Cd25 bv605

A panel of monoclonal antibodies was developed to enable phenotypic characterization of B and T lymphocyte subpopulations: CD3-FITC, CD4-APC, CD25-BV421, FoxP3-PE, CD19-PeCy7, CD9-BB700 (BD Bioscience, France), CD24-BV510, and CD38-APC-Cy7 (Sony Biotechnology, UK); hematopoietic stem and progenitor cells: CD34-BV421 (Biolegend, France), cKit-APC-H7, CD135-PE, lineage-FITC, Sca-1-BV510, CD16/32-PerCPCy5.5, CD127-APC (BD Bioscience), and TLR-4-PeCy7 (Biolegend); and dendritic cells: CD11c-PeCy7 (eBioscience, France), CMH-II-FITC, Siglec-H-BV510, CD11b-APC-H7, and CD103-PerCP5. For intracellular staining, cells were fixed and permeabilized using a Cytofix/Cytoperm kit (BD Biosciences). For in vitro human B cell differentiation experiments, cells were stained with Fixable Viability Dye eFluor 450 to identify dead cells, followed by staining with CD25-BV605 (Biolegend), CD19-BUV395, CD9-FITC, CD27-BUV737, CD38-BV711 and intracellular IL-10-PE (BD Bioscience). Cells were analyzed on a Canto II flow cytometer (BD Biosciences). Data were acquired using Diva 8.0 software and analyzed with FlowJo X (TreeStar, Williamson Way, Ashland, USA). Fluorescence minus one staining controls were used for all panels, and dead cells were removed using viability staining.

GICs and PBMCs were stained for CD3-PE-Cy5.5 (eBioscience), CD4-BV711, CD8-PerCP, CD25-BV605, CD45RA-BV570, CCR7-BV650, CD39-BV421, PD-1-APC-Cy, BTLA-PE, Tim-3-PE-Cy7 (all Biolegend), LAG3 (Enzo Life Sciences, Lörrach, Germany), CTLA4-PE-CF594 (BD) and Live/dead-Aqua dye (Life technologies, Carlsbad, CA) or with isotype controls. Cells were fixed and permeabilized, followed by ICS using Foxp3-FITC (eBioscience) and Ki67-Alexa Fluor 700 (BD) and measured on an LSR Fortessa (BD).

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).

Out of the 104 mBC patients included in the mass cytometry analysis, tumor biopsies were obtained from 63 patients (HR⁺ BC, n = 42; TNBC, n = 21) and were subjected to flow cytometry. Briefly, freshly obtained tumor biopsies were mechanically chopped and then dissociated in DMEM F12 medium containing 10% FBS, collagenase-IV (1 mg/mL), hyaluronidase (1 mg/mL), & DNase-I (50 µg/mL) in a 37 °C water bath for 45 min. Single cell suspensions were then filtered, washed with PBS, and stained with antibodies CD45-A488, CD3-Pacific blue, CD4-PE, CD25-BV605, CD14-PE-Cy7, CD33-BV711, HLA-DR-BV650 (Biolegend), CD127-BV786 (BD Biosciences), and Fixable Viability Dye (eFluor780, ThermoFisher). Samples were acquired using LSR-II/FACSymphony A5 flow cytometer (BD Biosciences), and the data analyzed with FlowJo.

5×10⁵ PBMCs from each patient/donor were stained with antibodies CD3-BUV395 (RRID:AB_2744382), CD4-BV510 (RRID:AB_2869877) (BD Biosciences, NJ, USA), CD25-BV605 (RRID:AB_2800963, BioLegend) and Fixable Viability Dye eFluor780 (ThermoFisher). After fixation and permeabilization using Foxp3 /Transcription Factor Staining Buffer Set (ThermoFisher), PBMCs were incubated with antibody Foxp3-PE (RRID:AB_1944444, ThermoFisher). Samples were acquired using BD FACSymphony A5 flow cytometer (BD Biosciences), and the data analyzed with FlowJo.

Prior to antibody incubation, cells were stained with viability dye (Zombie™ dye, 1:500, Biolegend) for live cell/dead cell discrimination and incubated with Fc receptor blocking solution (Human TruStain FcX, BioLegend, CA) to prevent unspecific antibody binding. Extracellular antigens were stained using anti-human cluster of differentiation (CD)4-PE-Cy7, CD8-PE, CD14-PE-CF594 and CD19-FITC/PerCP-Cy5.5, CD20-APC-Cy7, CD25-BV605, CD27-PacificBlue, CD38-FITC, CD80-PE-Cy7, CD150-BV-421, major histocompatibility complex class II (MHC-II)-APC (all Biolegend, CA), CD19-PerCp-Cy5.5, CD40-PE-Dazzle, CD69-FITC, CD86-BV421, and CD95-PE (all BD Biosciences, NJ) antibodies. For analysis of intracellular cytokines, cells were permeabilized by adding fixation/permeabilization solution (Cytofix/Cytoperm, BD Biosciences, NJ) and stained with anti-human interleukin (IL)-6-FITC, IL-10-PE/CF594, and tumor necrosis factor (TNF)-Alexa Flour 700 (all BD Biosciences, NJ) antibodies. Apoptosis was evaluated using propidium iodide-PE and annexin V-FITC (both BioLegend, CA). Samples were analyzed using a LSRII Fortessa; FACS Diva (BD Biosciences) and FlowJo software were used to quantify flow cytometric data.