PMC

(a) ELISA to detect NP-specific IgM and IgG present in the serum of Foxo1^+/+Cd21^Cre (open circles) and Foxo1^L/LCd21^Cre (filled circles) mice before and 7 or 14 days after IP immunization with 100 μg of NP-KLH in alum. b) Splenic GL7⁺Fas⁺ germinal center B cells were quantified by FACS, and c) visualized by fluorescent imaging of frozen sections by PNA (green) and B220 (red) (D14 only). FACS plots and histology are representative of 3 mice/group.

(a) Representative FACS profiles of n = 3–5 mice per group of spleen, peripheral blood, and lymph node from Foxo1^+/+mb1^Cre, Foxo1^L/Lmb1^Cre, and Foxo3^−/− mice indicating B cell (CD19⁺) percentages (left) and percentage of cells expressing IgM and IgD (right). (b) Absolute numbers (symbol) and averages number (dash) of splenocytes and splenic B cells from n=3–5 mice per group. (c) Representative FACS profiles of n = 3–5 mice per group showing percentages of bone marrow B cells, and subsets of immature and mature B cells, pro-B cells, pre-B cells, and subcompartments of pre-pro-B (CD19⁻BP1⁻), early-pro-B (CD19⁺BP1⁻) and late-pro-B (CD19⁺BP1⁺) cells. (d) Absolute number (symbols) and average numbers (dashes) of bone marrow cells and B lineage cells subcompartments. (e) Representative semi-quantitative PCR of 3 independent experiments of cDNA obtained from Foxo1^+/+mb1^Cre pro B cells (IgM⁻IgD⁻CD2⁻CD25⁻B220⁺) and Foxo1^L/Lmb1^Cre pro B cells (B220⁺).

(a) IL-7Rα expression on pro-B cells from Foxo1^+/+mb1^Cre (red) and Foxo1^L/Lmb1^Cre (blue) mice. (b) Ann V expression on late-pro-B cells from Foxo1^+/+mb1^Cre (red) and Foxo1^L/Lmb1^Cre (blue) mice. (c) Intracellular Bim expression in pro-B (line) and non-B (shaded) cells and MFI analysis of Bim expression from pro-B and non-B cells (5 mice of each group indicated with symbols, and average MFI indicated with dashes). (d) Intracellular Bcl-x_L expression of pro-B (line) and non-B (shaded) cells and MFI of Bcl-x_L expression of pro-B and non-B cells (3–4 mice of each group). (e) Foxo1^+/+ and Foxo1^L/L B220⁺ bone marrow cells were grown in culture with IL-7 for 2 days, infected with MSCV-Bcl-x_L or left uninfected, followed by infection with MIT or MIT-Cre. MIT-Cre-transduced (Thy1.1⁺) B cells were enumerated by FACS on day 2 and day 5. (f) Bcl-x_L infected cells superinfected with either MIT or MIT-Cre were labeled with CFSE and cultured for 4 days. FACS analysis of proliferation was determined by CFSE division. FACS profiles are representative of 3–5 independent experiments.

(a) Representative FACS profile of n=3 mice per group of intracellular μ heavy chain expression in pro-B cells from Foxo1^+/+mb1^Cre (red), Foxo1^L/Lmb1^Cre (blue), and Rag^−/− (shaded gray) bone marrow. (b) Southern blot analysis of distal V_H-DJ_H rearrangements (V_HJ558), proximal V_H-DJ_H rearrangements (V_H7183), D-J rearrangements, and control (C_μ) in pro-B cells. (c) Pro-B cells were analyzed by semi-quantitative RT-PCR for expression of Rag1, Rag2, Foxo1, and Actb expression. (d) Foxo1 binding to the Erag enhancer region was analyzed by ChIP on chromatin from pre-B cells from IL-7 cultures using antibodies for Foxo1, E47, and an isotype control, followed by PCR of three regions of the Erag enhancer region. All 3 regions contain putative forkhead binding sites while Erag1 and Erag2 regions contain E-box binding sites. Southern blot, RT-PCR, and ChiP analyses are representative of 3 independent experiments.

