B cell development in G5pr^−/−. (A) Flow cytometric analysis of cells from the bone marrow, spleen, axillary lymph node, peritoneal cavity, and peripheral blood. The numbers indicate the relative percentages of cells within the indicated gates. Bone marrow contains (top) pro–B cells (B220⁺ CD43⁺), pre–B cells and immature B cells (B220^Low CD43⁻), and mature recirculating B cells (B220^High CD43⁻) and (middle) pro–B cells and pre–B cells (B220⁺ IgM⁻ IgD⁻), immature B cells (B220⁺ IgM⁺ IgD^Low/−), and mature recirculating B cells (B220⁺ IgM⁺ IgD^High). The spleen contains (top) B cells (B220⁺) and T cells (CD3⁺); (middle) transitional type-1 B cells (IgM^High IgD^Low/−), transitional type-2 B cells (IgM^High IgD^High), and mature B cells (IgM^Low IgD^High); and (bottom) marginal zone B cells (CD21^High CD23^Low/−) and follicular B cells (CD21⁺ CD23⁺). The lymph node contains (top) B cells (B220⁺) and T cells (CD3⁺) and (bottom) transitional type-2 B cells (IgM^High IgD^High) and mature B cells (IgM^Low IgD^High). The peritoneal cavity contains B-1a cells (CD5⁺ IgM⁺) and the peripheral blood contains B cells (B220⁺) and T cells (CD3⁺). (B) Ig levels of various classes were measured by ELISA with the serum of nonimmunized mice. Specific Ab responses were measured after immunization with either TI-II Ag (C) or TD Ag (D). TNP-specific Abs were measured, and the results for TI-II Ag (IgM and IgG3) and TD Ag (IgM and IgG1) are shown. Horizontal lines represent mean Ab titers. (E) Immunization with TD Ag and SRBCs created GCs in the spleen comparable with those in control mice. The spleen sections were studied with G5PR2/2 mice (b, d, and f) in comparison with those of control mice (a, c, and e). Higher magnification images, outlined by boxes in panels a and b, show control mice (c and e) and mutant mice (d and f). PNA⁺ GCs (brown), IgD⁺ B cells (blue), B220⁺ B cells (brown), and CD4⁺ T cells (blue) are shown. Arrows indicate typical GCs, and asterisks indicate extrafollicular regions. TZ, T cell zone; FO, follicular region.

BCR-mediated signaling in G5PR-deficient B cells. (A) The activation of G5pr^+/− and G5pr^−/− B cells in response to 10 μg/ml anti-IgM F(ab')₂ Ab as assessed by the up-regulation of CD25 and CD69. Histograms represent CD25 or CD69 expression with (black plot) and without (white plot) stimulation. (B) A Western blot analysis for tyrosine-phosphorylated proteins. The isolated splenic B cells from G5pr^+/− and G5pr^−/− littermates were stimulated with anti-IgM F(ab')₂ Ab. Cell lysates were subjected to Western blotting with antiphosphotyrosine Ab (4G10) and reprobed with anti–β-actin Ab for controls. (C) A calcium Flux analysis in response to BCR cross-linking. A flow cytometry analysis depicting calcium flux (y axis) as a function of time (x axis) in the cells stimulated with 10 μg/ml anti-IgM F(ab')₂ Ab. The arrow indicates the time point of stimulation. (D) BCR-induced Erk phosphorylation was monitored by a Western blot analysis using phosphorylated Erk-specific Ab. The entire Erk expression was also monitored as an internal control. Erk1 (p44) and Erk2 (p42) were indicated. (E) Time course of Akt phosphorylation and the degradation of IκBα. (F) BCR cross-linking–dependent expression of cyclin D2 and phosphorylation of Rb. The phosphorylated Rb was detected as shifted bands because of a slower mobility than nonphosphorylated Rb. 10⁶ B cells were stimulated with 10 μg/ml anti-IgM F(ab')₂ Ab. Each membrane was reprobed with anti–β-actin Ab as control.

Mitochondrial apoptotic machinery in G5PR-deficient B cells. (A) 10⁶ B cells stimulated either with 10 μg/ml anti-IgM F(ab')₂ Ab or 10 μg/ml LPS for 24 h were lysed in hypotonic buffer containing propidium iodide, and nuclear DNA contents were analyzed. The number in each box indicates the percentage of cells in that box based on the gated sample. (B) For the quantitation of ΔΨm, 10⁶ splenic B cells were stimulated either with 10 μg/ml anti-IgM F(ab')₂ Ab or 10 μg/ml LPS for 24 h and subjected to staining with the ΔΨm-sensitive dye DiOC₆. The proportion of depolarized cells is shown by the percentage in the histogram. (C) 10⁶ splenic B cells were stimulated with anti-IgM F(ab')₂ Ab or LPS and were harvested and characterized by TUNEL assay. FITC-dUTP–incorporated cells were measured by flow cytometry. The number in each box indicates the percentage of cells in that box based on the gated sample.

Generation of a CD19-Cre–mediated G5PR-deficient mouse. (A) Targeting of the G5pr gene. (top model) Genomic organization of the G5pr gene locus (WT). H, HindIII; S, SalI. The bar indicates the probe used for Southern blots. The loxP sites are indicated by triangles. (bottom models) The targeting vector, the targeted allele (F), and the deleted allele (Δ). The G5pr-deleted allele was generated by the Cre-mediated deletion of loxP-flanked exons I and II. Correct targeting was verified by a Southern blot analysis of HindIII-digested DNAs with the indicated probe. Lengths of the respective HindIII fragments are shown. DT-A represents a selection cassette for diptheria toxin-A. (B) Germ line transmission of a G5pr-targeted allele. Tail DNAs from wild-type and G5pr^F/wt littermates were digested with HindIII, followed by Southern blot analysis. (C) CD19-Cre–mediated deletion of exons I and II flanked with loxP sites. DNAs of splenic B cells purified from G5pr^+/− and G5pr^−/− littermates were digested with HindIII, followed by a Southern blot analysis. (D) CD19-Cre–mediated disruption of the G5pr gene. RNA was extracted from splenic B cells isolated from G5pr^+/− and G5pr^−/− littermates and subjected to RT-PCR using exons spanning specific primers designed from exons II and IV of the G5pr gene. (E) Spleens from G5pr^+/−and G5pr^−/−mice. (F) Columns show mean cell numbers of total nucleated cells, B cells, and T cells in the spleens. The number of cells was calculated based on the proportion of B cells and T cells determined as B220⁺ and CD3⁺, respectively. Error bars represent SEM. A significant difference in cell numbers between G5pr^+/− and G5pr^−/− mice was indicated (P < 0.01).

B cell proliferation in response to the mitogenic stimuli in vitro. (A) Splenic B cells were cultured for 48 h in the presence of medium alone, anti-IgM F(ab')₂ Ab, or 100 U/ml IL-4 (left) or LPS (right). B) Kinetics of the responses of WT, G5pr^+/−, and G5pr^−/− B cells to anti-IgM Ab indicated a similar response curve with the peak at 48 h after stimulation. [³H]thymidine was added for the final 6 h. The results are representatives of three experiments performed in triplicate. The incorporation of [³H]thymidine is shown as counts per min (cpm) with the SD of the triplicate culture samples per each.

Cell death of G5PR-deficient B cells. Splenic B cells from either G5pr^+/− or G5pr^−/− mice were stimulated with 10 μg/ml anti-IgM F(ab')₂ Ab. (A) Living cells were identified with forward scatter (FSC) and side scatter (SSC) on the FACS profile. (B) Apoptotic cells were monitored as annexin V–FITC positive cells. Lymphoid gating was set by the parameters of SSC and FSC. The number in each box indicates the percentage of cells in that box based on the gated sample.

BCR-mediated regulation of apoptotic and survival signaling molecules in G5PR^−/− B cells. Expression of Bcl-XL or Bcl-2, expression and phosphorylation of Bim, the phosphorylation of JNK or p38, or expression and phosphorylation of Bim in the presence of JNK inhibitor were studied. (A) The membrane was probed with anti–Bcl-XL or anti–Bcl-2 Ab. (B) Three major isoforms of Bim—short type (S), large type (L), and extra-large type (EL)—were detected with anti-Bim Ab. The relative intensities of the bands are shown based on the controls. 10⁶ splenic B cells from both mutant and control mice were stimulated with 10 mg/ml anti-IgM F(ab')2 Ab for the time depicted in the figures. To show the loading amounts, each membrane was reblotted with anti–β-actin Ab. (C) 10⁶ splenic B cells were stimulated with 10 mg/ml anti-IgM F(ab')2 Ab, and total RNAs were isolated, converted to cDNA, and subjected to measure BimEL expression by RT-PCR. (D) BCR-induced JNK and p38 phosphorylation was monitored with phospho-JNK or phospho-p38–specific Ab. The entire JNK expression was monitored as an internal control. JNK1 (p46) and JNK2 (p54) are indicated with arrows. An asterisk indicates a nonspecific band. The relative intensities of bands were shown based on the controls. (E) Splenic B cells were stimulated with 10 mg/ml anti-IgM F(ab')2 Ab for 24 h after incubation with or without 2 mM SP600125 for 30 min. Bim induction and its phosphorylation were detected (B).