PTPN22 R620W gene editing in T cells enhances low avidity TCR responses
- Seattle Children's Research Institute, United States
- Benaroya Research Institute, United States
- University of Washington, United States
Abstract
A genetic variant in the gene PTPN22 (R620W, rs2476601) is strongly associated with increased risk for multiple autoimmune diseases and linked to altered TCR regulation and T cell activation. Here, we utilize Crispr/Cas9 gene editing with donor DNA repair templates in human cord blood-derived, naive T cells to generate PTPN22 risk edited (620W), non-risk edited (620R) or knock out T cells from the same donor. PTPN22 risk edited cells exhibited increased activation marker expression following non-specific TCR engagement, findings that mimicked PTPN22 KO cells. Next, using lentiviral delivery of T1D patient-derived TCRs against the pancreatic autoantigen, islet-specific glucose-6 phosphatase catalytic subunit-related protein (IGRP), we demonstrate that loss of PTPN22 function led to enhanced signaling in T cells expressing a lower avidity self-reactive TCR, but not a high avidity TCR. In this setting, loss of PTPN22 mediated enhanced proliferation and Th1 skewing. Importantly, expression of the risk variant in association with a lower avidity TCR also increased proliferation relative to PTPN22 non-risk T cells. Together, these findings suggest that, in primary human T cells, PTPN22 rs2476601 contributes to autoimmunity risk by permitting increased TCR signaling and activation in mildly self-reactive T cells, thereby potentially expanding the self-reactive T cell pool and skewing this population toward an inflammatory phenotype.
Data availability
Prism files containing the numerical data and statistical tests used to generate all figures has been provided
Article and author information
Author details
Funding
National Institute of Diabetes and Digestive and Kidney Diseases (DP3DK111802)
- Warren Anderson
- Fariba Barahmand-pour-Whitman
- Peter S Linsley
- Karen Cerosaletti
- Jane H Buckner
- David J Rawlings
Seattle Children's Foundation (Children's Guild Association Endowed Chair in Pediatric Immunology)
- David J Rawlings
Seattle Children's Research Institute (Center for Immunity and Immunotherapies)
- David J Rawlings
Seattle Children's Research Institute (Program for Cell and Gene Therapy)
- David J Rawlings
Seattle Children's Research Institute (Hansen Investigator in Pediatric Innovation Endowment)
- David J Rawlings
Benaroya Family Gift Fund
- David J Rawlings
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Bernard Malissen, Centre d'Immunologie de Marseille-Luminy, Aix Marseille Université, France
Ethics
Human subjects: Study Approval (Human Subjects)For gene editing experiments using adult PBMCs, human donor leukopaks were purchased from the Fred Hutchinson Cancer Research Center, which were obtained from consenting donors under an IRB-approved protocol and cryopreserved. For gene editing experiments using umbilical cord blood derived PBMCs, cord units were purchased from the Bloodworks Northwest, which were obtained with consent under an IRB-approved protocol and cryopreserved. After collection, all samples were de-identified for the protection of human blood donors.
Version history
- Received: July 4, 2022
- Preprint posted: July 22, 2022 (view preprint)
- Accepted: March 21, 2023
- Accepted Manuscript published: March 24, 2023 (version 1)
- Version of Record published: March 31, 2023 (version 2)
Copyright
© 2023, Anderson 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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PTPN22 R620W gene editing in T cells enhances low avidity TCR responses
eLife 12:e81577.
https://doi.org/10.7554/eLife.81577
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:
