An extrachromosomal switch recombination substrate reveals kinetics and substrate requirements of switch recombination in primary murine B cells

Katja Petry1, Gregor Siebenkotten1,3, Rainer Christine1,2, Katharina Hein1,2 and Andreas Radbruch1,2,4

1 Institut für Genetik der Universität zu Köln, 50931 Köln, Germany
2 Deutsches Rheumaforschungszentrum, Hannoversche Strasse 27, 10115 Berlin, Germany
3 AMAXA GmbH, 10117 Berlin, Germany
4 Medizinische Fakultät (Charité), Humboldt-Universität, 10098 Berlin, Germany

Correspondence to: A. Radbruch, Deutsches Rheumaforschungszentrum, Hannoversche Strasse 87, 10115 Berlin, Germany


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Ig class switch recombination occurs in B lymphocytes upon activation, and is targeted to distinct switch (S) regions by cytokine-mediated induction of switch transcripts spanning the entire S region and the adjacent constant region gene segments. Using a novel type of switch recombination substrate, constructed according to the intron–exon structure of the IgH locus, but with heterologous elements, we here have tested the structural requirements for targeting and the kinetics of switch recombination in activated primary murine B cells. When transfected at various times after activation, up to 10% of the transfected B cells perform recombination of the substrate within 12 h. Switch recombination in primary B cells is restricted to the first 72 h after onset of activation, then rapidly decreases to background levels, as obtained in plasmacytoma cells or with substrates carrying no S region sequences. In terms of structural requirements, switch recombination is targeted to any transcription unit that contains an intronic S region and depends on processing of the primary transcript by splicing.

Keywords: B lymphocytes, class switch recombination, Ig, lipopolysaccharide, primary cells, transient transfection


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Activation of primary murine B cells leads to proliferation, differentiation and antibody class switching. About 25% of the cells activated in vitro by lipopolysaccharide (LPS) switch the expressed isotype, predominantly from IgM to IgG2b and IgG3 (13). By now, it is clear that the molecular basis of antibody class switching in B cells is an intrachromosomal, loop-out and deletion-type of recombination (2,4,5). Regulation and kinetics of expression of the switch recombination activity during B cell differentiation are less clear.

Recombination occurs between highly repetitive sequences, the switch (S) regions that are located in the intron upstream of each constant region (CH) gene (2,68). By recombination, the expressed CH gene is replaced by one of the CH genes located further downstream on the chromosome. The S regions differ in length and sequence, but all are characterized by the repetition of the more or less conserved motif (GAGCT)1–7(GGGGT) (9,10). Since break points in recombined S regions are scattered all over the sequence, switch recombination is a region-specific event rather than a sequence-specific one (11). Despite the similarity in sequence between the different S regions, switch recombination is targeted to distinct S regions, as is evident from the fact that usually the S regions of the same CH genes are recombined on both IgH loci of an individual switched B cell (12,13), even though one of the IgH loci is allelically excluded.

It has been speculated that switch recombination is targeted to distinct S regions by transcription of that S region prior to recombination (14,15). Indeed, transcription of the S regions, as induced by mitogens and cytokines, is correlated with subsequent switch recombination of those S regions (16,17). `Switch' transcription starts from unconventional promotors 5' of a pseudo-exon (I exon), which is located upstream of the S region, includes the S region and the entire adjacent CH gene, terminating at the transcriptional termination sites of that CH gene. The primary transcript is spliced and polyadenylated to give a non-coding `switch transcript'.

Evidence for the relevance of switch transcription in directing switch recombination to distinct S regions has been obtained by targeted mutation of the murine germline IgH locus, i.e. removing and replacing the transcriptional control elements for switch transcription. Deletion of the DNA region carrying the promoter of switch transcription results in inhibition of class switching (1820). Switch recombination can be targeted to distinct S regions by heterologous promoters, that replace the endogenous switch transcription promoters (2123), but only if the replacing sequences contain exon-like fragments 5' of the S region (22,23). This suggests that not switch transcription but rather the switch transcript itself or its processing may be required to target switch recombination to the transcribed S region.

Using a novel type of switch recombination substrate (SRS), we show here that insertion of S region sequences into an intron of any transcription unit, generating a processed transcript, targets this S region for switch recombination. Moreover, the rapid readout of recombination of the SRS by expression of a cell surface reporter gene allowed us to determine the time window in B cell differentiation during which switch recombinase is active.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Vector construction
pSRSori-Sµ/S{gamma}2 was subcloned from pSRS (Christine et al., in press), by exchange of the pSRS backbone for that of pcDNAmp. pcDNAmp is a derivative of pcDNA I (Invitrogen, Groningen, The Netherlands) in which the NruI–SfiI fragment was exchanged for the ampicillin resistance fragment of pUC18. pcDNAmp carries a Polyoma virus origin of replication, replication being dependent on Polyoma virus large T antigen expression. The construction of pSRS is described in detail elsewhere (Christine et al., in press). Briefly, Sµ is a 1.7 kb HindIII fragment of BALB/c Sµ, largely composed of (GAGCT)n(GGGGT) sequences, and S{gamma}2b is a 3.3 kb PstI–HindIII fragment of BALB/c S{gamma}2b. Both S region fragments are oriented as in the IgH locus. The splice acceptor site 3' of Sµ originates from the murine C{kappa} gene segment, it is followed by 48 bp of coding sequence from C{kappa} and a synthetic translational stop oligonucleotide. The transcriptional stop element is composed of a strong synthetic poly(A) site and the transcriptional `pause' site of the human {alpha} globin gene, taken from pSPAIPI+ (24).

In pSRSori-{lambda}1/S{gamma}2 and pSRSori-{lambda}1/{lambda}2, the S regions were replaced by a 1.8 kb BssHII fragment or a 3.4 kb BsaAI fragment [{lambda}1: bp 14815–16649; {lambda}2: bp 17250–20653 of the {lambda}(c1857 Sam 7) genome; Boehringer, Mannheim, Germany]. In pSRSori-{Delta}sas, the 3' Sµ splice acceptor site was deleted as a 0.3 kb PstI fragment from pSRSori-Sµ/S{gamma}2. phCD4ori was generated from pSRSori-{lambda}1/{lambda}2 by deletion of the region spanning 5' Sµ to 3' S{gamma}2b by a HindIII digestion. Construction of pH-2Kk and pCMV LT2 are described elsewhere (25,26).

