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The Journal of Immunology, 1998, 161: 1336-1347.
Copyright © 1998 by The American Association of Immunologists

Efficient Recombination of a Switch Substrate Retrovector in CD40-Activated B Lymphocytes: Implications for the Control of CH Gene Switch Recombination1

Jack Ballantyne2,*, Diane L. Henry2,*, Jurgen R. Muller2,*, Francine Briere{dagger}, Clifford M. Snapper{ddagger}, Marilyn Kehry§ and Kenneth B. Marcu3,*

* Department of Biochemistry and Cell Biology, Institute for Cell and Developmental Biology, State University of New York, Stony Brook, NY 11794; {dagger} Laboratory for Immunological Research, Schering-Plough, Dardilly, France; {ddagger} Department of Pathology, F. Edward Hebert School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, MD 20814; and § Department of Immunological Diseases, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, CT 06877


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Maturing B lymphocytes possess a recombination activity that switches the class of heavy chain Ig. The nature of the recombination activity, its molecular requirements and regulation remain elusive questions about B lymphocyte biology and development. Class switch recombination is controlled by cytokine response elements that are required to differentially activate CH gene transcription before their subsequent recombination. Here, we show that cultures of purified murine and human B cells, stimulated only by CD40 receptor engagement, possess a potent switch recombination activity. CD40 ligand-stimulated murine and human B lymphocytes were infected with recombinant retroviruses containing Sµ and S{gamma}2b sequences. Chromosomally integrated switch substrate retrovectors (SSRs), harboring constitutively transcribed S sequences, underwent extensive recombinations restricted to their S sequences with structural features akin to endogenous switching. SSR recombination commenced 4 days postinfection (5 days poststimulation) with extensive switch sequence recombination over the next 2 to 3 days. In contrast, endogenous S{gamma}2b and S{gamma}1 sequences did not undergo appreciable switch recombination upon CD40 signaling alone. As expected, IL-4 induced endogenous Sµ to S{gamma}1 switching, while endogenous Sµ to S{gamma}2b fusions remained undetectable. Surprisingly, IL-4 enhanced the onset of SSR recombination in CD40-stimulated murine B cells, with S-S products appearing only 2 days postinfection and reaching a maximum within 2 to 3 days. The efficiency of switch recombination with SSRs ressembles that seen for endogenous CH class switching.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Antibody class (or isotype) switching, by which Ig heavy chains of a given Ag specificity acquire different effector functions, is principally mediated by class switch recombination. Switch recombination has been documented as an illegitimate, nonhomologous DNA rearrangement event juxtaposing a functionally assembled VDJ gene segment (initially 5' of the Cµ gene in an IgM-bearing B cell) in close proximity to a 3' distal CH gene ({gamma}3,{gamma}1,{gamma}2b,{gamma}2a,{epsilon} or {alpha} in the murine nomenclature) (1-5). Switch recombination occurs by a looping out and deletion mechanism resulting in the loss of all intervening CH genes in the form of a circular byproduct (2-5). Recombinations are mediated by switch (S)4 target sequences that are positioned 1 to 4 kb upstream of each CH gene segment. S regions consist of short, tandemly repetitious sequences of DNA, each possessing a characteristic unique sequence but having an overall similarity to all other S regions (1).

Induction of Ig secretion and class switching can be mediated by both T cell-dependent and -independent (T-dependent and -independent) Ags that operate via different signaling mechanisms. Whereas T-dependent signaling between cognate B and T cells is mediated through the CD40R:CD40L system, T-independent Ags (as exemplified by bacterial polysaccharides) extensively cross-link surface Ig receptors (sIg) to deliver activation signals (6), and simultaneous activation through both pathways may occur in vivo (7, 8). Interestingly, the use of multivalent sIg cross-linking by dextran-conjugated anti-IgD ({alpha}{delta}-dex) or anti-IgM ({alpha}µ-dex) was found to be strongly synergistic with CD40 signaling for cellular proliferation, viability, class switch recombination, and Ig secretion (9). In vivo switching to specific IgCH isotypes in response to T-dependent Ags involves the costimulation of CD40 along with one of a number of cytokines (IL-4, IL-5, IL-10, IFN-{gamma}, and TGF-ß) (10). The isotype switch requires cell division and occurs in the S phase of the cell cycle (11-16). Isotype switching was shown to be dependent on cell division number rather than the length of time the cells have been stimulated (16).

Extensive evidence favors a model of directed class switching that is in part manifested by the differential transcriptional competence of the CH genes. Switching to a particular CH gene segment is preceded by the expression of its "sterile" or so-called germline transcript (2-5), and a firm correlation has been established for the cytokine-mediated induction of such germline CH RNAs and the subsequent switch recombination (4). Cytokine and mitogen-inducible germline CH RNAs contain a short IH exon (residing 5' of each S region) that is spliced to the 3' adjacent CH region (4). Since most I exons possess translation stop signals in all three reading frames, it is unlikely that germline CH RNAs encode functional protein products (4), although a single report has demonstrated that a truncated Cµ protein can be generated in vitro from CH germline RNA (17). The transcriptional activity of the donor and acceptor CH gene segments remain prerequisites for switch recombinase targeting. This transcribed status is believed to engender a particular CH gene with the "accessibility" to undergo switch recombination (hence the so-called accessibility model for switch recombination) (4). Alterations in chromatin configuration induced by transcription of the transcripts themselves may be responsible for the increased accessibility of the S sequences for switch recombination, but the role(s) of sterile CH transcription in class switch recombination remain unknown.

The loss of sterile CH transcripts produced by homologous gene targeting in mice is generally correlated, both in vitro and in vivo, with the absence or reduction of class switch recombination to that CH gene. Mutation or deletion of the I regions and associated promoters of the C{gamma}1 (18) and C{gamma}2b (19) genes resulted in the cis-acting suppression of their recombination. Similarly, deletion of some of the JH segments, all of Eµ, and part of the Iµ exon abrogated switch recombination at the Cµ locus (20). Replacement of the IL-4-inducible I{epsilon} promoter and exon with a highly transcriptionally competent VH promoter/Eµ enhancer in normal B cells and in the 18.81A20 pre-B cell line results in switching to IgE but at a greatly reduced frequency (only about 1-10% of the frequency seen upon IL-4 stimulation in the wild type) (21, 22). These and findings with other cytokines indicate that accessibility involves more than the transcriptional process itself (5).

It remains unknown how the tandem repetitive S regions mediate switching and how switching is molecularly regulated. S sequences may represent a structural motif that is directly recognized by the DNA-binding domain of a common switch recombinase; and S region-binding proteins may act as accessory molecules to recruit and target the recombination machinery by specific protein-protein interactions (23). Alternatively, independent isotype-specific switch recombinases may exist with differential substrate specificities (24). Recently, evidence for a sequence-specific double strand break in the S{gamma}3 region was proposed as an initiating step for the recombination process (25). An error prone DNA repair activity is also involved in the switch recombination process, as evidenced by the accumulation of mutations nearby switch recombination sites (12, 13). The recent identification of a B cell-specific exonuclease, in which activity is modulated by germline CH switch region transcripts, implies a more direct role for switch region transcripts in the recombination mechanism (26).

In vivo measurements of endogenous class switch recombination do not distinguish regulation of S sequence accessibility from recombinase activity itself or its targeting. Defining the class switch recombinase mechanism and its regulation by necessity requires the separation of accessibility control from the direct control of the recombinase activity itself. The switch recombination field has been hampered by the lack of an efficacious, robust assay for switch recombinase activity in normal B lymphoctyes. By employing retroviruses to stably transduce S region sequences into cells, we previously demonstrated that constitutively transcribed S sequences are sufficient for mediating S sequence-specific recombination in several immortalized lines of late stage pre-B and mature B cells but were recombinationally inactive in non-B cell lines (23, 27-29). An independent study with a minichromosome substrate demonstrated that S region transcription only targeted switch recombination when the S sequences were in their normal physiologic orientation (30), implying that strand-specific RNA produced from the downstream S region may also play a role in switch recombination.

Here, chromosomally integrated switch substrate retrovectors (SSRs) are employed to detect switch recombinase activity in cultures of normal B cells. We show that CD40-stimulated mature B cells have the capacity to recombine the majority of constitutively accessible SSR targets within several days of in vitro culture and that IL-4 acts in concert with CD40L to potentiate their recombination. Switch recombination of the endogenous CH locus follows a time course that is similar to the switch substrate retrovector but is dependent on both CD40L and an exogenous cytokine.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture

All growth media (GM) were supplemented with penicillin (100 U/ml) and streptomycin (100 mg/ml). Murine splenic B cells were cultured in RPMI 1640 supplemented with 5% FBS, 2-ME (50 µM), and L-glutamine (2 mM). Human tonsillar naive B cells were cultured in Iscove’s medium supplemented with 5% FBS, human transferrin (50 mg/ml), bovine insulin (5 mg/ml), and 2-ME (50 µM). Ltk- cells stably expressing human cell surface CD40L (Ltk-hCD40L) (31, 32) were cultured in RPMI 1640 supplemented with 10% FBS and L-glutamine (2 mM).

Isolation of resting murine splenic B lymphocytes

Spleens were removed from DBA/2 mice and washed several times in GM, and the cells were dissociated by mechanical agitation. RBC were removed by suspension in 5 ml ACK lysing buffer (0.15 M NH4Cl, 1 mM KHCO3, 0.1 mM Na2EDTA, pH 7.2-7.4) and incubation at ambient temperature with occasional agitation. T cells were removed next by complement-mediated immune lysis with anti-Thy-1.2-specific mAb. Briefly, 107 cells were suspended in 1 ml GM and CD90 Thy-1.2 anti-mouse Ab (PharMingen, San Diego, CA) added to 10 µg/ml and incubated at 37°C for 30 min. The unbound mAb was removed by centrifugation and the cell pellet resuspended in filter-sterilized, 10-fold-diluted rabbit serum complement (Low Tox-M rabbit complement; Cedarlane Laboratories, Hornby, Ontario, Canada) in GM to 107 cells/ml. After incubation at 37°C for 30 min, 10 ml of GM was added, the resulting cell suspension centrifuged, and the supernatant (containing the unbound complement) discarded. The resulting T cell-depleted B cell pellet from each spleen was resuspended in 2.5 ml of ice cold HBSS (Life Technologies, Gaithersburg, MD). Total B cells were layered on a step gradient consisting of 50, 60, 66, and 70% Percoll (Pharmacia, Piscataway, NJ) and separated by centrifugation for 13 min at 1900 x g. The HBSS and 50% Percoll layers were aspirated and the small resting B cells at the 66/70% interface collected. The small resting B cells were washed three times with HBSS before further treatment with polyclonal activators and/or cytokines. Percoll gradient-purified cells were judged to be highly purified populations of resting, mature B cells by staining with IgM and B220 cell surface markers and by the absence of T cell-specific markers such as Thy1.2, CD4 and CD8 (data not shown).

Isolation of sIgD+ B cells from human tonsils

Naive human tonsillar B cells were isolated as previously described (33). In brief, tonsils taken from patients during routine tonsillectomy were finely minced and the resulting cell suspension subjected to T cell depletion by rosetting with SRBC and standard Ficoll gradient separation. Isolation of sIgD+ populations was performed using a preparative magnetic cell separation system (MACS; Becton Dickinson, Mountain View, CA) (33). Briefly, unfractionated tonsil B cells suspended in PBS/1% BSA/0.01% sodium azide/5 mM EDTA, were labeled by incubation with biotinylated goat anti-human IgD Abs. After washing in PBS/sodium azide/EDTA, B cells were resuspended at 2 x 108 cells/ml in the same buffer and incubated with streptavidin microbeads (5 µl of the solution provided by the manufacturer per 108 cells). Then, cells were deposited on specially designed columns and separated into IgD-positive and -negative preparations using a high-gradient magnetic field. Unlabeled sIgD- cells were eluted from the column during the application of the magnetic field, while the sIgD+ B cells coated with magnetic beads were then recovered by vigorous washing of the column matrix after removal from the magnetic field.

Culture in the CD40 system

Resting murine splenic B cells were activated with murine CD40L (mCD40L) or with Ltk-hCD40L. Naive human sIgD+ tonsillar B cells were activated with Ltk-hCD40L.

mCD40L. Murine B cells (2 x 105/ml) were stimulated with 2.7 µg/ml of plasma membranes prepared from Sf21 insect cells that had been stably infected with a mCD40L-expressing recombinant baculovirus and harvested 66 h after infection (34).

Ltk-hCD40L. Adherent Ltk-hCD40L cells (31, 32) were cultured in RPMI 1640 supplemented with 5% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were removed from tissue culture dishes by treatment with Versene (Life Technologies) for 2 to 5 min at 37°C. Cells were washed two to three times in GM before incubation in 50 µg/ml mitomycin C for 30 min at 37°C to inhibit DNA synthesis, followed by three to four washes in GM. Approximately 1 x 105 Percoll gradient-purified B cells were mixed with ~5 x 104 mitomycin C-treated Ltk-hCD40L cells in a final volume of 1 ml/well of a 24-well plate.

In some of the murine B cell experiments, {alpha}{delta}-dex (3 ng/ml) was added as a coactivator together with CD40L. If required, murine rIL-4 (3000 U/ml) was added at the inception of stimulation. In human tonsillar B cells, human rIL-4 was added at 50 U/ml. Twenty-four hours later, SSR infection was performed as described below at a multiplicity of infection of 1:1. Cells were subsequently harvested 2, 4, and 6 days postinfection and stored at -70°C until needed, or their DNA was directly isolated.

SSR preparation and B cell infection

The retroviral switch substrate LNSL(Sµ/S{gamma}2b)HyTk was constructed as previously described (23). Bing (CAK8) cells were transfected with linearized switch plasmid DNA precisely adhering to the calcium phosphate protocol of Pear et al. (35). Filtered viral supernatants were immediately concentrated four- to sixfold with Centriprep-500 centrifugal concentration units (Amicon, Beverly, MA) yielding viral titers of 5 x 105 to 2 x 106 G1071 Neo focus-forming units/ml.

Purified B cells (1 x 105) were incubated with an appropriate volume of Bing (CAK8) viral supernatant yielding a multiplicity of infection of 1:1 along with 2 µg/ml polybrene in 5% CO2 at 37°C. After 3 h, the GM was replaced and the B cells cultured for 2 to 6 days postinfection before harvesting and DNA isolation.

Genomic DNA isolation

A rapid lysis technique, suitable for small quantities of cells, was employed (36). Briefly, cell pellets were suspended in 500 ml TEN (100 mM Tris pH 8, 5 mM EDTA, 0.2% SDS, 200 mM NaCl, 100 mg/ml proteinase K) and incubated at 37°C for 1 to 2 h. An equal volume of isopropanol was added to precipitate high m.w. genomic DNA at room temperature. After centrifugation and a 70% ethanol wash, the DNA pellet was allowed to air dry before resolubilization in 10 mM Tris-Cl, pH 8.0, 1 mM EDTA at 56°C. The DNA was quantified by UV spectrophotometry and the quantity and quality verified by agarose gel electrophoresis.

DNA probes

The Sµ probe comprised a 1.2-kb SacI-HindIII fragment consisting of an uninterrupted block of Sµ tandem repeats isolated from the plasmid m2-20 (37). The S{gamma}2b probe comprised a 700-bp PCR-derived fragment of S{gamma}2b tandem repeats generated from pLNSL(Sµ/S{gamma}2b)HyTk. The Neo probe comprised a 440-bp PCR fragment of Gag and Neo gene sequences also derived from the SSR. The HyTk probe comprised a 200-bp NaeI/NruI fragment from the plasmid tgCMV/HyTK (38). DNA probes were labeled with 32P using a Random-Primed DNA Labeling Kit (Boehringer Mannheim, Indianapolis, IN).

Membrane hybridization

Acrylamide gels were electroblotted on filter membranes, essentially as previously described (39), which were subsequently prehybridized and hybridized according to standard protocols. After hybridization, membranes were washed sequentially in 2x SSC, 0.1% SDS for 5 min and 30 min at room temperature, followed by 0.2x SSC, 0.1% SDS at 65°C for 30 to 60 min. Subsequent analyses were performed with a Molecular Dynamics Storm 860 PhosphorImager (Sunnyvale, CA).

PCR assays

Direct PCR for Sµ-S{gamma}2b recombination. For standard PCR analysis, 50 to 100 ng of genomic DNA was used for the first round of amplification in a 40-µl reaction containing 0.5 mM of 5' Sµ primer p3 (5'-TGGCTTAACCGAGATGAGCC-3') and 3' S{gamma}2b primer p4 (5'-CTGTTACAGTTGCACCACAG-3') in 5 mM KCl, 1 mM Tris-HCl, 1.5 mM MgCl2, 200 mM each dNTP and 2.5 U AmpliTaq DNA polymerase (Perkin-Elmer, Foster City, CA). A hot start protocol was used, which consisted of a separation of the primers MgCl2 and dNTPs from the other reaction components in a lower chamber created by wax beads (Perkin-Elmer) before amplification. The cycling conditions were: 94°C for 4 min initial denaturation; 32 cycles of 94°C for 1 min, 60°C for 1 min and 72°C for 1 min; and a final extension at 72°C for 5 min. The second-round nested reaction consisted of the same components as the first round except for the use of nested 5' Sµ primer p5 (5'-AACTCTACTGCCTACACTGG-3') and nested 3' S{gamma}2b primer p6 (5'-CTATGAACCATAGTTCCTCC-3') along with 2 µl of amplified product from the first-round PCR (5% of total) as input DNA. The first-round and nested Sµ and S{gamma}2b primers were derived from 5' and 3' nonrepetitive sequences of Sµ (40) and S{gamma}2b (41) within the SSR.

For long template PCR, first-round amplification of 100 ng of genomic DNA was performed in a 40-µl reaction containing 0.5 µM of 5' Neo primer p9 (5'-CGGGGAAGGGACTGGCTGCTATT-3') and 3' S{gamma}2b/pSP72 primer p10 (5'-GGTACCCGGGGATCCTTGTCACTT-3') along with 200 µM each dNTP and 4 U of rTth DNA Polymerase XL according to the provider’s recommended conditions (Perkin-Elmer). PCR reactions were assembled according to the hot start protocol (Perkin-Elmer) before amplification. The cycling conditions were: 94°C for 4 min, 32 cycles of 94°C for 30 s, 65°C for 5 min, and a final extension at 72°C for 5 min. The second-round nested reaction consisted of the same components and reaction conditions as the first round except for the use of nested 5' Neo primer p11 (5'GCATCGCCTTCTATCGCCTTCTTG-3') and nested 3' S{gamma}2b flanking sequence primer p12 (5'-ACATGCCGCTCTCCCCAGGTATCC-3') along with 2 µl of amplified product from the first-round PCR (5% of total) as input DNA. The first-round Gag/Neo and S{gamma}2b primers and nested Neo and S{gamma}2b primers were derived from retroviral Gag and Neo sequences and 3' nonrepetitive S{gamma}2b flanking sequences (41, 42).

Direct PCR for Hytk, Neo, and Myc sequences. The same components and reaction conditions as described above were used in a single round of multiplexed nonnested PCR with the use of primer set p1 (5'-CGTACACAAATCGCCCGCAGAAGC-3') and p2 (5'-CTCCGAAAGGGCCCCCAACACGAT-3') to detect HyTk (38) as a 659-bp fragment (Tanneal = 64°C) and primer set p7 (5'-CCTCCGCCTCCTCTTCCTCCATC-3') and p8 (5'-CTTCGCCCAATAGCAGCCAGTCC-3') to detect a 439-bp fragment of fused retroviral Gag/Neo sequences (Tanneal = 62°C) (42). An evolutionarily conserved c-myc primer pair (5'-TGGAAACCCCGGTAAGCACAGA-3' and 5'-CCGGTTTTCCCTTCCCCTTTCCT-3') produced a 206-bp fragment (43), which verified the relative integrity of the murine and human genomic DNAs.

PCR products were separated on 5% polyacrylamide gels and stained with ethidium bromide (EtBr) or electroblotted and hybridized to appropriate 32P-labeled probes.

Digestion-circularization (DC) PCR for endogenous Sµ>S{gamma}1 and Sµ>S{gamma}2b switching and ß2-microglobulin sequences. Genomic DNAs were cut with EcoRI and ligated under dilute conditions as described by Chu et al. (44). One hundred nanograms of ligated DNAs was submitted to standard PCRs. Sµ>S{gamma}1 recombinations were detected as a 219-bp band by a primer set complementary to sequences located near the EcoRI site 5' of the genomic Sµ region (5'-GCGGGAGACCAATAATCAGAGGGAAG-3') and just upstream of the EcoRI site 3' of the genomic S{gamma}1 region (5'-GATGGAGAGCAGGGTCTCCTGGGTAGG-3') as described (44). Sµ>Sg2b recombinations were visualized as a 475-bp product with the same 5' Sµ primer and a different second primer complementary to sequences located near the EcoRI site 3' of the S{gamma}2b region (5'-GAGATGAGAGCAGAGAACAGGACA-3') (41). The completeness of EcoRI digestions and ligations was verified by PCR with a primer set complementary to first intron (5'-GCCACAGGGGAAGACAGACGAAAAC-3') and second exon (5'-TGGGAAGCCGAACATACTGAACTGC-3') sequences of the murine ß2-microglobulin gene producing a 1.8-kb product (45). All PCRs were performed at a 64°C annealing temperature with the same cycling conditions as described above, except that the ß2-microglobulin extension step was for 2 min at 72°C.

DNA sequencing

Products of PCR reactions were size fractionated by electrophoresis on 1.5% agarose gels and stained with EtBr. Visible DNA products were excised, eluted, and sequenced by the dideoxynucleotide chain termination method using the fmol cycle sequencing system (Promega, Madison, WI).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The switch substrate retrovirus recombination assay

The LNSL(Sµ/S{gamma}2b)HyTk SSR was prepared by transient transfection into the Bing in vitro packaging line to produce a high titered, amphotyped viral stock capable of infecting both rodent and primate cells (23). As shown in Figure 1Go, the switch cassette comprises segments of the BALB/c mouse Sµ and S{gamma}2b regions separated by the bifunctional HyTk gene driven by the strong CMV promoter (38). The Sµ donor sequence comprises 1470 bp of tandem repeats and 330 bp of 5' flanking sequence (37, 40), and the S{gamma}2b acceptor sequence comprises 735 bp of 49-bp tandem repeats and 565 bp of 3' flanking nonrepetitive sequence (41, 46). HyTk consists of a hygromycin resistance gene conjoined to the herpes simplex virus thymidine kinase (HSVtk) gene (38). The presence of multiple strong promoters (LTR, SV40, and CMV) ensures the constitutive transcription of the integrated provirus; its Neo and HyTk expression casettes have been shown to be simultaneously expressed in a variety of B and non-B cell lines (23, 29). In addition, the Neo gene serves as a stable, proviral integration marker, while loss of the HyTk gene occurs as a consequence of SSR switch recombination.



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  		FIGURE 1. The LNSL(Sµ/S{gamma}2b)HyTk SSR (23) and its PCR readout assays. The locations and sizes of Gag, Neo, SV40, Sµ, CMV/HyTk, and S{gamma}2b sequences are indicated. R represents tandemly repetitive blocks of S region sequences and nr designates unique, nonrepetitive sequences directly flanking the S region tandem repeats. The relative locations of PCR primer pairs and the predicted structures and sizes of their amplification products with and without Sµ>S{gamma}2b switch recombination are shown. The sequences of all PCR primers are provided in Materials and Methods.

 
To measure the recombination status of the chromosomally integrated SSR, several direct PCR strategies were developed (summarized in Fig. 1Go). Gag/Neo sequences were amplified as a 439-bp fragment with primers within the retroviral Gag and Neo sequences (primer pair p7/p8 in Fig. 1Go). The relative percentage of B cells harboring stably integrated SSRs was determined by limiting dilution, a semiquantitative analysis of the latter Gag/Neo PCR product in comparison with B cell lines harboring a single copy of the SSR per genome equivalent; and ~1 to 2% of B cells were found to harbor the SSR provirus (23) (data not shown). The HyTk gene is detected as a 659-bp PCR product using primers within the hygro and tk sequences respectively (primer pair p1/p2 in Fig. 1Go). Progressive reduction of the HyTk PCR product as a function of in vitro B cell culture incubation time would represent one indicator of S-S recombination. If S-S-mediated recombination occurs between the Sµ and S{gamma}2b tandem repeats of the SSR, a range of possible products can be formed depending upon the location of the recombination breakpoints within each switch sequence. The primary switch recombination assay employs nested PCR primers within nonrepetitive Sµ and S{gamma}2b flanking sequences, which detect S-S recombination events exclusively occurring within the Sµ and S{gamma}2b tandem repeats (Fig. 1Go, p3/p4 and p5/p6). Sµ/S{gamma}2b hybrid products ranging in length from ~40 to 2290 bp are visualized in EtBr-stained gels and by sequential hybridizations with Sµ and S{gamma}2b repetitive sequence probes. Finally, long template, nested PCR with SSR-specific primers can independently determine whether recombinations are restricted to the repetitive S sequences of the SSRs (primer pairs p9/p10 and p11/p12 in Fig. 1Go).

CD40 ligation of resting murine B cells is sufficient to cause SSR switch recombination

It is known that CD40 receptor multimerization is important for effective signal transduction, and this may be mediated by CD40L binding (34). Resting murine splenic B cells were stimulated with mCD40L as an Sf21 plasma membrane fraction (34). After 24 h, cells were infected with the SSR and parallel cultures were allowed to proliferate for an additional 2, 4, or 6 days before harvesting their genomic DNAs for direct PCR assays. Control cells without SSR infection were similarly treated and their genomic DNAs harvested on the 4th or 5th day of in vitro culture. Multiple independent experiments were performed with essentially the same observations; the results of one such experiment are shown in Figure 2Go. Switch recombination was revealed, by both EtBr fluorescence and hybridization to an Sµ repetitive sequence probe, 4 and 6 days postinfection (Fig. 2Go, d4, d6) but not after 2 days (Fig. 2Go, d2). Switch recombination bands seen in day 4 are not observed in day 6, because genomic DNAs were routinely isolated from parallel cultures of CD40-stimulated infected cells. The day 2 time point was reproducibly negative, while independent samplings at days 4 and 6 produced unique banding patterns implying that the majority of recombination products represent single molecular events in individual cells. Switch recombination products were not detectable in the uninfected but similarly stimulated control cells (Fig. 2Go, U). c-Myc sequences were present in the SSR-infected and uninfected samples, verifying the quality of the genomic DNAs (Fig. 2Go, lower panel).



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  		FIGURE 2. SSR recombination in murine splenic B cells stimulated with mCD40L. Resting murine splenic B cells (2 x 105) were stimulated with 2.7 mg/ml of a preparation of plasma membranes from Sf21 cells that had been stably infected with mCD40L. Twenty-four hours later, the cells were infected with the SSR LNSL(Sµ/S{gamma}2b)HyTk. Two (lane d2), four (lane d4), and six (lane d6) days postinfection, S sequence recombination was assessed in parallel cultures by the direct PCR method as described in the text, using 100 ng genomic DNA as template. DNA from uninfected cells stimulated with mCD40L for 7 days was used as a negative control (lane U). The upper panel shows the PCR products after electrophoretic separation and subsequent EtBr staining. The middle panel illustrates probing with a repetitive Sµ sequence probe. A direct PCR assay with c-mycprimers verifies the presence of added genomic DNA template (lower panel). M, m.w. marker, {phi}X174 HaeIII.

 
To investigate the role of CD40 receptor engagement in SSR recombination by an alternative means of CD40L presentation, murine splenic B cells were stimulated with a nondividing feeder layer of Ltk- cells harboring a stably transfected cell surface human CD40L (Ltk-hCD40L) (31, 32). Twenty-four hours post-CD40R stimulation, cells were infected with the SSR and allowed to proliferate in culture for an additional 2 to 6 days before genomic DNAs were harvested for direct PCR analyses. As with the mCD40L stimulation experiments, uninfected control cells were treated in a similar fashion and their DNAs harvested after 4 days in culture. The PCR analysis was conducted in triplicate using three different template inputs comprising 50, 100, and 200 ng of DNA with essentially the same results. Results for the 100-ng input DNA are shown in Figure 3Go. Stimulation of murine B cells with hCD40L resulted in detectable S-S recombination, by both EtBr fluorescence and hybridization to an S{gamma}2b repetitive sequence probe, 4 and 6 days postinfection (Fig. 3Go, upper panels d4 and d6). In contrast, 2 days after infection (3 days after stimulation), no S-S recombination was detected (Fig. 3Go, d2) even when the DNA input was increased to 200 ng (>30,000 genomic equivalents). Results with an Sµ repetitive sequence probe were analogous to those shown for S{gamma}2b (data not shown). Thus, the 3-day lag in switch recombination subsequent to mCD40L stimulation of resting murine splenic B cells was also observed by activation with hCD40L. Significantly, the HyTk signal in the unrecombined day 2 time point was reproducibly greater than in the day 4 and day 6 samples, which had both undergone extensive S-S recombination (Fig. 3Go, middle panels). The latter result concurred with the first appearance of switch recombination products. Uninfected B cells stimulated for 7 days (equivalent to 6 days postinfection) (lane U in Fig. 3Go) displayed no evidence of S-S recombination and, as expected, exhibited no HyTk signal. C-myc sequences were present in all of the SSR-uninfected and infected samples (Fig. 3Go, lower panel). The absence of similar bands in the S-S product arrays at 50-, 100-, and 200-ng DNA input sampled from day 4 and day 6 parallel cultures (Fig. 3Go and data not shown) supports the view that these recombination products represent rare, stochastic events occurring in single cells (23). This is in contrast to the more constant pattern of recombination products seen in a stably infected BCL1B1 neor control displaying recombination events which "jackpot out" during cellular expansion (Figs. 6Go and 7Go) (23).



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  		FIGURE 3. SSR recombination in murine splenic B cells stimulated with hCD40L. Resting murine splenic B cells were stimulated with hCD40L presented on the surface of Ltk- cells (Ltk-hCD40L) and infected 24 h later with the LNSL(Sµ/S{gamma}2b)HyTk SSR. PCR analyses were performed on parallel cultures over a 2- to 6-day time course as indicated in Figure 2Go. "U" represents a 7-day stimulated, uninfected control as described in the Figure 2Go legend. The upper panel shows the products after electrophoretic separation and subsequent EtBr staining (left) or after electroblotting and probing with an S{gamma}2b repetitive sequence probe (right). Direct PCR assays with HyTk and c-myc primers verify the presence of the SSR (middle panels) and genomic DNA (lower panel), respectively. Increased specificity of the HyTkassay is provided by using an HyTk-specific probe. M, m.w. marker, {phi}X174 HaeIII.

 


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  		FIGURE 6. DC-PCRs for endogenous Sµ>S{gamma}1 and Sµ>S{gamma}2b recombinations. DC-PCRs were performed as described in Materials and Methods. Genomic DNA samples (100 ng) correspond to days 4 and 5 postinfection (5 and 6 days poststimulation) with B cells cultured with either Ltk-hCD40 alone or Ltk-hCD40L + IL-4. Samples (50 ng) of genomic DNAs of MPC-11, a {gamma}2b-expressing plasma cell tumor with a Sµ>S{gamma}2b switch (46), and HyB147, a {gamma}1 expressing hybridoma with a µ>{gamma}1 switch (K.B.M., unpublished observation) were included as postive controls. EtBr-stained gels (left) were electroblotted and hybridized (right) with a ~100 bp 5' Sµ sequence located in between the EcoRI site 5' of the Sµ region and the 5' Sµ primer. The low m.w. marker is a {phi}X174 HaeIII digest. The high m.w. marker employed for the analysis of the ß2-microglobulin control PCR (lower panel) is a 1-kb ladder (Life Technologies).

 


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  		FIGURE 7. SSR-specific primed PCRs reveal that internal SSR recombinations are targeted to the Sµ and S{gamma}2b switch sequences. Long template PCR was performed on genomic DNAs at days 3, 4, and 5 postinfection (from Expt. II in Fig. 5Go) with primers located in Neo and vector sequences 3' of the S{gamma}2b genomic sequence (see primer pairs p9/p10 and p11/p12 in Fig. 1Go). The gel was electroblotted and hybridized to Sµ + S{gamma}2b repetitive sequence probes. Essentially all EtBr-staining bands, which are found uniquely in the infected samples, hybridize to the switch region probes, and their size distribution indicates that the zone of recombination resides with the Sµ and S{gamma}2b sequences. Lane U is an uninfected negative control (4 days poststimulation). Lane C is a stably infected population of neor BCL1B1 cells revealing several predominant switch recombination products and the adjacent - lane is a "no DNA" control consisting of amplification reagents without genomic DNA. Direct PCR assays with c-myc primers verify the presence of added genomic DNA template (lower panel).

 
We also attempted to sample LPS stimulated B cells for recombination activity. However, after 1 day of LPS treatment, the number of cells that entered their division cycle and proliferated was insufficient for an adequate amount of SSR infection. Consequently, we performed these experiments with CD40-stimulated B cells, which proliferated vigorously.

IL-4 stimulates the onset of switch recombination activity in CD40-activated murine B cells

To study the effect of other activators and cytokines, resting murine splenic B cells were costimulated with Ltk-hCD40L and high m.w. {alpha}{delta}-dex (9). Multivalent {alpha}{delta}-dex has been demonstrated to be strongly synergistic with CD40L for induction of B cell proliferation, viable cell outgrowth, isotype switching, and maturation to Ig secretion (9). Twenty-four hours after costimulating murine B lymphocytes with Ltk-hCD40L and {alpha}{delta}-dex, cells were infected with the SSR, and parallel cultures were allowed to proliferate for an additional 2, 4, or 6 days before harvesting their genomic DNAs for PCR assays. Uninfected B cell cultures were treated in an identical manner. One representative example of several independent experiments is shown in Figure 4Go. Costimulation with hCD40L and {alpha}{delta}-dex yielded detectable recombination activity revealed with an S{gamma}2b probe at 4 days postinfection and still apparent after 6 days. As in the previous experiments employing CD40L alone, no recombination was detectable at 2 days postinfection. As before, uninfected control B cells displayed no recombination under any of the above-described stimulation regimes (lane U in Figs. 4Go and 5Go).



