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1 Department of Microbiology, The Mount Sinai School of Medicine, New York, NY 10024, USA
2 Department of Biology, Occidental College, Los Angeles, CA, USA
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Abstract |
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Introduction |
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Somatic mutation of rearranged V genes takes place in germinal centres (GC) and is confined to GC-derived B cells (Jacobs et al. 1991). After antigen stimulation and selection, the majority of mutations are preferentially targeted to the complementary determining regions (CDR) (Wu et al. 1979). The frequency of somatic mutation appears to vary during ontogeny. However, the results obtained from analyzing the structure of V genes expressed in lymphocytes from neonates, adults or aged animals and humans are variable. For example, Press (2000) demonstrated that neonatal murine B cells are capable of activating the somatic mutation machinery, as mutations in the rearranged V genes have be seen in lymphocytes from mice immunized 1 day after birth. In contrast, somatic mutations were not found in the GC of aged mice immunized with a hapten-conjugate T-dependent antigen (Miller & Kelsoe 1995). In humans, van Dijk-Hard et al. (1997) found a decreased frequency of mutations in the VH4 gene in individuals older than 50 years. Conversely, frequent mutations were observed in both VH and V
genes expressed in lymphocytes isolated from elderly individuals (Klein et al. 1993; Rosner et al. 2001). Yet, it is likely that somatic mutations observed in VH or V genes in the latter study were as a result of the persistence of B cell clones that accumulated mutations during the long life of the animals.
In this study, we investigated the frequency and mechanisms of reverse-point mutations in a transgenic (Tg) mouse model designed to be inert to the affinity selection imposed on rearranged V genes. The Tg mouse (HuG-X) was generated using a chimeric Ig heavy chain gene composed of a murine VH anti-dansyl (DNS) gene linked to a reporter human IgG1 constant region gene. A TAG stop codon was introduced in the D segment of the VH gene by site-specific mutagenesis. Somatic mutation of the TAG stop codon allows translation of the chimeric gene, resulting in expression of murine VH and human IgG1 constant region heavy chains. B cells displaying reverse-point mutations were detected by two-colour staining with anti-murine B220 and anti-human IgG1 antibodies coupled to different fluorochromes. Our Tg model provides a view of the frequency of mutation intrinsic to the mutational process rather than that caused by antigen selection used in studies aimed at determining the frequency of B cells exhibiting mutations (Clarke et al. 1985). Our results show that the rate of frequency of spontaneous reverse mutation is very low in naïve animals, yet it is significantly increased after stimulation with anti-IgD antibodies. The frequency of point mutation is age dependent and correlates with increased double-strand DNA breaks (DSB).
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Results |
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The construct used to generate the HuG-X transgenic mice was based on the pSV2gpt anti-DNS
1 plasmid (Oi & Morrison 1986). It contains a murine VDJ linked to the human 1 constant region. The rearranged DNA contains a VHIII gene segment (by the Kabat nomenclature), which is a member of the J606 family, a shortened D segment (6 amino acids) derived from DSP2.2 and a JH3 segment. The plasmid was modified to add a TAG stop codon in the D region. Three mutations were introduced into the construct by PCR based site-specific mutagenesis, to create the TAG stop codon and a SpeI restriction site (Fig. 1A). The SpeI restriction site is centered on the TAG stop codon; somatic mutation of the TAG stop codon thus destroys the SpeI site. The genotyping of the Tg mice was carried out by PCR using a pair of primers corresponding to the murine VH (forward) and the human 1 CH1 domain (reverse). As can be seen in Fig. 1(B), HuG-X Tg mice display a PCR product of 340 bp, while wild-type mice do not show a PCR amplification product. RNA extracted from the spleen of HuG-X Tg mice and littermates tested negative by PCR for the HuG-X transgene was used in RT-PCR assay to determine the transcription of VH HuG-X gene. The data presented in Fig. 1(C) demonstrate that HuG-X Tg mice, with the exception of one line, did transcribe the Hug-X transgene.
