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Deletion of the immunoglobulin heavy chain 3′ regulatory region super-enhancer affects somatic hypermutation in B1 B cells

Cellular & Molecular Immunology volume 16, pages 195–197 (2019)Cite this article

Mouse B1 B cells originate in the embryonic liver and are the major B-cell population in the peritoneal and pleural cavities.1,2,3,4,5 By contrast, mouse B2 B cells originate in the bone marrow and are the major B-cell population in the bone marrow, spleen, and blood. B1 and B2 B cells differ not only in their origin and locations, but also in their antigen specificity, cell surface markers, capacities for class-switch recombination (CSR) and somatic hypermutation (SHM). IgH cis-regulatory regions and, particularly, transcriptional super-enhancers are major locus regulators under both normal and pathological conditions.6,7 Important differences have been found regarding the ability of the IgH 3′ regulatory region (3′RR) super-enhancer to control the B1 and B2 B-cell fate.8,9 Thus, the 3′RR super-enhancer controls the IgA CSR in B2 B cells,10,11 but not in B1 B cells.12 On the other hand, the 3′RR super-enhancer controls the late VDJ repertoire diversity in B1 B cells,9 but not in B2 B cells.13,14 Taken together, these data indicate similarities and differences in the 3′RR control of B1 and B2 B-cell maturation. Similar to CSR, SHM requires activation-induced cytidine deaminase (AID) activity.15 While AID targets the switch region during CSR, generating double-strand DNA breaks to change the Ig heavy-chain isotype for new effector functions, the enzyme targets VDJ regions during SHM to improve the Ig affinity for antigens.15 Previous studies have highlighted the role of the 3′RR super-enhancer as the master control element of SHM in B2 B cells.16,17 We thus investigated whether the 3′RR super-enhancer also controls SHM in B1 B cells.

Our research was approved by our local ethics committee review board (Comité Régional d'Ethique sur l'Expérimentation Animale du Limousin, Limoges, France) and carried out according to the European guidelines for animal experimentation. The disruption of the 3′RR was carried out in the Sv/129 embryonic stem cell line.10 Mice were bred and maintained under specific pathogen-free conditions. Sv/129 wt mice were used as control mice. AID-deficient mice were used as a negative control (to correct for baseline noise due to PCR errors and mutations in the VDJ segment, induced during the generation of the VDJ repertoire). B1 B cells were isolated from the peritoneal cavity of mice immunized orally with sheep red blood cells for 2 weeks and intraperitoneally with 10 μg of LPS (Invivogen, Toulouse, France) for 3 days. B1 B cells were sorted using a BD FACSAria III sorter (BD Biosciences, Franklin Lakes, NJ) (Flow cytometry and cell sorting platform of the University of Limoges, France). The following antibodies were used: anti-B220-BV510 (Biolegend, San Diego, CA), anti-CD23-PC (Biolegens) 7, anti-IgM-FITC (eBioscience, San Diego CA), anti-CD11b-eF780 (eBioscience), anti-IgD-BV421 (Biolegend), and anti-CD19-PE (Biolegend). Mouse B1 B cells are distinguished based on cell membrane surface markers (CD19+B220lowIgMhighIgDlowCD23-CD11b+/low).9,12 RNA was extracted from sorted B1 B cells, and 250 ng of RNA was used for sequencing. Transcripts were amplified by 5′ Rapid Amplification of cDNA-ends by polymerase chain reaction (5'RACE PCR) using a reverse primer hybridizing within the µ CH1 exon as described.18 Sequencing adapter sequences were thus added by primer extension, and resulting amplicons were sequenced on a MiSeq system (Illumina, Evry, France). Sequence analysis was performed using the IMGT/High-V-Quest19 and R programs.

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Acknowledgements

This work was supported by grants from Ligue Contre le Cancer (Equipe labellisée LIGUE 2018) and Agence Nationale de la Recherche (ANR: projet EpiSwitch-3′RR 2016). N.G. was supported by a grant from Association de Spécialisation et d’Orientation Scientifique (Lebanon), the municipality of Khiam (Lebanon) and the Société Française d’Hématologie. H.I. is supported by a fellowship from the University of Limoges. F.B. is supported by Fondation Partenariale de l′Université de Limoges and ALURAD. We thank the flow cytometry facility of the Limoges University for B-cell sorting.

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  1. Saintamand Alexis

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

  2. These authors contributed equally: Issaoui Hussein, Ghazzaui Nour.

Authors and Affiliations

  1. Centre National de la Recherche Scientifique Unité Mixte de Recherche 7276, Institut National de la Santé et de la Recherche Médicale U1262, CBRS, rue du Pr. Descottes, 87025, Limoges, France

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

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H.I., N.G., A.S., F.B., and Y.D. designed and performed the experiments and wrote the manuscript. Y.D. obtained financial grants.

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Correspondence to Denizot Yves or Saintamand Alexis.

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Hussein, I., Nour, G., François, B. et al. Deletion of the immunoglobulin heavy chain 3′ regulatory region super-enhancer affects somatic hypermutation in B1 B cells. Cell Mol Immunol 16, 195–197 (2019). https://doi.org/10.1038/s41423-018-0091-2

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