Characterisation of the immune repertoire of a humanised transgenic mouse through immunophenotyping and high-throughput sequencing
- University of Oxford, United Kingdom
- Kymab, United Kingdom
- University of Zurich, Switzerland
- Alchemab Therapeutics Ltd, United Kingdom
Abstract
Immunoglobulin loci-transgenic animals are widely used in antibody discovery and increasingly in vaccine response modelling. In this study, we phenotypically characterised B-cell populations from the Intelliselect® Transgenic mouse (Kymouse) demonstrating full B-cell development competence. Comparison of the naïve B-cell receptor (BCR) repertoires of Kymice BCRs, naïve human, and murine BCR repertoires revealed key differences in germline gene usage and junctional diversification. These differences result in Kymice having CDRH3 length and diversity intermediate between mice and humans. To compare the structural space explored by CDRH3s in each species’ repertoire, we used computational structure prediction to show that Kymouse naïve BCR repertoires are more human-like than mouse-like in their predicted distribution of CDRH3 shape. Our combined sequence and structural analysis indicates that the naïve Kymouse BCR repertoire is diverse with key similarities to human repertoires, while immunophenotyping confirms that selected naïve B-cells are able to go through complete development.
Data availability
The processed paired sequencing data is available at doing 10.5281/zenodo.7474232 and the processed bulk VH sequence data has been deposited in the Observed Antibody Space (http://opig.stats.ox.ac.uk/webapps/oas/oas). Immunophenotyping data is available in the FlowRepository (https://flowrepository.org/) under IDs FR-FCM-Z5LB (bone marrow samples) and FR-FCM-Z5LC (splenocyte and lymph node samples).
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Paired VH:VL IGM sequences from 22 naïve KymiceZenodo, 10.5281/zenodo.7474232.
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Pre-processed IgH repertoire sequencing data from BioProject PRJNA748239Zenodo, doi:10.5281/zenodo.5155565.
Article and author information
Author details
Valentin von Niederhäusern
Aleksandr Kovaltsuk
Funding
Bill & Melinda Gates Foundation (OPP1159947)
- Paul Kellam
Medical Research Council (MR/R015708/1)
- Eve Richardson
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Ranjan Sen, National Institute on Aging, United States
Ethics
Animal experimentation: This study was carried out under Project Licenses 70/8718 issued by the UK Government Home Office under Animal (Scientific Procedures) Act (A(SP)A), 1986, incorporating Directive 2010/63/EU of the European Parliament, and with the approval of the Sanger Institute Animal Welfare and Ethical Review Body. The Institute complied with the Code of Practice issued by the UK Government which aids compliance with the A(SP)A. The Institute has a PHS assurance F16-00128 (WTSI).
Version history
- Preprint posted: June 29, 2022 (view preprint)
- Received: July 5, 2022
- Accepted: March 26, 2023
- Accepted Manuscript published: March 27, 2023 (version 1)
- Version of Record published: April 19, 2023 (version 2)
Copyright
© 2023, Richardson 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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Characterisation of the immune repertoire of a humanised transgenic mouse through immunophenotyping and high-throughput sequencing
eLife 12:e81629.
https://doi.org/10.7554/eLife.81629
Further reading
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- Cell Biology
- Immunology and Inflammation
Cytokine polyfunctionality is a well-established concept in immune cells, especially T cells, and their ability to concurrently produce multiple cytokines has been associated with better immunological disease control and subsequent effectiveness during infection and disease. To date, only little is known about the secretion dynamics of those cells, masked by the widespread deployment of mainly time-integrated endpoint measurement techniques that do not easily differentiate between concurrent and sequential secretion. Here, we employed a single-cell microfluidic platform capable of resolving the secretion dynamics of individual PBMCs. To study the dynamics of poly-cytokine secretion, as well as the dynamics of concurrent and sequential polyfunctionality, we analyzed the response at different time points after ex vivo activation. First, we observed the simultaneous secretion of cytokines over the measurement time for most stimulants in a subpopulation of cells only. Second, polyfunctionality generally decreased with prolonged stimulation times and revealed no correlation with the concentration of secreted cytokines in response to stimulation. However, we observed a general trend towards higher cytokine secretion in polyfunctional cells, with their secretion dynamics being distinctly different from mono-cytokine-secreting cells. This study provided insights into the distinct secretion behavior of heterogenous cell populations after stimulation with well-described agents and such a system could provide a better understanding of various immune dynamics in therapy and disease.
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- Immunology and Inflammation
- Medicine
Background:
Prinflammatory extracellular chromatin from neutrophil extracellular traps (NETs) and other cellular sources is found in COVID-19 patients and may promote pathology. We determined whether pulmonary administration of the endonuclease dornase alfa reduced systemic inflammation by clearing extracellular chromatin.
Methods:
Eligible patients were randomized (3:1) to the best available care including dexamethasone (R-BAC) or to BAC with twice-daily nebulized dornase alfa (R-BAC + DA) for seven days or until discharge. A 2:1 ratio of matched contemporary controls (CC-BAC) provided additional comparators. The primary endpoint was the improvement in C-reactive protein (CRP) over time, analyzed using a repeated-measures mixed model, adjusted for baseline factors.
Results:
We recruited 39 evaluable participants: 30 randomized to dornase alfa (R-BAC +DA), 9 randomized to BAC (R-BAC), and included 60 CC-BAC participants. Dornase alfa was well tolerated and reduced CRP by 33% compared to the combined BAC groups (T-BAC). Least squares (LS) mean post-dexamethasone CRP fell from 101.9 mg/L to 23.23 mg/L in R-BAC +DA participants versus a 99.5 mg/L to 34.82 mg/L reduction in the T-BAC group at 7 days; p=0.01. The anti-inflammatory effect of dornase alfa was further confirmed with subgroup and sensitivity analyses on randomised participants only, mitigating potential biases associated with the use of CC-BAC participants. Dornase alfa increased live discharge rates by 63% (HR 1.63, 95% CI 1.01–2.61, p=0.03), increased lymphocyte counts (LS mean: 1.08 vs 0.87, p=0.02) and reduced circulating cf-DNA and the coagulopathy marker D-dimer (LS mean: 570.78 vs 1656.96 μg/mL, p=0.004).
Conclusions:
Dornase alfa reduces pathogenic inflammation in COVID-19 pneumonia, demonstrating the benefit of cost-effective therapies that target extracellular chromatin.
Funding:
LifeArc, Breathing Matters, The Francis Crick Institute (CRUK, Medical Research Council, Wellcome Trust).
Clinical trial number:
