Polyclonal hyper-IgE mouse model reveals mechanistic insights into antibody class switch recombination

Significance

Switch (S) regions are repetitive DNA sequences. During an immune response, one of several S regions recombine with a donor switch (Sμ) that is constitutively “on,” resulting in the production of antibodies with new functions. Donor Sμ is large and very repeat-rich, while another switch, Sε, is less than half its size with a low density of repeats. We replaced Sε with Sμ in mice. These mice switch to Sε more effectively and produce high levels of IgE antibodies implicated in asthma, making this a useful model to study disease. In addition, placing Sμ outside of its native context revealed insights into how switches work.

Abstract

Preceding antibody constant regions are switch (S) regions varying in length and repeat density that are targets of activation-induced cytidine deaminase. We asked how participating S regions influence each other to orchestrate rearrangements at the IgH locus by engineering mice in which the weakest S region, Sε, is replaced with prominent recombination hotspot Sμ. These mice produce copious polyclonal IgE upon challenge, providing a platform to study IgE biology and therapeutic interventions. The insertion enhances ε germ-line transcript levels, shows a preference for direct vs. sequential switching, and reduces intraswitch recombination events at native Sμ. These results suggest that the sufficiency of Sμ to mediate IgH rearrangements may be influenced by context-dependent cues.

Switch (S) regions are essential and specialized targets of activation-induced cytidine deaminase (AID) (1–3) that are ordered 5′-Sμ-Sγ3-Sγ1-Sγ2b-Sγ2a-Sε-Sα-3′ (4) in the mouse IgH locus (Fig. 1A). Joining of distant dsDNA breaks (DSBs) between donor Sμ and any downstream S region constitutes class switch recombination (CSR). CSR to specific constant heavy-chain genes is subject to tight transcriptional regulation, which increases the accessibility of a given S region before CSR (5, 6). The primary role of S regions is to seed DSBs (7), which are repaired by nonhomologous end joining (8) predominately during the G1 phase of the cell cycle (9), whereas homologous recombination is dispensable in CSR (8, 10, 11). AID initiates CSR by targeting cytidines in transcribed repeat-rich S regions (12–14). Limited amounts of active AID in B cells can be inferred from recent studies that showed that AID heterozygous mice have reduced levels of somatic hypermutation (SHM) and CSR (15–18). Epigenetic modifications during CSR are emerging as an important regulatory mechanism to influence CSR (19). Thus, complex regulatory mechanisms act in concert to ensure appropriate AID regulation, likely to limit its off-target activity (20).

Fig. 1.

Targeting strategy and generation of SμKI mice. B, BamHI; E, EcoRI; H, HindIII. (A) Schematic representation of the IgH constant region in mice. (B) Schematic representation of Sμ, Sε, and SμKI within the IgH locus. Sμ, Cμ, Iε, and Cε probes are depicted. Estimated sizes of Sμ vs. SμKI after HindIII digestion and SμKI vs. Sε after BamHI digestion are indicated.

S regions have acquired intrinsic properties to make them the ultimate substrate for AID within the genome (21). Ancient S regions resemble SHM substrates, except they have a higher density of hotspots. The density of hotspots in S regions is significantly higher than in V regions (4), potentially creating areas highly susceptible to DSBs (4). In mammals, S regions appear to have further diverged by incorporating features such as the ability to form R-loops, which are single-stranded DNA loops formed by the association of an RNA transcript with a DNA template (22) and G-quartets, which are four-stranded structures of guanine-rich DNA (23), to maximize them as targets for AID (24). S region length enhances CSR (25), and there is an inverse correlation between the distance of DSBs and recombination frequency (7). In mice, Sε is one of the shortest and least repetitive S regions, and, with the exception of Sα, it is the farthest from Sμ (4). CSR to Sε involves sequential CSR between Sμ and Sγ1 before combining with Sε (26–29); however, a sequential pathway is not required, as direct CSR between Sμ and Sε occurs when Sγ1 is genetically ablated (30, 31). It is possible for multiple DSBs to occur within a single S region, which leads to intraswitch recombination (ISR). This phenomenon is seen more frequently in Sμ (28, 32, 33) than in other S regions in the context of the normal IgH locus, possibly because it is enriched in AID target motif sites (4) or because context-dependent cues regulate the targeting of the donor Sμ region. ISR in downstream acceptor S regions are more abundant in Sμ^−/− (28) or transcriptionally inactive Sμ mutants (34), suggesting acceptor S regions are able to mount ISR in the absence of Sμ.

To generate a polyclonal “hyper-IgE” mouse model and to gain insights into how S regions work outside their native context, we created a mouse model in which the weakest S region, Sε, was replaced with the strongest AID hotspot, Sμ. Sμ knock-in (SμKI) mice produce abundant IgE at the expense of other isotypes. SμKI IgE is antigen (Ag)-specific and produced in response to a variety of local and systemic stimuli. On a mechanistic level, the presence of Sμ in place of Sε enhances ε germ-line transcript (GLT), suggesting its presence influences accessibility of the locus. Circle transcript studies reveal a preference for direct CSR vs. sequential in SμKI mice. The knocked-in switch also negatively affects ISR of endogenous Sμ. Taken together, these results suggest the Sμ sequence has properties that are at least in part context-dependent.

Results

SμKI Modified ε Allele Outcompetes Sγ1 to Produce High Amounts of IgE in Stimulated B Cells.

An overview of the IgH locus is shown in Fig. 1A. A diagram of probes used in Southern blotting analyses is shown in Fig. 1B. Gene targeting was used to replace murine Sε (∼2 kb) with Sμ (∼5 kb) in C57BL/6 ES cells (Fig. S1 A and B). Homozygous knock-in mice are referred to hereafter as SμKI mice, and all animals were maintained in a C57BL/6 background. A phenotypic analysis of the SμKI mice did not reveal any defects in lymphocyte development (Fig. S1C). The effect of inserted Sμ on CSR was assessed by FACS and ELISA analysis of splenocytes stimulated with LPS plus IL-4 or LPS alone for 4 d (Fig. 2). In cells stimulated with LPS and IL-4 (Fig. 2A), WT and SμKI cultures contained similar numbers of IgM⁺ B cells. However, in the SμKI cultures, IgG1⁺ B cells were decreased ∼11-fold, whereas the level of IgE⁺ B cells in the SμKI cultures increased approximately sixfold relative to WT. As expected, this effect was AID-dependent, as AID-deficient splenocytes (35) yielded negligible IgG1⁺ or IgE⁺ B cells. In response to LPS stimulation, WT and SμKI cells produced comparable percentages of IgM⁺ B cells (Fig. 2B). A ∼50% decrease in IgG3 levels was seen in the SμKI, whereas an approximately fourfold increase in IgE⁺ cells was seen in SμKI cultures relative to WT cultures. ELISAs conducted 6 d after stimulation showed similar tradeoffs in secreted IgG and IgE (Fig. 2 C and D). With LPS/IL-4 stimulation (Fig. 2C), IgM levels were comparable among WT, heterozygous, and SμKI cultures; however, IgE was far more abundant when cells carried an SμKI allele. With LPS treatment (Fig. 2D), a ∼50% decrease in secreted IgG3 was observed in the KI, as well as an enhanced level of secreted IgE.

Fig. 2.

In vitro stimulated SμKI B cells produce higher levels of IgE and lower levels of IgG1 antibodies compared with WT B cells. (A) Splenocytes were stimulated 4 d with LPS/IL-4 and the indicated antibody isotypes measured by FACS. B220 was used as a B-cell marker, and AID^−/− mice were used as negative control. (B) Splenocytes were stimulated 4 d with LPS, and the indicated antibody isotypes measured by FACS. B220 was used as a B-cell marker. (C) Splenocytes from WT, heterozygous, and SμKI mice (n = 5) were stimulated with LPS/IL-4 for 6 d, and the indicated antibody isotypes measured by ELISA. (D) Splenocytes from WT, heterozygous, and SμKI mice (n = 5) were stimulated with LPS for 6 d, and indicated antibody isotypes measured by ELISA.

DNA rearrangements of spleen cell-derived IgH loci in hybridomas reflect CSR events in normal splenic B cells at a single-cell level. To quantitate CSR, hybridomas were generated from splenocytes stimulated for 2 and 4 d with LPS/IL-4 (Table 1). On day 4, IgG1⁺ clones were reduced ∼10-fold in the KI relative to WT, and IgE⁺ clones increased approximately sevenfold. Heterozygous hybridomas showed an intermediate phenotype, with threefold fewer IgG1⁺ clones and fivefold more IgE⁺ clones relative to WT. The increased number of IgE⁺ clones in the KI does not appear to result from sequential switching, as more IgE⁺ B-cells are already evident in SμKI compared with WT only 2 d after stimulation. PCR and sequencing analysis confirmed that IgE⁺ hybridomas contained Sμ–Sε (WT) or Sμ–Sμ (SμKI) junctions (Figs. S2 and S3). These results, together with the aforementioned FACS and ELISA data, are consistent with AID targeting to ε being enhanced substantially by substitution of Sμ for Sε.

Table 1.

Quantification of isotype switching by B-cell hybridomas

Day/hybridoma	No. of clones	IgM+, %	IgG1+, %	IgE+, %
Day 2
WT	562	95	3	2
KI	594	88	1	11
Day 4
WT (fusion 1)	1,440	32	57	11
WT (fusion 2)	946	21	68	11
HET	1,379	20	21	59
KI (fusion 1)	1,444	14	6	80
KI (fusion 2)	1,141	18	4	78

At 2 or 4 d after LPS/IL-4 stimulation, splenocytes from WT and SμKI (i.e., heterozygous or homozygous) mice were fused to the NS-1 host to create hybridomas. ELISAs were used to obtain percent IgM⁺, IgG1⁺, and IgE⁺ hybridomas.

SμKI Mice Produce Copious IgE in Response to a Variety of Systemic and Local Challenges.

We determined whether the IgE produced by SμKI mice is Ag-specific or merely reflects a nonspecific surge in CSR to the modified ε locus by applying several systemic and local challenges. Mice were immunized with T-cell–dependent antigen 2,4,6-trinitrophenyl ovalbumin (TNP-OVA) (31) and IgE titer was measured. No TNP-specific IgE was present before immunization (Fig. 3A), although slightly enhanced total serum IgE was seen in SμKI (Fig. S4A). On days 14 and 28 postimmunization, TNP-specific IgE titers in SμKI mice were approximately fivefold higher than in WT mice. SμKI mice were also locally challenged with various model allergens to see if they would produce serum IgE in excess. SμKI mice immunized with TNP-OVA were challenged intranasally with aerosolized TNP-OVA to model asthma (Fig. 3B). Challenged SμKI mice produced 8- to 10-fold higher TNP-specific serum IgE than their WT counterparts, whereas baseline TNP-specific IgE was undetectable. Remarkably, SμKI mice accumulated significant levels of Ag-specific IgE (∼100,000 ng/mL) within 4 d after challenge. Induction of IgE in SμKI mice was similarly robust following intranasal delivery of an Ag mixture in the absence of systemic sensitization (Fig. 3C and Fig. S4B), or following s.c. infection with Nippostrongylus brasiliensis larva (Fig. 3D). By contrast, IgE titers in WT mice postinfection barely reached the basal IgE titers seen in the SμKI mice. In sum, our in vivo data demonstrate that the SμKI allele supports robust IgE isotype switching in response to a variety of challenges.

Fig. 3.

SμKI mice express higher levels of IgE compared with WT mice in vivo. (A) WT (n = 3), heterozygous (Het; n = 3), and SμKI (n = 7) mice were immunized with TNP-OVA. TNP-OVA–specific serum IgE levels were measured by ELISA before and at 14 and 28 d postimmunization. (B) Mice (n = 5 per group) were immunized with TNP-OVA/alum and challenged with aerosolized 1% TNP-OVA. Baseline, prechallenge and postchallenge TNP-OVA–specific serum IgE levels were measured by ELISA. (C) WT, Het, or SμKI mice (n = 6 per group) were sensitized to multiple allergens as explained in Materials and Methods, and total serum IgE levels were measured by ELISA before and on days 21 and 42 postsensitization. (D) Total serum IgE levels in WT, Het, and SμKI mice (n = 5 per group) were measured by ELISA before and 4, 9, and 14 d postinfection with 500 N. brasiliensis larvae. P values were calculated between WT and SμKI samples by using a t test assuming unequal variances.

Replacement of Sε with Sμ Enhances Epsilon GLT.

