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, United States
- 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.
Reviewing Editor
- Tomohiro Kurosaki, Osaka University, Japan
Version history
- Received: October 19, 2018
- Accepted: December 31, 2018
- Accepted Manuscript published: January 16, 2019 (version 1)
- Accepted Manuscript updated: January 17, 2019 (version 2)
- 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.
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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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- Evolutionary Biology
- Immunology and Inflammation
CD4+ T cell activation is driven by five-module receptor complexes. The T cell receptor (TCR) is the receptor module that binds composite surfaces of peptide antigens embedded within MHCII molecules (pMHCII). It associates with three signaling modules (CD3γε, CD3δε, and CD3ζζ) to form TCR-CD3 complexes. CD4 is the coreceptor module. It reciprocally associates with TCR-CD3-pMHCII assemblies on the outside of a CD4+ T cells and with the Src kinase, LCK, on the inside. Previously, we reported that the CD4 transmembrane GGXXG and cytoplasmic juxtamembrane (C/F)CV+C motifs found in eutherian (placental mammal) CD4 have constituent residues that evolved under purifying selection (Lee et al., 2022). Expressing mutants of these motifs together in T cell hybridomas increased CD4-LCK association but reduced CD3ζ, ZAP70, and PLCγ1 phosphorylation levels, as well as IL-2 production, in response to agonist pMHCII. Because these mutants preferentially localized CD4-LCK pairs to non-raft membrane fractions, one explanation for our results was that they impaired proximal signaling by sequestering LCK away from TCR-CD3. An alternative hypothesis is that the mutations directly impacted signaling because the motifs normally play an LCK-independent role in signaling. The goal of this study was to discriminate between these possibilities. Using T cell hybridomas, our results indicate that: intracellular CD4-LCK interactions are not necessary for pMHCII-specific signal initiation; the GGXXG and (C/F)CV+C motifs are key determinants of CD4-mediated pMHCII-specific signal amplification; the GGXXG and (C/F)CV+C motifs exert their functions independently of direct CD4-LCK association. These data provide a mechanistic explanation for why residues within these motifs are under purifying selection in jawed vertebrates. The results are also important to consider for biomimetic engineering of synthetic receptors.
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- Genetics and Genomics
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