Brilliant violet buffer

For flow cytometry, cells were stained with saturating concentrations of surface antibodies and a fixable viability dye diluted in 1:1 PBS (Invitrogen): Brilliant Violet Buffer (BD Bioscience). Following surface staining, cells were either fixed with Cytofix (BD Bioscience) or fixed and permeabilized with Cytofix/cytoperm (BD Bioscience) where further intracellular staining was performed. Intracellular antibodies in saturating concentrations were diluted in a 0.1% saponin-based buffer (Sigma-Aldrich).
For the detection of GM1-enriched microdomains, cells were surface stained as described above and then stained with 25 μg/ml cholera toxin B-fluorescein isothiocyanate (CTB-FITC) at 4°C followed by fixation as above. For the detection of neutral lipid droplets, cells were surface stained and fixed as described above, followed by staining with HSC LipidTOX Neutral Lipid Stain according to the manufacturer’s instructions. For the measurement of membrane cholesterol, cells were surface stained and fixed as described above, followed by a staining with 50 μg/ml Filipin complex (Sigma-Aldrich) for 2 h at room temperature.
All samples were acquired on a BD Bioscience Fortessa-X20 and analysed using FlowJo v.10 (BD Bioscience). Full details on monoclonal antibodies and other fluorescent agents can be found in Supplementary Table 2.

Peripheral blood was collected at baseline (pre-treatment), C2D1, and post-cycle 3 (C4D1 or C6D1) for flow cytometry analysis. Peripheral blood was obtained using heparin collection tubes, and peripheral blood mononuclear cells (PBMCs) were isolated within 6 h using Ficoll-Paque Separation according to manufacturer’s instructions (GE Healthcare). PBMCs were cryopreserved in 10% DMSO, 90% FBS and thawed rapidly for flow cytometry analysis. Single-cell suspensions were prepared on ice in 2% FBS in PBS. Antibody cocktails were diluted in Brilliant Violet Buffer (BD Biosciences). Samples were acquired using a Cytek Aurora spectral cytometer with user settings established by Spectroflo QC beads. Unmixing and compensation were performed in Spectroflo software using a mix of reference controls from either single stained PBMCs or single stained OneComp compensation beads (eBioscience). Samples were stained with fluorescently tagged antibodies (Supplemental Table 4). Antibodies were titrated for optimal signal-to-noise ratio prior to use. Flow cytometry analysis was performed using Flowjo vX, and the CATALYST R package was used for FlowSOM analysis and UMAP projections [21 (link)]. All samples were gated on live, single cells.

Multiparametric flow cytometry was used for ex vivo analysis of B cells. For subset gating, combinations of the following mAbs were used: CD19-BV786, CD27-BUV395, CD20–Alexa Fluor 700, CD10-BV605, CD3-BV711, CD21-BV421, and CD45-BUV05. Full details of mAbs used, including those used for phenotypic characterization, are given in Supplemental Table 1.
Cells were stained with Fixable LIVE/DEAD Dye (Life Technologies, Thermo Fisher Scientific) before incubation with saturating concentrations of surface mAbs diluted in 50% Brilliant Violet Buffer (BD Biosciences) and 50% PBS for 30 minutes at 4°C. In all instances, cells were stained in the presence of FcR-blocking reagent (Miltenyi Biotec). Cells were fixed and permeabilized for further functional assessment with either Cytofix/Cytoperm (BD Biosciences) or FoxP3 Buffer Set (BD Biosciences) according to the manufacturer’s instructions for intracellular or intranculear staining respectively. Saturating concentrations of mAbs for 30 minutes at 4°C were diluted in 0.1% saponin (Sigma-Aldrich) for the detection of intracellular proteins or in 1× PBS for the detection of intranuclear proteins. All samples were acquired on a Fortessa-X20 (BD Biosciences) and analyzed using FlowJo (Tree Star).