Sγ3 switch sequences function in place of endogenous Sγ1 to mediate antibody class switching

We used our previously established strategy to replace the endogenous 10-kb Sγ1^a region of a γ1^a/γ1^b F1 embryonic stem (ES) cell line with a 2-kb portion of endogenous Sγ3 (2-Sγ3), a 2-kb synthetic Sγ3 (2-SSγ3), and a 2-kb synthetic Sγ1 (2-SSγ1; Fig. 1) (7, 9, 13). For generation of the 2-SSγ3 sequence, we used linkers to concatemerize 40 copies (∼2 kb) of the Sγ3 consensus sequence (Fig. S1). The orientation of tandem repeats in the SSγ3 is unidirectional, therefore mimicking the repeat structure and nucleotide content of the endogenous Sγ3 (26). The 2-Sγ3 sequence comprises 1946 bp of the endogenous Sγ3 region from nucleotides 646 to 2592 (available from GenBank/EMBL/DDBJ under accession no. M12182) (26). This sequence has been previously shown to mediate recombination in transient assays and has been used to assay for S region–specific factors (18, 20). The WT Sγ1 repeat is nearly identical to the synthetic Sγ1 region and functionally supports CSR in a linear fashion compared with the WT Sγ1 sequence (9). The purpose of testing synthetic Sγ1 and Sγ3 substrates was to determine if this approach would allow endogenous CSR assays of substrates in which the only variables were the few nucleotide differences within each repeat unit and also to be able to test potential functions of candidate motifs within a given S region by generation of synthetic sequences with differing repeat structures.

The F1 ES cell was derived from the hybrid 129Sv-C57BL/6 mice in which the two IgH alleles represent the IgH^a (from 129/Sv) or IgH^b (from C57BL/6) allotypes, respectively. The presence of sequence polymorphisms and allotypic markers (antibodies to IgG1^a) facilitates comparison of the level of CSR on modified alleles to the internal control of the unmodified IgH^b allele. After successful gene targeting, the inserted neo cassette was removed by loxP/Cre recombination (Fig. 1). To analyze the effect of transcription orientation, the two loxP sites flanking the insert were placed in inverted orientation, allowing Cre-mediated recombination to invert the test sequences. Southern blot analyses were performed to confirm the correct integration of the replaced sequences (Fig. 1 B) (9). The Sγ1-replaced mutant F1 ES cells were injected into RAG2-deficient blastocysts to generate chimeric mice to obtain mature B lymphocytes harboring the targeted mutation (27). Splenocytes from mutant and control mice were activated for 2 d with antibody against CD40 (anti-CD40) plus IL-4, a treatment that induces germline transcription of the Cγ1 gene and CSR to IgG1. Subsequently, we measured the relative levels of steady-state germline transcription from the WT γ1^b and targeted γ1^a alleles by RT-PCR (Fig. 2) (5, 9, 13). The level of the endogenous γ1^b transcript serves as an internal control, and could be distinguished by an MboI restriction site polymorphism only present in the C57B6 allele. The SSγ1, Sγ3, and SSγ3 replacements generated similar levels of germline transcripts to those from the WT γ1^b allele.

Transient studies suggested that Sγ3 has the potential to support CSR in B cells stimulated to undergo CSR to Sγ1 (21). To compare CSR efficiency of Sγ3- or SSγ3-replaced endogenous Sγ1 alleles with Sγ1 alleles harboring similar lengths of Sγ1 repeats, we performed ELISA to quantify IgG1^a versus total IgG1 in the anti-CD40 plus IL-4 culture supernatants (5, 7, 9, 13). The WT controls were splenic B cells derived from WT F1 ES cells using RAG2-deficient blastocyst complementation. The ratio of IgG1^a to total IgG1 in WT F1 cells was set at 100% (7). These experiments showed that in anti-CD40 plus IL-4–stimulated B cells, size-matched sequences of SSγ1, Sγ3, or SSγ3 all generated levels of IgG1^a secretion that ranged from 25–50% of those of WT alleles (Fig. 3). On the other hand, stimulation of 2-Sγ3 splenocytes for up to 6 d by treatment with LPS, conditions that normally induce germline Cγ3 gene transcription and IgG3 CSR, did not result in any significant increase in IgG1 production in either WT or mutant B cells (Fig. S2), consistent with the fact that the germline γ1 gene is not transcribed under LPS stimulation conditions.

