Notch2IC-expressing B cells are preactivated and hyperresponsive to LPS and α-CD40 stimulation in vitro. (A) Notch2IC-expressing B cells express similar levels of CD80, CD86, and ICAM-1 as wt MZ B cells. Splenocytes were stained with antibodies specific for the indicated surface markers. Histograms show overlays of surface expression of the indicated molecules on lymphocyte-gated, B220⁺, hCD2⁺ B cells from Notch2IC//CD19Cre^+/− (black line) and on lymphocyte-gated, B220⁺ B cells from control (gray line) mice (top). Histograms in the bottom row are additionally gated on CD21^high cells to compare Notch2IC-expressing MZ B/MZ B precursor cells with control MZ B/MZ B precursor cells. Data are representative for 3 independent experiments. (B) Notch2IC-expressing B cells display higher c-Myc levels in comparison to control B cells. Splenic B cells were purified from Notch2IC//CD19Cre^+/− and control mice, and whole-cell extracts from unstimulated cells were subjected to immunoblot analysis with the use of an antibody specific for c-Myc. Equal protein loading was controlled by α-tubulin staining. The result is representative for 5 independent experiments. (C) Slightly enhanced proliferation of Notch2IC-expressing B cells in comparison to control B cells directly after ex vivo isolation. Splenic B cells from Notch2IC//CD19Cre^+/− and control mice were stained with propidium iodide and were subjected to flow cytometry for cell-cycle analysis. Markers indicate cells in phase G₀+G₁ (left), S (middle), and G₂+M (right) of the cell cycle. Numbers indicate the mean percentages with SDs, and P values of 4 independent experiments. (D) The considerable higher proliferation rate of Notch2IC-expressing MZ B cells in comparison to control B cells is caused by their MZ B-cell phenotype. Splenic B cells or sorted MZ B cells of Notch2IC//CD19Cre^+/− and control mice were labeled with CFSE and cultured for 3 days with the indicated stimuli. After 3 days, proliferation profiles of propidium iodide–negative cells were assessed by flow cytometric analysis and are displayed in the histograms. The tables under each histogram show the proliferation index (average number of divisions of proliferating cells), percentage divided (percentage of cells that initially start to divide), and the division index (average number of divisions of all cells). Values were calculated with the FlowJo Version 8.8.6 software. Numbers indicate values of 1 representative of 4 (splenic B lymphocytes) or 3 (sorted splenic MZ B cells) independent experiments. MZ B cells were sorted with a FACS Aria after staining of the surface markers B220, CD21, and CD23. In the case of sorted MZ B cells as controls either wt or CD19Cre^+/− B cells were used. Both controls displayed the same proliferation abilities. Before cell sorting the cells were enriched by depleting CD43⁺ and CD23⁺ cells with magnetic beads. (E) After stimulation, switching to IgG1 is enhanced in Notch2IC-expressing B cells in comparison to control B cells. Splenic B cells of Notch2IC//CD19Cre^+/− and control mice were cultured with α-CD40 and IL-4, LPS and IL-4, or without stimulation (w/o). After indicated culture times, cells were stained for IgM, IgG1, and hCD2. Numbers indicate mean percentages and SDs of IgG1⁺ control B cells and IgG1⁺, hCD2⁺ Notch2IC-expressing B cells of 3 independent experiments. Only propidium iodide–negative cells were included in the analysis. (F) The switching activity is similar after stimulation of sorted MZ B cells from Notch2IC//CD19Cre^+/− and control mice. MZ B cells were sorted as described in panel C and cultured with α-CD40 or α-CD40+IL-4 for 3 days. Unsorted splenic B cells from control mice were included. Numbers indicate mean percentages of 3 independent experiments and SDs.