BCR-Induced long-term induction of c-Rel coincides with expression of A1 and Bcl-x_L. A and B, Splenic mature B cells were stimulated with anti-IgM F(ab′)₂ (10 μg/ml) for indicated times; nuclear (NE) and whole cell extracts (WCE) were prepared as described in methods. Three micrograms NE or 10 μg WCE were fractionated by SDS-PAGE and transferred to PVDF membranes, which were probed with Abs against NF-κB proteins RelA and c-Rel as indicated. TATA box binding protein and protein kinase C (PKC) μ were used as loading controls for NE and WCE, respectively. C, Analysis of BCR-induced c-Rel mRNA expression in naive splenic B cells. Cells were stimulated with 10 μg/ml anti-IgM F(ab′)₂ for the indicated time points and RNA levels of c-Rel were determined by qRT-PCR. Relative fold induction of each gene was normalized to GAPDH and calibrated to the corresponding nonstimulated sample. Data are mean with SD from three experiments. D and E, c-Rel regulates long-term expression of A1 and Bcl-x_L in mouse splenic B cells following BCR stimulation. Naive splenic B cells from WT and c-Rel deficient (c-rel^−/−) splenic B cells were stimulated for the indicated times with 10 μg/ml anti-IgM F(ab′)₂ and qRT-PCR was performed to determine the fold change in the steady-state levels of A1 and Bcl-x_L mRNA compared with the endogenous control GAPDH and calibrated to the nonstimulated sample. Data are mean with SD from three experiments. F, Western blot analysis was performed on total cellular extracts from WT and c-Rel deficient (c-rel^−/−) splenic B cells stimulated with anti-IgM F(ab′)₂ for the indicated times. Blot was probed with anti-Bcl-x_L, -A1, -c-Rel, and -β-Tubulin Abs. The data in all panels are representative of three independent experiments using different mice for each experiment.

BCR signaling induces cRel and anti-apoptotic genes preferentially in T2 and mature B cells. A, Purification of T1, T2 (CD21^int-T2), and mature Fo B cell populations. Freshly isolated splenocytes from 4-wk-old C57BL/6 mice were depleted of RBCs. T1 and T2 B cells were obtained by MACS enrichment of AA4.1⁺ transitional B cells, followed by FACS purification using Abs directed against CD23, CD24 and CD21 to sort AA4.1⁺C23⁻CD24⁺CD21^low (T1) and AA4.1⁺ CD23⁺CD24⁺CD21⁺ (T2) B cells. T2-PreMZ (AA4.1⁺C23⁺CD24⁺CD21^high) B cell subset was excluded from sorted T1 and T2 B cells. Thus, the T1 and T2 B cells we have used here have previously been described by Hoek et al. and Meyer-Bahlburg (3, 4). The purity of T1 and for T2 was determined by postsort FCM analysis. Mature Fo B cells were MACS enriched from the AA4.1 negative fraction by CD43 depletion followed by FACS purification using the same Ab scheme as for T1 and T2 sorts; AA4.1⁻CD23⁺CD24^lowCD21⁺ (Fo B). The purity of the mature Fo B cells was determined by postsort FCM analysis. B, Analysis of BCR-induced steady-state levels of c-Rel mRNA in FACS-purified T1, T2, and mature Fo B cells from C57BL6 mice. Cells were stimulated with 10 μg/ml anti-IgM F(ab′)₂ for 4 h and analyzed for c-Rel mRNA by qRT-PCR. Relative fold levels of c-Rel was normalized to 18S and calibrated to the nonstimulated sample for each population. Data are mean with SD of five experiments using five to ten mice as a source of B cells for cell sorting. *, p = 0.0036; **, p = 0.0103. Levels of c-Rel protein in freshly isolated T1, T2, and mature B cells were similar (data not shown). C and D, Expression profile of NF-κB dependent A1 and Bcl-x_L genes in transitional and mature Fo B cells. mRNA levels for A1 and Bcl-x_L were determined by qRT-PCR in T1, T2 and mature Fo (M) B cells stimulated with 10 μg/ml anti-IgM F(ab′)₂ for 4 h. Relative fold expression of each gene was normalized to 18S and calibrated to the nonstimulated sample for each population. Data are mean with SD of five experiments using five to ten mice as a source of B cells for FACS sorting as described in A. p values for A1 gene in anti-IgM treated samples; *, p = 0.051; **, p = 0.047. p values for Bcl-x_L gene in anti-IgM treated samples; *, p = ≪0.000; **, p = 0.0024. D, Increased BCR-induced NF-κB DNA binding activity in T2 and mature Fo B cells relative to T1 cells. EMSA analysis for NF-κB DNA binding activity in FACS-sorted T1, T2, and mature Fo B (M) cells following stimulation with anti-IgM F(ab′)₂ or 5 mg/ml LPS (as a positive control) or left nonstimulated. Equal amounts (2.0 μg) of nuclear extracts per lane were used in each DNA binding reaction. The NF-Y band was used as loading control. These experiments are representative of three independent experiments using five to ten mice as a source of B cells for FACS sorting. E, Fold change for total NF-κB activity from D normalized to NF-Y and calibrated to the nonstimulated sample. F, Cytoplasmic extracts were prepared from equivalent number of cells (1 × 10⁶) and analyzed by immunoblotting for A1, Bcl-2, Bcl-x_L, and Mcl-1. Blot was stripped and probed for A1, Bcl-2, Bcl-x_L, Mcl-1, and anti-p38 sequentially as a loading control. Data are representative of at least three experiments.

