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. 2017 Apr 3;214(4):1181–1198. doi: 10.1084/jem.20161263

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© 2017 Inoue et al.

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Figure 1. — Hyperexpansion of preGC B cells with Foxo1 ablation. (A) Left, flow cytometry of intracellular Foxo1 protein expression in naive B cells at day 0 (CD45.1⁺B220⁺NP⁺CD38⁺), activated B cells on day 4 (CD45.1⁺B220⁺NP⁺CD38⁺GL7⁻ or CD45.1⁺B220⁺NP⁺CD38⁺GL7⁺), LZ (CD45.1⁺B220⁺NP⁺CD38⁻CD86^hiCXCR4^lo), and DZ (CD45.1⁺B220⁺NP⁺CD38⁻CD86^loCXCR4^hi) GC B cells on day 10. Wild-type mice were transferred with B1-8^hi CD45.1⁺ B cells and then immunized i.p. with NP-CGG/alum on day 0. Foxo1 KO staining controls (gray histograms) were prepared as previously described in Figs. 1 C and 2 A. (right) Histograms indicating the geometric mean fluorescence intensity (gMFI) of each population. n = 4 biological replicates. (B) Analysis of Foxo1 protein and mRNA expression in DZ and LZ GC B cells by Western blot (top) and real-time qPCR (bottom). Foxo1-proficient LZ, DZ, and Foxo1-deficient GC B cells were sorted from mice prepared as described in Fig. 2 A. Actin, loading control. n = 3 biological replicates. (C, top) Schematic illustration of the experimental protocol. (bottom left) Flow cytometry of NP-specific donor B cells (CD45.1⁺B220⁺NP⁺). (bottom right) Histograms representing the cell number of preGC (Donor B220⁺NP⁺GL7⁺CD38⁺IgD⁺CCR6^hi) and early GC (Donor B220⁺NP⁺IgD^loCCR6^lo) B cells in 10⁶ splenocytes. n = 3 biological replicates. (D) Real-time qPCR analysis of Foxo1 mRNA expression in Foxo1^+/+ERT2cre B1-8^hi and Foxo1^f/f ERT2cre B1-8^hi preGC B cells. Error bars represent SD. Data are representative of three (A) or two (B and C) independent experiments, and from one experiment with three biological replicates (D). *, P < 0.05; **, P < 0.01; ***, P < 0.001; unpaired Student’s t test (A, B, and D) and paired Student’s t test (C).