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. Author manuscript; available in PMC: 2014 Jun 1.
Published in final edited form as: J Immunol. 2013 Apr 29;190(11):5559–5566. doi: 10.4049/jimmunol.1102503

Regulation of VH Replacement by B Cell Receptor (BCR)-mediated Signaling in Human Immature B Cells

Jing Liu *,, 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 *,‡,**
PMCID: PMC3660396  NIHMSID: NIHMS464732  PMID: 23630348

Abstract

VH replacement provides a unique RAG-mediated recombination mechanism to edit non-functional IgH genes or IgH genes encoding self reactive B cell receptors (BCRs) and contributes to the diversification of antibody repertoire in mouse and human. Currently, it is not clear how VH replacement is regulated during early B lineage cell development. Here we show that crosslinking 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 co-stimulation, presumably through activation of the PI3 kinase pathway. These results show for the first time that VH replacement is regulated by BCR-mediated signaling in human immature B cells, which can be modulated by physiological and pharmacological treatments.

Introduction

The variable region exons of immunoglobulin (Ig) genes are assembled in developing B lineage cells by recombination activating gene products (RAG1 and RAG2) mediated recombination to join previously separate variable (V), diversity (D) (for heavy chain only), and joining (J) gene segments1–4. The specific rearrangement of different V, D, J gene segments is directed by the recombination signal sequences (RSS) flanking each rearranging gene segment3. This random V(D)J recombination process is essential for the generation of a highly diversified antibody repertoire, however, it also produces a large number of non-functional Ig gene rearrangements or Ig genes encoding autoreactive antibodies5–7. These non-functional or self reactive Ig rearrangements must be changed through RAG-mediated secondary recombination, a process known as receptor editing. Otherwise, B cells carrying defective Ig genes cannot development further along the B lineage pathway and B cells expressing autoreactive BCRs will be eliminated by clonal deletion or silenced by anergy6–9.

Most of the previous works on receptor editing focused on the Ig light chain genes6,7. The organizations of the Ig κ and λ gene loci allow continuous editing by joining any upstream Vκ or Vλ gene with a downstream Jκ or Jλ gene, respectively, until there are no available VL or JL genes or the recombination machinery is inactivated10,11. Through the analysis of an engineered mouse with one Cκ allele marked by the human Cκ region, it has been estimated that about 25% of peripheral B cells have edited their Igκ genes12. Upon BCR stimulation in vitro, up to 70% of murine immature B cells altered their light chain genes13, indicating that light chain editing is regulated by BCR signaling. Moreover, it has been shown that light chain gene editing is confined to the bone marrow early immature B cells, followed by a strict negative selection step14.

Accumulating studies have shown that IgH genes can also be edited by a unique VH replacement process15–17. VH replacement occurs through RAG-mediated secondary recombination between a cryptic recombination signal sequence (cRSS) embedded within the framework 3 region of a previously rearranged VH gene and the 23-bp RSS of an upstream germline VH gene18. VH replacement was first observed in murine pre B leukemia cell lines that initially harbored non-functional IgH rearrangements but later generated functional IgH genes and express μ heavy chains15,16. Later studies using various knock-in mouse models demonstrated that VH replacement is employed to edit IgH genes encoding anti-DNA antibodies19–21; change the knocked-in IgH rearrangement encoding monoclonal anti-NP antibodies and diversify the antibody repertoire22; and rescue B cells carrying two non-functional IgH alleles to generate an almost normal B cell compartment23,24. Potential VH replacement products can be identified from mouse IgH gene sequences through searching for the pentameric VH replacement footprints within the VH-DH junctions25,26, indicating that VH replacement products contribute to the diversification of the mouse antibody repertoire. Analyses of RAG-mediated double stranded DNA breaks (DSBs) corresponding to the VH cRSS sites in early murine B lineage cells indicated that VH replacement occurred at the pro B cell stage and less frequently in the immature B cells27,28.

In human early B lineage cells, ongoing VH replacement was documented in a differentiating human B lineage leukemic cell line, EU12, and in human bone marrow immature B cells18. Purified RAG1 and RAG2 core proteins efficiently bind to and cleave DNA substrates containing the cRSS motifs derived from different families of human VH genes, confirming that VH replacement is a RAG-mediated recombination process18. Moreover, by searching for the VH replacement footprints within the VH-DH junctions, it was estimated that VH replacement products contribute to about 5% of the human primary antibody repertoire in healthy donors17,18. Recent studies showed that the frequencies of VH replacement products are dramatically increased in IgH genes derived from HIV patients or IgH genes encoding anti-HIV antibodies29, suggesting the abnormal regulation of VH replacement during chronic HIV infection.