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In preliminary experiments we studied the occurrence of cells expressing B220 and human IgG1 in Ficoll-gradient separated mononuclear blood cells harvested monthly over 1 year. Among the 77 HuG-X Tg mice studied, we found two mice, one 4 weeks old and one 9 weeks old, with 2.84% and 4.54% B220+ HuIgG1+ B cells. The other 75 Tg mice and Balbc mice had 0.77 ± 0.4% (SD) double positive cells. Because of the low frequency of spontaneous mutation, we further studied the percentage of cells expressing the reverse mutation in HuG-X Tg mice injected with anti-IgD antibody, as the occurrence of somatic mutation requires DNA transcription (Peters & Storb 1996). Anti-IgD antibodies crosslink the IgD receptor, inducing a strong DNA synthesis and proliferation of mature B cells (Finkelman et al. 1982).
To prove the stimulatory effect of anti-IgD antibodies on the HuG-X mice, we investigated the activation of B cells in vivo by studying cell cycle distribution, measuring the DNA content of splenic cells stained with propidium iodide. The data depicted in Table 1 show a significant increase of cells in the S and G2 + M phases in the spleen from 2-, 12- and 24-month-old HuG-X Tg mice injected with anti-IgD antibodies as compared with age-matched littermate control mice.
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Localization of cells exhibiting somatic reverse mutations
We then studied the localization of cells synthesizing chimeric molecule on sections from the spleens of 2-month-old HuG-X Tg mice harvested 6 days after injection with anti-IgD antibodies or PBS. Serial sections were stained with PE-anti-peanut agglutinin (PNA) antibodies, FITC-rabbit anti-human IgG antibodies or FITC-rabbit IgG. The data presented in Fig. 3 show that GCs were strongly stained with anti-PNA mAb (Fig. 3A) and a significant number of cells in GC and a few cells in the mantle zone were stained with anti-human IgG antibodies (Fig. 3B). HuIgG+ cells were rarely observed in splenic red or white pulp outside of the GC and no staining was observed with the FITC-rabbit IgG isotype control (Fig. 3C). Additionally, no HuIgG+ cells were observed in follicles or nodules of the white pulp in HuG-X Tg mice injected with PBS (Fig. 3D,E). These results suggest that most cells exhibiting reverse mutations were localized in the GC.
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As the highest numbers of B cells expressing reverse mutation were identified in 2-month-old HuG-X Tg mice injected with anti-IgD antibody, we studied reverse mutation at the TAG stop codon by sequencing the HuG-X VH gene from these mice. The TAG codon is located in the SpeI restriction site of the HuG-X VH construct and a mutation within the TAG codon destroys this site. We took advantage of this information to clone and sequence VH cDNA obtained from the amplification of splenic RNA from three different 2-month-old Hug-x Tg mice injected with anti-IgD antibodies. Figure 4 shows that cDNA fragments amplified from the plasmid containing the HuG VH construct with VH forward and JH3 reverse primers were completely digested by Spe1 (143 and 203 bp bands), while the cDNA fragments obtained from RNA extracted from anti-IgD injected mice showed three distinct bands (two bands similar to the plasmid control and a 346-bp band which was resistant to SpeI digestion). The RT-PCR products that were resistant to SpeI digestion were purified and cloned into the pGEM-T vector, then sequenced. Figure 4 shows an example of reverse mutation in the stop codon of Hug-X transgene. Table 4 shows the sequences of cDNA of 12 clones obtained from the SpeI-undigested 346-bp band. As can be seen, 10 clones exhibited base-pair substitution mutations in the TAG stop codon, keeping the open reading frame and allowing the transcription of this gene. Seven of these 10 clones showed a reverse mutation in which the ‘A’ from the TAG stop codon was replaced by a ‘G’, while in the other three clones the ‘T’ in the TAG stop codon was replaced by an ‘A.’ Four clones showed additional point mutations around the mutated stop codon and two clones exhibited a single base pair deletion in the stop codon.
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The molecular mechanisms behind somatic mutation still remain elusive. Several mechanisms have been proposed and were recently extensively reviewed (Harris et al. 1999; Diaz & Casali 2002; Honjo et al. 2002). Brenner & Milstein (1966) hypothesized that somatic mutation is a two-step process consisting of cleavage of DNA resolved by a repair process involving error-prone polymerases. This concept is strongly supported by recent evidence of DNA double-strand breaks (DSB) in somatically mutated V genes. Thus, we investigated DSB in HuG-X Tg mice by a linker-ligation assay for broken-ended DNA using ligation mediated-PCR (LM-PCR) as previously described (Schlissel et al. 1993).