Plasmids were purified after propagation in Escherichia coli DH5{alpha} by commercial DNA preparation kits (Qiagen, Hilden, Germany).

Cell culture
Spleens were prepared from 8- to 12-week-old CB20 mice (from our own breeding facility), disaggregated and washed in PBS. T cells were depleted by MACS using anti-Thy-1.2 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Efficiency of depletion was controlled by immunofluorescence, using a FACScan and CellQuest research software (Becton Dickinson, Mountain View, CA), and was usually >97%. Remaining cells were cultured at 37°C/7% CO2at a concentration of 1.5x106/ml in RPMI 1640 medium with 10% FCS, 50 µM 2-mercaptoethanol, 100 µg/ml streptomycin, 50 µg/ml penicillin and 40 µg/ml bacterial LPS (Sigma, St Louis, MO). Cells were fed after 48 h by adding half a volume of medium. Cell composition of the cultures was controlled by immunofluorescence, which indicated that usually after depletion >90% of the cells were B cells, with their frequency further increasing with time. Non-B cells were mainly macrophages, but almost no T cells (<0.4%) were present. On day 3 ~8% of the B cells were surface positive for IgG3 or IgG2b. X63Ag8.653 were cultured in the same way, except that the medium was not supplemented with LPS.

Transfection
B cells were harvested by centrifugation (180 g) after various lengths of incubation and resuspended at a concentration of 2.5x107/300 µl, or 2.5x107/600 µl for X63Ag8.653, in RPMI 1640 without phenol red (Life Technologies, Eggenstein, Germany), buffered with 40 mM HEPES (pH 7.9; Life Technologies) and transferred to electroporation cuvettes containing the DNA. Before 2.5 µg pH-2Kk and 2.5 µg pCMVLT2 had been premixed, and 5 µg pSRSori vectors was supplemented per probe, which corresponds to a molar ratio of ~1:1:1. Cells were incubated on ice for 10 min, transfected with a BioRad Genepulser (B cells: 300 V; X63Ag8.653: 240 V; both 960 µF) and again incubated at 37°C for 10 min. Cells were then resuspended at a concentration of 1.25x106/ml in prewarmed medium containing 50% of supernatant from the cell culture before transfection. Samples of the cultures were analyzed cytometrically by FACScan before transfection to determine the ratio of high-density resting B cells and low-density activated B cells.

Analyses of switch recombination activity
After 15 h of incubation cells were recovered for analysis, and depleted of dead cells and debris by centrifugation over Ficoll-Paque. After washing twice in PBS/0.5% BSA, cells were stained with anti-H-2Kk microbeads in PBS/BSA/0.02% NaN3 and incubated at 7°C for 20 min. Anti-hCD4–phycoerythrin (PE) and anti-H-2Kk–FITC mAb were added, and cells were incubated on ice for another 10 min. Cells were washed once with and resuspended in PBS/BSA/NaN3. H-2Kk-positive cells were enriched by MiniMACS (Miltenyi Biotec, Bergisch Gladbach, Germany) and the enriched cell fraction as well as cells of the negative fraction and unseparated cells were analyzed cytometrically.

Flow cytometry
For flow cytometry a FACScan and FACScan research software were used (Becton Dickinson). Propidium iodide (PI) (1 µg/ml) was used for exclusion of dead cells. A lymphocyte gate was set by forward and sideward light scattering parameters. Cytometric gating of the analyzed cell fraction is described in Fig. 2Go. CellQuest software (Becton Dickinson) was used for evaluation of data.



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Fig. 2. Flow cytometric analysis of substrate recombination in LPS-activated primary B cells. Transfected and MACS enriched cells were stained with anti-hCD4, anti-H-2Kk antibodies and propidium iodide. (A) Resting B cells and B cell blasts were distinguished according to forward light scatter and further analysis was restricted to (B) living cells, and (C) living and transfected (H-2Kk-positive) cells. (D) Switch recombination frequencies within the population of resting B cells or B cell blasts are given as the percentage of hCD4+ cells among H-2Kk-positive cells.

 
Antibodies
The following antibodies were used. mAb: anti-Thy 1.2–PE (HO-13) (27), anti-hCD4–PE (Leu3a; Becton Dickinson), anti-H-2Kk–FITC (Miltenyi Biotec), anti-B220–PE (Ra3-3A1/6.1) (28), anti-IgM–PE (R33-24) (29) and anti-{kappa}–FITC (R33-18) (30); polyclonal antibodies: goat anti-mouse IgG2b–FITC and goat anti-mouse IgG3–FITC (Southern Biotechnology Associates, Birmingham, AL).


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
SRS
pSRSori-Sµ/S{gamma}2b (Fig. 1Go) is a derivative of pSRS (Christine et al., in press). The design of the SRS is based on the molecular structure of the endogenous IgH locus. However, except for the S region fragments themselves, all fragments are not derived from the IgH locus. Sµ and S{gamma}2b are each located in introns of adjacent transcription units, which are separated by efficient translational and transcriptional stop sequences. Transcription of each unit is driven by constitutively active heterologous promoter/enhancer elements. Recombination between the S regions deletes the stop between the two transcription units, and the hCD4 extracellular and transmembrane/intracellular exons become part of one transcript. Recombination is thus rapidly reported by expression of hCD4 on the cell surface and can be analyzed on the single-cell level by immunofluorescence. This direct readout facilitates analysis of episomal recombination in primary B cells.