Currently, it is not known how VH replacement is regulated. The efficient replacement of the knocked-in 3H9 IgH transgene encoding anti-DNA antibodies suggested that VH replacement occurs upon antigen encounter19,22. The ongoing VH replacement in human bone marrow immature B cells also led to the speculation that VH replacement is regulated by BCR-mediated signaling18. In this study, we investigate how VH replacement is regulated by BCR-mediated signaling in human immature B cells.

Materials and Methods

Cell culture and reagents

Human B lineage EU12 cells were maintained as described previously18,30. EU12 μHC+ cells were purified from the parental EU12 cell culture by FACS sorting and maintained separately. For EU12 or EU12 μHC+ cells, cell surface μHC, Igλ and CD34 expression were monitored routinely by FACS analysis. The detailed information of all the antibodies used in this study is included in Supplementary Table 1. Genistein and PI3 Kinase inhibitor LY294002 were purchased from Sigma-Aldrich; Syk inhibitor II (2-(2-Aminoethylamino)-4-(3-trifluoromethylanilino)-pyrimidine-5-carboxamide, dihydrochloride, dihydrate; 574712) and Syk inhibitor III (3,4-methylenedioxy-β-nitrostyrene; 574713) were obtained from Calbiochem; Src kinase inhibitor PP1 was obtained from Biomol International.

Ca++ influx assay

Ca++ influx was measured by FACS analysis using the Fluo-3AM fluorescence dye according to the manufacturer's protocol. Briefly, EU12 μHC+ cells were washed with RPMI medium containing 1% FBS and pre-loaded with Fluo-3AM (1 μM)/Pluronic® F-127 at 1:1 ratio (Molecular Probes, Invitrogen) at 106 cells/ml for 30 min at 37°C in the dark. Different amounts of the F(ab')2 goat anti-human μHC fragments (2, 5, or 10 μg/ml) were added into each sample tube at 30 sec after starting analyzing the samples on a C6 Cytometer. Data was collected continuously for up to 4 min and analyzed using the CFlow Plus software package (BD, Accuri Cytometers Inc., MI).

Cell cycle and viability analysis

For cell cycle analysis, EU12 μHC+ cells (1×106 cells/ml) were stimulated with or without F(ab')2 goat anti-human μHC at different time intervals. Cells were fixed in 1 ml of ice-cold 70% ethanol overnight at 4°C. After washing with cold PBS and treatment with RNase (0.5 μg/ml) for 30 min at 37°C, cells were incubated with propidium iodide (PI, 20 μg/ml) for 30 min at 37°C in the dark. DNA content was analyzed on a FACScalibur. Results were analyzed using the WinMDI2.8 software (Stanford University, CA). For cell viability analysis after treatment with different kinase inhibitors, cells were stained with PI (2 μg/ml) and analyzed by FACS. PI+ cells were considered dead cells.

LM-PCR detection of RAG-mediated DSBs at VH cRSS sites

To directly analyze the ongoing VH replacement, LM-PCR was performed as described previously18. For analysis of VH replacement in EU12 cells, EU12 μHC+ cells (1×106 cells/ml) were treated for 24 hours with or without F(ab')2 goat anti-human μHC fragments (2 μg/ml). Genomic DNA was isolated using standard proteinase K digestion and phenol/chloroform extraction protocol31. For LM-PCR, 1 μg of DNA was ligated with 20 pmol of re-annealed double-stranded (DS) DNA linkers at 14°C overnight. The ligation reaction was terminated by the addition of an equal volume of stopping solution and denatured at 95°C for 15 min. For semi-quantative analysis of RAG-mediated double stranded DNA breaks (DSBs) at the VH3 cRSS sites, serial 5 fold dilutions of ligation samples were used as templates in two rounds (total 60 cycles) of semi quantitative LM-PCR with nested primer sets as previously described18. Second round PCR products (10 μl) were separated on 2% agarose gel and visualized under UV light after ethidium bromide (EtBr) staining. PCR products between 250–270 bp (from 6 donors) were subcloned into pCRII vectors and sequenced. The successful rate of LMPCR, treatment, cloning and sequencing results in all the studies are summarized in Supplementary Table 2.

