1. Immunology and Inflammation
  2. Microbiology and Infectious Disease
Download icon

Reprogramming the antigen specificity of B cells using genome-editing technologies

  1. James E Voss  Is a corresponding author
  2. Alicia Gonzalez-Martin  Is a corresponding author
  3. Raiees Andrabi
  4. Roberta P Fuller
  5. Ben Murrell
  6. Laura E McCoy
  7. Katelyn Porter
  8. Deli Huang
  9. Wenjuan Li
  10. Devin Sok
  11. Khoa Le
  12. Bryan Briney
  13. Morgan Chateau
  14. Geoffrey Rogers
  15. Lars Hangartner
  16. Ann J Feeney
  17. David Nemazee
  18. Paula Cannon
  19. Dennis Burton  Is a corresponding author
  1. The Scripps Research Institute, United States
  2. Universidad Autónoma de Madrid (UAM), Spain
  3. Karolinska Institutet, United States
  4. University College London, United Kingdom
  5. University of Southern California, United States
Short Report
  • Cited 22
  • Views 6,867
  • Annotations
Cite this article as: eLife 2019;8:e42995 doi: 10.7554/eLife.42995

Abstract

We have developed a method to introduce novel paratopes into the human antibody repertoire by modifying the immunoglobulin (Ig) genes of mature B cells directly using genome editing technologies. We used CRISPR-Cas9 in a homology directed repair strategy, to replace the heavy chain (HC) variable region in B cell lines with that from an HIV broadly neutralizing antibody, PG9. Our strategy is designed to function in cells that have undergone VDJ recombination using any combination of variable (V), diversity (D) and joining (J) genes. The modified locus expresses PG9 HC which pairs with native light chains resulting in the cell surface expression of HIV specific B cell receptors (BCRs). Endogenous activation-induced cytidine deaminase (AID) in engineered cells allowed for Ig class switching and generated BCR variants with improved anti-HIV neutralizing activity. Thus, BCRs engineered in this way retain the genetic flexibility normally required for affinity maturation during adaptive immune responses. Peripheral blood derived primary B cells from three different donors were edited using this strategy. Engineered cells could bind the PG9 epitope by FACS and sequenced mRNA from these cells showed PG9 HC expressed as several different isotypes after culture with CD40 ligand and IL-4.

Data availability

Next generation sequencing data from RT-PCR amplicons have been deposited at Dryad:DOI: https://doi.org/10.5061/dryad.45j0r70.Amplification free whole genome sequencing reads mapped to the human reference genome have been deposited to NCBI with BioSample accession numbers SAMN09404498 and SAMN09404497

The following data sets were generated

Article and author information

Author details

  1. James E Voss

    Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, United States
    For correspondence
    jvoss@scripps.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4777-1596
  2. Alicia Gonzalez-Martin

    Department of Biochemistry, Universidad Autónoma de Madrid (UAM), Madrid, Spain
    For correspondence
    alicia.gonzalezm@uam.es
    Competing interests
    The authors declare that no competing interests exist.
  3. Raiees Andrabi

    Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Roberta P Fuller

    Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Ben Murrell

    Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Laura E McCoy

    Division of Infection and Immunity, University College London, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  7. Katelyn Porter

    Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Deli Huang

    Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, United States
    Competing interests
    The authors declare that no competing interests exist.
  9. Wenjuan Li

    Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, United States
    Competing interests
    The authors declare that no competing interests exist.
  10. Devin Sok

    Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, United States
    Competing interests
    The authors declare that no competing interests exist.
  11. Khoa Le

    Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, United States
    Competing interests
    The authors declare that no competing interests exist.
  12. Bryan Briney

    Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, United States
    Competing interests
    The authors declare that no competing interests exist.
  13. Morgan Chateau

    Keck School of Medicine, University of Southern California, Los Angeles, United States
    Competing interests
    The authors declare that no competing interests exist.
  14. Geoffrey Rogers

    Keck School of Medicine, University of Southern California, Los Angeles, United States
    Competing interests
    The authors declare that no competing interests exist.
  15. Lars Hangartner

    Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, United States
    Competing interests
    The authors declare that no competing interests exist.
  16. Ann J Feeney

    Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, United States
    Competing interests
    The authors declare that no competing interests exist.
  17. David Nemazee

    Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, United States
    Competing interests
    The authors declare that no competing interests exist.
  18. Paula Cannon

    Keck School of Medicine, University of Southern California, Los Angeles, United States
    Competing interests
    The authors declare that no competing interests exist.
  19. Dennis Burton

    Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, United States
    For correspondence
    burton@scripps.edu
    Competing interests
    The authors declare that no competing interests exist.

Funding

National Institutes of Health (5R01DE025167-05)

  • Dennis Burton

Bill and Melinda Gates Foundation (OPP1183956)

  • James E Voss

Ramón y Cajal Merit Award, Ministerio de Ciencia, Innovacion y Universidades (RYC-2016-21155)

  • Alicia Gonzalez-Martin

Marie-Curie Fellowship (FP7-PEOPLE-2013-IOF)

  • Laura E McCoy

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Tomohiro Kurosaki, Osaka University, Japan

Publication history

  1. Received: October 19, 2018
  2. Accepted: December 31, 2018
  3. Accepted Manuscript published: January 16, 2019 (version 1)
  4. Accepted Manuscript updated: January 17, 2019 (version 2)
  5. Version of Record published: January 31, 2019 (version 3)

Copyright

© 2019, Voss et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 6,867
    Page views
  • 1,088
    Downloads
  • 22
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, Scopus, PubMed Central.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)

Further reading

    1. Developmental Biology
    2. Immunology and Inflammation
    Lydia K Lutes et al.
    Research Article Updated

    Functional tuning of T cells based on their degree of self-reactivity is established during positive selection in the thymus, although how positive selection differs for thymocytes with relatively low versus high self-reactivity is unclear. In addition, preselection thymocytes are highly sensitive to low-affinity ligands, but the mechanism underlying their enhanced T cell receptor (TCR) sensitivity is not fully understood. Here we show that murine thymocytes with low self-reactivity experience briefer TCR signals and complete positive selection more slowly than those with high self-reactivity. Additionally, we provide evidence that cells with low self-reactivity retain a preselection gene expression signature as they mature, including genes previously implicated in modulating TCR sensitivity and a novel group of ion channel genes. Our results imply that thymocytes with low self-reactivity downregulate TCR sensitivity more slowly during positive selection, and associate membrane ion channel expression with thymocyte self-reactivity and progress through positive selection.

    1. Immunology and Inflammation
    Khursheed A Wani et al.
    Research Article

    The model organism Caenorhabditis elegans mounts transcriptional defense responses against intestinal bacterial infections that elicit overlapping starvation and infection responses, the regulation of which is not well understood. Direct comparison of C. elegans that were starved or infected with Staphylococcus aureus revealed a large infection-specific transcriptional signature, which was almost completely abrogated by deletion of transcription factor hlh-30/TFEB, except for six genes including a flavin-containing monooxygenase (FMO) gene, fmo-2/FMO5. Deletion of fmo-2/FMO5 severely compromised infection survival, thus identifying the first FMO with innate immunity functions in animals. Moreover, fmo-2/FMO5 induction required the nuclear hormone receptor, NHR-49/PPAR-α, which controlled host defense cell non-autonomously. These findings reveal an infection-specific host response to S. aureus, identify HLH-30/TFEB as its main regulator, reveal FMOs as important innate immunity effectors in animals, and identify the mechanism of FMO regulation through NHR-49/PPAR-α during S. aureus infection, with implications for host defense and inflammation in higher organisms.