Figure 1: In vitro induction and massive expansion of phenotypically GC B cells from naïve B cells.

(a) Schematic representation of the iGB cell culture system. (b) Cumulative fold increase in the number of live B cells cultured on 40LB with IL-4 (closed circles), on 3T3/BAFF with anti-CD40 Ab (1 μg ml⁻¹) plus IL-4 (open circles), or on 3T3/BAFF with LPS (10 μg ml⁻¹) plus IL-4 (shaded circles). Shown are mean values with s.d. at the indicated time points from three independent experiments, each done singly from an individual mouse. (c) B cells were cultured as in (b) for 3 days and pulsed with BrdU for the last 1 h. The cells were stained with anti-BrdU Ab and propidium iodide and analysed by flow cytometry. Numbers indicate the percentages of the cells in the G1 (lower left window), S (upper window), and G2-M (lower right window) phases of cell cycle. The data shown are representative of two independent experiments. (d) CFSE-labelled splenic naïve B cells were cultured for 3 or 4 days on 3T3/BAFF with anti-CD40 Ab (1 μg ml⁻¹) plus IL-4 (thick line) or without (shaded) ('3T3/BAFF'), or on 40LB with IL-4 (thick line) or without (shaded) ('40LB'), and then analysed by flow cytometry. Unlabelled B cells cultured on 40LB with IL-4 served as negative controls (thin line). Theoretical cell division numbers are noted on top of each histogram. The data shown are representative of three independent experiments. (e) Flow cytometric analysis for the indicated markers of iGB cells cultured with IL-4 for 4 or 6 days, or of splenic naïve B cells from a C57BL/6 mouse. Expression of Fas, GL7, CD38, and PNA ligand was analysed for CD138⁻ gated cells. Numbers indicate the percentages of cells in the indicated quadrants or windows. The data shown are representative of numerous independent experiments. (f) Flow cytometric analysis of iGB cells cultured with IL-4 for 4 days (day 4), and of pooled splenic B cells from three mice that had been immunized with 100 μg of NP-CGG/alum 10 days before (10 days after imm). Expression of CD38, PNA ligand, Fas and GL7 on CD19⁺ CD138^– gated cells is shown. (g) Western blot analysis was performed using the following cells purified by the FACSAria II cell sorter: IgG1⁺, IgM⁺ or IgE⁺, CD138⁻ populations from iGB cells 4 days after primary culture with IL-4 (iGB-4), and IgG1⁺ or IgE⁺, CD138⁻ populations from those after the subsequent culture for 2 more days with IL-21 (iGB-21), splenic GC B cells (GC: GL7⁺, Fas⁺, CD19⁺, CD138⁻) from 20 C57BL/6 mice immunized with BSA/CFA 2 weeks earlier, splenic naïve follicular B cells (NF: CD21⁺, CD23^high, CD19⁺, CD43⁻) from unimmunized C57BL/6 mice. IgG⁺ mouse B-lymphoma cell line A20 was used as a control. All samples consist of 2×10⁶ cells per lane, except the IgM⁺ iGB-4 ells (1×10⁶ cells). The blot was probed with antibodies against indicated proteins.