Cd19 bv785

All flow cytometry used appropriate isotype controls. Gating and compensation were aided by the performing fluorescence minus one controls. Cells isolated from biopsies or blood were stained with blue-fluorescent reactive dye (Life Technologies), CD19-BV785, CD27-APC (BD Biosciences) or PE or BV421, CD10-BV605 or APC, CD38-PerCp-ef710 (eBioscience), CD24-PE/Cy7 or BV605, IgD-APC/Cy7, IgM-V450 (BD Biosciences), IgG-PE/Cy7 and IgA-FITC (Miltenyi Biotec), in 2% FCS staining buffer with 1 mM EDTA for 15 min at 4 °C before analysis on the BD LSRFortessa (BD Biosciences). For high-throughput sequencing analysis, cells were stained with LIVE/DEAD Fixable Aqua (Life Technologies) or DAPI, CD19-BV785, CD27-FITC or APC, IgD-APC/Cy7, CD38-PerCp-ef710 (eBiosciences), CD10-BV605 in 2% FCS staining buffer with 1 mM EDTA for 15 min at 4 °C before sorted on the BD FACSAria (BD Biosciences). Four subsets from PBMC and three subsets from paired biopsy mononuclear cells from Donors 1 to 4. The numbers of cells used to generate sequences for each sample from Donors 1 to 4 are shown in Supplementary Fig. 14d.

Peripheral blood mononuclear cells (PBMCs) were obtained from healthy individuals homozygous for the FcγRIIB-T232 or FcγRIIB-I232 site, under appropriate ethics approval from the NIHR Cambridge Bioresource. Inclusion criteria for individuals were people aged between 44 and 77 years, with no serious co-morbidities, no direct family history of autoimmune disease, no use of immunosuppressants or steroids and no hospitalisation within the last 12 months. Individuals were age and sex matched (18 females and 11 males matched between genotypes). Flow sorting was performed using CD19-BV785, CD38-BV711, CD3-NC650, CD14-605NC, CD24-PerCP-Cy5.5, IgD-FITC, CD27-PE-Cy7 (all from BD Bioscience) and Aqua (for live-dead cell detection, Invitrogen), where flow protocol is outlined in Supplementary Fig. 7.

PBMCs were phenotyped using LEGENDScreen™ kits, as per the manufacturers instructions. Data was collected on the BD Verse flow cytometer and analyzed using FlowJo 8.7 software (TreeStar).
For multi-color flow cytometry samples were stained with live/dead fixable blue (Invitrogen), for 20 min at RT and antibodies for flow cytometry, for 30 min at 4°C as follows; CD19 BV785 (HIB19), CD24 PECy7 (ML5) (BD), CD38 BV605 (HIT2, BV605), to define B cells and subsets, and combinations of the following; SIRPα/β APC (SE5A5), CD324 APC-Fire750 (67A4), CD1c BV421 (L161), CD127 BV510 (AO19D5), CD1a PE-Dazzle594 (HI149), CD167a PE (S1D6), CD138 APC (DL-101) (all BioLegend unless stated otherwise). Appropriate FMO controls were used, and samples were fixed before acquiring on the flow cytometer. Data was collected on the BD LSR2 flow cytometer (BD), and analyzed using FlowJo version 8.7 or 10.5.