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  		FIGURE 4. SSR recombination in murine splenic B cells costimulated with hCD40L and {alpha}{delta}-dex. Resting murine splenic B cells were costimulated with hCD40L presented on the surface of Ltk- cells (Ltk-hCD40L) and {alpha}{delta}-dex (3 ng/ml) and infected 24 h later with the LNSL(Sµ/S{gamma}2b)HyTk SSR. PCR analyses were performed as indicated in Figure 2Go. DNA from uninfected B cells stimulated with hCD40L/{alpha}{delta}-dex for 7 days served as a negative control (lane U). The upper panel shows the products after electrophoretic separation and EtBr staining. The blot was hybridized to an S{gamma}2b probe. Direct PCR assays with c-myc primers verify the presence of added genomic DNA template (lower panel). M, m.w. marker, {phi}X174 HaeIII.

 


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  		FIGURE 5. IL-4 abrogates the 4-day lag in CD40L-induced SSR recombination. Percoll gradient-purified murine B cells from DBA/2 mice were stimulated with Ltk-hCD40L + {alpha}{delta}-dex + IL-4 in two independent experiments. Genomic DNAs (100 ng each) were prepared on days 2, 3, 4, and 5 (Expt. I) and days 3, 4, and 5 (Expt. II) postinfection and submitted to direct PCR analyses for Sµ-S{gamma}2b, HyTk, Gag/Neo, and c-myc sequences. Gels were electroblotted and hybridized to the indicated DNA probes. The control (C) lane contains a neor population of SSR-infected BCL1B1 cells (23). Direct PCR assays with c-myc primers verify the presence of added genomic DNA template (lower panel). The m.w. marker is a {phi}X174 HaeIII digest (the sizes of the bands are shown in the other figures).

 
Next, we investigated the effects of IL-4 on the switch recombination activity of CD40-stimulated murine B cells. Figure 5Go depicts two representative experiments in which SSR-infected B cells were stimulated with a combination of hCD40L, {alpha}{delta}-dex, and IL-4 for either 2 to 6 or 3 to 5 days. Most notably, complete abrogation of the recombination lag was observed in each case (Fig. 5Go, Expt. I and II). Interestingly although the Neo signal intensity (indicative of the presence of the retrovector) remained similar at all time points, the HyTk signal dramatically dropped off 5 days poststimulation with hCD40L/{alpha}{delta}-dex/IL-4 (Fig. 5Go, see Neo and HyTk panels, respectively). Furthermore, PCRs of limiting dilutions of template DNAs with uninfected genomic DNA revealed that the relative quantities of the Neo sequences were comparable throughout the time course (data not shown). Intensely hybridizing recombination products in one time point were not seen in others, since, as in the above analyses, all PCRs were performed with DNAs from parallel cultures. The latest time points (5 days postinfection) in the presence of IL-4 + CD40 did not generally display strongly hybridizing bands but only a faint smear of recombination products, indicating that recombination was active throughout the time course. Long template PCRs performed on the experiment II samples in Figure 5Go also revealed a complex array of recombination products, which were visible as long as 5 days postinfection (see Fig. 7Go). Uninfected hCD40L/{alpha}{delta}-dex/IL-4-stimulated B cells were, as noted previously, S-S recombination negative. These results are consistent with the majority of chromosomally integrated SSRs undergoing continuous S-S-mediated recombination.

IL-4 + CD40 signaling induces endogenous Sµ>S>{gamma}1 switching but not Sµ>S{gamma}2b in contrast to the SSR recombinations

The recombination status of the endogenous Sµ, S{gamma}1, and S{gamma}2b sequences was examined by DC-PCR in SSR infected B cells as first described by Chu et al. (44). Genomic DNA is digested with EcoRI in known sequences flanking the Sµ, S{gamma}1, and S{gamma}2b regions. The resultant fragments are ligated under diluted conditions, generating circles and regions encompassing the "circle joints," and are amplified with primers complementary to sequences near the EcoRI sites 5' of Sµ and 3' of either S{gamma}1 or S{gamma}2b, respectively. A primer pair specific for Sµ/S{gamma}1 recombinations was employed as described (44). The same 5' Sµ primer was matched to another primer located near the first EcoRI site 3' of the S{gamma}2b repeats to score for Sµ/S{gamma}2b switch products. Ab-secreting cell lines harboring endogenous Sµ/S{gamma}1 (MAZ hybridoma 147) (K.B.M., unpublished observations) or Sµ/S{gamma}2b (MPC-ll plasma cell tumor (46)) recombinations served as positive controls. To confirm that EcoRI digestions and ligations were successful, PCR assays were also performed on the same EcoRI-digested/ligated genomic DNAs with a primer pair residing on an EcoRI fragment spanning intron 1 and exon 2 sequences of the ß2-microglobulin gene that generates an ~1.8-kb DC-PCR product (45). The DC-PCR assay revealed that the endogenous S{gamma}1 region became available for switch recombination only in the hCD40L/{alpha}{delta}-dex/IL-4-stimulated B cells but not with hCD40L/{alpha}{delta}-dex stimulation alone, while endogenous Sµ/S{gamma}2b recombination was not detected under either culture condition (Fig. 6Go). The endogenous Sµ to S{gamma}1 switches accumulated in the 4- to 5-day postinfected samples, which also possessed the highest degree of SSR-specific Sµ/S{gamma}2b recombinations.

Sequence specificity of the SSR recombination product array

An important concern underlying the use of switch sequence substrates to detect putative switch recombinase activities has been one of sequence specificity (4, 5). However, our previous experience with switch substrate retrovectors in B cell lines had consistently revealed a high degree of switch sequence-specific recombination (27-29). To address this issue, CD40L/{alpha}{delta}-dex/Il-4-stimulated murine B cells were harvested 3 to 5 days after SSR infection and analyzed for retrovector switch recombination by nested, long template PCR (illustrated by primer pairs 9/10 and 11/12 in Fig. 1Go). First-round p9/p10 primers anneal to Neo and retroviral vector-specific sequences and nested p11/p12 primers are specific for Neo and 3' nonrepetitive S{gamma}2b flanking sequences (see Fig. 1Go). Consistent with recombinations being focused within the two S regions, PCR products dramatically shifted to higher m.w., producing an SSR-specific S-S product array from ~800 to 3500 bp (see Fig. 7Go). Significantly, Sµ + S{gamma}2b-positive PCR products below ~800 bp were not apparent. Minor invariant EtBr-staining bands below 800 bp also failed to hybridize to a retrovector-specific SV40 sequence probe; direct sequence analysis also revealed them to be PCR artifacts (data not shown). In addition, restriction digestions revealed that 330 bp of retrovector-specific SV40 sequences, residing between Neo and Sµ sequences (see Fig. 1Go), remained intact within the high m.w. recombination products (data not shown). These observations all support the view that SSR recombinations are essentially confined to the repetitive switch sequences.

