Autoantibody discovery across monogenic, acquired, and COVID19-associated autoimmunity with scalable PhIP-Seq
- University of California, San Francisco, United States
- National Institute of Allergy and Infectious Diseases, United States
- Karolinska Institute, Sweden
- Rockefeller University, United States
- Stanford University, United States
- University of California, San Diego, United States
- Seattle Children's Research Institute, United States
Abstract
Phage Immunoprecipitation-Sequencing (PhIP-Seq) allows for unbiased, proteome-wide autoantibody discovery across a variety of disease settings, with identification of disease-specific autoantigens providing new insight into previously poorly understood forms of immune dysregulation. Despite several successful implementations of PhIP-Seq for autoantigen discovery, including our previous work (Vazquez et al. 2020), current protocols are inherently difficult to scale to accommodate large cohorts of cases and importantly, healthy controls. Here, we develop and validate a high throughput extension of PhIP-seq in various etiologies of autoimmune and inflammatory diseases, including APS1, IPEX, RAG1/2 deficiency, Kawasaki Disease (KD), Multisystem Inflammatory Syndrome in Children (MIS-C), and finally, mild and severe forms of COVID19. We demonstrate that these scaled datasets enable machine-learning approaches that result in robust prediction of disease status, as well as the ability to detect both known and novel autoantigens, such as PDYN in APS1 patients, and intestinally expressed proteins BEST4 and BTNL8 in IPEX patients. Remarkably, BEST4 antibodies were also found in 2 patients with RAG1/2 deficiency, one of whom had very early onset IBD. Scaled PhIP-Seq examination of both MIS-C and KD demonstrated rare, overlapping antigens, including CGNL1, as well as several strongly enriched putative pneumonia-associated antigens in severe COVID19, including the endosomal protein EEA1. Together, scaled PhIP-Seq provides a valuable tool for broadly assessing both rare and common autoantigen overlap between autoimmune diseases of varying origins and etiologies.
Data availability
Full PhIP-Seq data for all cohorts presented is available for download at Dryad at https://doi.org/10.5061/dryad.qfttdz0k4. All available deidentified clinical data for this study is available in Supplemental Table 1.
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Autoantibody discovery across monogenic, acquired, and COVID19-associated autoimmunity with scalable PhIP-SeqDryad Digital Repository, doi:10.5061/dryad.qfttdz0k4.
Article and author information
Author details
Funding
National Institute of Allergy and Infectious Diseases (5P01AI118688)
- Mark S Anderson
UCSF-CTSI TL1 (TR001871)
- Zoe Quandt
Division of Intramural Research, National Institute of Allergy and Infectious Diseases (1 ZIA AI001222)
- Luigi D Notarangelo
National Institute of Child Health and Development (1R61HD105590)
- Adriana Tremoulet
- Jane C Burns
Multiple sources**
- Jean-Laurent Casanova
FRM (EA20170638020)
- Paul Bastard
MD-PhD program of the Imagine Institute
- Paul Bastard
National Institute of Allergy and Infectious Diseases (1ZIAAI001175)
- Michail S Lionakis
National Institute of Diabetes and Digestive and Kidney Diseases (1F30DK123915)
- Sara E Vazquez
Chan Zuckerberg Biohub
- Joseph L DeRisi
Parker Institute for Cancer Immunotherapy
- Mark S Anderson
Juvenile Diabetes Research Foundation United States of America
- Mark S Anderson
Helmsley Charitable Trust
- Mark S Anderson
National Institute of General Medical Sciences (5T32GM007618)
- Mark S Anderson
American Diabetes Association (1-19-PDF-131)
- Zoe Quandt
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.**multiple sources includes: The Laboratory of Human Genetics of Infectious Diseases is supported by the Howard Hughes Medical Institute, the Rockefeller University, the St. Giles Foundation, the National Institutes of Health (NIH) (R01AI088364 and R01AI163029), the National Center for Advancing Translational Sciences (NCATS), NIH Clinical and Translational Science Award (CTSA) program (UL1TR001866), the Fisher Center for Alzheimer's Research Foundation, the Meyer Foundation, the JPB Foundation, the French National Research Agency (ANR) under the Investments for the Future" program (ANR-10-IAHU-01)
Reviewing Editor
- Antony Rosen, Johns Hopkins University School of Medicine, United States
Ethics
Human subjects: Detailed information on consent, where applicable, is available in the methods section of the manuscript.
Version history
- Received: March 23, 2022
- Preprint posted: March 25, 2022 (view preprint)
- Accepted: October 10, 2022
- Accepted Manuscript published: October 27, 2022 (version 1)
- Version of Record published: November 30, 2022 (version 2)
Copyright
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
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Autoantibody discovery across monogenic, acquired, and COVID19-associated autoimmunity with scalable PhIP-Seq
eLife 11:e78550.
https://doi.org/10.7554/eLife.78550
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:
