich) for 4 hr prior to flow staining (Lu et al., 2015 (link)). For intracellular staining, cells were fixed and permeabilized using the Mouse FOXP3 Buffer Set (BD) per the manufacturer’s protocol. The fluorophore-conjugated antibodies used in this study were as follows: Live/Dead Fix Blue (Invitrogen), CD3 PerCPCy5.5 (Biolegend), TCRb PE/Cy7 (Biolegend), CD4 PB (Biolegend), CD4 AF700 (Biolegend), CD8 BV650 (Biolegend), CD25 BV421 (Biolegend), FOXP3 AF488 (Biolegend), ROR gamma T PE (Invitrogen), TCF1 AF647 (Cell Signaling Technologies), TCF1 PE (Biolegend), IFNγ AF647 (Biolegend), IL17A FITC (Biolegend), and IL17A PE (ebioscience). Samples were analyzed using BD LSR II flow cytometer (BD Biosciences) and FlowJo software (TreeStar).

To analyze cell surface activation markers, PBMCs were stimulated by 1 μM TLR7/8 agonists or DMSO (as a negative control) for 24 h at 37°C. Then cells were collected and washed in FACS buffer (PBS + 2%FBS). Cells were incubated by fluorescence antibodies at 4°C for 30 min. For T cells, CD3-PE-Cy7 (Biolegend), CD4-PE (BD), CD8-FITC (Biolegend), CD25-BV421 (Biolegend), CD69-BV510 (Biolegend). For NK cells, CD16-PE (Biolegend), CD56-APC (Biolegend), CD69-BV510 (Biolegend), NKG2D-BV421 (Biolegend). For monocytes, CD14-PE-Cy7 (Invitrogen), CD40-APC-Cy7 (Biolegend) HLR-DR-BV510 (Biolegend). For dendritic cells, anti-human lineage cocktail-APC (Biolegend), CD11c-PE-Cy7 (Biolegend), CD80-BV510 (Biolegend), CD86-AF488 (Biolegend).

The immunochemical reagents used were as follows: anti-human IgG, Fcγ-PE (1/200 dilution, #109-116-170; Jackson Immunoresearch, West Grove, PA, USA), CD3-BV711 (1/250 dilution, #300463; BioLegend, San Diego, CA, USA), CD4-BV510 (1/100 dilution, #344633; BioLegend), CD25-BV421 or CD25-BV605 (1/40 dilution, #302629 or #302631; BioLegend); CD127-PerCP-Cy5.5 (1/40 dilution, #560551; BD Biosciences, Franklin Lakes, NJ, USA), TNFR2-PE (1/70 or 1/100 dilution, #22235; R&D Systems, Minneapolis, MN, USA), and Foxp3-AlexaFluor 647 (1/100 dilution, #320213; BioLegend). Cells were treated with PBS containing 0.2% sodium azide and 5% FBS. For the antibody-binding analysis of TNFR2-expressing Ramos-Blue and HEK293T cells, primary antibodies were first incubated in a dilution series for 30 min on ice and then labeled with anti-human IgG-PE. For quantitation, BD Quantibrite™ PE Phycoerythrin Fluorescence Quantitation Kit (BD Biosciences, lot #76536) was used. For the multi-color labeling experiment, fluorescent intensities were compensated using BD™ CompBeads Anti-Mouse Ig, κ/Negative Control Compensation Particles Set (BD Biosciences), and the performance of two anti-CD25 antibodies was confirmed to be equivalent. The cells were analyzed using a BD LSRFortessa Cell Analyzer (BD Biosciences) and data was analyzed using FlowJo_v10.8.1 (FlowJo, LLC).

PBMCs (7 × 10⁵ cells) were stained with certain antibodies against L/D-FVS510, CD45-FITC, CD4-PE, CD25-BV421 (all from Biolegend, United States) to analyse the Treg frequency, and FOXP3-APC (EB). The impacted cells were run with BD FACSCalibur and BD FACSAria III flow cytometers (BD Biosciences, United States). Using positive gates and gating controls by Fluorescence minus one (FMO) controls. For Th17 cell analysis, cells were incubated with phorbol-12-myristate-13-acetate (PMA) (50 ng/mL), ionomycin (1 μg/mL), and monesin (1.7 μg/mL; Sigma-Aldrich, United States) at 37°C for 6 h to activate T cells and stimulate the accumulation of intracellular IL-17. Then, the cells were stained with specific antibodies against L/D-FVS510, CD45-FITC, CD4-PE (all from Biolegend, San Diego, California, United States), and IL-17A-APC (EB). The FACSCalibur flow cytometer (BD Biosciences) was used to analyse the stained cells.

Single cells suspensions from spleens were prepared as described above. Samples were incubated in RPMI 1640 + 10% FBS at 37 °C for 2 h in the presence of Click-it EdU reagent (ThermoFisher #C10419) at a 1:1000 dilution. The remainder of the procedure was followed as per the manufacturer’s recommendations. Cells were stained for 1 h with viability dye eFluor780 (ThermoFischer L34961) at 1:100 dilution, and surface antibodies CD3e AF488 (1:400, Biolegend 100321), CD4 BV510 (1:1600, Biolegend 100449), CD8a BV570 (1:400, Biolegend 100740), NK1.1 AF700 (1:800, Biolegend 108730), CD25 BV421 (1:400, Biolegend 102043). Samples were acquired on a Cytek Aurora flow cytometry instrument.