Profiling B Cell Subsets and Receptor Expression

We processed PBMCs of the entire study population in four different batches. Frozen PBMCs were thawed, washed twice with PBS (Fisher Scientific) and stained with live/dead marker (Zombie Yellow™ Fixable Viability dye, eBioscience) and fluorochrome-conjugated antibodies against surface markers: anti-CD19 BV510, anti-CD24 BV421, anti-IgD APC-Cy7 (all BioLegend); anti-CD27 AF-700, anti-CD38 APC, anti-CD40 PE-Cy7, anti-BAFF-R FITC (all eBioscience); anti-CD86 PE-CF594, and anti-TACI BV650 (all BD Biosciences). A total of five B cell subpopulations could be measured using the following gating strategy as previously reported (18 (link)): total (CD19⁺), transitional (CD19⁺CD24^hiCD38^hi), naïve (CD19⁺CD27^-), unswitched memory (CD19⁺CD27⁺IgD⁺) and switched memory B cells (CD19⁺CD27⁺IgD^-). We measured expression of CD40, BAFF-R and TACI as percentage of positive cells for all five B cell subsets and as mean of fluorescence intensity (MFI) across positive cells for each cell type. The absolute cell counts of plasmablasts (CD19⁺CD24^-CD38^hi) were too low (< 100 cells) to reliably determine expression levels. Data were collected on BD Symphony flow cytometer (BD Biosciences). For data analysis, we used FlowJo (LLC, V10). Supplementary Figure 1 shows representative FACS plots.