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

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.

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

  • 11,229
    views
  • 1,643
    downloads
  • 76
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

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)

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

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

  1. James E Voss
  2. Alicia Gonzalez-Martin
  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
(2019)
Reprogramming the antigen specificity of B cells using genome-editing technologies
eLife 8:e42995.
https://doi.org/10.7554/eLife.42995

Share this article

https://doi.org/10.7554/eLife.42995

Further reading

    1. Genetics and Genomics
    2. Immunology and Inflammation
    Patsy R Tomlinson, Rachel G Knox ... Robert K Semple
    Research Article

    PIK3R1 encodes three regulatory subunits of class IA phosphoinositide 3-kinase (PI3K), each associating with any of three catalytic subunits, namely p110α, p110β, or p110δ. Constitutional PIK3R1 mutations cause diseases with a genotype-phenotype relationship not yet fully explained: heterozygous loss-of-function mutations cause SHORT syndrome, featuring insulin resistance and short stature attributed to reduced p110α function, while heterozygous activating mutations cause immunodeficiency, attributed to p110δ activation and known as APDS2. Surprisingly, APDS2 patients do not show features of p110α hyperactivation, but do commonly have SHORT syndrome-like features, suggesting p110α hypofunction. We sought to investigate this. In dermal fibroblasts from an APDS2 patient, we found no increased PI3K signalling, with p110δ expression markedly reduced. In preadipocytes, the APDS2 variant was potently dominant negative, associating with Irs1 and Irs2 but failing to heterodimerise with p110α. This attenuation of p110α signalling by a p110δ-activating PIK3R1 variant potentially explains co-incidence of gain-of-function and loss-of-function PIK3R1 phenotypes.

    1. Cancer Biology
    2. Immunology and Inflammation
    Sofia V Krasik, Ekaterina A Bryushkova ... Ekaterina O Serebrovskaya
    Research Article

    The current understanding of humoral immune response in cancer patients suggests that tumors may be infiltrated with diffuse B cells of extra-tumoral origin or may develop organized lymphoid structures, where somatic hypermutation and antigen-driven selection occur locally. These processes are believed to be significantly influenced by the tumor microenvironment through secretory factors and biased cell-cell interactions. To explore the manifestation of this influence, we used deep unbiased immunoglobulin profiling and systematically characterized the relationships between B cells in circulation, draining lymph nodes (draining LNs), and tumors in 14 patients with three human cancers. We demonstrated that draining LNs are differentially involved in the interaction with the tumor site, and that significant heterogeneity exists even between different parts of a single lymph node (LN). Next, we confirmed and elaborated upon previous observations regarding intratumoral immunoglobulin heterogeneity. We identified B cell receptor (BCR) clonotypes that were expanded in tumors relative to draining LNs and blood and observed that these tumor-expanded clonotypes were less hypermutated than non-expanded (ubiquitous) clonotypes. Furthermore, we observed a shift in the properties of complementarity-determining region 3 of the BCR heavy chain (CDR-H3) towards less mature and less specific BCR repertoire in tumor-infiltrating B-cells compared to circulating B-cells, which may indicate less stringent control for antibody-producing B cell development in tumor microenvironment (TME). In addition, we found repertoire-level evidence that B-cells may be selected according to their CDR-H3 physicochemical properties before they activate somatic hypermutation (SHM). Altogether, our work outlines a broad picture of the differences in the tumor BCR repertoire relative to non-tumor tissues and points to the unexpected features of the SHM process.