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Fig. 1. pSRSori-Sµ/S{gamma}2b (top line) and its transcripts (bottom line). S regions (Sµ, S{gamma}2b; black boxes), inserted in their physiological orientations, are flanked by the exons coding for the extracellular (hCD4ec) or transmembrane/intracellular domains (hCD4t/i; grey boxes) of the hCD4 protein. The two transcription units, driven by the CMV promoter/enhancer (open triangle) and Thy-1.2 promoter/SV40 enhancer (filled triangle), are separated by a transcription stop sequence (grey octagon) that terminates transcription starting at the 5' promotor/enhancer. By RNA splicing, the transcribed intronic S regions are excised. The hCD4ec exons of the upstream transcript are spliced to the splice acceptor site of the C{kappa} exon (horizontally striped box), which is followed by a synthetic translational stop oligonucleotide (black stroke). For the downstream transcript, exons of the murine Thy-1.2 gene (open boxes) are spliced to the hCD4t/i exons. Polyadenylation of the transcripts is driven by a synthetic (upstream transcript) poly(A) site (black diamonds) or one derived from the bovine growth hormone gene (downstream transcript). An integrated Polyoma virus origin of replication (hatched, thick black box) allows for autonomous replication of the substrate after co-transfection of a Polyoma virus large T antigen expression vector. An ampicillin resistance gene and a bacterial ColE1 origin of replication (thin, hatched black boxes) permits substrate propagation in bacteria. If recombination between S regions takes place, the intervening sequences, including the transcriptional stop sequence, are deleted and the complete hCD4 gene becomes part of one transcript, which results in cell surface expression of hCD4.

 
For analysis of switch recombination, cells were cotransfected by electroporation with SRS, a second vector, constitutively expressing the Polyoma virus large T antigen (pCMV LT2) and a third vector (pH-2Kk), coding constitutively for a protein expressed on the cell surface, the murine MHC I antigen H-2Kk.

Co-transfection of pCMV LT2 was used to facilitate autonomous replication of SRS, as replication may be required for switch recombination (31,32).

The H-2Kk protein served as an indicator for transfected cells and to separate them from non-transfected cells by high gradient MACS and/or electronic gating in cytometric analyses (Fig. 2Go). To analyze the kinetics of expression and degree of co-expression, B cells were co-transfected with equimolar amounts of pH-2Kk, preswitched SRS (phCD4ori) and pCMV LT2. Both reporter genes, H-2Kk and hCD4, are expressed within 3 h after transfection. However, hCD4 expression lasted longer than H-2Kk expression, because of the autonomous replication of the plasmid phCD4ori. In the experiments described below, analyses were carried out at the peak of H-2Kk expression, i.e. 15 h after transfection. At this time, plasmid DNA recovered from MACS-purified, H-2Kk-positive and -negative cells was probed for the first hCD4 exon of the SRS. More than 96% of the recovered hCD4 DNA was contained within the H-2Kk-positive cells, indicating nearly complete co-transfection. Thus, by co-transfection, the analysis became independent of variable transfection rates. SRS transfection was established, optimized and controlled for several aspects. First, conditions were optimized until transfection efficiencies of >10% were routinely obtained. By cytometric analysis for expression of B220, IgM or {kappa} light chains, transfection of B cells was confirmed. To ensure that transfection does not influence B cell differentiation, especially with regard to Ig class switching or proliferation, B cells transfected on day 2 and non-transfected B cells were analyzed for endogenous class switching. They were stained for IgG3 and IgG2b surface expression, after activation of the cells with LPS. Frequencies of IgG3+ and IgG2b+ cells were identical in all cases.

Specificity of the staining for hCD4 was controlled in each experiment, by using a sample transfected only with pH-2Kk and pCMV LT2, but not a hCD4-expressing vector. The percentage of unspecific staining, usually <0.2%, was subtracted from the experimental values given in the tables.

Finally, we controlled our analysis for the existence of recombined SRS in the DNA used for transfection. Bacterial recombination during plasmid preparation was assessed by Southern blot analysis of each vector preparation before transfection. Vector preparations with <0.05% of recombined SRS were used for transfection. In addition, the direct comparison of frequencies determined for the different types of cells confirms that we have analyzed recombination of the substrate within eukaryotic cells.

Figure 2Go illustrates the cytometric analysis of a transient recombination assay. In this experiment, the substrate is recombined in 4.4% of the B cell blasts, activated with LPS for 2 days. Only 0.11% of small, LPS unresponsive B cells are stained for hCD4, which corresponds to the level of background staining.

Switch recombination activity is restricted to a short time interval in the course of B cell differentiation
The possibility to transfect primary murine B cells with SRS with good efficiencies and rapidly read out recombination, enabled us to analyze the kinetics of switch recombination activity in the course of B cell activation. Splenic murine B cells were polyclonally activated with LPS, and transfected 24, 48, 72 and 96 h after onset of stimulation. Transfected cells were cultured for another 15 h and analyzed as described above for the frequencies of hCD4-positive cells among H-2Kk-positive B cell blasts. Figure 3Go(A) shows a typical experiment. Results of three independent experiments are listed in Table 1Go. Among the B cells activated for 24 h, ~8% of the transfected cells have recombined the SRS, a frequency that increases to ~10% of the B cells activated for 48 h. Among B cells activated for 72 h, only 6% have recombined SRS, and even less, 2%, of the 96 h LPS blasts. SRS transfected X63Ag8.653 myeloma cells and B cells activated for 6 days have recombined the SRS on average with a frequency of 0.5%, which is about the same as obtained with a SRS without S region sequences (see below). X63 cells spontaneously switch their expressed endogenous IgH locus at frequencies of 10–6 to 10–7/cell/generation (33), i.e. they essentially do not perform Ig class switching. The data demonstrate that switch recombinase is active in LPSstimulated, primary B cells within the first 2–3 days after onset of activation and not longer.




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Fig. 3. Kinetics of switch recombination activity in primary B cell blasts in the course of LPS activation. (A) The efficiency of recombination of pSRSori-Sµ/S{gamma}2b was determined for the first 4 days after LPS stimulation. Flow cytometry was performed as described above (see Fig. 2Go; control: B cells transfected only with pH-2Kk). (B) Recombination activity (left y-axis) is compared to the total number of resting B cells and activated B cell blasts (right y-axis). Two lines delineate the window of recombination activity, reflecting the time span between transfection and analysis (15 h), minus 3 h to account for lag before expression of hCD4, i.e. 12 h.

