View larger version (41K): [in this window] [in a new window] [Download PPT slide]   Figure 2. Reduced AID activity in AID+/– B cells. (A) AID mRNA (graph, AID+/+ and AID+/–; n = 5) and protein (AID+/+, AID+/–, and AID–/–), as measured by real-time PCR and Western blotting. Error bars represent the mean ± SD. (B, left) IgG1 switching (pseudocolor plot) profiles in wild-type and AID+/– B cells activated ex vivo, as determined by flow cytometry. (right) Mean switching to IgG1, IgG3, and IgG2a. Values represent the mean ± SD (n = 5). Antibodies were B220-PercP-Cy5.5 and IgG1-PE. Dead cells were gated out using DAPI. (C) Flow cytometric measurement of cell proliferation and IgG1 switching on CFSE-labeled B cells activated for 96 h with LPS + IL-4. The graph represents percentages of 1 switching at each cell division (n = 2). Error bars represent the mean ± SD. (D) The percentage of intra-switch deletions as determined by Southern blotting on hybridomas generated from AID+/+ and AID+/– lymphocytes activated for 96 h in the presence of LPS + IL-4. Intact Sµ domains result in a 6.9-kb band, whereas smaller bands (stars) represent intra-switch deletions. (E) Analysis of AID gene expression by single-cell RT-PCR (137 bp) from sorted LPS + IL-4–activated B cells. (F) Sµ mutation frequency in AID+/+, AID+/–, and AID–/– activated B cells. Pie chart segments are proportional to the number of sequences carrying an equal number of mutations (indicated on the periphery). The total number of sequences is portrayed in the middle of each chart, and mutation frequency (calculated as the total mutations per total base pairs sequenced) and p-values are shown below the charts.

Reduced inter- and intra-switch recombination in AID^+/– cells could conceivably result from monoallelic expression of Aicda. Under this scenario, up to 50% of AID^+/– B cells would transcribe the neomycin-deleted allele, leading to a complete absence of AID expression and activity in 50% of cultured cells. To directly evaluate this idea, we monitored AID gene transcription by single-cell RT-PCR (23). By this assay, only the untargeted Aicda allele is amplified using PCR primers specific for AID exons 2 and 3, which are deleted by neomycin insertion in the targeted allele (3). We detected AID in 185 out of 192 single AID^+/– B cells assayed (Fig. 2 E), a result that clearly demonstrates Aicda transcription to be biallelic. We conclude that CSR defects in AID^+/– cultures result from a reduction in overall AID expression levels.

AID deamination leads to nucleotide substitutions at S domains (24, 25), primarily as a result of DNA replication over uracils and error-prone repair activity (6). To measure S mutation frequency, we cloned and sequenced the 5' end of Sµ from AID^+/– and wild-type lymphocytes stimulated with LPS and IL-4 for 96 h. Sequencing analysis showed a striking reduction in Sµ mutations in heterozygous cells (P < 0.0005; Fig. 2 F). The calculated mutation frequency in AID^+/– cells was in fact not significantly higher than the PCR error rate determined using AID^–/– controls (P = 0.2; Fig. 2 F). Collectively, our data reveal that the frequency of S mutation and recombination in stimulated B cells is determined, at least in part, by AID gene dosage.

Reduced AID activity in AID^+/– mice during the immune response
We next examined AID activity during the humoral immune response. AID^+/– and control BALB/c littermates were immunized in the front footpads by subcutaneous injection of 50 µg of chicken globulin (CGG) in the presence of complete adjuvant. CSR levels were then monitored in germinal centers (GCs) from axillary and brachial lymph nodes 7 d after immunization. In agreement with the B cell culture results, we found fewer IgG1 B220⁺CD95^high GC cells in AID^+/– mice relative to controls (P = 0.04; Fig. 3 A). Notably, the disparity in CSR levels between the two strains of mice was not as striking as that seen in ex vivo cultures. This might be explained by the combined facts that switched cells accumulate over time as a function of the cell cycle (Fig. 2 C), and that B cell recruitment to and proliferation in GCs is extensive and asynchronized (26).

View larger version (22K): [in this window] [in a new window] [Download PPT slide]   Figure 3. Reduced AID activity in AID+/– mice during the immune response. (A) Measurement of IgG1 levels in GCs from AID+/+ and AID+/– mice. Values represent the mean ± SD (n = 5). (B) Somatic hypermutation as determined by sequencing the JH4 intron from AID+/+, AID+/–, and AID–/– GC cells. (C) ELISA analysis of serum from five AID+/+ and AID+/– mice immunized with NP-CGG. IgG1 antibodies displaying low affinity for NP were captured in this assay using NP30-BSA, whereas high affinity antibodies were monitored with NP7-BSA, as described in Results and discussion. Horizontal bars represent the mean.

