Sequential class switching is required for the generation of high affinity IgE antibodies

Abstract

IgE antibodies with high affinity for their antigens can be stably cross-linked at low concentrations by trace amounts of antigen, whereas IgE antibodies with low affinity bind their antigens weakly. In this study, we find that there are two distinct pathways to generate high and low affinity IgE. High affinity IgE is generated through sequential class switching (μ→γ→ε) in which an intermediary IgG phase is necessary for the affinity maturation of the IgE response, where the IgE inherits somatic hypermutations and high affinity from the IgG1 phase. In contrast, low affinity IgE is generated through direct class switching (μ→ε) and is much less mutated. Mice deficient in IgG1 production cannot produce high affinity IgE, even after repeated immunizations. We demonstrate that a small amount of high affinity IgE can cause anaphylaxis and is pathogenic. Low affinity IgE competes with high affinity IgE for binding to Fcε receptors and prevents anaphylaxis and is thus beneficial.

Figure 1.

A simple PCR assay to detect sequential switching to IgE. (A) Schematic representation of direct and sequential switching together with the chromosomal products. Sγ1 remnants are always indicative of sequentially switched Sμ-Sε junctions. (B and C) Molecular characterization of the Sµ-Sε junction from IgE-producing hybridomas. (B) Sµ-Sε PCR on the DNA of hybridomas SE1.3, Boettcher, 15.3, and b4. (C) Sγ1 qPCR on miniprepped Sµ-Sε junctions. The abundance is calculated as the relative transcription normalized to neomycin resistance gene expression fixed at 10,000.

Figure 2.

Sequential switching is correlated with increased IgE affinity for antigen. (A) TBmc mice were immunized with 100 µg OVA-PEP1 for one, two, and four times as indicated in the x axis. Serum was taken for ELISA to determine the IgE anti-PEP1 titer, and IgE⁺ cells were sorted to prepare DNA, Sμ-Sε PCR, cloning, and Sγ1 qPCR assay. Blue bars indicate the anti-PEP1 IgE titer (right y axis), and the red line indicates the Sγ1 ratio of Sμ-Sε junctions (left y axis). Three mice from each group and at least 24 clones/mouse were randomly chosen for Sγ1 qPCR. SD is indicated. (B) Sγ1 ratio in A of each individual mouse by single colony qPCR.

Figure 3.

Sequentially switched Sμ-Sε junctions harbor a large number of mutations. (A) TBmc mice were immunized two or four times with OVA-PEP1. The DNA was extracted, and the Sμ-Sε junctions were amplified. 33 clones from each group were sequenced. Diamonds show the approximate position of the mutations at the junction. (B) Sγ1⁻ and Sγ1⁺ junctions from mice immunized four times were sequenced and analyzed using pies (left) to show the distribution of point mutation frequency and columns (right) to show the overall mutation frequency. Error bars indicate SEM. (C) Mean number of point mutations at the junctions after two and four immunizations. Sγ1⁺ junctions were split into two junctions, Sμ-Sγ1 and Sγ1-Sε, and counted separately.

Figure 4.

IgE affinity is impaired in IgG1-deficient mice. (A) WT and hMT mice were immunized with NP₁₉KLH four times. Serum was analyzed by ELISA with NP₄- and NP₂₃-coated plates. (left) NP₄/NP₂₃ ratio. (right) Absolute absorbance value. Error bars indicate SEM. Each dot represents one mouse. (B) Same sera used in A were subjected to total IgE ELISA. (C) IgG1 and IgG2a ELISA with NP₄/NP₂₃ plates. (D) Sγ1 ratio in WT and hMT mice using the Sγ1 qPCR assay. Each dot represents one animal (four animals per group). The number of Sγ1⁺ junctions in each mouse is shown in the right panel, divided by the total number of Sμ-Sε sequences. (A–D) Horizontal bars indicate the mean.

Figure 5.

Mutations in the VDJ region of IgG1-deficient mice and their WT littermates. (A) Total splenocytes from the same hMT and WT littermate mice used in Fig. 4 A were prepared for cDNA. PCR was performed using a common V_H1.1 forward primer and Cε and Cγ2a reverse primers. NP^high and NP^low IgG1⁺ cells were sorted by staining with NP-PE. cDNA was prepared, and V_H1.1- Cγ1 PCR was performed. After sequencing, the NP affinity–enhancing mutations were tabulated. (B and C) Total nucleotide mutation analysis of the same sequences described in A (B) and the analysis of total mutation frequency (C). Error bars indicate SEM.

Figure 6.

Antigen-independent IgE responses lack Sγ1 remnants. (A) BALB/c mice were injected intravenously with 100 µg of goat anti-IgD serum or NP₁₉KLH. Total IgE and IgG1 levels were determined by ELISA. Error bars indicate SEM. (B) Approximately 10⁴ purified IgE⁺ cells from anti-IgD–treated mice were used for Sμ-Sε PCR. Amplified Sμ-Sε junctions from three different mice are shown. Arrowheads indicate the dominant Sμ-Sε bands. (C) Sγ1 ratio measured by the Sγ1 qPCR assay. Each dot represents one mouse (four animals per group; 20 clones per mouse). The overall Sγ1 ratio among the 80 junctions is shown in the pie chart (bottom). (D) Sμ-Sε junctions from anti-IgD– or NP₁₉KLH-treated mice were sequenced, and the number of mutations of each junction was plotted. Each dot represents a junction (13 junctions from three mice per group). (C and D) Horizontal bars indicate the mean.

Figure 7.

Anaphylaxis caused by IgE of high affinity to its antigen can be competed by IgE of low affinity for the same antigen. TBmc mice were immunized with OVA-HA or OVA-PEP1 two times, and sera were collected and depleted of IgG and IgA. IgE concentration was measured by ELISA. 25 ng of total IgE was injected intradermally into the ear of recipient BALB/c mice. 24 h later, 50 µg OVA-HA, OVA-PEP1, or unconjugated PEP1 was injected intravenously together with 1% Evans blue. (A and B) 30 min later, mice were sacrificed, and the extent of Evans blue leakage was determined either visually (A) or spectrophotometrically (B; three mice per group). (C) Before injecting into the ears, sera were diluted as indicated. (D and E) Similar PCA assay was performed using sera from TBmc mice immunized with OVA-PEP1 one, two, or four times. The PEP1-specific IgE titer is shown above the photos. A total of 25 ng IgE was injected per ear. 24 h later, recipient BALB/c mice were injected intravenously with OVA-PEP1 and Evans blue. The ears are shown in D, and Evans blue was measured spectrophotometrically in E. (F) 6 ng of high affinity anti-PEP1 IgE was mixed with 100 times more PBS, anti-HA IgE, IgG1, or IgM to perform the PCA assay mediated by OVA-PEP1. The extent of Evans blue leakage was determined visually. (G) Quantification of Evans blue leakage by spectrophotometry in the ears injected with 1, 20, 100, and 500 times more IgM, IgG, or low affinity IgE (three mice each group). (B, C, E, and G) Error bars indicate SEM.