Anti cd127 clone a019d5

Foxp3/Transcription Factor Staining Buffer Set, True‐Phos™ Perm Buffer, Cell Staining Buffer, and recombinant human cytokines for in vitro stimulation (IFN‐gamma, interleukin (IL)‐6, IL‐2, IL‐4; all carrier‐free) were purchased from Biolegend.
The following antibodies were used to analyse Tregs via flow cytometry: anti‐CD4 (clone OKT4; APC‐Cy7‐labeled), anti‐CD3 (clone HIT3a; PE‐Cy7) anti‐CD25 (clone M‐A251; PerCP‐labeled), anti‐CD127 (clone A019D5; BV‐421‐labeled), anti‐Foxp3 (clone 206D; AlexaFluor647‐labeled), anti‐CD39 (clone A1; FITC‐labeled), and isotype controls (all from Biolegend). STAT activation was studied using commercially available PE‐labeled antibodies against phospho‐STAT1 (clone A17012A.Rec), phospho‐STAT3 (clone 13A3‐1), phospho‐STAT5 (clone A17016B.Rec), and phospho‐STAT6 (clone A15137E) (all Biolegend).

PBMCs and plasma sample were isolated from blood of donors at the Cardiac Catheterization laboratory in University of Virginia, Charlottesville. Enrolled participants included males and females of different ages (for human subjects characteristics, see Supplementary Table 1) and informed consent was obtained from all subjects. Studies were performed with the approval of the Institutional Review Board of Health Science Research at the University of Virginia. Blood was drawn into vacutainer EDTA (BD Bioscience), spun and the top plasma layer was collected and frozen at −80 °C until analyzed. Thawed plasma samples were tested for the levels of ApoAI and IL-21 using human ApoAI (ABCAM, Cambridge, MA) and IL-21 ELISA (ebioscience) kits, respectively, according to manufacturer’s instruction. Peripheral blood mononuclear cells (PBMC) were isolated from blood that was depleted from plasma with Ficoll-Paque™PLUS (GE Healthcare) and SepMate™ tubes (STEMCELL Technologies, Inc.). For Treg percentages, Treg were identified as CD4⁺CD25⁺CD127⁻ [and/or Foxp3⁺] cells by flow cytometry. Antibodies used were anti-CD4 (clone OKT4; 1 test per sample, Tonbo Bioscience), anti-CD25 (clone BC96; 1 test per sample, Tonbo Bioscience), anti-CD127 (clone A019D5, 1 test per sample, Biolegend), and anti-Foxp3 (clone PCH101, 0.5 μg per test ebioscience).