Downstream class switching leads to IgE antibody production by B lymphocytes lacking IgM switch regions

Abstract

Ig heavy chain (IgH) class-switch recombination (CSR) replaces the IgH C mu constant region exons with one of several sets of downstream IgH constant region exons (e.g., C gamma, C epsilon, or C alpha), which affects switching from IgM to another IgH class (e.g., IgG, IgE, or IgA). Activation-induced cytidine deaminase (AID) initiates CSR by promoting DNA double-strand breaks (DSBs) within switch (S) regions flanking the donor C mu (S mu) and a downstream acceptor C(H) (e.g., S gamma, S epsilon, S alpha) that are then joined to complete CSR. DSBs generated in S mu frequently are joined within S mu to form internal switch region deletions (ISD). AID-induced ISD and mutations have been considered rare in downstream S regions, suggesting that AID targeting to these S regions requires its prior recruitment to S mu. We have now assayed for CSR and ISD in B cells lacking S mu (S mu(-/-) B cells). In S mu(-/-) B cells activated for CSR to IgG1 and IgE, CSR to IgG1 was greatly reduced; but, surprisingly, CSR to IgE occurred at nearly normal levels. Moreover, normal B cells had substantial S gamma1 ISD and increased mutations in and near S gamma1, and levels of both were greatly increased in S mu(-/-) B cells. Finally, S mu(-/-) B cells underwent downstream CSR between S gamma1 and S epsilon on alleles that lacked S mu CSR to these sequences. Our findings show that AID targets downstream S regions independently of CSR with Smu and implicate an alternative pathway for IgE class switching that involves generation and joining of DSBs within two different downstream S regions before S mu joining.

Fig. 1.

IgG1 and IgE CSR in Sμ^−/− B cells. (A) FACS analysis on days 2, 3, and 4 of anti-CD40/IL-4–stimulated splenic B cells from WT, Sμ^−/−, and AID^−/− mice by surface staining with anti–IgG1-FITC and anti-B220-PE-Cy5 antibodies. The percentage of IgG1⁺B220⁺ cells represents CSR level to IgG1 at each time point. (B) Statistical analysis of three independent FACS analysis experiments for IgG1 switching of anti-CD40/IL-4–stimulated B cells from WT, Sμ^−/−, and AID^−/− mouse spleens at day 2, 3, and 4. (C) Hybridoma analysis by supernatant ELISA on clones generated from day 4 anti-CD40/IL-4–stimulated B-cell fusion with NS-1 fusion partner cell line. IgM, IgG1, and IgE single-positive clones were counted and analyzed for percentage in total single positive clones for each IgH isotype in three independent fusions on WT and Sμ^−/− B cells. SDs calculated from the three experiments are shown. Detailed numbers are listed in Table S1.

Fig. 2.

Sγ1 rearrangements on unswitched alleles in WT and Sμ^−/− IgM-producing hybridomas. (A Top) Map of the Cγ1 gene. The EcoRI sites are indicated (RI), and the Iγ1 probe is indicated. (Middle and Bottom) Southern blotting analysis of EcoRI-digested genomic DNA extracted from IgM-producing hybridomas generated from day 4 anti-CD40/IL-4–stimulated WT (Middle) and Sμ^−/− (Bottom) B cells for hybridization to an Iγ1 probe (18). Iγ1-hybridizing germline bands of EcoRI-digested genomic DNA from WT (129/SvJ background) mouse kidneys are indicated with an arrow head and labeled as GL. Lanes with – on top show no bands hybridizing to the Iγ1 probe or a J_H4 or Cμ probe (used to show that assayed hybridomas had rearranged J_H alleles and to confirm their genotype as WT or mutant, respectively) (Fig. S2). These lanes were not counted in the calculations shown in Fig. S2B, whereas lanes with * on top contain one or two unswitched alleles that have undergone rearrangements of the Iγ1-hybridizing fragment. (B) Percentage of total unswitched Sγ1 alleles in WT and Sμ^−/− IgM-producing hybridomas that contain rearrangements of the Iγ1-hybridzing fragments (presumed Sγ1 rearrangements). SDs calculated from the three experiments are shown. Detailed numbers are listed in Table S1.

Fig. 3.

Mutations accumulate in the Iγ1/Sγ1 region in WT and Sμ^−/− B cells. (A) Mutation frequency in the Iγ1/Sγ1 region of WT and Sμ^−/− B cells. SDs are calculated from three independent experiments. Detailed numbers are listed in Table S3. (B) The total number of Iγ1/Sγ1 sequences from WT and Sμ^−/− B cells analyzed is indicated in the center of each pie chart. The percentage of individual sequences containing different numbers of mutations is listed in different segments of the pie charts and is proportional to the segment sizes in the pie charts.

Fig. 4.

Sμ^−/− IgM-producing hybridomas contain Sγ1–Sε junctions on IgH alleles that have not undergone CSR with Sμ. (A) Diagram of the Southern blotting and PCR strategies to detect Sγ1–Sε junctions. (A) Upper diagram shows germline Cγ1 and Cε genes, which are separated by 60 kb, and Lower diagram shows putative CSR product between the two genes. RI, EcoRI sites. The Cμ and Iγ1 Southern blot probes and the PCR primers used in the assay are also indicated. The PCR primers would only generate a band in the rearranged allele (Lower) because of the long distance of their separation in the germline configuration (Upper). (B Upper and Lower) Southern blot with Cε and Iγ1 probes, respectively. Rearranged bands that cohybridize with the Cε and Iγ1 probe are indicated with an asterisk. Digested NS-1 fusion-partner genomic DNA hybridizes to the Cε probe at the same size as germline genomic DNA, which is shown as GL/NS-1 (NS-1). (C) PCR analyses of genomic DNA from a set of WT (Left) and Sμ^−/− IgM⁺ hybridomas (Right). The same-sized bands observed in the Sμ^−/− lines were observed in repeat analyses. In analyses of three separate sets of WT hybridomas no PCR bands were observed in 24 samples analyzed, whereas in analyses of three separate sets of Sμ^−/− hybridomas, we observed bands in 15 of 42 samples analyzed, with 10 representative samples illustrated here.