To explore the mechanistic basis for enhanced IgE production in SμKI mice, we analyzed GLT levels under various stimuli. Germ-line transcripts are non-coding RNAs that begin at a short exon upstream of each S region termed the I exon, and proceed through the constant regions. Purified B cells were activated 2 d with LPS or LPS plus IL-4 (Fig. 4 A and B and Fig. S5A), LPS plus IFN-γ, or IL-4 plus αCD-40 and TGF-β (Fig. S5 B and C). The LPS-treated samples showed comparable levels of GLT induction for μ, γ2b, and γ3 between WT and SμKI B cells. As expected, WT cells did not produce ε transcripts before or after LPS treatment. In contrast, SμKI cells showed enhanced basal ε transcription without stimulation, and with LPS treatment this was slightly enhanced (Fig. 4A). Likewise, in independent stimulations that used LPS plus IL-4 (Fig. 4B), ε GLT levels were again high in resting SμKI cells and were induced by approximately twofold in response to activation. GLT levels for μ and γ1 appeared unaltered by the Sμ insertion. Northern blot analysis consistently showed enhanced ε GLT (Fig. S5A) (36). GLT levels for μ, γ2a, and α appeared comparable between WT and SμKI (Fig. S5 B and C). The enhanced ε GLT seen in the knock-in suggests that, in addition to providing a longer and more motif-rich region for CSR, the insertion alters ε locus accessibility.

Fig. 4.

GLT levels in WT vs. Sμ-KI mice. At least three independent mice per genotype were tested with similar results. (A) Semiquantitative RT-PCR with RNA from WT or Sμ-KI B cells that were freshly isolated or stimulated with LPS for 2 d. Starting amounts of RNA were 2 ng, 10 ng, and 50 ng. A fixed cycle number in the linear range for the specific product was used. Gel images are shown for μ (25 cycles) and β-actin (25 cycles). Southern blot autoradiographs are shown for γ2b (30 cycles), γ3 (30 cycles), and ε (45 cycles for WT, 30 cycles for SμKI). (B) Similar to A but with LPS/IL-4 stimulation. Gel images are shown for μ, γ1 (30 cycles), and β-actin. A Southern blot autoradiograph is shown for ε.

CSR to the SμKI Locus Is Largely Direct Instead of Sequential.

The enhanced CSR to the SμKI locus raises the question of whether switching occurs sequentially via Sγ1 or is direct. Sequential switching is a two-part mechanism in which CSR first occurs between Sμ and Sγ1, which subsequently switches to Sε, or between Sγ1 and Sε, which then switches to Sμ (27, 28). To address this question, we looked for the presence of circle transcripts in LPS/IL-4 activated B cells over time (Fig. 5). Circle transcripts are produced when the intervening genomic DNA between two connecting S regions is looped out, forming an episome in which the I promoter of the downstream S region ends up driving transcription of the constant (C) region adjacent to the upstream S region.

Fig. 5.

Contribution of direct vs. sequential switching to IgE. (A) Diagram of the IgH locus and relevant features and primers indicated. (B) RNA was isolated from WT or SμKI B cells at the indicated time points after LPS/IL-4 stimulation. RT-PCR was performed by using primers specific to Iε and Cμ (result of circles formed from Sμ–Sε junctions, lanes 1–5), and primers specific to Iε and Cγ1 (result of circles formed from Sγ1:Sε junctions, lanes 6–10). The resulting products were hybridized to probes corresponding to Cμ and Cγ1. (C) β-Actin at 20 cycles is shown as a loading control.

We analyzed RNA from resting and activated WT and SμKI B cells by using RT-PCR and primer sets capable of detecting direct and sequential switch products (Fig. 5A). The product of primers IεFW and CμRV (Fig. 5B, lanes 1–5) displays switching to Sε, which could be direct or sequential via an Sμ/Sγ1 or Sγ1/Sε intermediate. Visualized with a probe to Cμ, this product appears after 48 h and appears significantly more robust in SμKI cells, consistent with their enhanced IgE production. The combination of primers IεFW and Cγ1RV identifies CSR between Sγ1 and Sε, which is a unique sequential switch product (Fig. 5B, lanes 6–10). Highlighted by a Cγ1 probe, this product appears after 72 h in WT cells, becoming more abundant by 96 h. Its levels are significantly lower in SμKI cells, suggesting the cells favor a more direct mechanism of CSR over a sequential pathway. Equivalent RNA inputs were used in this experiment, as shown by β-actin RT-PCR (Fig. 5C).

To determine what fraction of B cells undergo sequential switching, we generated a panel of IgM⁺ and IgE⁺ hybridomas from WT and SμKI B cells stimulated with LPS/IL-4 (Fig. S6). Genomic DNA from the IgM⁺ hybridomas was digested with EcoRI to release a fragment containing Sε/SμKI (Fig. S6 A and B; see also Fig. 1B) and hybridized to the Cε probe. In the event of recombination between Sγ1 and Sε/SμKI, the size of the EcoRI fragment would be altered (28). We did not detect changes in band size in hybridomas from 2- or 4-d stimulated B cells, suggesting the frequency of sequential switching is low in both genotypes. We also used a nested PCR strategy to amplify Sμ–Sε junctions from IgE⁺ hybridomas and probed for the presence of Sγ1 in Southern blots (Fig. S6 C and D). We detected seven Sμ-Sε junctions containing Sγ1 among 57 WT hybridomas (12.2%), vs. 1 of 58 hybridomas containing Sγ1 for SμKI (1.7%), suggesting sequential switching is less favored in SμKI B cells.

Endogenous Sμ ISR Is Reduced in the Presence of Knocked-In Sμ.

To view the impact of the SμKI on ISR, IgM⁺ hybridomas were analyzed for deletions within endogenous Sμ by Southern blot (Fig. 6 A and B). ISR was detected in 60% of WT hybridomas, which decreased to 38% and 25% of heterozygous and homozygous SμKI hybridomas, respectively. The observation that SμKI negatively impacts ISR at upstream endogenous Sμ implies that the insertion competes away factors necessary for ISR or that SμKI channels endogenous Sμ region breaks into a productive CSR reaction with higher frequency. It should be noted that Southern blotting is a low-resolution assay, and so may reflect only a fraction of total events (32).

Fig. 6.

ISR frequency in B-cell hybridomas by Southern blot. Restriction fragments and probes are depicted in Fig. 1B. (A) IgM⁺ hybridomas derived from LPS/IL-4 stimulated B cells were digested with EcoRI and probed with Cμ to detect ISR within endogenous Sμ. (B) Table summarizing the number of endogenous Sμ ISR events in IgM⁺ hybridomas in WT, heterozygous (HET), and SμKI. (C) IgM⁺ hybridomas derived from LPS/IL-4–stimulated B cells were digested with BamHI and probed with Iε. Note that an ∼3-kb increase in size is observed when Sε is replaced with Sμ (i.e., SμKI). ISR of Sε or SμKI was absent in all 48 WT hybridomas screened, and only one ISR event was detected in the SμKI (asterisk). The Sε band in WT samples may originate from both the B-cell and NS-1 fusion partner, whereas, in KI samples, it stems only from the fusion partner.

We next examined whether SμKI could undergo ISR in the context of the ε locus. The Iε probe was used to analyze ISR within Sε and inserted Sμ in hybridomas created from IgM⁺ B cells (Fig. 6C). Among 96 hybridomas, Sε did not display any internal recombination events and SμKI displayed only one event, suggesting the primary sequence of Sμ is not efficient at ISR. Instead, context-dependent cues such as the presence of regulatory elements (e.g., enhancer Eμ or Iμ), rate of transcription, or local chromatin modifications may have influence.

The differences in mechanism by which ISR, CSR, and SHM occur are not well understood and the processes can be temporally distinct (37). We hypothesized that, if SμKI impacts other S regions in the context of CSR, it might also affect SHM in SμKI B cells. We sorted IgM⁺ or IgG1⁺ germinal center B cells in response to TNP-OVA to scan for mutations downstream of JH4 and upstream of endogenous Sμ (Fig. S7A) (31). Our analysis revealed that the rate of SHM in SμKI B cells was not significantly different from WT. Rather, there was a slight trend toward lower mutation frequency in the SμKI allele. To document the impact on SHM directly in V regions, a panel of IgG1-producing TNP-OVA specific hybridomas was generated after TNP-OVA immunization, and heavy chain variable region (VH) gene sequences were aligned with germ-line counterparts to locate mutations (Fig. S7B). We did not detect a major difference in mutation frequency between WT and SμKI. Thus, alteration of the IgH locus in SμKI impacts CSR and ISR, but does not appear to impact SHM.

