BCL6 is critical for the development of a diverse primary B cell repertoire

Abstract

BCL6 protects germinal center (GC) B cells against DNA damage-induced apoptosis during somatic hypermutation and class-switch recombination. Although expression of BCL6 was not found in early IL-7-dependent B cell precursors, we report that IL-7Ralpha-Stat5 signaling negatively regulates BCL6. Upon productive VH-DJH gene rearrangement and expression of a mu heavy chain, however, activation of pre-B cell receptor signaling strongly induces BCL6 expression, whereas IL-7Ralpha-Stat5 signaling is attenuated. At the transition from IL-7-dependent to -independent stages of B cell development, BCL6 is activated, reaches expression levels resembling those in GC B cells, and protects pre-B cells from DNA damage-induced apoptosis during immunoglobulin (Ig) light chain gene recombination. In the absence of BCL6, DNA breaks during Ig light chain gene rearrangement lead to excessive up-regulation of Arf and p53. As a consequence, the pool of new bone marrow immature B cells is markedly reduced in size and clonal diversity. We conclude that negative regulation of Arf by BCL6 is required for pre-B cell self-renewal and the formation of a diverse polyclonal B cell repertoire.

Figure 1.

Regulation of BCL6 during inducible pre–B cell differentiation. (A) IL-7–dependent and BCR-ABL1–transformed pre–B cells were induced to differentiate by withdrawal of 10 ng/ml IL-7 and ABL1 kinase inhibition (2 µmol/liter STI571), respectively. Cell size (FSC) and κ light chain surface expression were monitored by flow cytometry (n = 5). Numbers indicate percentages. (B) To identify genes that are differentially regulated during induced pre–B cell differentiation, we studied pre–B cells stimulated to differentiate in a microarray analysis. Genes were sorted based on the ratio of gene expression values observed upon withdrawal of IL-7 from IL-7–dependent pre–B cells. (C) Likewise, protein lysates from pre–B cells in the presence or absence of induced differentiation (treatment with 10 µmol/liter STI571 or withdrawal of IL-7 for 24 h) were analyzed by Western blotting using antibodies against STAT5, phosphorylated STAT5 at Y694, BCL6 (clone N3), MYC, and an ACTB antibody as loading control (n = 6). The asterisk denotes a nonspecific band that is consistently observed with the N3 BCL6 antibody. Of note, BCR-ABL1 kinase signaling results in stronger STAT5 tyrosine phosphorylation at Y694 and detection of two phosphoproteins compared with IL-7–dependent STAT5 phosphorylation, where only one band is detected (see D). (D) To directly compare BCL6 protein expression levels in pre–B cells upon IL-7 withdrawal and DLBCL cells and tonsillar GC B cells, Western blot analyses were repeated with cell lysates from these cell populations (cell lysates from three IL-7 withdrawal experiments). FSC, forward scatter.

Figure 2.

The balance between MYC and BCL6 regulates Vκ-Jκ light chain gene recombination. (A) HPCs, pro–B cells (fractions B and C), large cycling pre–BII cells (fraction C’), small resting pre–BII cells (fraction D), and immature B cells (fraction E) were sorted from normal mouse bone marrow (n = 3) and subjected to quantitative RT-PCR to measure mRNA levels of BCL6 and MYC relative to Hprt. Mean values ± SD of three experiments are given. Numbers in the bar chart denote the ratios of BCL6 versus MYC mRNA levels. (B) Recruitment of BCL6 to the MYC promoter was identified by ChIP-on-chip analysis and confirmed by single locus QChIP analysis. ChIP-on-chip analysis was performed for three BCR-ABL1–transformed pre–B cell lines (each two replicates) under control conditions or after treatment with 10 µmol/liter STI571 for 24 h. Two replicate experiments for one BCR-ABL1–transformed pre–B ALL cell line are shown. (C and D) IL-7–dependent (C) and BCR-ABL1–transformed (D) pre–B cells were induced to differentiate by IL-7 withdrawal or inhibition of BCR-ABL1 kinase activity. To test the function of MYC and BCL6 during induced pre–B cell differentiation, pre–B cells were transduced with retroviral vectors encoding BCL6-IRES-GFP, MYC-IRES-GFP, or an IRES-GFP empty vector control. Percentages of κ light chain⁺ GFP⁺ cells are indicated (mean values of three experiments ± SD). (E) IL-7–dependent bone marrow pre–B cells from MYC^fl/fl and MYC^fl/fl × mx-Cre mice were transduced with retroviral vectors encoding BCL6-IRES-GFP or an IRES-GFP (empty vector control) and cultured in the presence of 10 ng/ml IL-7. 2,500 U/ml IFN-β was added to all cultures to induce expression of Cre and MYC deletion in Mx-Cre × MYC^fl/fl but not MYC^fl/fl pre–B cells (mean values ± SD; n = 3).

Figure 3.