For quantative analysis of the relative levels of VH replacement, real-time LMPCR was performed with a slightly modified protocol as described previously 32. Briefly, DS-DNA linkers were labeled with biotin-dCTP through a Klenow enzyme fill-in reaction. Purified genomic DNA from control or treated EU12 μHC+ cells was ligated to biotin labeled DS-DNA linkers overnight at 14°C and a mock ligation reaction without T4 ligase was set up as a negative control. The ligation samples were digested with BamHI/HindIII (5U each) at 37°C for 2 h. Biotin-tagged DNA was enriched using Streptavidin conjugated magnetic beads (Promega). After washing twice with 1 ml of TE buffer (pH 8.0), the enriched DNA samples were eluted by incubation at 75°C for 15 min and subjected to real time PCR analyses using the VH1n or VH3n sense primers and the LinkcRSS2 primers (Supplementary Table 1). Real time PCR was performed on an ABI PRISM® 7900HT Sequence Detection System using the SYBR GREEN PCR Master Mix (Applied Biosystems). The results obtained for DSBs at cRSS sites were normalized to GAPDH or ACTB genomic DNA level in each sample.

Detection of VH replacement excision circles

VH replacement excision circle was analyzed by PCR as previously described 18. Briefly, cellular DNA was extracted from control or treated EU12 μHC+ cells (1×106 cells). For kinase inhibitor treatment, cells were pre-treated with different inhibitors (1 μM) for 1 hours followed by 24 hours BCR stimulation. Cell viability was monitored by FACS analysis using PI staining. One tenth of the cellular DNA samples were analyzed by two rounds of semi-nested PCR amplification to detect VH replacement excision circles. The primer sequences are listed in Supplementary Table 1. The second round PCR products (10 μl) were separated on 2% agarose gel electrophoresis and visualized under UV light with EtBr staining.

RT-PCR analysis of RAG1 and RAG2 gene expression

Total RNA was purified from control or anti-IgM antibody treated EU12 μHC+ cells or purified primary immature or mature naïve B cells from healthy donors using Trizol according to the manufacturer's protocol. To specifically detect RAG1 and RAG2 cDNA but not genomic DNA, we used a modified approach for the first strand cDNA synthesis 33. Briefly, 0.5 μg of total RNA was used as template in reverse transcription reaction using the (dT)17-adapter oligonucleotide (Supplementary Table 1) and the high capacity cDNA reverse transcription kit (Applied Biosystems). The cDNA was then amplified in separate first-round PCR reactions using sense primers specific for RAG-1 (RAG1F1) or RAG-2 (RAG2F1) in conjunction with the antisense primer (adapter) hybridized with the adapter region of the (dT)17-adapter primer (Supplementary Table 1). The first-round PCR conditions were 94°C for 5 m, followed by 20 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 30 s, with no final extension at 72°C. The second-round PCR was performed using 2 μl of the first-round PCR product as template and a set of nested primers specific for RAG-1 (RAG1F1 and RAGR1), RAG-2 (RAG2F1 and RAG2R1). The PCR conditions were the same as those used in the first-round PCR with 10 cycles performed. ACTB was amplified using ACTB1 and ACTB2 primers for one-round of PCR under the following conditions: 94°C for 5 m, followed by 15 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 30 s, with no final extension at 72°C. PCR products were separated on 2% agarose gels and visualized under UV light after EtBr staining. The sequences of all the primers used in this study are listed in Supplementary Table 1.

Western blot analysis

Western blot analyses were performed to analyze the effects of different kinase inhibitors on BCR-mediated signaling events. Briefly, cells (10×106) were washed twice with cold PBS (Gibco) and cultured for 2 h in GIBCOTM Opti-MEM I reduced-serum medium (Invitrogen). Cells were pretreated with different inhibitors for 30 min and stimulated with F(ab')2 goat anti-human μHC antibody fragments (2 μg/ml) at different time intervals. For short time treatment, different inhibitors were used as Genistein (5 μM), Syk kinase Inhibitor Syk II (5 μM) or Syk III (5 μM), or Src kinase inhibitor PP1 (5 μM). For Western blot analyses, cells were harvested after 5 min of antibody stimulation, washed with cold PBS, and lysed with a lysis buffer containing 1% Nonidet P-40, 50 mM Tris.HCL (pH 7.5), 5 mM EDTA, 150 mM NaCl, with 1 tablet of protease inhibitor mix, PMSF (40 μM), and phosphatase inhibitor Na3VO4 (0.2 mM), Na2MoO4 (1 mM), and β-glycerol-phosphate (5 mM). Equal amounts of protein were resolved on 10% SDS/PAGE gels, transferred onto nitrocellulose membranes (Amersham Biosciences), and probed with indicated antibodies. After 4 times washing, bound antibodies were detected with HRP-conjugated secondary antibodies followed by ECL reagents. Results were analyzed using a FluorChemQ Multi Imager III system (Alpha Innotech).