In a pilot experiment, a partially double-stranded oligonucleotide linker (BW linker) was ligated to genomic DNA purified from spleen cells of 2-month-old HuG Tg mice injected or not with anti-IgD. Genomic DNA from HuG-X Tg mice injected with anti-IgD antibodies was used as a negative control. We used a 5'-phosphorylated BW linker which can ligate to both 5'-phosphorylated blunt genomic DNA and blunt genomic DNA which has lost its 5' phosphate. A pair of nested PCR assays with HuG-X sequence specific primers and a primer identical to the ligated strand of the linker detected DSB associated with the HuG-X locus. The PCR products were used for Southern blot hybridization with the VH HuG sequence as a probe. Using the VH HuG probe, we detected an intense smear of 100 bp to 1 kbp in the DNA sample from anti-IgD injected HuG Tg mice, suggesting that DSB occurred at high frequency around HuG-X gene. This band was not observed using DNA extracted from the tail of Tg mice injected with anti-IgD antibodies or with DNA extracted from the spleen of naïve HuG-X Tg mice (data not shown).
In order to detect whether or not DSB occurs at the TAG stop codon, specific primers for the TAG site (TAG1–6) which contain 4 bp specific for the TAG site and part of the BW-1 primer were generated. The initial PCR products obtained with the linker and p418 primers were re-amplified with the VH forward primer and one of the TAG1–6 reverse primers. Figure 6 shows that a 180 bp band corresponding to the TAG stop codon is observed when the TAG1 or TAG4 primers were used for the second amplification, suggesting that at least two breaks occurred around the TAG stop codon. Figure 6 also shows much more intense bands around 400 bp in TAG4 and TAG5, and around 1 kb in TAG1. This may indicate that the homologous 4-bp sequence in the TAG stop codon may also be located outside the TAG stop codon expressing the reverse mutation. No PCR products were obtained using as a template genomic DNA extracted from Balb/c mice (data not shown).
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Discussion |
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transgene can hypermutate (O’Brien et al. 1987). An inherent difficulty in studying somatic mutation in Tg mice is that several copies of the transgene can be randomly integrated into the genome. We believe that this drawback is not relevant to our study as we have determined that the number of B220+ cells exhibiting a reverse mutation is independent of the number of copies of the transgene inserted into the genome of a single B cell. A longitudinal study of spontaneous reverse mutation in HuG-X mice found that only two of 77 mice had an increased number of cells exhibiting a reverse mutation. The low frequency of spontaneous reverse mutation was probably because of a lack of activation of mutational machinery. It was suggested that the targeting step of the somatic mutation process is linked to transcription (Peters & Storb 1996; Fukita et al. 1998) as the heavy chain promoter can be replaced with a heterologous promoter without affecting the somatic hypermuation process (Fukita et al. 1998).
To overcome this problem, we reasoned that polyclonal stimulation in vivo resulting in the activation of replicative machinery transcription might lead to increased reverse somatic mutation in the VH HuG-X transgene. Therefore, we studied the reverse mutation process during ontogeny in HuG-X mice injected with goat anti-murine IgD antibodies. Anti-IgD antibody induces a T-dependent activation and proliferation of mature, resting B cells leading to DNA synthesis (Finkelman et al. 1992) and induces formation of GCs (Flotte et al. 1984), which provides the microenvironment required for the occurrence of somatic mutation (Berek et al. 1991). These features are particularly important as it was demonstrated that the somatic mutation process is T-dependent (Yang et al. 1996), and that the mutation occurs during transition of IgD+ CD23+ B cells to IgD– IgG+ CD23– in GC centroblasts (Pascual et al. 1994) which can mature and display class switching.
The results reported herein show a significant increase of number of B cells in the S or G2 + M phase in HuG-X Tg mice injected with anti-IgD antibodies, which was paralleled by an increased number of B cells exhibiting reverse mutation of the transgene. Our most striking observation is the significant variation in the per cent of cells exhibiting reverse mutation of the transgene throughout life. The percentage of B cells displaying a reverse mutation was low in 1-month-old mice, in agreement with a previous report implicating that stimulation with anti-IgD has a weak effect in young mice (Muul et al. 1983). A sharp increase in reverse mutation frequency was observed in 2-month-old mice, followed by a slight decrease that plateaus in 3- to 12-month-old mice. A significant decrease was observed in 24-month-old HuG-X Tg mice. These results show that there is a genuine age-dependent decrease of the number of cells displaying reverse mutation.
Additionally, ontogenic variation of reverse mutation was not related to phenotypic and functional differences of B cells. This is in agreement with data indicating that in humans B cells are generated throughout life (Nunez et al. 1996), and that B cells are maintained because of increased longevity despite a several-fold decrease in population of precursors in bone marrow (Kline et al. 1999). As predicted from the studies carried out in normal mice, an increased number of GC was observed in HuG-X Tg mice injected with anti-IgD antibodies. While an increased number of cells displaying a reverse mutation were identified in GC, a few cells were also observed in the mantle zone, probably representing mature B cells which have emigrated from the GC. This explanation is supported by an increased number of syndecan+ cells and is in agreement with the observation showing an increased number of plasma cells in mice injected with anti-IgD antibodies (Finkelman et al. 1982). In addition to detecting B cells exhibiting a reverse mutation by staining with a reagent for the reporter gene, the reverse mutations were confirmed by sequence analysis of cDNA obtained by amplification of splenic RNA extracted from HuG-X mice injected with anti-IgD antibodies. Our results showed that the ‘A’ of the TAG stop codon was mutated more often the ‘T’ in agreement with results previously reported (Golding et al. 1987). No mutations involving the ‘G’ were observed in the 10 individual clones sequenced. It is noteworthy that point mutations, deletion or insertions were observed not only within or around stop codon but also along entire VH Hug-X transgene as assessed by sequencing 39 clones containing cDNA from the spleen of several 2-month-old HuG-X Tg mice injected with anti-IgD antibodies.
Brenner & Milstein's (1996) hypothesis of a two-step process of somatic mutation, namely breaking of DNA followed by an error-prone polymerase that introduces and fixes mutations, is strongly supported by recent data. Studies carried out on Ramos B-cell cells, which constitutively mutate their V gene at a rate of 2 x 10–5 bp/generation, showed abundant DSB in their V gene (Papavasiliou & Schatz 2000; Zang et al. 2001; Bross et al. 2000). Based on this information, we reasoned that the reverse mutation in the D segment of the VH HuG-X gene should be intimately associated with DSB occurring within or not far away from the TAG site. We detected DSB associated with the VH HuG locus by nested PCR using a VH HuG specific primer and a primer identical to the ligated linker. Southern blot analysis of PCR products probed with a VH HuG-X probe showed an intense smear of 100 bp–1 kb in the DNA sample from anti-IgD injected HuG-X Tg mice corresponding to DSB in the VH HuG gene. The lack of DSB in DNA from uninjected control HuG-X mice suggests that the initiation of transcription induced by anti-IgD antibodies generated both DSB and reverse mutation. We also showed by PCR, using a VH HuG primer and specific primers for the TAG codon containing 4 bp specific for the TAG site and part of the BW1 primer, that DSB occurred in the TAG stop codon. PCR amplification with these primers showed that two primers, TAG1 and TAG4, generated a 180-bp band corresponding to the stop codon, suggesting that at least two breaks occurred around the stop codon. A straightforward interpretation of these data is that DSB which occur in and near the TAG stop codon are tightly linked to the somatic mutation process. However, we cannot rule out a role for activation-induced cytidine deaminase (AID), a novel member of the RNA-editing deaminase family, in the decreased rate of mutations in 24-month-old mice. AID plays an important role in both class switching and somatic mutation (Muramatsu et al. 1999). Recently, we have shown a significant decrease of the transcription of AID gene with ageing, which was not related to variation of phenotype and functional properties of B cells in aged mice (Radu et al. 2003).
In conclusion, our data demonstrate ontogenic variation of the number of B cells exhibiting reverse mutation of the HuG-X reporter transgene. These variations are independent of phenotypic and functional differences of B cells, and are associated with DSB around the TAG stop codon, which in our model is the substrate for reverse mutation.
