Anti cd25 fitc

FCM analysis was performed as we recently reported [20 (link), 22 (link)]. In brief, the fresh-enriched MNCs (day 0) and the MNC-derived cells (day 14) were harvested by centrifugation at 300×g for 5 min and resuspended by 1 × PBS (Solarbio, China) for twice. After that, the cells were incubated in 1 × PBS (Solarbio, China) with 2% fetal bovine serum (FBS) (Australia) and the fluorescence-conjugated antibodies such as anti-CD3-PE (BioLegend, USA), anti-CD3-APC (BioLegend, USA), anti-CD4-PE (BioLegend, USA), anti-CD8-PE-Cy7 (BioLegend, USA), anti-CD56-APC (BioLegend, USA), anti-CD16-FITC (BioLegend, USA), anti-CD25-FITC (BioLegend, USA), anti-NKG2D-perCP-Cy5.5 (BioLegend, USA), anti-NKp44-APC-Cy7, anti-NKp46-PE-Cy7 (BD Biosci, USA), anti-NKG2A-PE (BD Biosci, USA), anti-CD107a-PE (BD Biosci, USA), 7-AAD (BD Pharmigen), Propidium iodide (PI) (BD Pharmigen, USA) or Annexin V-FITC (Tianjin Sungene Biotech, China) in dark for 30 min. Finally, the cells were washed and turned to FACS Canto II (BD Biosci, USA) and FlowJo 10.0 software (Tree Star, USA) for analysis. The list of the indicated antibodies was available in Additional file 1: Table S3.

Phenotypic characteristics of immune cells were assessed by flow cytometry (FACSVerse cytometer; BD, USA) using monoclonal antibodies: anti-CD25-FITC, anti-CD45R0-FITC, anti-CD19-PE-Cy7, anti-CD4-PE-Cy7, anti-CD3-PE-Cy7, anti-CD8-PE-Cy7, anti-CD127-PE-Cy7, anti-CD14-PerCP, and anti-CD45RA-Pacific Blue (Biolegend, USA); and anti-TNFR1-PE, anti-TNFR2-PE, anti-TNFR1-APC, and anti-TNFR2-APC (R&D Systems, USA). Data processing and calculation of fluorescence intensity parameters were performed in the FacsDiva software (BD, USA). To calculate the number of receptor molecules per cell, the BD QuantiBRITE PE Kit (BD Biosciences, USA) was used according to the method described earlier [22 (link)].

Following stimulation for flow cytometry experiments, cells were fixed with 4% para-formaldehyde for 10 min before being washed 3 times with PBS and blocked/quenched with 5% bovine serum + 0.1 mM glycine overnight at 4°C. Cells were stained with 1 μg/ml of anti-BTLA AlexaFluor 647 (RRID AB_2650979; BioLegend Cat. No. 344519), anti-CTLA4 PE (RRID AB_10645522; BioLegend Cat. No. 349905), anti-CD86 Brilliant Violet 421 (RRID AB_10899582; BioLegend Cat. No. 305425), anti-CD69 APC (RRID AB_314844; BioLegend Cat. No. 310909), or anti-CD25 FITC (RRID AB_314273; BioLegend Cat. No. 302603) for 1 h at room temperature then washed 3 times with PBS before being analysed using a FACSCanto II™ flow cytometer (BD Biosciences). Data were analysed using FlowJo version 8.8.7. Staining was performed in three independent experiments with cells from different donors.

T cells were stained with the following fluorochrome-conjugated mouse monoclonal anti-human antibodies for 20 minutes at room temperature: anti-CD25 FITC (BioLegend, catalog no. 302604, RRID:AB_314274), anti-CD38 FITC (BioLegend, catalog no. 303504, RRID:AB_314356), anti-TIM3 PerCP-Cy5.5 (BioLegend, catalog no. 345015), anti-CD4 Pacific Blue (BioLegend, catalog no. 317429), anti-CD45RO Brilliant Violet 605 (BioLegend, catalog no. 304238), anti-TIGIT Brilliant Violet 605 (BioLegend, catalog no. 372711), anti-PD1 Brilliant Violet 650 (BioLegend, catalog no. 329949), anti-CD3 Brilliant Violet 711 (BioLegend, catalog no. 317328), anti-CD101 PE (BioLegend, catalog no. 331011, RRID:AB_2716106), anti-LAG3 PE Dazzle (BioLegend, catalog no. 369331), anti-CD62 L PE Dazzle (BioLegend, catalog no. 304842), anti-CD8 PE-Cy7 (BioLegend, catalog no. 301012), and the anti-BCMA CAR idiotype APC (Allogene Therapeutics). T cells were then washed with PBS and stained with the e780 fixable viability dye (Thermo Fisher, catalog no. 65–0865–14) for 30 minutes at 4°C. All the samples were fixed using BD stabilizing fixative (BD Biosciences, catalog no. 339860) for further FACS analysis.