Purification of primary immature B cells from human PBMC or tonsil samples

All protocols involving human samples have been approved by the Institutional Review Boards (IRB) at the University of Alabama at Birmingham (UAB) and at the University of Nebraska Medical Center (UNMC). Peripheral blood samples (10 to 20 ml) were obtained from healthy donors with informed consent. Mononuclear cells (PBMCs) were isolated using density gradient centrifugation with lymphocyte separation medium (Mediatech, Herndon, VA). Immature B cells (IgM+CD27−CD10+) or mature naïve B cells (IgM+CD27−CD10−) were purified by FACS sorting using a MoFlow cell sorter (Dako Cytomation, Fort Collins, CO) at the UAB FACS core facility or a BD AIRE II at the UNMC FACS core facility. The purity of isolated cells was monitored on a FACScalibur or a C6 plus FACS analyzer (BD Bioscience). For in vitro culture, purified human immature or mature naïve B cells (103 cells) were seeded into 96 well-plates with 100 μl medium with or without F(ab')2 goat anti-human μHC fragments (2 μg/ml). Cells were collected after 24 hr and genomic DNA was purified and subjected to LM-PCR analysis to detect ongoing VH3 cRSS DSBs. The successful rate for LM-PCR analysis of cRSS DSBs in primary immature B cells with or without antibody treatment was summarized in Supplementary Table 2.

Tonsillar lymphocytes were prepared by tissue mincing and filtration through a 70-mm wire mesh followed by density gradient centrifugation. Tonsillar immature B cells (IgM+CD24hi) cells and mature B cells (IgM+CD24Med) were purified using a MoFlow cell Sorter at the UAB FACS core facility. The purity of sorted cells was monitored by analysis on a FACS Calibur (BD Bioscience). Purified tonsillar immature or naïve mature B cells (1000 cells) were seeded into 96 well plates with 100 μl medium with or without F(ab')2 goat anti-human μHC antibody fragments (2 μg/ml) and incubated at 37°C for 24 h. Genomic DNA was prepared and subjected to LM-PCR analysis to detect VH3 cRSS DSBs.

Results

Crosslinking BCRs on EU12 μHC+ cells results in BCR internalization and growth arrest

Our previous studies have shown that VH replacement occurs spontaneously in human EU12 cells under normal cell culture conditions 18. The μHC+ subpopulation in the EU12 cell culture is phenotypically similar to human bone marrow immature B cells (IgM+CD10+CD24high) (Supplementary Figure 1A). As an initial assessment of the BCR signaling capacity in EU12 μHC+ cells, crosslinking BCRs with different concentrations of F(ab')2 goat anti-human μHC antibody fragments quickly induced Ca++ influx (Figure 1A). We have observed that after overnight culturing the EU12 parental cells with F(ab')2 goat anti-human μHC fragments (2 μg/ml), the μHC+ subpopulation disappeared (Figure 1B, top panel). The failure to detect cell surface BCRs was not due to anti-μHC antibodies masking the epitopes, because the anti-Igλ antibodies also failed to detect cell surface BCRs (Figure 1B, bottom panel). After purification of the EU12 μHC+ cells and culturing them overnight with the same amount of F(ab')2 goat anti-human μHC antibodies (2 μg/ml), the majority of the cells remained viable, but they lost cell surface μHC and Igλ expression (Figure 1C). Further analyses showed that treatment of EU12 μHC+ cells with anti-IgM antibodies quickly induced internalization of BCRs, which could be seen as soon as 1 min after the addition of the crosslinking antibodies. After 30 min of treatment, almost all of the EU12 μHC+ cells became negative for cell surface BCRs (Figure 1D). The complete internalization of BCR upon anti-IgM antibody treatment observed with the EU12 μHC+ cells is totally different from the responses observed with other human B cell lines representing mature B cells, such as Daudi or Ramos, in which only a fraction of the BCRs are internalized under the same conditions (Supplementary Figure 1B). Moreover, crosslinking of BCRs on the EU12 μHC+ cells inhibits cell proliferation, especially after 2 days of treatment (Figure 1E). Cell cycle analyses showed that BCR crosslinking resulted in reduced number of cells at the G2/M phase and increased apoptosis (Figure 1F). These results demonstrate that BCRs on EU12 μHC+ cells are functional to transduce signals and the EU12 μHC+ cells are highly sensitive to BCR crosslinking.