 

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Table 1. Substrate recombination of pSRSori-Sµ/S{gamma}2b in the course of LPS activation
 
As a parameter for cell proliferation, the total numbers of resting versus activated B cells in the cultures were determined and related to substrate recombination efficiencies (Fig. 3BGo) (34). The number of B cell blasts increased exponentially until day 3, with no further increase thereafter, indicating that the vast majority of activated cells had finished exponential cell division 3 days after onset of activation (35,36). Switch recombination activity thus occurs before and during the phase of extensive proliferation of activated B cells.

Nevertheless, recombination of the SRS itself does not require DNA replication. Although in most experiments we have co-transfected SRS with a vector coding for Polyoma large T antigen (pCMV LT2), thus inducing replication of SRS, and facilitating readout and recovery, recombination of SRS was also observed in cells transfected with SRS alone, which would then not replicate. The results of a direct comparison are shown in Table 2Go. In all experiments, the frequencies of switched cells transfected with non-replicating SRS were lower than those of cells carrying replicating SRS, but significantly higher than those of control cells (Tables 3 and 4GoGo).


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Table 2. Recombination frequencies of replicating versus non-replicating pSRSori-Sµ/S{gamma}2b
 

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Table 3. Recombination frequencies of modified substrates in primary B cells
 

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Table 4. Recombination of the substrates in non-switching cells
 
SRS recombination is S region dependent
In activated primary B cells, recombination of SRS is strictly dependent on the presence of S region sequences in the substrate. This was shown by replacing the Sµ or the Sµ and S{gamma}2b components of the SRS for phage {lambda} sequences. The fragments of {lambda} DNA are not related to S regions, they are neither repetitive nor G/C-rich. They are not homologous to each other and are of about the same length as the S regions they replace. The vectors have been designated pSRSori-{lambda}1/S{gamma}2b and pSRSori-{lambda}1/{lambda}2 (Fig. 4AGo). B cells, stimulated with LPS for 2 days, were transfected with pSRSori-Sµ/S{gamma}2b or its modified derivatives. Recombination frequencies of the substrates in several independent experiments are listed in Table 3Go. The exchange of both S regions in the SRS for phage {lambda} sequences (pSRSori-{lambda}1/{lambda}2) reduces the frequency of SRS-switched cells by a factor of 9–14, to frequencies of cellular controls, i.e. late plasma blasts or the plasmacytoma line X63Ag8.563 (see below, Table 4Go).



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Fig. 4. Derivatives of the SRS. (A) In pSRSori-{lambda}1/S{gamma}2b and pSRSori-{lambda}1/{lambda}2 the S regions of pSRSori-Sµ/S{gamma}2b are replaced by phage {lambda} sequences (white hatched boxes). (B) In pSRSori-{Delta}sas the splice acceptor site located 3' of Sµ in pSRSori-Sµ/S{gamma}2b is deleted. The designation of other elements is as indicated in Fig. 1Go.

 
For the plasmid in which only Sµ was replaced by a {lambda} sequence, but not S{gamma}2b (pSRSori-{lambda}1/S{gamma}2b), recombination frequencies were also reduced, by a factor of 1–2, but still were considerably higher than in controls. Thus, an individual S region is apparently sufficient to initiate switch recombination, which can then progress into the 5' direction.

Splicing of the Sµ transcript enhances SRS recombination
Previously we had shown by replacement of the I{gamma}1 promoter and I{gamma}1 exon with the heterologous human metallothionein IIA (hMT) promoter that switch recombination is targeted to distinct S regions by the induction of switch transcripts. We had obtained preliminary evidence that splicing of the I exon to the heavy chain constant region exons might be crucial for this targeting (22). Here, we confirm this hypothesis by deleting the splice acceptor site of the last exon of the extracellular part of the hCD4 gene, 3' of Sµ, in pSRSori-{Delta}sas (Fig. 4BGo). In contrast to pSRSori-Sµ/S{gamma}2b, where the Sµ region is spliced out of the primary transcript (Fig. 1Go), it is not in pSRSori-{Delta}sas. Recombination of pSRSori-{Delta}sas was analyzed together with pSRSori-Sµ/S{gamma}2b and the vectors containing {lambda} sequences (Table 3Go). Compared to the original SRS, recombination frequencies of pSRSori-{Delta}sas were reduced ~2- to 3-fold in activated primary B cells, showing that recombination of SRS is indeed enhanced by processing of the primary switch transcript.

Substrate recombination in late B cell blasts and plasmacytoma cells
To analyze to what extent recombination of SRS at the low frequencies observed in late plasmablasts, activated for 6 days, and plasmacytoma cells is mediated by the class switch recombination machinery, we transfected X63Ag8.653 plasmacytoma cells and primary B cells after 6 days of LPS stimulation with all the SRS variants we had obtained (Table 4Go and Fig. 5Go). Except for one case, the frequencies of hCD4-expressing cells were <1%. On average we found 0.51% of the plasmacytoma cells and 0.53% of the LPS-activated day 6 B cell blasts expressing hCD4, irrespective of whether the transfected SRS contained S regions and splice signals or not. The average frequencies with which the substrates are recombined in the plasmacytoma cells and late B cell blasts match the frequencies (0.4%) we found for recombination of the substrate carrying no S region (pSRSori-{lambda}1/{lambda}2) in freshly activated B cells (2 days). This confirms the notion that switch recombinase is active in a short time window of 2–3 days after onset of activation in B lymphocytes.



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Fig. 5. The average recombination frequencies for all substrates and all analyzed cell types are depicted. The figure is based on the data listed in Tables 3 and 4GoGo.

 

    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
So far the analysis of the molecular requirements and kinetics of switch recombination by means of extrachromosomal recombination substrates has been hampered by either the low transfection efficiency of primary B cells or the low recombination activity of cells from B cell lines. Here, we describe an extrachromosomal SRS, recombination of which leads to the expression of a cell surface marker that can be analyzed cytometrically on viable cells. A second surface marker is used for identification and positive selection of transfected cells. Analysis by immunofluorescence offers the option to include additional parameters for analysis at the single cell level. We have used this approach to analyze kinetics and sequence requirements of switch recombination in primary murine B lymphocytes, activated with LPS in vitro.

