Gitr bv605

For surface staining, PBMCs were incubated with directly conjugated antibodies for 30 min at 4°C. Antibodies used included anti-human CD3-BUV737 (clone VCHT1), CD3-BV786 (clone SK7), CD4-BUV395 (clone RPA-T4), CD8-BUV395 (clone RPA-T8), GITR-BV605 (clone V27-580), CD38-BUV737 (clone HB7), CD25-PE-CF594 (clone M-A251, BD Biosciences, San Diego, CA, USA), CD4-APC-fire750 (clone SK3), CD8-BV421 (clone RPA-T8), αβTCR-BV421 (clone IP26), CD56-AF700 (clone 5.1H11), CD56-APC (clone 5.1H11), FasL-PE-Cy7 (clone NDK-1), CTLA-4-BV786 (clone BNI3), CD73-BV711 (clone AD2), HLA-DR-AF700 (clone L243, BioLegend, San Diego, CA, USA), LAG3-APC (clone 3DS223H, Invitrogen, Carlsbad, CA, USA) and the corresponding isotype controls. For intracellular staining of Foxp3 (clone 25901C7, BD Biosciences), Granzyme A-PE-cy7 (clone CB9), granzyme B-APC-fire750 (clone A16A02), perforin-PE-CF594 (clone dG9), Ki-67-BV711 (clone Ki-67, BioLegend) and IDO-APC (clone eyedio, Invitrogen) cells were fixed and permeabilized using Foxp3 Staining Buffer Set (BD Biosciences) according to the manufacturer’s recommendations. A fixable viability dye eFluor 506 (Ebioscience, San Diego, CA, USA) was used to assess cell viability. Data were acquired on a BD LSR Fortessa flow cytometer (BD Biosciences) and analyzed with FlowJo software (Tree Star, Ashland, OR, USA).

PBMCs were incubated with directly conjugated antibodies for 30 min at 4°C. The cells were then washed before flow cytometric analysis. Antibodies used were anti-human CD3-BUV737, CD4-BUV395, PD-1-BV711, CD38-FITC, GITR-BV605 (BD Biosciences, San Diego, CA, USA), CD8-BV510, CTLA-4-BV786, OX40-APC-Fire750, 4-1BB-BV421, HLA-DR-AF700 (BioLegend, San Diego, CA, USA), TIGIT- PE-Cy7, LAG-3-APC, ICOS-PE, (Ebioscience, San Diego, CA, USA), and the corresponding isotype controls. Data acquisition was performed on an LSR Fortessa flow cytometer (BD Biosciences), and data analysis was performed using FlowJo Software (Tree Star, Ashland, OR, USA).