Figure 4: iMB cells rapidly differentiate into plasma cells in vivo on immunization.

(a) Control C57BL/6 mice (αHEL-iGB cells: −) or mice that received Hy10 Ly5.1⁺ iGB cells 50 days before (αHEL-iGB cells: +) were injected i.v. with 2×10⁷ pooled splenocytes from mice primed with OVA with CFA 2 months before, and immunized i.v. with OVA-HEL (20 μg) in PBS (Ag:OVA-HEL: +) or not (Ag:OVA-HEL: −) 12 h later. Five days after immunization, spleen cells were analysed by flow cytometry. Each histogram shows the staining profile of the indicated marker in Ly5.1⁺ HEL^high or HEL^low donor B cells and CD19⁺ Ly5.1⁻ recipient B cells defined in the dot plots. (b–g) Photomicrographs of sections of the same spleens of the mice receiving iGB cells in (a), unimmunized (b–d) or immunized (e–g). Sections were stained with anti-B220 (red), HEL (green), and anti-Thy1 (blue) (b,d,e,g); anti-MAdCAM-1 (red), HEL (green), and anti-B220 (blue) (c); or HEL (red), anti-IgG1 (green), and anti-B220 (blue) (f). Data are representative of three independent experiments. Magnification is ×20 (b,c,e,f) or ×4 (d, g). Bars: 100 μm (b,c,e,f) or 1 mm (d,g). (d,g) show low power views of the images in (b) and (e), respectively. (h) Splenocytes (2×10⁷) from OVA-primed mice (T_mem) were transferred i.v. into non-irradiated C57BL/6 mice with the HEL⁺ iMB cell-containing splenic B cells shown in Figure 3d (M) (n=5), with sorted HEL⁺ follicular B cells (Fo) (n=2) or MZ B cells (MZ) (n=2) from naïve Hy10 mice, or without B-cells (−) (n=2). All donor B cells contained 1.2×10⁵ HEL⁺ B cells. After i.v. immunization with (+) or without (−) 20 μg OVA-HEL, serum titres of anti-HEL IgG1 in individual mice were determined on the indicated days. Horizontal bars indicate the average value in each group. Data shown are representative of three independent experiments performed with two-to-five mice per group.