American Society of Hematology

Figure 3

$Figure 3. Characterization of Notch2IC-expressing B cells as MZ B cells. (A) Characteristic MZ B-cell surface marker expression on Notch2IC+ B cells. Histograms show overlays of cell size (forward scatter) or 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 CD21high 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-D) Notch2IC-expressing B cells are mainly located in the splenic MZ. Immunohistochemical stainings of splenic cryosections for (B) MOMA-1+ metallophilic macrophages (α-MOMA1; blue), lining the MZ at the sinus, IgM+ B cells (α-IgM; red), and CD3+ T cells (α-CD3; blue). The MZ is indicated by arrows. Scale bar: 250 μm. (C) MOMA-1+ metallophilic macrophages (α-MOMA-1; marine blue), CD3+ T cells (α-CD3; pigeon blue), and hCD2+ (α-hCD2; red). An arrowhead indicates hCD2+ B cells within the follicle. Scale bar: 250 μm. (D) MOMA-1+ metallophilic macrophages (α-MOMA1; blue) and CD1d+ MZ B cells and MZ B-cell precursor cells (α-CD1d; red) and CD3+ T cells (α-CD3; blue). CD1d+ cells within the follicle are indicated by an arrow. Panels C and D are serial sections stained with different antibodies. The arrows indicate MZ B cells or MZ B-cell precursors located inside the primary follicles. Scale bar: 250 μm. (E) The numbers of MZ B-cell precursors are not increased in Notch2IC//CD19Cre+/− mice. Splenic B cells isolated by CD43+ depletion were stained with antibodies specific for CD21/35, IgM, CD23, and AA4.1 and were analyzed by flow cytometry. Plots in the left panel are lymphocyte-gated and display percentages of cells belonging to fraction I to III (Fr. I, II, III) according to Allman and colleagues21,22; IgMhighCD21/35low/int cells (Fr. I); IgMlow/intCD21/35int cells (Fr. II); IgMhighCD21/35high cells (Fr. III). Numbers in the right panel display the frequency of events as a function of the indicated parent gate, which is stated above. Fr. I harbors T1 and T2 B cells; Fr. II, T3 and Fo B cells as well as potential MZ B-cell precursors* (MZ Pre*); and Fr. III, MZ B cells and conventional MZ B-cell precursors. Data are representative for 3 independent experiments.$

Characterization of Notch2IC-expressing B cells as MZ B cells. (A) Characteristic MZ B-cell surface marker expression on Notch2IC⁺ B cells. Histograms show overlays of cell size (forward scatter) or 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 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-D) Notch2IC-expressing B cells are mainly located in the splenic MZ. Immunohistochemical stainings of splenic cryosections for (B) MOMA-1⁺ metallophilic macrophages (α-MOMA1; blue), lining the MZ at the sinus, IgM⁺ B cells (α-IgM; red), and CD3⁺ T cells (α-CD3; blue). The MZ is indicated by arrows. Scale bar: 250 μm. (C) MOMA-1⁺ metallophilic macrophages (α-MOMA-1; marine blue), CD3⁺ T cells (α-CD3; pigeon blue), and hCD2⁺ (α-hCD2; red). An arrowhead indicates hCD2⁺ B cells within the follicle. Scale bar: 250 μm. (D) MOMA-1⁺ metallophilic macrophages (α-MOMA1; blue) and CD1d⁺ MZ B cells and MZ B-cell precursor cells (α-CD1d; red) and CD3⁺ T cells (α-CD3; blue). CD1d⁺ cells within the follicle are indicated by an arrow. Panels C and D are serial sections stained with different antibodies. The arrows indicate MZ B cells or MZ B-cell precursors located inside the primary follicles. Scale bar: 250 μm. (E) The numbers of MZ B-cell precursors are not increased in Notch2IC//CD19Cre^+/− mice. Splenic B cells isolated by CD43⁺ depletion were stained with antibodies specific for CD21/35, IgM, CD23, and AA4.1 and were analyzed by flow cytometry. Plots in the left panel are lymphocyte-gated and display percentages of cells belonging to fraction I to III (Fr. I, II, III) according to Allman and colleagues^21,22; IgM^highCD21/35^low/int cells (Fr. I); IgM^low/intCD21/35^int cells (Fr. II); IgM^highCD21/35^high cells (Fr. III). Numbers in the right panel display the frequency of events as a function of the indicated parent gate, which is stated above. Fr. I harbors T1 and T2 B cells; Fr. II, T3 and Fo B cells as well as potential MZ B-cell precursors* (MZ Pre*); and Fr. III, MZ B cells and conventional MZ B-cell precursors. Data are representative for 3 independent experiments.

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