To quantify CSR at a single-cell level, we generated hybridomas from activated B cells. Hybridomas represent fusions of individual B cells to the myeloma partner cell line. We selected IgG1-producing hybridomas by ELISA and compared the level of IgG1^a with IgG1^b to score for recombination efficiency between WT and mutated alleles. The IgH locus is subject to allelic exclusion, and only one of the two IgH alleles in a given B cell is recombined functionally into a V(D)J coding region. Therefore, in an F1 control, half of the activated B cells should produce IgH^a allotype antibodies, and the other half should produce IgH^b allotype antibodies. Relative CSR frequency is defined by the ratio of IgG1^a- to IgG1^b- producing hybridomas (IgG1^a/IgG1^b) and is arbitrarily set as 100% for the F1 control. In these experiments, the WT F1 B cell population stimulated with anti-CD40/IL-4 yielded 63 IgG1^a- and 42 IgG1^b-producing hybridomas (Table I). In the absence of Sγ1, we did not find any IgG1^a-producing hybridomas (0 IgG1^a and 160 IgG1^b hybridomas), as expected (7). Compared with the control, the relative ratio of IgG1^a/IgG1^b hybridomas was 31% in 2-SSγ1, 30% in 2-SSγ3, and 47% in 2-Sγ3, respectively. Therefore, the levels of CSR to alleles in which Sγ1 sequences were replaced with endogenous or synthetic Sγ3 sequences was at least as high as those to alleles in which Sγ1 was replaced with synthetic Sγ1.

Although the levels of CSR supported by the 2-Sγ3 replacement alleles appeared slightly higher in both the ELISA and hybridoma analyses, the differences in the ELISA analyses were not statistically significant. Determining whether or not there is a slight preference for the 2-Sγ3 sequences would require further study. However, inversion of each type of inserted S region sequence clearly resulted in substantially decreased IgH class switching to IgG1^a (Fig. 3 and Table I), potentially caused by decreased R loop formation in the inverse direction (7, 9, 13). Finally, we used a nested PCR approach to map Sμ to SSγ3 junctions in IgG1^a-producing hybridomas. Junctions occurred throughout the SSγ3 repeat, similar to WT Sγ3 (Fig. S3) (10). Although four out of eight junctions (50%) fell into a GAGCT motif surrounded by G nucleotides, further mutational studies on synthetic S regions would be required to identify the motifs that are preferentially targeted during CSR in vivo.

Given the central role of IgH isotype class switching in the humoral immunity, it is of significant interest to identify the mechanisms that contribute to the specificity of this process. Past studies have led to the view that S region–specific factors may be required for targeting CSR to Sγ3 versus Sγ1, and vice versa (24). In this report, we show that synthetic or WT Sγ3, or size-matched Sγ1 sequences, when inserted in place of the endogenous Sγ1, can mediate roughly similar levels of CSR under B cell activation conditions in which CSR to the endogenous Sγ1, but not the endogenous Sγ3, is induced. We have previously shown that the Xenopus Sμ sequence, when substituted for the mouse Sγ1 region, can mediate CSR at substantial levels, even though it is AT rich and lacks the ability to form an R loop structure in vitro (13) and in vivo (Leiber, M., personal communication). Thus, our previous Xenopus Sμ replacement study (13) complements our current findings; collectively, these studies strongly indicate that nucleotide sequence differences between Sγ1 and other S regions are not likely to be major determinants of endogenous IgG1 CSR targeting. Correspondingly, our current findings support germline transcriptional activation of the Sγ1 region as the primary mechanism for targeting CSR to IgG1 (28, 29). In this context, the finding that Sγ3 sequences in place of Sγ1 sequences do not support CSR to IgG1 under conditions (LPS activation) in which IgG3 CSR is induced would reflect the fact that LPS fails to induce germline transcription of the Cγ1 gene promoter. Finally, we note that our approach now can be used to test for potential roles of putative S region–specific factors in mediating specific CSR events to other S regions under other stimulation conditions (21, 24).

To generate synthetic Sγ3, the consensus Sγ3 (5′-GGATCCGGGGAGCTGGGGTAGGTTGGGAGTGTGGGGACCAGGCTGGGCAGCTCTGAGATCT-3′; BamHI and BglII sites are underlined; reference 26) was oligomerized by sequential cloning into the BamHI site of S85 vector to generate 2-SSγ3 (Fig. S1). After each cloning step, the insert orientation was confirmed by sequencing and restriction endonuclease digestion. The consensus repeats were confirmed to be unidirectional. The 2-SSγ3 sequence was excised as a NotI and SalI fragment and cloned into the targeting construct previously described (9). The endogenous Sγ3 region was excised as a BamHI/NotI fragment from pSV5 plasmid (provided by A. Kenter, University of Illinois at Chicago, Chicago, IL) and cloned into pBluescript. Subsequently, the NotI/SalI fragment was ligated into the targeting vector as previously described (9). 2-SSγ1 has been previously described (9).

