Immunophenotyping of Lymphocytes and B-cells

Blood samples from the baseline visit were processed within 4 hours for analysis by flow cytometry. For lymphocyte phenotyping, ethylenediaminetetraacetic acid whole blood was stained for CD3, CD4, CD8, CD45, CD16, CD56, and CD19, as previously described (30 (link)). For B-cell phenotyping, PBMCs were isolated from lithium heparin whole blood by Ficoll gradient density centrifugation. One million PBMCs were incubated with the following antibodies: CD19-VioGreen, anti-IgD-VioBlue, CD24-PerCP-Vio700, CD38-FITC, CD27-APC, CD86-PE-Vio770, CD21-APC-Vio770, and anti-IgM-PE (Miltenyi Biotec, Bergisch Gladbach, Germany). Samples were measured using a FACSLyric flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Data were analyzed using the FACSSuite (BD Biosciences). The gating strategy is shown in Supplemental Figure 1.