BCR signaling induces sustained c-Rel expression in T2 and mature, but not in T1 B cells. A, Purification of T1, T2, and mature CD23⁺ B cell populations by FACS. Splenocytes from six to eight spleens from 7- to 12-wk-old Bcl-2 Tg mice were depleted of RBCs. T1 and T2 B cells were obtained by MACS enrichment of AA4.1⁺ transitional B cells, followed by FACS-sorting as described in Fig. 2A. B, Analysis of BCR-induced steady state levels of c-Rel mRNA in FACS-purified T1, T2, and mature B cells from Bcl-2 Tg mice. Cells were stimulated with 10 μg/ml anti-IgM F(ab′)₂ for 16 h and analyzed for c-Rel mRNA by qRT-PCR. Relative fold levels of c-Rel was normalized to GAPDH and calibrated to the nonstimulated sample for each population. *, p = 0.0279; **, p = 0.0043. Data are mean with SD calculated from five experiments using five to ten mice as a source of B cells for cell sorting. C, BCR signaling induces accumulation c-Rel in T2 and follicular mature B cells (CD23⁺) but not in T1 B cells. Immunoblot analysis of total cellular extracts from FACS-sorted T1, T2, and mature CD23⁺ B cells from Bcl-2 transgenic mice following stimulation with anti-IgM F(ab′)₂ for 16 h. Blot was probed sequentially with anti-cRel and –p38 Abs and fluorescently labeled secondary Abs. Data were collected and quantified by Odyssey. Data are representative of three experiments. D, FCM analysis of c-Rel expression in MACS enriched splenic B cells after 16 h culture with or without anti-IgM F(ab′)₂ (10 μg/ml) Abs. Cells were stained for B cell subset identification (T1, T2, and mature Fo B cells) as in A, fixed, permeabilized, and intracellularly labeled with Abs against c-Rel as described in Materials and Methods. c-Rel levels in nonstimulated and stimulated cells (open solid line histograms) and competition with unlabeled anti-c-Rel Abs (filled gray histograms) are shown for each splenic B cell subset. Data are displayed as mean fluorescence intensity (MFI). E, Fold change for c-Rel protein levels over nonstimulated samples from D and two additional experiments. *, p = 0.05.

