DOI: 10.5061/dryad.9cnp5hqmn
title: Integrated plasma proteomic and single-cell immune signaling network signatures demarcate mild, moderate, and severe COVID-19

description: The biological determinants underlying the range of COVID-19 clinical
 manifestations are not fully understood. Here, over 1400 plasma proteins
 and 2600 single-cell immune features comprising cell phenotype, endogenous
 signaling activity, and signaling responses to inflammatory ligands are
 cross-sectionally assessed in peripheral blood from 97 patients with mild,
 moderate, and severe COVID-19 and 40 uninfected patients. Using an
 integrated computational approach to analyze the combined plasma and
 single-cell proteomic data, we identify and independently validate a
 multivariate model classifying COVID-19 severity (multi-class
 AUCtraining = 0.799, p-value = 4.2e-6; multi-class
 AUCvalidation = 0.773, p-value = 7.7e-6). Examination of
 informative model features reveals novel biological signatures of COVID-19
 severity, including the dysregulation of JAK/STAT, MAPK/mTOR, and NF-κB
 immune signaling networks in addition to recapitulating known hallmarks of
 COVID-19. These results provide a set of early determinants of COVID-19
 severity that may point to therapeutic targets for prevention and/or
 treatment of COVID-19 progression.
description: Mass cytometry: Blood was collected in EDTA
 tubes. Anticoagulated blood was processed into peripheral blood
 mononuclear cells (PBMC) by density gradient centrifugation using
 published methods (Syed et al., 2014). PBMC were stored in 10% DMSO and
 frozen in liquid nitrogen until thawing and
 staining. <br>Cryopreserved PBMC were quickly thawed, washed two
 times with supplemented medium, and rested for 1h at 37°C in RPMI 1640
 medium supplemented with 10% fetal bovine serum, 1%
 Penicillin-Streptomycin, and 1% L-Glutamine. PBMC were counted and checked
 for viability. 0.5-1x10<sup>6</sup> cells were either
 stimulated with lipopolysaccharide (LPS; 1?g/ml) and CL097 (TLR7/8
 agonist; 1?g/ml), interleukin-2 (IL-2), IL-4, IL-6 and interferon-? (IFN?;
 all 100 ng/ml), a cocktail of phorbol 12-myristate 13-acetate (PMA),
 ionomycin, brefeldin A and monensin (1x; PI cocktail), or left
 unstimulated for 15 minutes at 37°C. After stimulation, samples were fixed
 with Proteomic Stabilizer (SmartTube) and stored at -80°C until further
 processing for mass cytometry analysis.  Plasma
 proteomics: Plasma was isolated from EDTA-collected
 blood within 4hrs of collection centrifuging tubes at 500 x g for 10
 minutes at room temperature (RT). Plasma was transferred into fresh
 conical tubes and centrifuged again (500 x g, 10 minutes, RT) before
 aliquoting into 500 µL cryovials and transferred to -80°C for long-term
 storage. <br>Plasma protein levels were quantified using Olink
 multiplex proximity extension assay (PEA) panels (Olink Proteomics; www.olink.com)
 according to the manufacturer’s instructions. Plasma was treated with 1%
 Triton X-100 for 2 hours at room temperature to inactivate any potential
 virus, before shipping to Olink Proteomics. <br>The raw expression
 values obtained with the Olink assay are provided in the arbitrary unit
 Normalized Protein Expression (NPX), where high NPX values represent high
 protein concentration. Values were log2-transformed to account for
 heteroskedasticity. Proteins close to the limit of detection are flagged
 in the raw data.
description: The data files contain:<br>- FCS files of mass cytometry
 measurements<br>- PatientCharacteristics: file containing clinical
 information regarding the patients. Column "Cohort" indicates
 which samples were used for training and which for validation.<br>-
 Penalization matrix: penalization matrix used to penalize the mass
 cytometry data<br>- Olink_NPXvalues: data from the Olink plasma
 proteomic assay. Proteomic values are provided in the arbitrary unit
 NPX<br>- CyTOF_FrequencySignaling_preprocessed: Frequencies and
 processed phosphosignaling responses of the mass cytometry
 dataset<br>- Correlation_matrix: Absolute spearman correlation
 coefficients of the chord diagram depicted in Figure 3D.
 Further information and requests for resources and
 reagents should be directed to an will be fulfilled by the lead contact,
 Brice Gaudillière (gbrice@stanford.edu)