The targeting constructs were transfected into ES cells in which the Sγ1^a was deleted (7). The targeted ES cells were identified by Southern blotting as described in Fig. 1 B (13). The deletion of the neo^r gene was achieved by infecting ES cells with cre-expressing adenovirus. Targeted ES cells were subcloned and injected into RAG2-deficient blastocysts to produce mature lymphocytes that all harbored the mutant allele (27). Splenic B cells from 6–8-wk-old chimeras were used in our experiments. Mouse protocols were approved by the Institutional Animal Care and Use Committee of Children's Hospital.

ELISA and hybridoma analysis were performed as previously described (13). Spleen cells from 6–8-wk-old chimeras were stimulated in vitro with 1 μg/ml anti-CD40 (HM40-3; BD Biosciences) plus 25 ng/ml IL-4, or 20 μg/ml LPS alone. 1.5 × 10⁶ cells were seeded in one well of a sixwell plate (0.5 × 10⁶ cells/ml) in RPMI 1640 media supplemented with 10% FBS, 2 mM glutamine, 100 U/ml penicillin–streptomycin, and 100 μM β-mercaptoethanol. Stimulated B cells were used to generate hybridomas (after 4 d) or ELISA (after 6 d), as previously described (7, 13). Monoclonal anti–mouse IgG1^a (Igh-4a; BD Biosciences) was used to detect IgG1^a (from the mutated allele). Alkaline phosphotase–conjugated goat anti–mouse IgG1 (SouthernBiotech) was used as the detection antibody. Purified mouse IgG1^a (BD Biosciences) was used as the standard. Because an antibody specific for IgG1^b is not available, we normalized the production of IgG1^a against IgG1 total for ELISA assays on splenic B cell stimulations. The ratio of IgG1^a/IgG1 total of the WT F1 chimeras was defined as 100% CSR efficiency for the WT γ1a allele. We measured the ratio of IgG1^a to IgG1 for different chimeras. Relative CSR efficiency was calculated by the ratio of IgG1^a- to IgG1^b-producing hybridomas. Hybridomas that produced only IgG1 and not IgG1^a were considered to produce IgG1^b. We defined the numbers of cells that switched γ1 on the a or b allele as γ1a and γ1b, respectively, and the numbers of total Ig⁺ cells for the two alleles as Ig^a and Ig^b, respectively. The switching efficiency to γ1 was given as Sa = Sa/Sb = (γ1a/γ1b)/(Ig^b/Ig^a). The ratio of Ig^a/Ig^b was determined by the relative ratio of productive V(D)J recombination on the two alleles and was expected to be close to 1. Thus, Ra can be simplified as γ1^a/γ1^b. After mutation of Sγ1, Ra′ = γ1a′/γ1b′, and Ra′/Ra = (γ1a′/γ1b′)/γ1^a/γ1^b) (7). For example, 2-SSγ1 produced 38 IgG1^a-producing and 81 IgG1^b-producing hybridomas. We normalized this ratio by dividing (38/81) to (63/42 = 1.5) to determine the CSR frequency of the mutated allele (31%).

CSR junctions were amplified from hybridomas by nested PCR (13). Nested mouse Iμ primers were 5′-CTCTGGCCCTGCTTATTGTTG-3′ followed by 5′-AGACCTGGGAATGTATGGTT-3′. The reverse nested primers were located in exon1 of Cγ1, and were 5′-CAATTTTCTTGTCCACCTTGGTGCTG-3′ followed by 5′-GTGTGCACACCGCTGGACAGG-3′. PCR products were gel purified and sequenced. S junctions were analyzed with the SeqMan program (DNAStar Lasergene) and the MEGABLAST program (National Center for Biotechnology Information; Fig. S3).

Fig. S1 shows the nucleotide alignment of the synthetic Sγ3 and endogenous Sγ3. Fig. S2 shows ELISA of LPS-stimulated splenocytes from 2-Sγ3. Fig. S3 provides an analysis of CSR junctions.

We thank Yuko Fujiwara and Nicole Stokes for generating RAG chimeras and Ming Tian for technical advice. We are grateful to Cherry Lei and Bill Forrest for statistical analyses.

This work was supported by National Institutes of Health grant AI31541 (to F.W. Alt). F.W. Alt is an Investigator of the Howard Hughes Medical Institute.

The authors have no conflicting financial interests.