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Experimental procedures |
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Goat anti-murine IgD serum prepared as previously described (Finkelman et al. 1982) was kindly donated by Dr Finkelman (University of Cincinnati, Cincinnati, OH). Purified FITC-rabbit anti-human IgG1 antibodies; R-PE anti-mouse CD45/B220, R-PE anti-mouse B7.1, FITC anti-mouse I-A d/I-Ed, R-PE anti-mouse CD138 (syndecan-1), R-PE anti-PNA, FITC anti-mouse IgD monoclonal antibodies, anti-mouse FcR (2.4G2) monoclonal antibody and FITC- goat IgG and mouse IgG were purchased from Pharmingen (San Diego, CA). Propidium iodide was purchased from Sigma (St Louis, MO).
Mice
The plasmid pSV2gpt anti-DNS h G1 (Oi & Morrison 1986), a gift from Dr Sherrie Morrison (UCLA, Los Angeles, CA) was modified for these experiments. The plasmid was modified to add a TAG stop codon in the D region. Three mutations were introduced into the construct by PCR-based site-specific mutagenesis, to create the TAG stop codon and a SpeI restriction site. For generating transgenic mice, a 15.7-kb KpnI–PvuI fragment was used. The precipitated DNA fragments were re-suspended in 10 mM Tris pH 7.4, 0.1 mM EDTA and injected into (B6C3) F1 x C57BL/6 fertilized eggs. The transgenic mice were generated using conventional methods. Offspring were backcrossed to BALB/c AnNTacfBr mice (Taconic Farms, Germantown, NY). HuGX-24.48-64 mice are from the third backcross and HuGX24.83-110 mice are from the fourth generation backcross. These mice were used to generate a colony of Tg mice. By RT-PCR it was shown that in Tg mice the HuG-X transgene is transcribed. The genotyping was carried out by PCR using one pair of primers: one specific for murine VH forward: 5'-GAGGATCCATGAAACTCC-3' and the second reverse primer for human 1 5'-GTAGGTCTGGGTGCCCAAGGT-3'.
Immunization of animals
1-, 2-, 3-, 6-, 12- and 24-month-old Hug Tg mice were injected interperitoneally with 0.2 mL goat anti-murine anti-IgD serum or goat IgG. Single cell suspension was prepared from mice injected with goat IgG or goat-anti-murine IgD serum 6 days after injection. Spleen single cell suspensions were used for flow cytometry and to measure the cellular DNA content.
Cytometry analysis
The number of B cells synthesizing chimeric HuG protein was determined by surface staining with R-PE anti-mouse B220 monoclonal Ab and intracellular staining with FITC-rabbit anti-human IgG1 antibodies. For intracellular staining for human IgG1, 106 cells were suspended in 1% bovine serum albumin (BSA) and incubated for 6 h in the presence of 2 mM monesin. At the end of the incubation, cells were washed three times with phosphate-buffered saline (PBS) and incubated for 30 min with 1 mg/mL 2.4 G2 mAb and then incubated for 30 min at 4 °C with PE-anti B220 mAb. The cells were paraformaldehyde fixed; saponin permeabilized and stained for 30 min with FITC rabbit anti-human IgG antibodies. The percentage of plasma cells bearing reverse mutation was determined by staining with PE-anti-CD138 and FITC rabbit anti-human IgG1 antibodies. Cells (5 x 105) per sample from a population gated on a window encompassing lymphocytes and large mononuclear cells were analyzed using a FacsCalibur cytometer with CellQuest software manufactured by Becton Dickison (San Diego, CA).
Tissue sections and immunofluorescence staining
Spleen specimens from 3-month-old HuG-X Tg mice injected with saline or goat anti-murine IgD serum were embedded in cryomolds (Fisher Scientific, Springfield, NJ). Serial 7-µm sections were fixed in 1 : 1 methanol : acetone for 10 min at –20 °C and air dried. The sections were then re-hydrated in 1% BSA and incubated for 1 h in a moist chamber with r-PE anti-PNA, FITC-rabbit anti-human IgG1, FITC-rabbit IgG isotype control. After washing three times in 1% BSA/PBS, sections were coverslipped with Vectashield (Vector Laboratories, Burlingame, CA) and examined in a Zeiss Axiophot microscope according to a previously described technique (Stan et al. 2001).
Analysis of DNA content and DNA synthesis
Cells from HuG-X Tg mice injected with saline or goat anti-murine anti-IgD serum were analyzed for DNA content 6 days after immunization using the propidium iodide method as previously described (Allen & Newland 2001).