Figure 1. The EU12 μHC+ cells are sensitive to BCR crosslinking.

Figure 1

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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.

BCR crosslinking induces VH replacement in the EU12 μHC+ cells

We next performed semi-quantative ligation mediated (LM)-PCR to determine if crosslinking BCR affects VH replacement in the EU12 μHC+ cells. Treatment with the F(ab')2 antiμHC antibodies resulted in an elevated level of LM-PCR products corresponding to the DSBs at the VH3 cRSS sites, indicating the induction of VH replacement (Figure 2, A and B). Previous studies showed that the majority of EU12 μHC+ cells expresses rearranged VH3-7DH3-10JH4 genes30. Analyses of the sequences of the LM-PCR products confirmed that the DSBs occurred at the VH3-7 cRSS sites (data not shown), indicating that the BCR signaling induced VH replacement targets the functionally expressed IgH genes in these cells. Crosslinking BCRs on the EU12 μHC+ cells also resulted in an accumulation of the VH1 to VH3 VH replacement excision circles, which can be detected by PCR reactions (Figure 2C). To quantitatively determine the relative levels of VH replacement, we developed a real time LM-PCR approach to analyze the DSBs at the VH cRSS borders (Figure 2D). Ligation of genomic DNA samples with biotin-labeled DS DNA linkers followed by Streptavidin magnetic bead purification resulted in a 70 fold enrichment of the VH1 cRSS DSBs and nearly 30 fold enrichment of the VH3 cRSS DSB LM-PCR signals over that observed with DNA samples without ligation reaction (Figure 2E). As measured by realtime LM-PCR, BCR stimulation resulted in a 14–15 fold induction of VH replacement in EU12 μHC+ cells (Figure 2F). Previous studies showed that the EU12 μHC+ cells express both RAG1 and RAG2 genes30. Crosslinking cell surface BCRs only slightly enhances the level of RAG1 gene expression, but does not affect RAG2 gene expression (Supplementary Figure 2A). Taken together, these results show that crosslinking cell surface BCRs induces VH replacement in the EU12 μHC+ cells.

Figure 2. Crosslinking BCR induces VH replacement in the EU12 μHC+ cells.

Figure 2

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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.

VH replacement occurs in the newly emigrated immature B cells in the peripheral blood and is enhanced by BCR crosslinking

We have shown previously that VH replacement occurs in human bone marrow immature B cells18. The newly emigrated immature B cells (IgM+CD27−CD10+) in the peripheral blood display similar features to their bone marrow counterparts34. To determine if VH replacement occurs in these cells, we purified newly emigrated immature B cells (IgM+CD27−CD10+) and naïve mature B cells (IgM+CD27−CD10−) from peripheral blood samples of 11 healthy donors (Figure 3A) and performed LM-PCR to detect DSBs at VH3 cRSS sites (Figure 3A). With two rounds of nested primer LM-PCR (total 60 cycles of PCR amplification), we were able to detect DSBs at VH3 cRSS borders in the newly emigrated immature B cells, but not in the naïve mature B cells from all 11 donors (Figure 3B and Supplementary Table 2). Moreover, treatment of purified immature or naïve mature B cells with F(ab')2 goat anti-human μHC fragments induces VH replacement in the newly emigrated immature B cells, but not in the naïve mature B cells (Figure 3C). The obtained LM-PCR products from 6 donors were cloned into pCRII vector and sequenced. Analysis of these sequences showed that the DSBs occurred at cRSS sites from different VH3 genes (Figure 3D). These results show for the first time that VH replacement occurs in the newly emigrated immature B cells from peripheral blood of healthy donors and can be further enhanced by BCR stimulation.

Figure 3. VH replacement occurs in newly emigrated human immature B cells from peripheral blood and is induced by BCR stimulation.

Figure 3

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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.

The relative levels of ongoing VH replacement in freshly isolated primary immature B cells were also analyzed by real-time LM-PCR. The level of real-time LM-PCR signal detected in the immature B cells is much higher than that in the mature naïve B cells in all the three donors, although the relative levels of LM-PCR signal at VH3 or VH1 cRSS sites are different from donor to donor (Supplementary Figure 2C).