Direct sequencing of individual PCR products from CD40L (alone or with {alpha}{delta}-dex) and CD40L/IL-4 stimulated cells provided further evidence for the sequence specificity of the switch recombinase activity. Eighteen S-S recombination products ranging in length from 125 to 820 bp, which were obtained after nested PCR with primer pairs p5 and p6 (see Fig. 1Go), were sequenced (Fig. 8Go). The structures were entirely consistent with that expected for bona fide S-S recombination products. In all instances, it was possible to determine unambiguously the Sµ and S{gamma}2b breakpoints within the retrovector recombination cassette (Fig. 8Go). Point mutations were observed in 10 of the 18 fragments (55%), and all were confined to only one side of the recombination breakpoint, either in the Sµ or S{gamma}2b sequence (see recombination sites a, d, e, g, h, j, l, o, p, and r in Fig. 8Go). In addition, S{gamma}2b sequence deletions (325 and 512 bp deletions, respectively) were found in two products that otherwise displayed no evidence of point mutations (Fig. 8Go, sites p, f, and k). The existence of multiple events within the same recombination product further substantiates the continuous nature of the process. Interestingly, while the S{gamma}2b breakpoints were found in the entire 735 bp of 49-bp tandem repeats, distinct clustering of the Sµ breakpoints within the 5' end of the Sµ tandem repeats was observed (Fig. 8Go). Breakpoints were localized to the 5' 340 bp of the 1470-bp Sµ repeats. Breakpoint clustering within the 5' portion of the Sµ repeats conceivably could be explained by extensive, ongoing recombinations preferentially occurring within the Sµ repeat block or the existence of recombination hot spots at the 5' end of the Sµ tandem repeats.



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  		FIGURE 8. Nucleotide sequences of switch recombination products. DNA sequences (about 20 bp) 5' and 3' of 18 independent recombination events under various in vitro conditions are shown. Internal deletions in S{gamma}2b sequences are indicated; sequence differences with the germline Sµ and S{gamma}2b sequences in the SSR are underlined. Sequences not present in either the Sµ or S{gamma}2b sequences but residing precisely at the recombination breakpoints were affixed to the Sµ sequence. The relative locations of each breakpoint are shown in the vector diagram, and the precise locations and sizes of the PCR products are provided below and to the right of each recombination event, respectively. In most cases, a larger portion of the Sµ tandem repeat block is deleted, but as shown in Figures 2 through 4GoGoGo, most PCR products strongly hybridize to either Sµ or S{gamma}2b probes due to the repetitive nature of the S region DNA probes. Nested, direct PCR primers 5 and 6 were used to generate the PCR products (shown in Fig. 1Go), which were purified via low melting agarose gel electrophoresis before fmol cycle sequencing (Promega).

 
CD40 triggering alone is also sufficient to produce SSR recombination in naive human tonsillar B cells

Naive sIgD+ human tonsillar cells were stimulated on a nondividing feeder layer of Ltk-hCD40L cells in the presence or absence of human IL-4 (31, 32). Twenty-four hours later, B cells were infected with the SSR. Six days postinfection (i.e., 7 days poststimulation), the nonadherent B cells were harvested, DNA isolated, and SSR recombination determined by direct PCR analysis (Fig. 9Go). SSR recombination products were clearly evident in the hCD40L-stimulated human B cells, both in the presence and absence of IL-4 (see Fig. 9Go). The S-S recombination product arrays were visualized by both EtBr fluorescence and hybridization to a mixture of Sµ and S{gamma}2b-repetitive sequence probes (Fig. 9Go). DNA from uninfected tonsillar B cells similarly stimulated by hCD40L and IL-4 revealed no S-S PCR products (lane U in Fig. 9Go). Amplified human c-myc sequences verified the presence of genomic DNAs in all PCR reactions (Fig. 9Go, lower panel).



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  		FIGURE 9. SSR recombination in human tonsillar sIgD+ cells stimulated with hCD40L in the presence or absence of IL-4. Purified human tonsillar sIgD+ cells were stimulated with Ltk-hCD40L cells, in the presence or absence of IL-4, and infected 24 h later with the LNSL(Sµ/S{gamma}2b)HyTk SSR. Six days postinfection, S sequence recombination was assessed by the direct PCR method as described in the text, using 100 ng of genomic DNA as input. Results are shown for SSR-infected cells in the absence or presence of IL-4. Uninfected cells stimulated with Ltk-hCD40L and IL-4 served as a negative control. The upper panel shows the PCR products after electrophoretic separation and subsequent EtBr staining. The middle panel is the electroblot from the EtBr gel, which has been hybridized with a mixture of Sµ and S{gamma}2b repetitive sequence probes. A direct PCR assay with c-mycprimers verifies the presence of genomic DNA (lower panel). The control is a stable population of neor BCL1B1 cells harboring the same SSR. The "no DNA" control consists of amplification reagents without genomic DNA. The m.w. marker is a {phi}X174 HaeIII digest.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
S sequences alone are sufficient for efficient switch recombination in CD40-activated B cells

Activation of resting B cells with CD40L serves as an experimental model for studying the effects of cognate interactions between B cells and activated T cells during the T-dependent immune response; these interactions are prerequisites for isotype switching in the germinal centers of secondary lymphoid organs. Employing constitutively transcribed SSRs and a direct PCR assay for switch recombinations, we identified a switch recombinase activity within several days post-SSR integration in established B cell lines (23). Here, unfettered by the regulated constraints of accessibility control, the same constitutively active SSRs have revealed the independent effects of CD40R engagement alone and IL-4 in the context of CD40 signaling on the switch recombinase activity of normal mature B lymphocytes. Three different experimental systems were employed with similar significant observations. Resting murine splenic B cells were activated either with Ltk-hCD40L cells or with an Sf21 insect cell membrane preparation containing mCD40L (34), while sIgD+-sorted human tonsillar cells were activated with Ltk-hCD40L (47). IL-4 is known to synergize with CD40L for B cell activation, proliferation, and isotype switching (32, 48). Similarly, multivalent Ag receptor cross-linkers such as {alpha}{delta}-dex also synergize with CD40L to increase cellular proliferation, isotype switching, and Ig secretion (9). Interestingly, and somewhat surprisingly, engagement of the CD40R in the absence of added exogenous cytokine resulted in significant SSR recombination in both murine and human B cells.

Direct sequencing of some of the PCR-generated S-S recombination products indicated that their structures were consistent with bona fide switch recombination. Interestingly, recombination breakpoints were identified throughout the entire 735-bp stretch of 49-bp repeats present in the S{gamma}2b acceptor sequence, whereas Sµ breakpoints were clustered within a 5' 350-bp segment of 1460-bp Sµ tandem repeats. Point mutations, insertions, and deletions were present, similar to the types of mutation previously observed subsequent to switch recombination, which are indicative of an error-prone DNA repair process (12-14). These mutations were probably not introduced by Taq polymerase, since all sequencing was directly performed on the PCR products without cloning.

The kinetics of switch recombination was investigated with murine B cells triggered by human or murine CD40L via multiple direct PCR assays. Switch recombinase activity appeared 4 to 6 days post-SSR infection, which is equivalent to 5 to 7 days post-CD40 stimulation. In addition, the kinetics of loss of HyTk gene sequences, positioned between the Sµ and S{gamma}2b sequences, paralleled the appearance of switch recombinase activity. Therefore, the kinetics and degree of SSR recombination is remarkably comparable to that reported for the endogenous IgCH locus after costimulation of in vitro cultures of human and murine B lymphocytes with CD40L and appropriate cytokines (49, 50). The 4-day lag in the appearance of SSR recombination products may be caused, in part, by the markedly asynchronous entry of B cells into the first cell division cycle and the delayed onset of class switching (16). Three days after CD40L stimulation, some B cells remained undivided, while others had divided as many as five or six times (16). Switching became detectable after the third division and was much more significant after the fourth, fifth, and sixth division cycles (16). Furthermore, appreciable numbers of cell subpopulations undergoing the fourth, fifth, and sixth division cycles become apparent only at 4 days poststimulation (16).

IL-4 in conjunction with CD40 stimulation can enhance the onset of switch recombination

IL-4 is known to enhance the proliferative potential of CD40L-stimulated B cells in addition to precipitating a switch to IgG1 and IgE (4, 5, 10, 51). As anticipated, only the endogenous Sµ and S{gamma}1 regions were shown to recombine upon engagement of the B cell CD40 and IL-4R, while no appreciable recombination between the endogenous Sµ and S{gamma}2b sequences was observed with CD40 signaling alone or in combination with IL-4 (Fig. 9Go). In sharp contrast to the endogenous CH locus, stimulation of murine B cells with IL-4 and CD40L dramatically enhanced the manifestation and extent of Sµ-S{gamma}2b recombination within the SSR. Recombination was easily detectable only 2 days postinfection and by 4 to 5 days appeared to be virtually complete as revealed by the extensive loss of the intervening HyTk gene. The more rapid onset and completeness of the SSR recombination reaction caused by IL-4 stimulation may result, in part, from the enhanced B cell proliferation also induced by this cytokine. We conclude that switch recombinase activities capable of recombining several if not all S regions are present in CD40-activated normal B cells.

Implications for the molecular requirements of the CH gene switch recombinase

Our findings suggest that mature naive B cells possess an intrinsic switch recombinase activity, which may be activated either by their developmental programming or by CD40R engagement. CD40R engagement could conceivably activate the recombinase complex or a limiting component thereof. A number of studies have shown that CD40L activation of murine and human B cells induces CH germline transcripts without exogenous cytokine supplementation, but no evidence of bona fide switch recombination has been noted (52-56). CD40 engagement alone, in both T cell- and IL-4-deficient mice, can result in class switching to IgE (57, 58), and earlier reports described IL-4-independent modes of class switching to IgE and IgG1 (59-61). However, it was not possible in each of these in vivo studies to preclude the possibility that other cytokines such as IL-10 (50) are wholly or partly required to synergize with CD40 triggering in the absence of IL-4 to effect switch recombination. In addition, no studies to date have been able to demonstrate effects on switch recombinase activity as opposed to other indirect effects on CH gene segment accessibility or targeting for recombination.

Here, we have shown that retrovirally transduced Sµ and S{gamma}2b sequences, integrated at random genomic sites, are alone sufficient to mediate extensive, switch sequence-specific recombination in CD40-activated resting B lymphocytes (Figs. 2Go-8). Since the integration of proviral sequences requires DNA synthesis (62), the intrinsic features of this assay system precludes our ability to examine the recombinase activity of resting mature B cells. Nevertheless, our findings show that CD40R engagement of B lymphocytes reveals that they possess a significant switch recombinase activity. The Sµ and S{gamma}2b sequences in the LNSL(Sµ/S{gamma}2b)HyTk retrovector are portions of their endogenous counterparts that are maintained in a transcriptionally active context by a combination of three strong, ubiquitous viral promotors and enhancers (LTR, SV40, and CMV). We previously reported that Sµ and S{gamma}2b sequences in the same SSR underwent switch recombination in a stochastic fashion in several late stage pre-B and mature B cell lines (23). However, in contrast to the extensive SSR recombinations shown here in short term cultures of stimulated normal B lymphocytes, our former study of B cell lines revealed that only up to 25% of integrated SSRs had recombined after 2 wk of continuous culture (i.e., at least 14 cell generations) (23). Therefore, our results favor the interpretation that normal mature B lymphocytes costimulated by IL-4 and CD40L possess a greater intrinsic switch recombinase activity than most established B cell lines. One previous study concluded that the I{epsilon} exon and its promoter may confer more than transcriptional accessibility per se for S region-specific switch recombination (22). Another group reported that sequences encompassing the splice donor signal of the I{gamma}1 exon were essential for switching to C{gamma}1, while the remainder of the I{gamma} exon may be dispensable given a high enough level of transcriptional activity (63). Since the SSR described here lacks these and all other CH locus control elements, but still recombines at frequencies comparable to the endogenous CH locus (in the presence of the appropriate cytokine), we are left to conclude that negative as well as positive cis-acting control elements may reside in the natural CH locus to direct the developmental timing and differential targeting of CH genes for switch recombination.

CD40L and IL-4 cytokine receptor triggering pathways operate by distinct mechanisms including, inter alia, the induction of the NF-{kappa}B/Rel (64-66) and STAT6 (67-70) transcription factors, respectively. Given our results, it is possible that the recombinase or a component thereof is regulated, in part, by NF-{kappa}B- and/or STAT 6-responsive elements. Synergy between DNA-bound NF-{kappa}B and STAT6 factors has been demonstrated for the I{gamma}1 and I{epsilon} promoters (56, 71). It will be interesting to determine whether other cytokines in the context of CD40 signaling also have the capability of enhancing the onset of switch recombination, as shown here with IL-4.

In summary, a retrovirally transduced switch recombination substrate convincingly demonstrates that a pair of Sµ donor and S{gamma}2b acceptor S sequences (in the absence of all known CH locus cis-acting promoters, enhancers, and RNA maturation signals) are themselves sufficient for mediating extensive, sequence-specific switch recombination in the context of normal B lymphocytes. SSRs harboring IgH locus control elements introduced into B cells stimulated with CD40L and other switch-mediating cytokines should facilitate a complete evaluation of switch recombinase targeting as a function of normal B cell development.


   Acknowledgments
 
We gratefully acknowledge the technical assistance of Ms. Darlene Balzarano for the performance of the endogenous DC-PCR assays.


   Footnotes
 
1 This work was supported by U.S. Public Health Service Research Grant GM26939 (awarded to K.B.M.). Back

2 These authors contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Kenneth B. Marcu, Department of Biochemistry and Cell Biology, SUNY at Stony Brook, Stony Brook, NY 11794-5215. E-mail address: Back

4 Abbreviations used in this paper: S, switch; SSR, switch substrate retrovector; Sµ, switch µ region; S{gamma}2b, switch {gamma}2b region; DC-PCR, digestion-circularization PCR; {alpha}{delta}-dex, dextran conjugated with anti-IgD; sIg, surface Ig; CD40L, CD40 ligand; hCD40L, human CD40L; mCD40L, murine CD40L; Ltk-hCD40L, human CD40L presented on the surface of Ltk- cells; GM, growth media; HyTk, hygromycin thymidine kinase gene; Neo, neomycin gene; neor, neomycin resistant; Gag, gene encoding retroviral structural proteins; EtBr, ethidium bromide; bp, base pair. Back

Received for publication December 30, 1997. Accepted for publication March 26, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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