Diminished chromosome 12 breaks and Igh-cMyc translocation–bearing cells in AID^+/– mice
Plasmacytomagenesis in pristane-treated mice is a multistep process initiated by the deregulation of cMyc upon translocation to the Igh loci. Fully malignant monoclonal tumors are thought to arise from a polyclonal pool of premalignant B cells bearing cMyc translocations (27). We reasoned that delayed tumor development in AID^+/– mice might result from a reduction in the number of clones carrying cMyc translocations. To investigate this, Bcl-xL transgenic AID^+/+ and AID^+/– mice were injected with pristane, and the presence of translocation-bearing cells before the onset of malignancy (approximately day 45; Fig. 1 B) was determined by long-distance PCR using nested primers specific for Sµ, S2a, S, and cMyc intron 1 (Fig. 4 A, diagram). At days 25 and 35 after pristane treatment, canonical translocations were amplified from DNA samples representing 5 x 10⁵ mononuclear cells isolated from Peyer's patches, mesenteric lymph nodes, or oil granuloma. DNA from three (day 35) or five (day 25) AID^+/+ or AID^+/– mice was analyzed. The specificity of the PCR was evaluated by Southern blotting using both Igh- and cMyc-specific probes, and unique T(12;15)-positive clones were distinguished by their size (from 1–4 kb; Fig. 4 A) and sequencing analysis (Fig. 4 B; and Table S1, available at http://www.jem.org/cgi/content/full/jem.20081007/DC1). At day 25, an analysis of 57 PCR samples from AID^+/+ mice revealed nine distinct translocations (16%, or 1 T(12;15)/10⁵ cells; Fig. 4 A). In contrast, only 2 out of 67 AID^+/– samples (3%, or 0.2 T(12;15)/10⁵ cells) showed bona fide Igh-cMyc translocations (P = 0.01; Fig. 4 A and Table S1). Similarly, 22 out of 69 (30%) AID^+/+ samples showed translocations at day 35 after pristane treatment, whereas we detected only 7 translocations in 72 (10%) AID^+/– samples assayed (2 vs. 0.6 T(12;15)/10⁵ cells, respectively; P = 0.001; Fig. 4 A). In both groups of mice, the majority of the translocations were mapped to S (70%; Table S1), a feature that suggestively reflects the prevalence of IgA CSR in gut-associated lymphoid tissues (23). Thus, compared with wild-type controls, AID^+/– mice display a substantial reduction in the number of translocation-bearing cells emerging upon tumor induction.

View larger version (37K): [in this window] [in a new window] [Download PPT slide]   Figure 4. Paucity of translocation-bearing clones and CSR-induced DNA breaks in AID+/– mice. (A) Igh-cMyc chromosomal translocations in BALB/c-Bcl-xL AID+/+ and BALB/c-Bcl-xL AID+/– mice (three mice per time point) were amplified by PCR 25 or 35 d after pristane injection using nested primers (arrows) specific for cMyc intron 1 and Igh Cµ, C2b, and C constant domains. PCR products were analyzed by Southern blotting using cMyc- and Igh-specific probes. (B) PCR products appearing multiple times were sequenced and counted once if they represented a single clone. (C) Igh FISH in metaphase spreads from AID+/+H2AX–/– and AID+/–H2AX–/– B cells activated for CSR. (left) The loss of the red VH signal with an intact green C signal (micrograph) denotes a chromosome 12 break. (right) Total number of breaks observed for each strain (metaphases analyzed are indicated on top of each bar).

We have shown that the absence of a single copy of Aicda affects the rate of intrachromosomal recombination and interchromosomal translocations involving the Igh loci. This haploinsufficiency for AID is likely explained by the marked reduction in the frequency of formation and availability of S-DNA lesions in AID^+/– cells. The strong inference is that AID protein content falls below a critical threshold in lymphocytes carrying a single copy of Aicda. Such a threshold might be imposed by mechanisms controlling nuclear AID (30, 31), which represents a minor fraction of total AID protein in activated B cells (32). Alternatively, error-free repair pathways, which have been shown to inhibit both CSR and SHM (17, 33), may compromise AID activity to a greater extent under conditions of limited AID transcription. Regardless of the mechanism at work, our findings demonstrate that AID expression levels directly determine the extent of AID-dependent DNA remodeling events, including chromosomal translocations. Thus, AID expression levels appear to determine the incidence of plasmacytomagenesis by specifying the number of cMyc translocation–bearing clones emerging during tumor induction. Overt transformation of such clones may subsequently rely on a mistargeted SHM, which as we have shown is also dependent on the amount of AID.