Up to 10% of transfected B lymphocytes perform recombination of the substrate after activation by LPS. However, recombination activity is restricted to the first 72 h after onset of mitogenic stimulation and then rapidly decreases. In B cells stimulated for 6 days and cells of the plasmacytoma line X63Ag8.653, recombination frequencies are reduced to background levels, as obtained with a substrate carrying no S region sequences. Recombination frequencies were also reduced when splicing of the primary Sµ transcript was blocked or when the Sµ region was replaced by phage {lambda} DNA.

An extrachomosomal SRS mimicking the endogenous IgH locus
In contrast to substrates used in previous analyses (3740), the SRS used in the present analysis matches the structure of the endogenous IgH locus with respect to the location of S regions in introns of processed transcripts. Suggestive evidence has been obtained by targeted mutation of the endogenous murine IgH locus that induction of a processed switch transcript may be necessary for recombination of the intronic S region (18,22). For SRS used here, two such transcription units are located adjacent to each other, separated by efficient transcriptional and translational stop sequences. The generation of two separate processed transcripts has been confirmed by Northern blotting (Christine et al., in press). Thus, the structural requirements of switch transcripts to target switch recombination, like promoter, exon–intron structure, splice signals, presence and location of S regions, can readily be tested by modification of the substrate elements.

Apart from providing for the first time a structural image of the endogenous S region environment, our SRS concept has the fundamental advantage to report successful recombination by display of a novel cell surface marker, hCD4. After recombination of the two intronic S regions on the SRS, the 5' and 3' exons of hCD4, which had been separated by the transcriptional stop element, now form one transcription unit, and the membrane form of hCD4 is expressed on the cell surface, without interfering with cell viability. Cells containing switched SRS can be analyzed cytometrically and isolated alive for further analysis. After transfection with preswitched SRS (phCD4ori), the cytometric reporter molecule is expressed on the cell surface of the transfected cells within 3 h (unpublished data). This rapid display allows us to read out the kinetics of switch recombination activity in B cells activated in vitro.

We have used the SRS to analyze switch recombination in the pre-B cell line 18.81 (Christine et al., in press), the myeloma cell line X63Ag8.653 and primary murine splenic B lymphocytes, activated for switch recombination by LPS (41,42).

Switch recombinase is only active early in B cell activation
In LPS-activated B lymphocytes, recombination of endogenous IgH loci so far has been analyzed on the level of expressed switched Ig, by plaque assay, ELISA and intracellular or surface immunofluorescence, and on the level of genomic DNA, by restriction endonuclease analysis and digestion–circularization PCR (2,4346). The six murine acceptor S regions S{gamma}3, S{gamma}1, S{gamma}2b, S{gamma}2a, S{varepsilon} and S{alpha} are involved in LPS-driven switch recombination with different frequencies, with those for S{gamma}3 and S{gamma}2b being the highest (1,3,47). Co-stimulation by IL-4 changes switch frequencies dramatically, shutting off switching to S{gamma}3 and S{gamma}2b, and inducing switching to S{gamma}1 and S{varepsilon} (12,4853). The frequency of switched cells among activated cells reaches a maximum of ~25% within a few days of in vitro culture (43). It has not been clear whether this maximum reflects transient activity of the switch recombinase or a subpopulation of B cells, with a long-lasting activity of the switch recombinase, having been induced for class switching and finally having recombined all their IgH loci or both (13). On the molecular level, switch recombination has first been observed after 2–3 days (13,54), but attack of the S regions may begin earlier, since S region-specific double-strand breaks are detectable by LM-PCR within 4 h after onset of activation (55).

To determine the kinetics of switch recombination, in the present analysis primary murine B cells, activated with LPS in vitro, were transfected with the SRS at various times after onset of activation and substrate recombination was assayed within the next 15 h. The frequency of cells expressing the recombination reporter gene hCD4 peaks on day 2–3 after onset of activation and decreases rapidly thereafter. In B cells stimulated with LPS for 6 days, the frequency was reduced >10-fold, reaching the same level as a control substrate without S regions, or the cellular control, X63Ag8.653 cells. These results clearly show for the first time that activity of switch recombination is restricted to a short time interval after activation of primary B cells. It remains obscure, however, whether this is the case in all B cells or just in a subpopulation.

The kinetics of switch recombination activity determined here coincides with proliferation of B cell blasts, corroborating the observation that switch recombination is restricted to proliferating cells, as has been noted before (45,56,57). Further, the low recombination activity in late plasma blasts, from day 6 onwards, is in line with earlier observations showing that plasmacytoma cells are not active for switch recombination, even though they proliferate (5860).

The timed expression of switch recombination activity in early activated B cells explains why the expression of switch transcripts may be a necessary but certainly is an insufficient condition of switch recombination. Switch transcripts were detectable several hours after onset of activation of B cells and the frequency of transcripts further increased till the end of the analysis on day 4 or 5 (52,61,62), even though transcription units were destroyed by switch recombination. In addition, since autonomous replication of SRS is similar at all times analyzed, as controlled by the preswitched SRS (phCD4ori, unpublished data), but recombination activity is restricted to a short time interval after stimulation, the data presented here imply that DNA replication is not a sufficient condition for switch recombination (31,32). Rather, expression of switch-relevant proteins may be cell cycle dependent. This assumption is supported by the present experiments indicating that a non-replicating SRS is recombined as well. The differences in frequencies of cells displaying switched non-replicating versus replicating SRS are most likely due to the fact that non-replicating recombined SRS is not amplified and has a shorter average persistence in the cell. Thus expression of the marker is lower (data not shown) and less cells are scored positive.

Analysis of substrate recombination described here was performed 15 h after transfection, which left the cells sufficient time for a complete cell cycle (35,36). The frequencies of cells expressing switched SRS, up to 10% within 12 h (15 minus 3 h to account for lag before expression of hCD4), are similar to the estimated frequency of cells per generation performing switch recombination of the endogenous IgH locus (2).

S regions render processable transcription units recombinogenic
Apart from the striking correlation between switch recombination on both IgH loci of individual B cells in isotype targeting (12), the correlation between transcription of S regions and their targeting for recombination (14,15) has led to the hypothesis that switch recombination is targeted to distinct S regions in the context of transcriptional accessibility. Targeted mutation of the transcription control elements in the murine germline and analysis of B cells from mutant mice added suggestive evidence to this concept (1823). Unexpectedly, transcription of S regions was found to be insufficient to target recombination to the transcribed region. Rather, switch recombination seemed to be dependent on processing of the primary switch transcripts (22,63).

The extrachromosomal substrates described here confirm and extend these results. Interfering with processing of the substrate transcript by deletion of a splice acceptor site results in a 2- to 3-fold reduction in the frequency of recombination, as compared to the original SRS. It is not entirely clear why this reduction is not more drastic. A possible explanation could be that deletion of the heterologous splice acceptor site 3' of Sµ did not completely abolish processing of transcripts, but rather was complemented for by alternative splice acceptor sites of Thy-1.2 exons. An alternative explanation could be that switch recombination extended from the S{gamma}2b transcription unit into the Sµ-transcription unit, an explanation that we also favor for the relatively high recombination frequencies upon replacement of Sµ, but not S{gamma}2b, by phage {lambda} sequences. In contrast to the substrate without S regions (pSRSori-{lambda}1/{lambda}2), pSRSori-{lambda}1/S{gamma}2b is recombined in activated B cells at significantly higher frequencies. This is in accord with previous evidence that switch recombination may consist of multiple recombination events within and between accessible S regions, and may extend into sequences located 5' of the S region (11,13,20,40,64,65). It is more of a challenge to explain why previous analyses of switch recombination with other substrates (3740) have not shown its dependency on processing of switch transcripts, as found for the endogenous IgH locus (22) and the substrates described here. However, for those previous analyses, a direct comparison of transcribed versus transcribed and processed transcripts is lacking. It remains to be determined to what extent recombination frequencies could have been enhanced through processing of switch transcripts from those substrates.

In general, these and our results point to the limitations of episomal SRS. They hardly can mimick the physiological distance between S regions nor the entire chromosomal context. Nevertheless, even in their relatively `open' constitution they show in principle the same requirements as endogenous Ig loci, analyzed by targeted mutation. The present SRS analysis, in accordance with the results from targeted mutation of the murine germline, shows that S regions can render recombinogenic for switch recombinase a heterologous transcription unit that is transcribed and processed by splicing. The high risk of switch recombination leading to deleterious or dangerous mutations is limited by the very restricted expression of switch recombinase in the lifetime of B cells.


    Acknowledgments
 
We thank Sigrid Irlenbusch and Gertrud von Hesberg for technical assistance, and Steffen Jung, Andreas Thiel and Alexander Scheffold for technical advice. For generous gifts of reagents we thank Stefan Miltenyi, Jürgen Schmitz, Walter Weichel and Mario Assenmacher. This work was supported by the Deutsche Forschungsgemeinschaft through SFB 243.


    Abbreviations
 
LPSlipopolysaccharide
PEphycoerythrin
PIpropidium iodide
SRSswitch recombination substrate

