Reprogramming the antigen specificity of B cells using genome-editing technologies
- The Scripps Research Institute, United States
- Universidad Autónoma de Madrid (UAM), Spain
- Karolinska Institutet, Sweden
- University College London, United Kingdom
- 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
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PG9HC(V434)Ramos-WGSNCBI SRA, SAMN09404498.
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PG9HC(V781)Ramos-WGSNCBI SRA, SAMN09404497.
Article and author information
Author details
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.
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Reprogramming the antigen specificity of B cells using genome-editing technologies
eLife 8:e42995.
https://doi.org/10.7554/eLife.42995
Further reading
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Diverse antibody repertoires spanning multiple lymphoid organs (i.e., bone marrow, spleen, lymph nodes) form the foundation of protective humoral immunity. Changes in their composition across lymphoid organs are a consequence of B-cell selection and migration events leading to a highly dynamic and unique physiological landscape of antibody repertoires upon antigenic challenge (e.g., vaccination). However, to what extent B cells encoding identical or similar antibody sequences (clones) are distributed across multiple lymphoid organs and how this is shaped by the strength of a humoral response remains largely unexplored. Here, we performed an in-depth systems analysis of antibody repertoires across multiple distinct lymphoid organs of immunized mice and discovered that organ-specific antibody repertoire features (i.e., germline V-gene usage and clonal expansion profiles) equilibrated upon a strong humoral response (multiple immunizations and high serum titers). This resulted in a surprisingly high degree of repertoire consolidation, characterized by highly connected and overlapping B-cell clones across multiple lymphoid organs. Finally, we revealed distinct physiological axes indicating clonal migrations and showed that antibody repertoire consolidation directly correlated with antigen specificity. Our study uncovered how a strong humoral response resulted in a more uniform but redundant physiological landscape of antibody repertoires, indicating that increases in antibody serum titers were a result of synergistic contributions from antigen-specific B-cell clones distributed across multiple lymphoid organs. Our findings provide valuable insights for the assessment and design of vaccine strategies.