RAG1 expression can be detected in purified immature B cells in 2 of the 4 analyzed samples and RAG1 expression is enhanced after anti-IgM antibody treatment in the immature B cells of all the 4 samples (Supplementary Figure 2B). RAG2 expression is relatively lower in the isolated immature B cells, which can be detected in the immature B cells of 2 donors without anti-IgM treatment and is up-regulated in the immature B cells of 3 donors after anti-IgM treatment (Supplementary Figure 2B). Compared to the RAG expression in immature B cells, there is almost no detectable RAG1 and RAG2 expression in the purified mature naïve B cells with or without anti-IgM antibody treatment in all the 4 donors (Supplementary Figure 2B). These results are consistent with the observed induction of VH replacement in the primary immature B cells.

Crosslinking BCR induces VH replacement in tonsillar immature B cells

The human tonsil contains a large percentage of B lineage cells. Among them a subset of CD24highIgM+ B cells represents the newly emigrant immature B cells34. Treatment of purified tonsillar immature B cells from different donors with F(ab')2 goat anti-human μHC fragments induces VH replacement, as indicated by the elevated levels of LM-PCR products corresponding to the DSBs at the VH3 cRSS sites (Figure 4, A and B). As negative controls, no LM-PCR product was detected in tonsillar mature B cells with or without anti-IgM antibody treatment. Sequence analyses of the LM-PCR products confirmed that the DSBs occurred at VH3-9, VH3-23, or VH3-64 cRSS sites (Figure 4C). These results demonstrate that BCR stimulation also induces VH replacement in tonsillar immature B cells.

Figure 4. BCR crosslinking induces VH replacement in tonsillar immature B cells.

Figure 4

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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.

BCR signaling mediated induction of VH replacement depends on the activation of Syk and Src kinases

Having evidence that BCR crosslinking induces VH replacement in the EU12 μHC+ cell and in purified human primary immature B cells, we continued to dissect the responsive BCR signaling events that induce VH replacement. It is well established that BCR signaling is transduced through a cascade of protein tyrosine phosphorylation events, which can be blocked by different protein kinase inhibitors at different steps35,36. Pretreatment of EU12 μHC+ cells with a general protein tyrosine kinase inhibitor, Genistein, prevents global cellular protein tyrosine phosphorylation induced by BCR stimulation (Figure 5A) and in particular, inhibits BCR signaling induced phosphorylation of ERK, AKT, and FOXO1 (Figure 5A, lanes 5 and 6). Genistein treatment inhibits BCR crosslinking induced VH replacement in the EU12 μHC+ cells, as indicated by the reduced levels of LM-PCR products corresponding to the VH3 cRSS DSBs and VH1 to VH3 replacement excision circles (Figure 5, A, B, and C, Lanes 5 and 6).

Figure 5. BCR signaling-induced VH replacement depends on Syk and Src kinase activation.

Figure 5

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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.

Upon BCR crosslinking, Syk and Src kinases are rapidly recruited to the BCR signaling complexes to activate downstream signaling events. Pretreatment of EU12 μHC+ cells with specific Syk kinase inhibitors, Syk II, inhibits BCR signaling induced ERK, AKT, and FOXO1 phosphorylation (Figure 5A, Lane 8) and completely blocks BCR stimulation induced VH replacement, as measured by the levels of VH3 cRSS DSBs by LM-PCR and VH1 to VH3 replacement excision circles (Figure 5, A, B, and C, Lane 8). Treatment of cells with Syk III blocks BCR singling induced protein tyrosine phosphorylation, but strongly enhanced ERK phosphorylation regardless of BCR crosslinking; it completely abolished BCR signaling induced VH replacement in the EU12 μHC+ cells (Figure 5, A, B, and C, Lanes 9 and 10). Treatment of cells with PP1 inhibits BCR signaling induced protein tyrosine phosphorylation, but with slightly enhancement of ERK phosphorylation regardless of BCR stimulation. PP1 treatment also inhibits anti-IgM antibody induced VH replacement, as measured by the levels of VH3 cRSS DSBs by LM-PCR and VH replacement excision circles (Figure 5, A, B, and 5C, Lanes 10 and 12). The inhibition of BCR signaling induced VH replacement by Genistein, Syk II, Syk III or PPI was not due to cytotoxicity of these inhibitors, because there were only about 10–20% dead cells after 24 hour treatment in most of the experiments (Supplementary Figure 1C). It should be pointed out that treatment with Genistein, Syk II, Syk III, or PP1 all inhibits the basal level of ongoing VH replacement in the EU12 μHC+ cells, suggesting that the spontaneous VH replacement in these cells is also regulated by BCR-mediated signaling. Taken together, these results indicate that BCR signaling induced activation of Syk and Src kinases is required for induction of VH replacement in EU12 μHC+ cells.

CD19 co-stimulation inhibits BCR signaling induced VH replacement

It has been well documented that BCR signaling can be modulated by different co-stimulatory or inhibitory receptors. For instance, CD19 is essential for B cell response to membrane bound antigens or antigen complexes; co-stimulation through CD19 amplifies BCR-mediated AKT phosphorylation37,38. Previous studies showed that light chain gene receptor editing was enhanced in Cd19−/− mice, suggesting that CD19 plays a negative regulatory role for receptor editing39,40. We therefore investigated the effect of CD19 costimulation on VH replacement in the EU12 μHC+ cells. Treatment of EU12 μHC+ cells with anti-CD19 antibodies (1 μg/ml) induced phosphorylation of ERK, AKT, and FOXO1 and slightly enhanced BCR signaling mediated AKT phosphorylation (Figure 6A). However, CD19 co-stimulation inhibits BCR signaling induced VH replacement, as measured by LM-PCR detection of DSBs at the VH3 cRSS borders (Figure 6B, Lanes 3 and 4). One of the major signaling events downstream of CD19 costimulation is activation of the PI3 kinase and AKT pathway. Pretreatment of EU12 μHC+ cells with the specific PI3 kinase inhibitor LY294002 inhibits AKT and FOXO1 phosphorylation (Figure 6C); Interestingly, LY294002 treatment augments BCR signaling induced V replacement in the EU12 μHC+ cells (Figure 6D, Lanes 3 and 4). These results demonstrate that CD19 co-stimulation negatively regulates BCR signaling induced V replacement in EU12 μHC+ cells, presumably through activation of the PI3K pathway.

Figure 6. BCR signaling-induced VH replacement is negatively regulated by CD19 costimulation.

Figure 6

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(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.

Discussion

Receptor editing provides an important mechanism to change unwanted Ig genes through RAG-mediated secondary recombination. It has been well documented that murine immature B cells have the capacity to re-express Rag genes and to edit their Ig light chain genes upon BCR stimulation13,41,42. Although light chain editing provides a powerful mechanism to neutralize autoreactivities contributed by the IgH chains, VH replacement provides a unique approach to delete non-functional or autoreactive IgH genes. Our previous studies showed that ongoing VH replacement occurs in human EU12 cells and in human bone marrow immature B cells18. In this study, we further investigated how VH replacement is regulated in human immature B cells.

Human leukemic EU12 cells undergo spontaneous differentiation from pro B cell to pre B cell, and then to immature B cell stages with ongoing VH replacement18,30 The μHC+ subpopulation of the EU12 cell culture has features similar to the human bone marrow immature B cells. Using the EU12 μHC+ cells as an experimental model system, we showed that crosslinking BCRs induced Ca++ influx, BCR internalization, inhibition of cell proliferation, and activation of SYK, ERK, and AKT kinases. Importantly, BCR stimulation resulted in a 14-fold induction of RAG-mediated DSBs at the VH3 cRSS borders and an accumulation of VH1 to VH3 replacement excision circles. These results show for the first time that VH replacement in human EU12 μHC+ cells is regulated by BCR-mediated signaling. Further analysis showed that the EU12 μHC+ cells express both RAG1 and RAG2 genes. However, there is no significant increase of RAG1 and RAG1 gene transcription in these cells upon BCR stimulation.

