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. 2013 Jun 1;190(11):5559-66.
doi: 10.4049/jimmunol.1102503. Epub 2013 Apr 29.

Regulation of VH replacement by B cell receptor-mediated signaling in human immature B cells

Jing Liu  1 , Miles D Lange, Sang Yong Hong, Wanqin Xie, Kerui Xu, Lin Huang, Yangsheng Yu, Götz R A Ehrhardt, Michael Zemlin, Peter D Burrows, Kaihong Su, Robert H Carter, Zhixin Zhang
Affiliations

Regulation of VH replacement by B cell receptor-mediated signaling in human immature B cells

Jing Liu et al. J Immunol. .

Abstract

VH replacement provides a unique RAG-mediated recombination mechanism to edit nonfunctional IgH genes or IgH genes encoding self-reactive BCRs and contributes to the diversification of Ab repertoire in the mouse and human. Currently, it is not clear how VH replacement is regulated during early B lineage cell development. In this article, we show that cross-linking BCRs induces VH replacement in human EU12 μHC(+) cells and in the newly emigrated immature B cells purified from peripheral blood of healthy donors or tonsillar samples. BCR signaling-induced VH replacement is dependent on the activation of Syk and Src kinases but is inhibited by CD19 costimulation, presumably through activation of the PI3K pathway. These results show that VH replacement is regulated by BCR-mediated signaling in human immature B cells, which can be modulated by physiological and pharmacological treatments.

