Cd32 fun 2

We used a combination of two antibodies directed to p24: p24 KC57-PE was obtained from Beckman Coulter (6604667) and p24 28B7-APC was obtained from MediMabs (MM-0289-APC). The following antibodies were used for staining: CD3 (UCHT-1), CD4 (SK3), CD8 (RPA-T8), CD45RA (HI100), CCR7 (3D12), PD-1 (EH12.1), β1 (MAR4), β7 (FIB504), CD69 (FN50), CD25 (M-A251), HLA-DR (G46-6), CD38 (HIT2), Ki67 (MOPC-21), CD95 (DX2), CD39 (Tu66), CD127 (HIL-7R-M21), CXCR3 (1C6), CCR4 (1G1), CCR6 (11A9) were purchased from BD Bioscience. LAG-3 (FAB2319) was obtained from R&D systems, and TIGIT (MBSA43) from eBioscience. CD27 (O323), Tim-3 (F38-2E2), α4 (9F10), CD28 (CD28.2) and CD32 (FUN-2) were purchased from BioLegend. Live/Dead Aqua Cell Stain (405nm) was obtained from ThermoFisher Scientific (L34957). Detailed panels of antibodies are reported in S2 Table.

After 24 h, 48 h, 72 h, and 6 d cultured cells were harvested, magnetically separated from stimulation beads and washed with FACS buffer (phosphate buffered saline with 2% fetal calf serum). Viability was determined by flow cytometry after 72 h and 6 d using Nicoletti buffer according to the protocol described elsewhere [47 (link)]. Surface markers were stained with anti-human antibodies against CD28 (CD28.2, BD, Heidelberg, Germany), CD137 (4B4, eBioscience, Frankfurt a.M., Germany), CD25 (M-A251, BD), CD69 (FN50, BD), CD16 (3G8, BD Bioscience), CD32 (FUN-2, BioLegend, San Diego, CA, USA), CD64 (10.1, BD Bioscience), CD152 (BN13, BD Bioscience), and CD279 (EH12.2H7, BioLegend). Isotypes and quiescent cells were stained as negative controls. The data were recorded on a FACS Calibur cytometer or an LSRFortessa flow cytometer (both BD), and analyzed using FlowJo software (Tree Star, Ashland, OR, USA).