Pd1 cd279 pe

The following anti-human monoclonal antibodies (mAbs) were used for flow cytometry: CD4 Alexa Fluor® 700, CD8 APC, CD39 PE-Cy7, CD73 FITC, and CD73 eFluor450, CCR7 (CD197) PE-Cy7, PD1 (CD279) PE (eBioscience); CCR7 PE-CF594, CD45 AmCyan and CD45 FITC (Becton Dickinson), CD3 APC-H7, CD25 FITC (BioLegend) and CD25 PE (MACS Miltenyi). All mAbs were titrated using normal PBL to establish optimal dilution. T_reg were defined by their ability to produce extracellular adenosine as previously described by Deaglio and Borselino [28 (link), 29 (link)]. We have previously demonstrated that these CD4⁺ CD39⁺ CD25⁺ T_reg are FOXP3⁺ CD127^neg [24 (link)].

The following fluorochrome-labeled monoclonal antibodies were used as described (22 (link), 33 (link)) for cell surface or intracellular staining of rhesus T cells: CD3 PerCP-Cy5.5, CD4 APC-H7, CD28 APC-H7, CD127 (IL-7Rα) PE, CD45RA PE-Cy7, CTLA4/CD152 APC, and CTLA4/CD152 VB450 (all from BD Biosciences, San Jose, CA, USA), CD8α AF700, CD25 AF700, and Foxp3 VB421 (all from Biolegend, San Diego, CA, USA), and PD1/CD279 PE (from eBioscience, San Diego, CA, USA). Following surface staining for CD3, CD4, CD8, CD25, CD28, CD127, and PD1, cells were fixed and permeabilized for 45 min at 4°C using Fixation/Permeabilization buffer (eBioscience™; ref 00-5123-43). After fixation/permeabilization, cells were stained for CTLA4 and Foxp3. No antibodies were added to the co-cultures.

The following anti-human monoclonal antibodies (mAb) were used for flow cytometry: CD4 AF700 (Cat 56-0048-82), CD73 FITC (Cat 11-0739-42), CD73 PE (Cat 12-0739-42), CD39 PE-Cy7 (Cat 25-0399-42), PD1 (CD279) PE (Cat 12-2799-42), and CCR7 (CD197) PE-Cy7 (Cat 25-1979-42) mAb (all eBioscience, San Diego, CA); CD5 AF700 (Cat 561159), CD19 PE-Cy7 (Cat 555414), and IgM PE (Cat 555783) (all BD Pharmingen, Heidelberg, Germany); CD26 APC (Cat 302710), CD25 FITC (Cat 356105), and CD40 PE-Cy7 (Cat 334322) (all Biolegend, San Diego, CA); CD25 PE (Cat 130-101-426) and IgA APC (Cat 130-093-113) (Milenty Biotec, Bergisch Gladbach, Germany). All mAb were titrated using normal PBMC to establish optimal staining dilutions.