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
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Abstract |
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Keywords: B lymphocytes, class switch recombination, Ig, lipopolysaccharide, primary cells, transient transfection
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Introduction |
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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)17(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.
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Methods |
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In pSRSori-1/S
2 and pSRSori-
1/
2, the S regions were replaced by a 1.8 kb BssHII fragment or a 3.4 kb BsaAI fragment [
1: bp 1481516649;
2: bp 1725020653 of the
(c1857 Sam 7) genome; Boehringer, Mannheim, Germany]. In pSRSori-
sas, the 3' Sµ splice acceptor site was deleted as a 0.3 kb PstI fragment from pSRSori-Sµ/S
2. phCD4ori was generated from pSRSori-
1/
2 by deletion of the region spanning 5' Sµ to 3' S
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 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-hCD4phycoerythrin (PE) and anti-H-2KkFITC 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. 2. CellQuest software (Becton Dickinson) was used for evaluation of data.
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Results |
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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. 2). 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
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 2 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 3(A) shows a typical experiment. Results of three independent experiments are listed in Table 1
. 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 106 to 107/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 23 days after onset of activation and not longer.
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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 2. 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 4
).
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Splicing of the Sµ transcript enhances SRS recombination
Previously we had shown by replacement of the I1 promoter and I
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-
sas (Fig. 4B
). In contrast to pSRSori-Sµ/S
2b, where the Sµ region is spliced out of the primary transcript (Fig. 1
), it is not in pSRSori-
sas. Recombination of pSRSori-
sas was analyzed together with pSRSori-Sµ/S
2b and the vectors containing
sequences (Table 3
). Compared to the original SRS, recombination frequencies of pSRSori-
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 4 and Fig. 5
). 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-
1/
2) in freshly activated B cells (2 days). This confirms the notion that switch recombinase is active in a short time window of 23 days after onset of activation in B lymphocytes.
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Discussion |
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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 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, exonintron 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 digestioncircularization PCR (2,4346). The six murine acceptor S regions S3, S
1, S
2b, S
2a, S
and S
are involved in LPS-driven switch recombination with different frequencies, with those for S
3 and S
2b being the highest (1,3,47). Co-stimulation by IL-4 changes switch frequencies dramatically, shutting off switching to S
3 and S
2b, and inducing switching to S
1 and S
(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 23 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 23 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 S2b 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
2b, by phage
sequences. In contrast to the substrate without S regions (pSRSori-
1/
2), pSRSori-
1/S
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.
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Acknowledgments |
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Abbreviations |
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LPS | lipopolysaccharide |
PE | phycoerythrin |
PI | propidium iodide |
SRS | switch recombination substrate |
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Notes |
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Received 9 October 1998, accepted 29 January 1999.
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References |
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