T1 B cells display reduced ability to up-regulate BAFF-R and p100 than T2 and mature B cells in response to BCR signaling. A, BCR signaling induces BAFF-R expression in T2 and mature but not in T1 B cells. FACS-sorted B cell subsets (as in Fig. 2A) were stimulated with 10 μg/ml anti-IgM F(ab′)₂ for the 4 h. BAFF-R RNA levels were determined by qRT-PCR analysis. Relative fold induction was normalized to 18S and calibrated to the nonstimulated samples. B, FACS-sorted splenic B cell subsets (as in Fig. 2A) from Bcl-2 transgenic (Bcl-2-Tg) mice were stimulated with 10 μg/ml anti-IgM F(ab′)₂ for the indicated time. RNA levels of p100 were determined by qRT-PCR analysis. Relative fold induction was normalized to GAPDH and calibrated to the nonstimulated sample for each population. Data are mean with SD calculated based on five independent experiments using more than five mice in each experiment. *, p = 0.0016; **, p = 0.0188. C, FACS-sorted splenic B cell subsets from Bcl-2 Tg mice (as in Fig. 3A) were stimulated with 10 μg/ml anti-IgM for 16 h and analyzed as in B. *, p = 0.0234; **, p = 0.0086. Mean data with SD calculated from five independent experiments using more than five mice in each experiment. D, BCR signaling results in accumulation of p100 in CD23⁺ (T2 + M) B cells but not in T1 B cells. Immunoblot analysis of total cellular extracts from MACS-enriched T1 and T2 + M B cells from Bcl-2 transgenic mice, as described in Materials and Methods following stimulation with anti-IgM F(ab′)₂ for 36 h. Blots were probed sequentially with anti-p100 and anti-β-tubulin Abs. Data are representative of three experiments.

BCR Induction of BAFF-R and its substrate p100 requires c-Rel. A–C, Kinetic analysis of BAFF-R and p100 in WT and crel^−/− B cells. Naive splenic B cells from WT and c-Rel deficient mice were stimulated with anti-IgM F(ab′)₂ 10 μg/ml for the indicated times. RNA levels of BAFF-R, p100 were determined by qRT-PCR analysis. Relative fold induction was normalized to GAPDH and calibrated to the nonstimulated sample for each population. Mean data and SD from three experiments. C, Western blot analysis of total cellular extracts from WT and c-Rel deficient splenic B cells stimulated as in A for the indicated times. Blot was probed with anti-p100 and β-tubulin Abs. Data are representative of three experiments.

BCR-induced c-Rel synthesis is mediated via a Btk dependent pathway. A, Analysis of BCR-induced c-Rel mRNA expression in WT and Btk-deficient (btk^−/−) B cells. Cells stimulated with 10 μg/ml anti-IgM F(ab′)₂ for the indicated times and analyzed for c-Rel mRNA expression by qRT-PCR. Relative fold induction of c-Rel was normalized to GAPDH and calibrated to the nonstimulated samples. Mean data and SD from three experiments. B, Immunoblot analysis of cellular extracts from AA4.1⁺ live immature B cells from WT and btk^−/− mice following stimulation with 10 μg/ml anti-IgM F(ab′)₂ or 5 μg/ml anti-CD40 for 14 h. Blot was probed sequentially with anti-c-Rel and β-Actin. Represents three independent experiments. C, Btk-deficient B cells display reduced c-Rel DNA binding in response to BCR stimulation. EMSA analysis for NF-κB binding activity in WT and btk^−/− splenic B cells stimulated with 10 μg/ml anti-IgM F(ab′)₂ for 14 h. Equal amounts (1.0 μg) of nuclear extracts per lane were used in each DNA binding reaction. The c-Rel and p50 subunits in NF-κB complexes were identified by addition of subunit-specific Abs (supershift) as indicated. D, Btk is required for BCR-induced p100 expression. Immunoblot analysis of cellular extracts from WT and btk^−/− splenic B cells following stimulation with anti-IgM F(ab′)₂ for 18 h. Blot was probed sequentially with p100 and p38. p38 was used as loading control. Data represent three or more experiments using multiple mice in all the above panels.