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Figures

Figure 1
Figure 1. The EU12 μHC+ cells are sensitive to BCR crosslinking
A) Crosslinking BCR induces Ca2+ influx in EU12 μHC+ cells. EU12 μHC+ cells were preloaded with Fluo3 dye and analyzed by FACS. F(ab')2 goat anti-human μHC antibodies (2 or 10 μg/ml) were added at the time point indicated by red arrows. Data was collected for 4 min. B, C) FACS analyses of the cell surface CD34 and μHC expression on the EU12 parental cells (B) or the μHC+ cells (C) after overnight treatment with F(ab')2 goat anti-human μHC antibodies (2 μg/ml). Numbers indicate the percentage of cells in each quadrant. D) FACS analyses of the cell surface μHC expression on the EU12 μHC+ cells after treatment with F(ab')2 goat anti-human μHC antibodies (2 μg/ml) for 0 min, 1 min, 5 min, 30 min, 1 h, or 4 h. Red line indicates the peak fluorescent intensity of μHC expression before treatment. E) Viable cell count of EU12 μHC+ cells after treatment with F(ab')2 goat anti-human μHC antibodies (2 μg/ml) for 1, 2, and 3 days. Results shown are means with standard deviation from triplicate experiments. F) Cell cycle analysis of EU12 μHC+ cells after treatment with F(ab')2 goat anti-human μHC antibodies (2 μg/ml) for 1 and 2 days. The percentage of hypodiploid cells, indicative of apoptosis, is indicated.
Figure 2
Figure 2. Crosslinking BCR induces VH replacement in the EU12 μHC+ cells
A) Diagram of the VH replacement process in EU12 cells. Double-stranded DNA breaks (DSBs) at the cRSS sites and excision circles were used as markers to analyze VH replacement. B) LM-PCR detection of RAG-mediated DSBs at the VH3 cRSS sites after treatment with or without F(ab')2 goat anti-human μHC antibodies (2 μg/ml). Serially diluted genomic DNA samples (1:5 dilution) were used as templates in the semi-quantative LM-PCR analyses. The CD19 promoter region was amplified by PCR to monitor the DNA input. C) Nested primer PCR detection of VH1 to VH3 excision circles. The identity of the excision circle PCR products was confirmed by DNA sequencing. D) Diagram showing the real time LM-PCR approach. The brown circle indicates the streptavidin magnetic beads. E) Real-time LM-PCR detection of the enrichment of DSBs at the VH1 and VH3 cRSS sites after ligation with the biotin labeled dsDNA linkers followed by purification with streptavidin magnetic beads. The + or − indicates with or without T4 DNA ligase in the ligation reaction, respectively. F) Real-time LM-PCR detection of the DSBs at the VH1 and VH3 cRSS sites in EU12 μHC+ cells with or without F(ab')2 goat anti-human μHC antibodies (2 μg/ml) stimulation. Results shown are mean values from triplicate reactions. The experiments were repeated twice.
Figure 3
Figure 3. VH replacement occurs in newly emigrated human immature B cells from peripheral blood and is induced by BCR stimulation
A) Identification and purification of newly emigrated immature B cells (i, IgM+CD27− CD10+) and mature naïve B cells (m, IgM+CD27−CD10−) from peripheral blood of healthy donors. B) Detection of double-stranded DNA breaks at the VH3 cRSS sites by LM-PCR in the newly emigrated immature B cells (i) but not in the mature naïve B cells (m) from 11 healthy donors (D1 to D11). The GAPDH or CD19 genomic DNA was amplified by PCR to monitor the DNA input. C) Crosslinking BCR induces VH replacement in the newly emigrated immature B cells (i) but not in the mature naïve B cells (m) from 5 healthy donors (D3-D5, D9, D10). The GAPDH or ACTB genomic DNA was amplified by PCR to monitor the DNA input. D) Sequence analysis of the LM-PCR products obtained from 6 healthy donors confirms that the DSBs occurred at the cRSS sites from different VH3 genes. Different donors, VH genes, cRSS, Linker, and numbers of sequences are indicated. The cRSS heptamer is highlighted in a red box.
Figure 4
Figure 4. BCR crosslinking induces VH replacement in tonsillar immature B cells
A) Purification of tonsillar immature B cells (i, CD24hiIgM+) and mature B cells (m, CD24+IgM+) by FACS. B) LM-PCR detection of DSBs at VH3 cRSS sites in tonsillar immature B cells (i) or mature B cells (m) with or without BCR crosslinking. Results shown are from three tonsillar samples. The GAPDH genomic DNA was amplified by PCR to monitor the DNA input. C) Sequence analysis of the LM-PCR products confirms that the DSBs occurred at different VH3 cRSS sites. The cRSS heptamer is highlighted with a red box. The linker primer sequence is indicated by the arrow.
Figure 5
Figure 5. BCR signaling-induced VH replacement depends on Syk and Src kinase activation
A) Western blot analyses of BCR signaling events upon treatment with different inhibitors. EU12 μHC+ cells (107 cells/ml) were pretreated with medium alone, DMSO, Genistein (50 μg/ml), Syk II (5 μM) or Syk III (5 μM), or PP1 (5 μM) for 30 min and stimulated with F(ab')2 goat anti-human μHC (2 μg/ml) for 3 min. Cell lysate was analyzed by Western blot using antibodies specific for the indicated BCR signaling components. Antibodies to β-ACTIN were used to control sample loading. B) LM-PCR detection of DSBs at the VH3 cRSS sites in EU12 μHC+ cells after treatment with different inhibitors, with or without BCR stimulation. The CD19 promoter region was amplified to monitor DNA input. C) PCR detection of VH1→VH3 excision circles in EU12 μHC+ cells after treatment with different inhibitors, with or without BCR stimulation. The CD19 promoter region was amplified to monitor DNA input. The results shown are representatives from more than three independent experiments.
Figure 6
Figure 6. BCR signaling-induced VH replacement is negatively regulated by CD19 costimulation
(A, C) Western blot analyses of BCR signaling events in EU12 μHC+ cells upon treatment with monoclonal anti-CD19 antibodies or PI3 kinase inhibitor LY294002. The level of β-ACTIN in each sample was monitored to control sample loading. (B, D) LM-PCR detection of DSBs at the VH3 cRSS sites in EU12 μHC+ cells after the indicated treatment. The CD19 promoter region was amplified to monitor DNA input. Exp 1, 2, and 3 indicate results from three independent experiments.
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