We have previously shown that VH replacement occurs in human bone marrow immature B cells18. The newly emigrated immature B cells in peripheral blood and human tonsil are phenotypically similar to the bone marrow immature B cells34. Using LM-PCR approaches, we were able to detect DSBs at the VH3 cRSS borders in the newly emigrated immature B cells purified from all the 11 analyzed healthy donors. Sequence analysis of the obtained LM-PCR products confirmed that the DSBs occurred at different VH3 genes. Moreover, stimulation of purified primary immature B cells from human peripheral blood or tonsillar samples with F(ab')2 goat anti-human μHC fragments further enhanced VH replacement. These results confirmed that VH replacement is regulated by BCR-mediated signaling in human primary immature B cells. RAG1 and RAG2 expression can be detected in purified immature B cells from healthy donors and their expression can be further enhanced with anti-IgM antibody treatment. Thus, the induction of RAG expression in primary immature B cells is correlated with the induction of VH replacement in these cells. Based on the results of BCR signaling mediated regulation of VH replacement and RAG expression in the EU12 μHC+ cells and in the primary immature B cells, we speculate that induction of RAG expression and VH replacement are regulated by separated mechanisms. Clearly, the RAG1 and RAG2 complexes are required for VH replacement recombination. However, expression of RAG1 and RAG2 may not be sufficient to initiate VH replacement in the EU12 μHC+ cells. Signaling from the BCR may also activate other factors to induce VH replacement.

BCR crosslinking induces a cascade of protein tyrosine phosphorylation events 36,43. Using different protein tyrosine kinase inhibitors, we further dissected the downstream BCR signaling events responsible for the induction of VH replacement in EU12 μHC+ cells. Upon BCR engagement, Syk and Src kinases are quickly recruited to the BCR signaling complexes and activated, which in turn initiate tyrosine phosphorylation of many cellular proteins44. Pretreatment of EU12 μHC+ cells with a general protein tyrosine kinase inhibitor, Genistein, blocked BCR signaling mediated global protein tyrosine phosphorylation; Genistein treatment marginally inhibited BCR signaling induced VH replacement. Pretreatment of the EU12 μHC+ cells with more potent and specific inhibitors Syk II and Syk III for Syk kinase or PP1 for Src kinase blocked BCR signaling induced protein tyrosine phosphorylation and completely blocked BCR stimulation induced VH replacement. These results indicated that BCR signaling induced Syk and Src kinases are required for induction of VH replacement. In comparison to Genistein, Syk II, Syk III, and PP1 are more potent inhibitors for Syk kinase, which completely inhibit BCR signaling induced VH replacement. Currently, several orally effective Syk kinase inhibitors are under clinical trials in treating different diseases. These Syk kinase inhibitors may offer specific approaches to prevent VH replacement.

CD19 is a co-stimulation receptor for BCR and is essential for B cell activation. However, lost of Cd19 enhances Igκ gene editing in murine immature B cells39,40,45, suggesting that Cd19 acts as a negative regulator for receptor editing. Here, our results showed that co-stimulation of EU12 μHC+ cells with anti-CD19 antibodies blocked BCR signaling induced VH replacement; conversely, pretreatment of EU12 μHC+ cells with LY294002 to inhibit PI3K and AKT activation enhanced BCR stimulation induced VH replacement. These results suggested that CD19 costimulation negatively regulated BCR stimulation induced VH replacement in human immature B cells through activation of the PI3K pathway. These results are consistent with the previous observation of intensive light chain editing in the immature B cells in Cd19−/− mouse. Previous studies have shown that elevated expression of FOXO1 induces Rag gene expression and induces Igκ gene recombination46. In our study, both BCR crosslinking and CD19 co-stimulation induced AKT and FOXO1 phosphorylation. However, these two treatments had totally opposite effects on the induction of VH replacement: co-stimulation with anti-CD19 antibodies inhibits BCR signaling induced VH replacement. Although co-stimulation with anti-CD19 antibody slightly enhances BCR-crosslinking induced AKT and FOXO1 phosphorylation. It is hard to believe that such changes will completely shut down the BCR signaling induced VH replacement. Further investigation is required to fully address how co-stimulation through CD19 inhibits BCR signaling induced VH replacement as well as light chain editing.

In summary, these results demonstrate that VH replacement is regulated by BCR-mediated signaling in human immature B cells, which can be further modulated by different pharmacological protein tyrosine kinase inhibitors or physiological costimulation through the cell surface CD19. Based on these results, we conclude that human immature B cells have the capacity to change unwanted IgH genes through VH replacement upon antigen encounter.

Supplementary Material

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2

Acknowledgments

The authors would like to thank Drs. Max D. Cooper, Harry Schroeder, Louis Justement, Hiromi Kubagawa, and John Kearney for scientific discussion and suggestions and Drs. Larry Gartland (UAB), Marion Spell (UAB), and Charles Kuszynski, Megan Michalak, and Victoria Smith (UNMC) for their help with cell sorting and FACS analysis.

This study is supported in part by NIH grants AR048592, AI073174, AI074948, and AI076475 to ZZ and AR059351 to KS.

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Supplementary Materials

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2
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