(a) Flow cytometric analysis of LPS + IL-4 stimulated (3 days) B cells from Foxo1^+/+Cd21^Cre and Foxo1^L/LCd21^Cre mice (2 mice per group repeated in 3 independent experiments). Proliferation was measured by CFSE partitioning on plasmablasts (Syn1⁺) undergoing CSR (IgG⁺). (b) Percentage of class switched cells generated in the absence or presence of the PI(3)Kδ inhibitor IC87114 (+IC8). Data is representative of 3 independent experiments. (c) Representative semi-quantitative PCR from two independent experiments to measure Aicda expression and the generation of γ1 germline transcripts (GLT), γ1 post-switch transcripts (PST), and γ1 circular transcripts (CT-lower band). (d) QT-PCR of Aicda expression of splenic germinal center B cells from Foxo1^+/+Cd21^Cre, Foxo3a^−/−, and Foxo1^L/LCd21^Cre mice (3 mice per group) 10 days after immunization with 200 μg of NP-KLH in alum.

(a) Representative FACS profiles of spleen and lymph node from Foxo1^+/+Cd21^Cre and Foxo1^L/LCd21^Cre indicating the percentages of B cells of total cells and the percentage of B cells that are IgM⁺IgD⁺ or CD9⁺CD21⁺ marginal zone B cells. Representative FACS profile of the expression level of CD62L on B cells in the spleen. (b) FACS profiles indicating the percentage of IgM⁺CD23⁻ B-1 cells and CD5⁺ B-1a vs CD5⁻ B-1b subsets from the peritoneal cavity of Foxo1^+/+Cd21^Cre (red) and Foxo1^L/LCd21^Cre (blue) mice. (c) Stimulation of purified splenic B cells from Foxo1^+/+Cd21^Cre (red) and Foxo1^L/LCd21^Cre (blue) mice with titrated amounts of anti-IgM F(ab′)₂ (48 h) and measurement of cell viability using 7AAD. Data is representative of triplicate stimulations done in 3 independent experiments (d) Immunoblot analysis for Bim expression in purified splenic B cells stimulated overnight with or without 10 μg/mL of anti-IgM F(ab′)₂. FACS plots are representative of 3 mice/group.

(a) Representative FACS profiles from Foxo1^+/+Cd19^Cre and Foxo1^L/LCd19^Cre mice indicating percentages of total B cells, immature and mature B cells, pro-B cells, and pre-B cells. (b) Quantitation of bone marrow B cell subsets from n=3 mice per group (indicated by symbols) and average numbers of each group (indicated by dashes). (c) Representative FACS profiles indicating percentage of B cells and IgM and IgD expression on B220⁺ B cells from the spleen, peripheral blood, and lymph node. (d) Quantitation of splenic B cell subsets from n=3 mice per group (indicated by symbols) and average numbers of each group (indicated by dashes). (e) Expression of CD21 and CD23 maturation markers on splenic B cells from Foxo1^+/+Cd19^Cre and Foxo1^L/LCd19^Cre mice. (f) Expression of intracellular μ heavy chain and κ light chain in splenic IgM⁺ (red), IgM⁻ (blue) B cells, and non-B cells (shaded gray). (g) Representative southern blot analysis of 3 independent experiments of proximal and distal V_H(D_H)J_H rearrangement, V_κ-J_κ rearrangement, V_κ to RS rearrangement, and c_μ control in IgM+ and IgM− B cells. FACS plots are representative of 3 mice/group.

(a) Representative FACS plots of proliferation measurements of purified splenic B cells from Foxo1^+/+Cd21^Cre (red) and Foxo1^L/LCd21^Cre (blue) mice following CFSE labeling and stimulation (3 days) with 1 μg/mL or 10 μg/mL of either anti-IgM F(ab′)₂ or intact anti-IgM in the presence or absence of IL-4; or stimulated with LPS and IL-4. CFSE partitioning was analyzed by FACS. (b) Immunoblot analysis of phospho-Akt and phospho-Erk from anti-IgM F(ab′)₂ stimulated splenic B cells. (c) Calcium flux by purified splenic B cells from Foxo1^+/+Cd21^Cre (red) and Foxo1^L/LCd21^Cre (blue) mice loaded with Fluo-4 and stimulated with 10 μg/mL anti-IgM F(ab′)₂. (d) ELISA to detect TNP-specific IgM and IgG3 present in the serum of Foxo1^+/+Cd21^Cre (open circles) and Foxo1^L/LCd21^Cre (filled circles) mice before and 5 days after IP immunization with 10 μg TNP-Ficoll in PBS. FACS profile and in vitro stimulation is representative of 3 independent experiments. ELISA data is representative of 2 independent experiments with 3 mice per group.