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IgD class switch recombination is not controlled through the immunoglobulin heavy chain 3′ regulatory region super-enhancer

Cellular & Molecular Immunology volume 14, pages 871–874 (2017)Cite this article

In secondary lymphoid organs, mature B cells express membrane immunoglobulin (Ig) of M and D isotypes (IgM and IgD, respectively) of the same specificity through alternative splicing of a pre-mRNA encompassing the VDJ variable region and Cμ and Cδ heavy chain constant exons.1 After encountering antigen, B cells undergo class switch recombination (CSR) by which the Cμ gene is substituted with Cγ, Cɛ or Cα, thereby generating IgG, IgE and IgA antibodies of the same antigenic specificity but with new effector functions. CSR requires the DNA-editing enzyme activation-induced deaminase (AID), which targets switch (S) regions preceding Cμ (namely, the Sμ donor region) and Cγ, Cɛ and Cα genes (namely, the Sγ,ɛ,α acceptor regions).1 CSR is controlled in trans through a wide spectrum of enzymes and proteins and in cis through the immunoglobulin heavy chain (IgH) 3′ regulatory region (3′RR) super-enhancer that specifically AID-poises the S acceptor but not the Sμ donor region.2 IgD CSR is a rare and enigmatic event restricted to a few B-cell subsets in specific lymphoid tissues (such as mesenteric lymph nodes, peritoneal cavity and mucosa-associated tissue) in both mice and humans.3, 4, 5 A recent study suggested that IgD CSR is initiated by microbiota,5 demonstrating a role for IgD in the homeostatic regulation of the microbial community. The mechanistic regulation of IgD CSR remains enigmatic, reflecting the difficulty to obtain a sufficient number of Sμ–σδ (σ for S-like) junction sequences for molecular analysis. We recently reported a new computational tool (CSReport) for the automatic analysis of CSR junctions sequenced using high-throughput sequencing,6 and we used this tool to analyze Sμ–σδ junctions in wild-type (wt) mice.7 Genomic deletion of the 3′RR super-enhancer abrogates CSR to IgG, IgE and IgA classes.2, 8 In contrast, a unique study, based on the analysis of a few Sμ–σδ junctions, reported the 3′RR super-enhancer as dispensable for IgD CSR.4 We thus used CSReport and high-throughput sequencing to conduct a more in depth analysis of Sμ–σδ junctions in 3′RR-deficient mice.

The present study was approved by our local ethics committee review board (Comité Régional d'Ethique sur l'Expérimentation Animale du Limousin, Limoges, France) and conducted according to the European guidelines for animal experimentation. Peritoneal cavity 3’RR-deficient mouse B cells were used for these experiments. In some experiments, 3′RR-deficient mice were pristane-treated (1 ml) for 2 months to induce inflammation prior to the recovery of peritoneal cavity cells. As previously described in detail,4 junctions were amplified using touchdown PCR followed by nested PCR. Libraries of 200 bp were prepared from the 1–2 kb PCR products of Sμ–σδ amplification for ion proton sequencing (‘GénoLim platform’ of the Limoges University, France). Sequenced reads were subsequently mapped to Sμ and σδ regions using the BLAST algorithm. The computational tool developed for experiments performs junction assembly, identifies break points in Sμ and σδ, identifies junction structures (blunt, micro-homology or junctions with insertions) and outputs a statistical summary of the identified junctions.

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Acknowledgements

This work was financially supported through a grant from the ANR (project EpiSwitch-3′RR 2016). NG was supported through a grant from the Association de Spécialisation et d’Orientation Scientifique (Lebanon) and the municipality of Khiam (Lebanon). HI was supported through a grant from CORC (FJA/NP 2015-109) and the University of Limoges. The authors thank the GénoLim platform and the Cytometry platform of the University of Limoges for sequencing and cell sorting. FB was supported through a grant from the Fondation Partenariale de l’Université de Limoges and ALURAD.

Author contributions

YD and AS designed the study. NG and HI performed the experiments. FB and YD analyzed the data. NG, HI, FB, YD and AS drafted the manuscript.

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  1. Alexis Saintamand

    Present address: 3Present address: INSERM U1236, Université Rennes 1, Rennes, France.,

  2. Hussein Issaoui and Nour Ghazzaui: These authors contributed equally to this work.

Authors and Affiliations

  1. UMR CNRS 7276, University of Limoges, CBRS, Limoges, 87025, France.

    Hussein Issaoui, Nour Ghazzaui, Alexis Saintamand, Yves Denizot & François Boyer

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  1. Hussein Issaoui
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Correspondence to Yves Denizot.

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Issaoui, H., Ghazzaui, N., Saintamand, A. et al. IgD class switch recombination is not controlled through the immunoglobulin heavy chain 3′ regulatory region super-enhancer. Cell Mol Immunol 14, 871–874 (2017). https://doi.org/10.1038/cmi.2017.81

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