Cd95 fitc

Whole blood was stained for flow cytometry as previously described (38 (link), 68 (link)). mAbs used were as follows: CD3- FITC (clone SP34) or CD3-PerCP (clone SP34-2); CD20-PE (clone 2H7); CD8-PErCP (clone SK1) or CD8-PE (clone RPA-T8); CD4-APC (clone L200) or CD4-PerCP (clone L200); HLA-DR-PerCP (clone L243); CD95-FITC (clone DX2) or CD95-APC (clone DX2); CD28-APC (clone CD28.2) or CD28-PE (clone CD28); Ki-67–FITC (clone B56); CD25-FITC (anti–IL-2 receptor) (clone 2A3); and FoxP3–Alexa Fluor 488 (clone 259D.C7) (all from BD Biosciences). All Abs were validated and titrated using AGM PBMCs. Data were acquired with an LSR II flow cytometer (BD Biosciences) and analyzed with CellQuest software (BD Biosciences). CD4⁺ and CD8⁺ T cell percentages were obtained by first gating on lymphocytes and then on CD3⁺ T cells. Memory, activation, and proliferation markers were determined by gating on lymphocytes, then on CD3⁺ T cells, and finally on CD4⁺CD3⁺ or CD8⁺CD3⁺ T cells. Gating strategy for Tregs is presented in Supplemental Figure 6, while the gating strategy for macrophages is presented in Supplemental Figure 7.

The detection of accessory molecule expression on RAW264.7 cells by FACS analysis was performed as follows. Briefly, RAW264.7 cells were harvested by accutase or EDTA dissociation buffer and washed with FACS buffer (3% bovine serum albumin (BSA) and 0.1% sodium azide in DPBS). The cells were stained with the optimal concentrations of fluorochrome-conjugated antibodies for 30 min. The antibodies used in these experiments were Fas ligand-APC (eBioscience, San Diego, CA), CD95-FITC (BD Pharmingen, San Diego, CA), and F4/80-FITC (clone: BM8; BioLegend, San Diego, CA). One hundred thousand cells per treatment were acquired and analyzed using a BD FACSVerse flow cytometer and FlowJo software (version 10.7, BD Ashland, OR). Cells were gated using forward scatter vs. sideward scatter properties to exclude debris and doublets. The mean fluorescence intensity (MFI) of the whole macrophage population was analyzed.

For surface staining, peripheral blood mononuclear cells (PBMCs) were incubated with directly conjugated mAbs for 30 min at 4 °C and then washed before analysis. Antibodies used were anti-human CD3-BV786 or CD3-APC, CD4-BV711, CD8-APC-H7, CD45RA-AF700, CD95-FITC, CD25 BV510, CD73 PE-CY7, CD11b AF700, CD45 BV786, CD14 BV711, CD56-PE-CF594, CD19-FITC, HLA-DR-PE, CD160-AF488 (BD Biosciences, San Diego, CA, USA), CCR7-BV421, PD-1-PE, 2B4-APC (BioLegend, San Diego, CA, USA), TIGIT-APC (Ebioscience, San Diego, CA, USA), LAG-3-AF700 (R&D Systems, Minneapolis, MN, USA) antibodies, and corresponding isotype controls. Data acquisition was performed on a LSR Fortessa flow cytometer (BD Biosciences), and data analysis was performed using FlowJo Software (Tree Star, Ashland, OR, USA).