Pre–B cell receptor activation induces expression of BCL6 via down-regulation of IL-7 responsiveness. IL-7–dependent pre–B cells were transduced with a retroviral vector encoding a constitutively active STAT5 mutant (STAT5-CA-GFP; Onishi et al., 1998) or an empty vector control (GFP). (A) GFP-expressing transduced cells were sorted, subjected to IL-7 withdrawal, and analyzed by Western blotting for expression of BCL6 and tyrosine phosphorylation of STAT5 using β-actin as a loading control (n = 3). (B) Mouse B cell precursors were isolated by B220⁺ MACS from freshly harvested bone marrow cells. Freshly isolated bone marrow B cell precursors and IL-7–dependent pre–B cell cultures were treated with 10 µg/ml of a neutralizing anti–IL-7 antibody overnight and were subjected to Western blot analysis (three experiments were performed). (C) Bone marrow B cell precursors from RAG2^−/− tTA/μ chain–transgenic mice are unable to express an endogenous μ chain but carry a functionally prerearranged μ chain under control of tetO sequences (Hess et al., 2001). These mice express a tTA under control of endogenous μ chain regulatory elements, and withdrawal of tetracycline results in activation of μ chain expression (routinely performed quality control). The effect of tetracycline-inducible activation of μ chain expression on BCL6 expression and STAT5 tyrosine phosphorylation was determined by Western blotting (n = 3; right). (D) IL-7–dependent pre–B cells lacking the pre–B cell receptor–related linker molecule SLP65 were transduced with retroviral expression vectors encoding either SLP65-GFP or GFP alone. Surface expression levels of IL-7Rα chain were measured by flow cytometry (the experiment was performed twice). The histogram shows the IL-7Rα levels in transduced GFP⁺ (green) and untransduced (gray) cells.

Figure 4.

Normal polyclonal B lymphopoiesis requires BCL6 survival signaling in late pre–B cells. Bone marrow mononuclear cells from BCL6^+/+ and BCL6^−/− mice were analyzed by flow cytometry using the indicated antibody combinations (numbers indicated percentages). (A) Bone marrow cells were analyzed using a FSC/SSC gate together with propidium iodide exclusion (viable lymphocytes). (B) Cells were analyzed using a B220⁺ gate. Four mice were studied in each group, and a detailed statistical analysis including absolute cell numbers is presented in Table S1. (C) To examine the clonal composition of κ⁺ immature B cells (fraction E), B220⁺ IgD⁻ κ⁺ cells were sorted and analyzed by Ig spectratyping. Spectratyping analysis separates Ig gene rearrangements based on the length of their V_H-DJ_H junction, and the height (fluorescence intensity) of each size peak indicates its relative representation within the B cell population analyzed. Individual size peaks are typically separated by 3 bp, reflecting the reading frame in functional V_H-DJ_H gene rearrangements. In total, four pairs of BCL6^+/+ and BCL6^−/− bone marrow–derived immature B cell samples were analyzed by spectratyping. FSC, forward scatter; SSC, side scatter.

Figure 5.

BCL6 is required for the development of a diverse primary B cell repertoire. (A) For two pairs of BCL6^+/+ and BCL6^−/− mice (I and II), in addition to bone marrow samples spleen tissue was also available. Immature B cells (fraction E) from bone marrow and mature B cells from spleens (fraction F) were sorted and subjected to spectratyping analysis to study length diversity of V_H-DJ_H junctions. In two BCL6^−/− mice, clonal expansions were identified that occurred both in the bone marrow and in the spleen with the same peak size, i.e., the same length of the V_H-DJ_H junction. Two such clonal expansions were found in BCL6^−/− mouse I (I.1 and I.2) and one expansion was found in BCL6^−/− mouse II (II.1). (B) To test whether these expansions with the same length of the V_H-DJ_H junction indeed belong to one clone, we performed a detailed sequence analysis of these V_H-DJ_H junctions. Alignments of V_H, D, and J_H gene segments are shown as well as N/P nucleotides in the junctional regions. Internal heptamers are highlighted in red, and sites at which the junctional homology with the parental V_H-DJ_H rearrangement ends are indicated by arrows.

Figure 6.

BCL6 promotes pre–B cell survival by negative regulation of ARF. (A) BCR-ABL1–transformed BCL6^+/+ and BCL6^−/− pre–B cells were induced to differentiate (10 µmol/liter STI571) and studied by microarray analysis. Genes were sorted based on the ratio of gene expression values in STI571-treated BCL6^+/+ compared with BCL6^−/− pre–B cells. (B) Differences in mRNA levels of checkpoint genes (ARF, p21, and p27) were verified by quantitative RT-PCR using HPRT as a reference (mean values ± SD; n = 3). (C) Recruitment of BCL6 to the genomic region of the CDKN2A locus (ARF) was verified by ChIP-on-chip analysis and confirmed by single locus QChIP analysis (n = 3). BCL6–DNA complexes were immunoprecipitated using an anti-BCL6 polyclonal antibody. ChIP products and their respective input genomic fragments were amplified by ligation-mediated PCR. QChIP was performed again at this stage for selected positive control loci to verify that the enrichment ratios were retained (not depicted). The genomic products of two biological ChIP replicates were labeled with Cy5 (for ChIP products) and Cy3 (for input) and cohybridized on genomic tiling arrays including the genomic region of the CDKN2A locus (Chr9:21,950629–22,003329). After hybridization, the relative enrichment for each probe was calculated as the signal ratio of ChIP to input. Peaks of enrichment for BCL6 relative to input were uploaded as custom tracks into the University of California, Santa Cruz genome browser and graphically represented as histograms. Peaks involving five or more oligonucleotide probes above threshold (2.5-fold SD above average enrichment) were considered positive. (D) To determine whether recruitment of BCL6 to the Arf (CDKN2A) promoter affects expression levels of proteins in the Arf–p53 pathway, BCR-ABL1–transformed BCL6^+/+ and BCL6^−/− pre–B cells were analyzed by Western blotting. (E) BCR-ABL1–transformed pre–B cells from ARF^+/+ and ARF^−/− mice were induced to differentiate (+STI571) or cultured under control conditions in the presence or absence of 5 µmol/liter of a peptidomimetic BCL6 inhibitor (RI-BPI; Cerchietti et al., 2009). Cell size (FSC) and viability (propidium iodide uptake) were measured by flow cytometry after 1 and 3 d (n = 3). Percentages of viable cells are indicated for each condition. FSC, forward scatter; PI, propidium iodide.