Our previous work revealed that noncanonical T(12;15) translocations can occur in the absence of AID (13). On the other hand, canonical translocations targeting S region DNA and cMyc intron 1 absolutely require AID enzymatic activity (12, 13). We now extend these results by showing that AID expression levels directly determine the rate of canonical translocations emerging during tumor induction in vivo. Ramiro et al. have similarly shown that cMyc translocations can be induced in an AID-dependent manner in vitro (28). In that study, translocations were rarely present under physiological AID levels in LPS- and IL-4–stimulated B cells, whereas AID overexpression dramatically increased their frequency (28). In like manner, Igh-cMyc translocations are produced to a disproportionately high degree in stimulated B lymphocytes deficient for microRNA 155 (34), which normally suppresses the steady state of Aicda mRNA and AID protein content (34, 35). We find it noteworthy that although the frequency of off-target translocations is highly sensitive to AID expression levels, in vivo on-target SHM and CSR are only mildly affected by AID overexpression (34) or underexpression (this report). It seems likely that AID enzymatic activity is differentially constrained at on and off targets. The unraveling of such regulatory mechanisms might help clarify how S DNA breaks promote physiological recombination or pathological translocations in the B cell compartment.

	MATERIALS AND METHODS

Top ABSTRACT RESULTS AND DISCUSSION MATERIALS AND METHODS REFERENCES

 
Tumor induction.
SV40-Eµ-Bcl-xL and AID^–/– (a gift from T. Honjo, Kyoto University, Kyoto, Japan) mice were backcrossed for 11 generations onto BALB/c mice in a conventional facility. Bcl-xL-AID^+/+ and Bcl-xL-AID^+/– mice were given 0.5 ml of pristane oil intraperitoneally at 8 wk of age, followed by a second injection of pristane 60 d later. BALB/c-Bcl-xL AID^+/+ and AID^+/– mice were intraperitoneally inoculated with 0.4 or 0.5 ml pristane (Aldrich Chemicals) at 2 mo of age. Using Wright-Giemsa–stained cytofuge preparations of ascites, plasma cell tumors were diagnosed by the presence of 10 or more tumor cells per field. All animal experiments were performed according to the NIH guidelines for laboratory animals and were approved by the Scientific Committee of the NIAMS and NCI Animal Facilities.

B cell culture.
To induce S recombination ex vivo, B lymphocytes were isolated from spleens by immunomagnetic depletion using CD43 MACS beads (Miltenyi Biotec). Purified cells were then activated in B cell media (RPMI 1640, 10% FCS, 1x antibiotic-antimycotic, 1% glutamine, 1x nonessential amino acids, 1% sodium pyruvate, 14.2 M 2-β-mercaptoethanol, and 10 mM Hepes) for 72 or 96 h in the presence of 25 µg/ml LPS (Escherichia coli 0111:B4; Sigma-Aldrich) and 5 ng/ml IL-4 (Invitrogen) to induce switching to IgG1, 2.5 ng/ml --dextran (Fina Biosolutions) for IgG3 CSR, or 2 ng/ml IFN- (PeproTech) for IgG2a switching. Cell proliferation was monitored by CFSE staining according to the manufacturer's instructions (Invitrogen).

Flow cytometry.
Unless otherwise stated, the following antibodies were obtained from BD Biosciences: B220-PercP-Cy5.5, Fab'2-IgM-Cy5 (Jackson ImmunoResearch Laboratories), IgG1-PE, IgG3-biotin, streptavidin-PE, and IgG2a-FITC. Apoptotic cells were gated out using TO-PRO-3 (Invitrogen) or DAPI (Sigma-Aldrich). Flow cytometers used were the FACSCalibur (BD Biosciences) and the Cyan (Dako), and a MoFlo instrument (Dako) was used for cell sorting. Flow cytometry data were analyzed with FlowJo software (Tree Star, Inc.).

qRT-PCR.
RNA was extracted with RNAqueous-Micro (Ambion) and treated with DNaseI. cDNA was synthesized, and the level of endogenous AID transcripts was monitored by real-time PCR with the following primers: PrimerBank-mAID5' (5'-gccaccttcgcaacaagtct-3') and PrimerBank-mAID3' (5'-ccgggcacagtcatagcac-3'). cDNA was normalized based on Ku80 mRNA with the following primers: QRT-mKu80-5' (5'-CTTCCTGGACGCCCTGATTG-3') and QRT-mKu80-3' (5'-GCTGCGGAGGTCAGTGAAC-3').