Preparation of VH HuG cDNA and sequencing
Two-month-old HuG-X Tg mice were injected with anti-IgD antibody and, 6 days later, total RNA was extracted from the spleen cells. Total RNA was treated with DnaseI (Gibco, Grand Island NY) to eliminate DNA contamination and 5 µg of total RNA was reverse-transcribed using Superscript II reverse transcriptase (Gibco) in 20 µL of solution according to the manufacturer's instructions. One microlitre of cDNA was used as template for PCR. The oligonucleotides sequence used for PCR are the p418 primer corresponding to the 5' end of the VH region and the p503 primer 5'-GAAGACTGGGCCCTTGG-3' corresponding to the human IgG1 constant region. For the second PCR, we used a VH forward primer 5'-CCAGAGTGAAGTCAAGCTTG-3' corresponding to 100 bp downstream of p418, and the JH3 reverse 5'-TGGCCCCAGTAAGCAAACCA-3' primer. PCR amplification was performed for 35 cycles, consisting of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s and extension at 72 °C for 30 s, in 50 µL reaction mixture containing 1 µL of cDNA, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 250 mM of each deoxynucleotide triphosphate, 15 pM of each primer and 1 unit of Plutinum Taq polymerase (Gibco). Amplification was accomplished using a DNA Thermal cycler 480 (Perkin-Elmer, Norwalk, CO). Control samples lacking reverse transcriptase did not show any amplified product (data not shown). A total of 39 PCR products were cloned into pGEM-T vector (Promega Madison, WI) and sequenced using an ABI3700 automated sequencer.
Linker-ligation PCR assay for broken-ended DNA
Ligation mediated PCR was performed as previously described (Schlissel et al. 1993), with minor modifications. High molecular weight DNA was extracted and purified by proteinase K digestion and phenol/chloroform extraction. The BW-1 5'-GCGGTGACCCGGGAGATCTGAATTC-3' and BW-2 5'-GAATTCAGATC-3' oligonucleotides were annealed by mixing 20 µM of each in a volume of 100 µL of 250 mM Tris (pH 7.7), heating the mixture to 90 °C for 5 min and gradually cooling to room temperature. The BW linker was phosphorylated by T4 polynucleotide kinase (Gibco) according to manufacture's instruction and 3 µg of genomic DNA was ligated to the BW linker in a reaction volume of 0.1 mL containing 0.2 µM linker and 2.5 units of T4 DNA ligase (Gibco) at 16 °C for 16 h. The reactions were then diluted with an equal volume of a buffer containing 10 mM Tris (pH 8.3), 50 mMKCl, 0.5% NP-40 and 0.5% Tween 20 and heated to 95 °C for 15 min to inactivate the ligase, dissociate the unligated strand of the linker and denature the DNA for subsequent PCR. A nested PCR strategy was used to identify the sites of linker ligation. The first PCR was performed using the p418 primer corresponding to the 5' region of VH and the BW-1 primer, as described above, except the annealing temperature was 57 °C. The BW-1 primer and the VH forward primer were used for the second PCR.
PCR products were separated in a 2% agarose gel and were transferred to nylon membrane and subjected to Southern blot analysis according to routine methods, using the VH HuG sequence as a probe. To detect the double-strand break specific to TAG stop codon, first PCR amplification was performed using linker and JH3 reverse primers. Of the first PCR product, 0.1 µL was used as template for re-amplification using the VH HuG forward and six primers around the stop codon (TAG1 to TAG6). TAG site-specific PCR products were subjected to Southern blot analysis as described above.
The sequences of TAG primers used in the experiments are illustrated below.
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Acknowledgements |
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Footnotes |
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Present addresses: aDepartment of Rheumatology and Hematology, Tohoku University School of Medicine, Sendai, Japan;
bDepartment of Immunology, Cantacuzino Institute, Bucharest, Romania;
cDepartment of Biology, The College of William and Mary, Williamsburg Va Hilde Cheroute;
dLa Jolla Institute for Allergy and Immunology, San Diego, CA, USA.
* Correspondence: E-mail: constantin.bona{at}mssm.edu |
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Received: 16 February 2004
Accepted: 29 July 2004
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