Anti cd3 fitc hit3a

Cell sorting was performed on a Sony SH800 Cell Sorter (Sony Corporation, Japan). Briefly, the PBMCs were incubated with the following Ab-conjugates for 30 min on ice in the dark: anti-CD3-FITC (HIT3a, 300305, 1:100, BioLegend), anti-CD8-PC5.5 (SK1, 344709, 1:100, BioLegend), and anti-CD57-PB (HCD57, 322315, 1:100, BioLegend). Following incubation, the stained PBMCs were washed and resuspended in FACS buffer. Propidium iodide (PI; Life Technologies) was added to the cells immediately before analysis. CD57⁺CD8⁺ and CD57^-CD8⁺ T cells were collected.

PBMCs from healthy donors were purified by density gradient centrifugation over lymphocyte separating medium (PAN Biotech). PBMCs were frozen in order to allow comparison with GPA patient probes that were transported deep-frozen to our collaboration partner. Thawed PBMCs from patients or healthy donors were incubated overnight in medium (IMDM with Glutamax™, 10% FCS, 1% Penicillin/Streptomycin, all from Gibco) with a final concentration of 10 μg/ml rituximab, infliximab, or obinutuzumab and without antibody as control. Next day, cells were washed and stained first with Zombie Yellow (BioLegend) in PBS for 15 min at room temperature, then stained with anti-CD56 Brilliant Violet 421 (NCAM16.2, BD), anti-CD3 FITC (Hit3a, BioLegend), anti-CD19 AlexaFluor700 (Hib19, BioLegend) or anti-CD19 AlexaFluor647 (SJ25-C1, Life Technologies), and anti-CD16 PE (3G8, BioLegend).