Western blotting.
To detect AID protein, cultured B cells in the presence of LPS and IL-4 were lysed with NP-40 lysis buffer (10 mM Tris-HCl [pH 8], 150 mM NaCl, 0.5% NP-40, 5 mM EDTA, 1 mM DTT, and protease inhibitor cocktail). 50 µg per well of lysates was separated using NuPAGE 4–12% Bis-Tris gels (Invitrogen). Gels were transferred to polyvinylidene fluoride membranes (Invitrogen) and analyzed by immunoblotting AID antisera (a gift from J. Stavnezer, University of Massachusetts, Worcester, MA) and anti–β-actin antibody (Millipore). For secondary antibodies, goat anti–mouse IgG (Millipore) and goat anti–rabbit IgG conjugated with horseradish peroxidase (HRP; Abcam) were used. Blots were visualized using the ECL-PLUS kit (GE Healthcare).

Sµ mutations.
Mutations at Sµ were determined by amplifying genomic DNA from LPS + IL-4–activated (96 h) B cells with primers Sµ(B) (5'-gtaaggagggacccaggctaag-3') and Sµ(D) (5'-cagtccagtgtaggcagtaga-3') at 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s (product = 650 bp). The final product was ligated into ZeroBlunt vector (Invitrogen), and positives clones were sequenced with M13 primers.

FISH analysis.
At 72 h, cultured B cells were arrested at mitosis with 100 ng/ml colcemid (Roche), swollen in prewarmed 0.075 M KCl for 15 min at 37°C and fixed by methanol/acetic acid treatment, and dropped in microscope slides using a humidity chamber (Thermotron). Slides were denatured in 75°C 70% formamide for 7 min; hybridized with following probes overnight at 37°C; washed, dehydrated, and stained with DAPI; and mounted with Vectashield (Vector Laboratories). BAC probes containing the IgH C and Vh regions were labeled by digoxigenin and a biotin nick translation kit (Roche), and were detected by antidigoxigenin FITC antibody (Roche), and Alexa Fluor 568–conjugated streptavidin (Invitrogen). FISH images were captured using an epifluorescence microscope (DMRB Leitz; Leica) and a charge-coupled device camera (model C4742-95; Hamamatsu Photonics).

Hybridoma analysis.
Analysis of CD43^– MACS-isolated B cells were stimulated with LPS + IL-4 for 96 h and fused to the SP2/0Ag-14 myeloma cell line. IgM-secreting clones, as determined by ELISA, were expanded in culture for further analysis. Genomic DNA was prepared and Southern blot analysis was performed using standard techniques.

Detection of Igh-cMyc translocations and plasma cell tumors.
Tissue samples were harvested 25 or 35 d after pristane injection. DNA samples from three (day 35) and five (day 25) mice were prepared as followed: Peyer's patches (three samples per mouse), mesenteric lymph nodes (two samples per mouse), and oil granuloma (two samples per mouse). Tissues were collected in separate tubes and DNA was isolated individually. The sensitivity of the translocation PCR was measured using serial dilution of positive control cells with wild-type mouse liver cells and calculated as >6.25 T(12;15) positive cells/10⁵ negative cells. PCR products were separated on 1% agarose gels and denatured for 15 min in the presence of 0.4 M NaOH before being transferred to nylon membranes. The Southern blot probe was amplified and labeled by the ECL Direct Labeling and Detection System (GE Healthcare). Probe primer sequences were as follows: c-myc forward, 5'-gctagcgcagtgaggagaag-3'; c-myc reverse, 5'-gacgccactgcaccagaga-3'; Cµ forward, 5'-agccagtgtctcagaggga-3'; Cµ reverse, 5'-cttaaggcaggcagggcta-3'; C2β forward, 5'-aagtcagaccgtcacctgca-3'; C2β reverse, 5'-cagaggatcatgtccttgagtg-3'; C forward, 5'-tgctggctgatcttcagtctc-3'; and C reverse, 5'-ttgaacatgacctgcaggtagtc-3'.

ELISA.
Five AID^+/+ and AID^+/– mice were immunized intraperitoneally with 100 µg NP₂₅-CGG precipitated in Alum (Thermo Fisher Scientific). Sera were collected before and at 7 or 28 d after immunization. Serum anti-NP levels were measured by standard ELISA. Plates were coated with 5 µg/ml NP₃₀-BSA or NP₇-BSA in PBS. Serum samples were diluted, and bound antibody was detected using HRP-conjugated anti–mouse IgG (Bethyl) followed by 3,3',5,5'-tetramethylbenzidine treatment.

Online supplemental material.
Table S1 shows the translocation PCR/Southern blot results from AID^+/+, AID^+/–, and AID^–/– mice carrying Bcl-xL. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20081007/DC1.

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