Discussion

Sμ is a constitutively transcribed donor S region that is unusually dense with AID target hotspots (4). S region size linearly correlates with enhanced CSR (25). In our model, we replaced the 2-kb Sε with a larger ∼5-kb core Sμ. The GLT of the SμKI locus is enhanced relative to unmodified Sε. Thus, increased CSR seen in the SμKI could be a result of the sequence of Sμ or the increased S region length combined with greater transcriptional accessibility. The result is a dramatic increase in the magnitude of IgE isotype switching, even exceeding the IgG1 levels associated with the largest S region, Sγ1.

It is not understood to what degree the context of Sμ influences its ability to undergo CSR and ISR. We provide data supporting the notion that Sμ inserted in place of Sε is insufficient to carry out certain functions it would have in its endogenous location. The knocked-in sequence is able to reduce ISR of endogenous Sμ from a distance, either by competing for limited factors or through providing DSBs leading to productive CSR with its upstream twin. However, despite the known recombinogenicity of Sμ, the KI is insufficient to generate levels of ISR comparable to its upstream counterpart, suggesting the sequence itself is insufficient or the ε locus is not conducive to ISR events (28, 31). This is also unusual given that ISR is found for endogenous Sε (28, 31) as well as other acceptor S regions (28, 32, 33). The robust targeting of native Sμ might facilitate productive CSR by making targeting of downstream S regions rate-limiting, which ultimately may limit the duration and production of unnecessary DSBs (32). Although AID is capable of initiating DSBs in all S regions, AID-mediated ISR may be influenced by regional DNA architecture and recruited DNA repair machinery. Our finding suggests that the frequent targeting of endogenous Sμ is context-dependent.

The mechanism of direct vs. sequential CSR to produce IgE is not well understood, and this process might be developmentally regulated (38). Our analysis of circle transcripts indicates that direct and sequential switching can mediate CSR. Sequential switching might involve Sμ/Sγ1 junctions joining to Sε or switching between Sγ1/Sε before joining Sμ (27, 39). In our assay, we observed reduced levels of Sγ1/Sε products in SμKI relative to WT. These data indicate that, in SμKI cells, switching is more direct. In addition, our ISR data suggests that a prominent, active downstream S substrate prefers direct switching vs. indirect with donor Sμ, in which there is a higher chance to generate two simultaneous DSBs (7). This interpretation agrees with the fact the sequential switching is mainly observed for Sε and not for other S regions (27, 39).

It is intriguing that steady-state GLT at the ε locus is positively affected by the Sμ insertion. This observation could explain the low but detectable levels of IgE observed in LPS-stimulated SμKI B cells. It could be that the ε locus is an inherently difficult substrate to transcribe and easily influenced by perturbations and/or the Sμ sequence somehow renders the locus transcriptionally open. In support of the latter, limited studies of marginal zone B-cell lymphomas suggest human Sμ sequence can act as a promoter to drive the expression of the Pax-5 gene in recurring (9:14) human chromosomal translocations (40). Sequence amplifications of Sμ and surrounding regulatory regions have been reported in mouse and human B-cell tumors that involve CSR (41). On the contrary, removal of core Sμ has not been shown to alter the GLT of endogenous Cμ (1), and substituting Sγ1 with Xenopus Sμ does not affect Iγ1 promoter activity in the context of the endogenous mouse IgH locus (3). Insertion of the heterologous promoter for Phosphoglycerate kinase 1 (Pgk) linked to neomycin within the IgH locus has been implied to affect GLT by isolating germ-line I promoters from the 3′ regulatory region, or by competing for access to this region (42, 43). In the context of the epsilon locus, replacement of Cε exons with a Pgk-neomycin cassette in an antisense orientation can inhibit the GLT of Cγ3, Cγ2b, and Cγ2a genes (36). According to our study, however, the enhanced activity of the Iε promoter at the SμKI allele does not appear to appreciably affect the GLT of other heavy chain constant (C_H) genes. In contrast to the Pgk-neomycin insertion model, the sense orientation of the endogenous I epsilon promoter in our SμKI model is conserved and does not include additional sequences such as a neomycin cassette. Our finding demands additional studies to elucidate the mechanism behind this observation.