    Notes
 
Transmitting editor: M. Neuberger

Received 9 October 1998, accepted 29 January 1999.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Kearney, J. F., Cooper, M. D. and Lawton, A. R. 1976. B cell differentiation induced by lipopolysaccharide. IV. Development of immunoglobulin class restriction in precursors of IgG-synthesising cells. J. Immunol. 117:1567.[Abstract]
  2. Radbruch, A. and Sablitzky, F. 1983. Deletion of Cµ genes in mouse B-lymphocytes upon stimulation with LPS. EMBO J. 2:1929.[ISI][Medline]
  3. Yuan, D. and Vitetta, E. 1983. Structural studies of cell surface and secreted IgG in LPS-stimulated murine B cells. Mol. Immunol. 20:367.[ISI][Medline]
  4. Irsch, J., Irlenbusch, S., Radl, J., Burrows, P. D., Cooper, M. D. and Radbruch, A. 1993. Switch recombination in normal IgA1+ B lymphocytes. Proc. Natl Acad. Sci. USA 91:1323.[Abstract]
  5. von Schwedler, U., Jäck, H. M. and Wabl, M. 1990. Circular DNA is a product of immunoglobulin class switch rearrangement. Nature 345:452.[ISI][Medline]
  6. Honjo, T. and Kataoka, T. 1978. Organization of the immunoglobulin heavy chain genes and allelic deletion model. Proc. Natl Acad. Sci. USA 75:2140.[Abstract]
  7. Kataoka, T., Kawakami, T., Takahashi, N. and Honjo, T. 1980. Rearrangement of immunoglobulin {gamma}1-chain gene and mechanism for heavy-chain class switch. Proc. Natl Acad. Sci. USA 77:919.[Abstract]
  8. Davis, M. M., Calme, K., Early, P. W., Livant, D. L., Joho, R., Weissman, I. L. and Hood, L. 1980. An immunoglobulin heavy-chain gene is formed by at least two recombination events. Nature 283:733.[ISI][Medline]
  9. Kataoka, T., Miyata, T. and Honjo, T. 1981. Repetitive sequences in class-switch recombination regions of immunoglobulin heavy chain genes. Cell 23: 357.[ISI][Medline]
  10. Gritzmacher, C. A. 1989. Molecular aspects of heavy-chain class switching. Crit. Rev. Immunol. 9:173.[ISI][Medline]
  11. Dunnick, W., Hert, G. Z., Scappino, L. and Gritzmacher, C. 1993. DNA sequences at immunoglobulin switch region recombination sites. Nucleic Acid Res. 21:365.[Abstract]
  12. Radbruch, A., Müller, W. and Rajewsky, K. 1986. Class switch recombination is IgG1 specific on active and inactive IgH loci of IgG1-secreting B-cell blasts. Proc. Natl Acad. Sci. USA 83:3954.[Abstract]
  13. Winter, E., Krawinkel, U. and Radbruch, A. 1987. Directed Ig class switch recombination in activated murine B cells. EMBO J. 6:1663.[Abstract]
  14. Alt, F. W., Blackwell, T. K., DePinho, R. A. and Reth, M. G. 1986. Regulation of genome rearrangement events during lymphocyte differentiation. Immunol. Rev. 89:5.[ISI][Medline]
  15. Stavnezer-Nordgren, J. and Serlin, S. 1986. Specificity of immunoglobulin heavy chain switch correlates with activity of germline heavy chain genes prior to switching. EMBO J. 5:95.[Abstract]
  16. Lorenz, M. and Radbruch, A. 1996. Developmental and molecular regulation of immunoglobulin class switch recombination. Curr. Top. Microbiol. Immunol. 217:151.[ISI][Medline]
  17. Stavnezer, J. 1996. Antibody class switching. Adv. Immunol. 61:79.[ISI][Medline]
  18. Jung, S., Rajewsky, K. and Radbruch, A. 1993. Shutdown of class switch recombination by deletion of a switch region control element. Science 259:984.[ISI][Medline]
  19. Zhang, J., Bottaro, A., Li, S., Stewart, V. and Alt, F. W. 1993. A selective defect in IgG2b switching as a result of targeted mutation of the I{gamma}2b promoter and exon. EMBO J. 12:3529.[Abstract]
  20. Gu, H., Zou, Y.-R. and Rajewsky, K. 1993. Independent control of immunoglobulin switch recombination at individual switch regions evidenced through cre-lox-P-mediated gene targeting. Cell 73:1155.[ISI][Medline]
  21. Bottaro, A., Lansford, R., Xu, L., Zhang, J., Rothman, P. and Alt, F. W. 1994. S-region transcription per se promotes basal IgE class switch recombination but additional factors regulate the efficiency of the process. EMBO J. 13:665.[Abstract]
  22. Lorenz, M., Jung, S. and Radbruch, A. 1995. Switch transcripts in immunoglobulin class switching. Science 267:1825.[ISI][Medline]
  23. Harriman, G. R., Bradley, A., Das, S., RogerS-Fani, P. and Davis, A. C. 1996. IgA class switch in I{alpha} exon-deficient mice. Role of germline transcription in class switch recombination. J. Clin. Invest. 97:477.[Abstract/Free Full Text]
  24. Enriquez-Harris, P., Levitt, N., Briggs, D. and Proudfoot, N. J. 1991. A pause site for RNA polymerase II is associated with termination of transcription. EMBO J. 10:1833.[Abstract]
  25. Weichel, W. 1989. Experimente zur Sekretion rekombinanter H-2Kk-Antigene. Doctoral thesis, University of Cologne, Germany
  26. Strauss, M., Hering, S., Lubbe, L. and Griffin, B. E. 1990. Immortalization and transformation of human fibroblasts by regulated expression of Polyoma virus T antigens. Oncogene 5:1223.[ISI][Medline]
  27. Marshak-Rothstein, A., Fink, P., Gridley, T., Rault, D. H., Bevan, M. J. and Gefter, M. L. 1979. Properties and application of monoclonal antibodies directed against determinants of they Thy-1 locus. J. Immunol. 122:2491.[Abstract]
  28. Coffman, R. L. and Weissman, I. L. 1981. B220: a B cell-specific member of the T200 glycoprotein family. Nature 289:681.[ISI][Medline]
  29. Grützman, R. 1981. Comparative idiotypic analysis of receptors for histocompatibility antigens. Doctoral thesis, University of Cologne, Germany.
  30. Reth, M., Hämmerling, G. J. and Rajewsky, K. 1978. Analysis of the repertoire of anti-NP antibodies in C57BL/6 mice by cell fusion. I. Characterization of antibody families in the primary and hyperimmune response. Eur. J. Immunol. 8:393.[ISI][Medline]
  31. van der Loo, W., Severinson-Gronowicz, E., Strober, S. and Herzenberg, L. A. 1979. Cell differentiation in the presence of cytochalasin B: studies on the `switch' to IgG secretion after polyclonal B cell activation. J. Immunol. 122:1203.[Abstract]
  32. Dunnick, W., Wilson, M. and Stavnezer, J. 1989. Mutations, duplications and deletions of recombinant switch regions suggest a role for DNA replication in the immunoglobulin heavy-chain switch. Mol. Cell. Biol. 9:1850.[ISI][Medline]
  33. Radbruch, A., Liesegang, B. and Rajewsky, K. 1980. Isolation of variants of mouse myeloma X63 that express changed immunoglobulin class. Proc. Natl Acad. Sci. USA 77:2909.[Abstract]
  34. Wetzel, G. D., Swain, S. L., Dutton, R. W. and Kettman, J. R. 1984. Evidence for two distinct activation states available to B lymphocytes. J. Immunol. 133:2327.[Abstract/Free Full Text]
