IgG⁺ MBCs display reduced expression of FOXP1 and an enhanced propensity to differentiate as compared with IgM⁺ MBCs. (A-B) IgM⁺ (IgG⁻IgA⁻) and IgG⁺ (IgM⁻IgA⁻) MBCs were sorted from human peripheral blood and cultured for 2 days after which FOXP1 gene expression levels in these cells, and in OCI-Ly7 and SKW6.4 cells, were compared by qRT-PCR (A) or by immunoblotting (B). (A) Expression levels are normalized to hypoxanthine guanine phosphoribosyl transferase (HPRT) and then to levels in IgM⁺ MBCs. Means ± SEM of 3 independent experiments are shown. (B) FOXP1 protein expression levels were determined by immunoblotting. β-tubulin levels were determined as loading control. FOXP1 and β-tubulin levels were quantified using Image Studio Lite software, and the ratio of FOXP1 over β-tubulin expression was determined for each lane. A representative blot of 2 independent experiments is shown. (C) PBMCs were isolated from human peripheral blood and stained for surface expression of CD19, CD27, IgM, and IgG, as well as intracellular expression of FOXP1. Intracellular FOXP1 expression in CD19⁺CD27⁺IgM⁺ MBCs and CD19⁺CD27⁺IgG⁺ MBCs was analyzed by flow cytometry. A histogram representative of 4 independent experiments (left), and the mean relative mean fluorescence intensity (MFI) (geometric mean) values ± SD of the IgM⁺ and IgG⁺ MBCs (n = 4; right) are shown. MFI values were normalized to values in IgM⁺ MBCs (1 sample t test, ***P < .001). A specificity control for the intracellular FOXP1 staining is shown in supplemental Figure 2B. (D-E) The sorted cells were cultured under conditions that promote PC differentiation. (D) Eight days after sorting, cells were analyzed for CD20 and CD38 surface expression by flow cytometry. Representative density plots of 1 out of 3 independent experiments are shown (left). Percentages of CD38⁺ cells were normalized to IgM⁺ MBCs. Means ± SD values of 3 independent experiments are shown (right). Levels were normalized to levels in IgM⁺ MBCs (1 sample t test, ***P < .001). (E) Gene expression levels of FOXP1, BCL6, PAX5 SPIB, BACH2, PRDM1, IRF4, and XBP1 were analyzed by qRT-PCR at the start (2 days after sorting) and end (8 days after sorting) of the PC differentiation assay. Expression levels were normalized to expression levels in IgM⁺ MBCs that had been cultured for 2 days. Means ± SEM of 2 independent experiments performed in triplicate are shown.