The SμKI hyper-IgE mouse model is unique for the study of IgE biology and its associated pathology. Allergen-bound IgE activates mast cells via the Fcε receptor and releases a spectrum of inflammatory mediators (44). Poor CSR potential and short IgE t_1/2 might have been selected during evolution to limit IgE abundance (4) and the danger of unwanted effector functions associated with anaphylaxis and allergies (44). Not only are acute asthmatic patients successfully treated with IgE neutralizing antibodies (45), new indications for this therapeutic intervention include allergic rhinitis, food allergy, atopic dermatitis, chronic urticaria, and allergic bronchopulmonary aspergillosis (45). IgE transgenic mice have the major shortcoming of being unable to mount a polyclonal Ag-specific IgE response (46–48). By altering de novo CSR, the resultant SμKI mice are able to mount diverse and polyclonal Ag-specific IgE responses to local or systemic challenges, thereby providing a unique experimental model for the accelerated assessment of future therapies in a facile system that more closely mimics the human condition.

Materials and Methods

CSR/ISR/SHM Assays.

Splenocytes were isolated and stimulated as previously explained (31). ELISAs and surface staining of B cells for IgM, IgG1, IgG3, and B220 were performed as explained (31). To visualize IgE, cells were blocked with anti-mouse IgE blocking antibody (eBioscience) and then stained with biotin-isotype or anti-mouse IgE-biotin (eBioscience) followed by streptavidin–phycoerythrin (Pharmingen). Southern blot methods were described previously (31). Southern probes included Cμ (XbaI-BamHI, 0.8 kb), Iε (BamHI-NcoI, 1 kb), Sμ (HindIII, 5 kb), and Cε (PstI-PvuI, 1 kb; Fig. 1B). For GLT and circle transcripts, RT-PCR reactions were carried out by using the Access RT-PCR kit (Promega). Cycling parameters were 45 °C for 45 min and 94 °C for 2 min, then repeating cycles of 94 °C for 30 s, 60 °C for 30 s, and 68 °C for 30 s. GLT amplification primers were reported elsewhere (24). Circle transcripts were detected with GLT primers except for CμRV 5′-AATGGTGCTGGGCAGGAAGT-3′.

Hybridomas.

The procedure for generating hybridomas have been described elsewhere (49). Briefly, splenocytes from 6- to 8-wk-old mice (WT, heterozygous, and KI) were stimulated in vitro with LPS (20 ng/mL) and IL-4 (25 ng/mL). After 4 d, 20 million B cells were fused with NS1 cells (American Type Culture Collection) at a 1:1 ratio by using the Cyto Pulse CEEF-50 apparatus (Cyto Pulse Sciences). After 7 to 10 d selection in hypoxanthine-aminopterin-thymidine (HAT) media (StemCell Technologies), the single hybridoma clones were screened for IgE-, IgM- or IgG1-specific hybridomas by Velocity 11 Biocel 1200 Automation System (Agilent Technologies). TNP-OVA specific hybridomas were generated from splenocytes at day 42 after repeated immunization (as described later) and screened by ELISA.

In Vivo Studies and Animal Care.

TNP-OVA immunization (T-cell–dependent immune response) was performed by i.p. injection of TNP-OVA/Alum adjuvant at day 0, followed by another injection of TNF-OVA at day 28. Blood samples were collected on days 3, 7, 14, 21, 28, 35, and 42 for antibody isotype measurements. For the asthma model, mice were sensitized with an i.p. injection of TNP-OVA/Alum on day 0. Mice were challenged with aerosolized 1% TNP-OVA on day 35 after sensitization, followed by seven consecutive days of challenging as indicated earlier. Blood samples for antibody isotype measurements were collected at day 2 (baseline bleed), day 34 (prechallenge), and day 42 (postchallenge). Mice infected with N. brasiliensis larvae s.c. were placed on polymyxin B (110 mg/L; Calbiochem) and neomycin (1.1 g/L; Sigma-Aldrich) medicated water for 5 d. Serum samples were collected before and 4, 9, or 14 d after infection for antibody isotype measurements. For the multiple Ag allergen model, mice were sensitized and exposed to multiple allergens intranasally twice per week for 6 wk. Each mouse was exposed intranasally to extracts of Dermatophagoides farinae (5 μg), Dermatophagoides pteronyssinus (5 μg), Ambrosia artemisiifolia (50 μg), and Aspergillus fumigatus (5 μg) (Greer Laboratories) mixed in 50 μL PBS solution. One week after the last exposure, total IgE and IgG1 were measured by ELISA. SμKI and WT mice were generated at Genentech and maintained in accordance with American Association of Laboratory Animal Care guidelines. The experiments were conducted in compliance with National Institute of Health Guide for the Care and Use of Laboratory Animals and were approved by the Genentech Institutional Animal Care and Use Committee.