  35. Kenter, A. L. and Watson, J. V. 1987. Cell cycle kinetics model of LPS-stimulated spleen cells correlates switch region rearrangements with S phases. J. Immunol. Methods 97:111.[ISI][Medline]
  36. Seyschab, H., Friedl, R., Schindler, D., Hoehn, H., Rabinovitch, P. S. and Chen, U. 1989. The effects of bacterial lipopolysaccharide, anti-receptor antibodies and recombinant interferon on mouse B cell cycle progression using 5-bromo-2'-deoxyuridine/Hoechst 33258 dye flow cytometry. Eur. J. Immunol. 19:1605.[ISI][Medline]
  37. Ott, D. E., Alt, F. W. and Marcu, K. B. 1987. Immunoglobulin heavy chain switch region recombination within a retroviral vector in murine pre-B cells. EMBO J. 6:577.[Abstract]
  38. Leung, H. and Maizels, N. 1992. Transcriptional regulatory elements stimulate recombination in extrachromosomal substrates carrying immunoglobulin switch-region sequences. Proc. Natl Acad. Sci. USA 89:4154.[Abstract]
  39. Lepse, C. L., Kumar, R. and Ganea, D. 1994. Extrachromosomal eukaryotic DNA substrates for switch recombination: analysis of isotype and cell specificity. DNA Cell Biol. 13:1151.[ISI][Medline]
  40. Daniels, G. A. and Lieber, M. R. 1995. Strand specificity in the transcriptional targeting of recombination at immunoglobulin switch sequences. Proc. Natl Acad. Sci. USA 92:5625.[Abstract]
  41. Severinson, E., Bergstedt-Lindqvist, S., van der Loo, W. and Fernandez, C. 1982. Characterisation of the IgG response induced by polyclonal B cell activators. Immunol. Rev. 67:73.[ISI][Medline]
  42. Cebra, J. J., Komisar, J. L. and Schweitzer, P. A. 1984. CH isotype switching during normal B-lymphocyte development. Annu. Rev. Immunol. 2:493.[ISI][Medline]
  43. Kearney, J. F. and Lawton, A. R. 1975. B lymphocyte differentiation induced by polysaccharide. I. Generation of cells synthesising four major immunoglobulin antibodies. J. Immunol. 115:671.[Abstract]
  44. Andersson, J., Coutinho, A. and Melchers, F. 1978. The switch from IgM to IgG secretion in single mitogen-stimulated B-cell clones. J. Exp. Med. 147:1744.[Abstract]
  45. Severinson-Gronowicz, E., Doss, C. and Schröder, J. 1979. Activation to IgG secretion by lipopolysaccharide requires several proliferation cycles. J. Immunol. 123:2057.[Abstract]
  46. Chu, C. C., Paul, W. E. and Max, E. E. 1992. Quantitation of immunoglobulin µ–{gamma}1 heavy chain switch region recombination by a digestion–circularization polymerase chain reaction method. Proc. Natl Acad. Sci. USA 89:6978.[Abstract]
  47. Coutinho, A. and Forni, L. 1982. Intraclonal diversification in immunoglobulin isotype secretion: an analysis of switch probabilities. EMBO J. 1:1251.[ISI][Medline]
  48. Isakson, P. C., Pure, E., Vitetta, E. S. and Krammer, P. H. 1982. T cell derived B cell differentiation factor(s). Effect on the isotype switch of murine B cells. J. Exp. Med. 155:734.[Abstract]
  49. Layton, J. E., Vitetta, E. S., Uhr, J. W. and Krammer, P. H. 1984. Clonal analysis of B cells induced to secrete IgG by T cell-derived lymphokine(s). J. Exp. Med. 160:1850.[Abstract]
  50. Coffman, R. and Carty, J. 1986. A T cell activity that enhances polyclonal IgE production and its inhibition by interferon-{gamma}. J. Immunol. 136:949.[Abstract/Free Full Text]
  51. Rothman, P., Lutzker, S., Cook, W., Coffman, R. and Alt, F. W. 1988. Mitogen plus interleukin 4 induction of C{varepsilon} transcripts in B lymphoid cells. J. Exp. Med. 168:2385.[Abstract]
  52. Lutzker, S., Rothman, P., Pollock, R., Coffman, R. and Alt, F. W. 1988. Mitogen- and IL-4-regulated expression of germline Ig {gamma}2b transcripts: evidence for directed heavy chain class switching. Cell 53:177.[ISI][Medline]
  53. Severinson, E., Fernandez, C. and Stavnezer, J. 1990. Induction of germ-line immunoglobulin heavy chain transcripts by mitogens and interleukins prior to switch recombination. Eur. J. Immunol. 20:1079.[ISI][Medline]
  54. Chu, C. C., Max, E. E. and Paul, W. E. 1993. DNA rearrangement can account for in vitro switching to IgG1. J. Exp. Med. 178:1381.[Abstract]
  55. Wuerffel, R. A., Du, J., Thompson, R. J. and Kenter, A. L. 1997. Ig S{gamma}3 DNA-specific double strand breaks are induced in mitogen-activated B cells and are implicated in switch recombination. J. Immunol. 159:4139.[Abstract]
  56. Lundgren, M., Strom, L., Bergquist, L. O., Skon, S., Heiden, T., Stavnezer, J. and Severinson, E. 1995. Cell cycle regulation of immunoglobulin class switch recombination and germ-line transcription: potential role of Ets family members. Eur. J. Immunol. 25:2042.[ISI][Medline]
  57. Hodgkin, P. D., Lee, J.-H. and Lyons, A. B. 1996. B cell differentiation and isotype switch is related to division cycle number. J. Exp. Med. 184:277.[Abstract]
  58. Sablitzky, F., Radbruch, A. and Rajewsky, K. 1982. Spontaneous immunoglobulin class switching in myeloma and hybridoma cell lines differs from physiological class switching. Immunol. Rev. 67:59.[ISI][Medline]
  59. Ott, D. E. and Marcu, K. B. 1989. Molecular requirements for immunoglobulin heavy chain constant region gene switch-recombination revealed with switch-substrate retroviruses. Int. Immunol. 1:582.[Medline]
  60. Klein, S. and Radbruch, A. 1994. Inhibition of class switch recombination in plasma cells. Cell. Immunol. 157:106.[ISI][Medline]
  61. Esser, C. and Radbruch, A. 1989. Rapid induction of transcription of unrearranged S{gamma}1 switch regions in activated murine B-cells by interleukin 4. EMBO J. 8:483.[Abstract]
  62. Rothman, P., Lutzker, S., Gorham, B., Stewart, V., Coffman, R. and Alt, F. W. 1990. Structure and expression of germline immunoglobulin {gamma}3 heavy chain gene transcripts: implication for mitogen and lymphokine directed class-switching. Int. Immunol. 2:621.[ISI][Medline]
  63. Hein, K., Lorenz, M., Petry, K. and Radbruch, A. 1998. Splicing of switch transcripts is required for targeting of antibody class switch recombination. J. Exp. Med. 188:2369[Abstract/Free Full Text]
  64. Zhang, K., Cheah, H. K. and Saxon, A. 1995. Secondary deletion recombination of rearranged switch region in Ig isotype-switched B cells. A mechanism for isotype stabilization. J. Immunol. 154:2237.[Abstract/Free Full Text]
  65. Leung, H. and Maizels, N. 1994 Regulation and targeting of recombination in extrachromosomal substrates carrying immunoglobulin switch region sequences. Mol. Cell. Biol. 14:1450.[Abstract]