science.sciencemag.org/cgi/content/full/science.

abc6261/DC1

Supplementary Materials for

Systems biological assessment of immunity to mild versus severe
COVID-19 infection in humans
Prabhu S. Arunachalam*, Florian Wimmers*, Chris Ka Pun Mok*, Ranawaka A. P. M.
Perera*, Madeleine Scott†, Thomas Hagan†, Natalia Sigal†, Yupeng Feng†, Laurel
Bristow, Owen Tak-Yin Tsang, Dhananjay Wagh, John Coller, Kathryn L. Pellegrini,
Dmitri Kazmin, Ghina Alaaeddine, Wai Shing Leung, Jacky Man Chun Chan, Thomas
Shiu Hong Chik, Chris Yau Chung Choi, Christopher Huerta, Michele Paine
McCullough, Huibin Lv, Evan Anderson, Srilatha Edupuganti, Amit A. Upadhyay, Steve
E. Bosinger, Holden Terry Maecker, Purvesh Khatri, Nadine Rouphael, Malik Peiris, Bali
Pulendran‡
*These authors contributed equally to this work. †These authors contributed equally to this work.
‡Corresponding author. Email: bpulend@stanford.edu

Published 11 August 2020 on Science First Release

DOI: 10.1126/science.abc6261

This PDF file includes:

Materials and Methods

Figs. S1 to S21
Tables S1 to S3
Caption for Table S4
References

Other Supplementary Material for this manuscript includes the following:

(available at science.sciencemag.org/cgi/content/full/science.abc6261/DC1)

Table S4 (.xlsx)
MDAR Reproducibility Checklist (.pdf)
Methods

Study design and patient characteristics

In the Atlanta cohort, 24 healthy controls, 40 patients with diagnosis of COVID-19 and 16 patients
with diagnosis of influenza and RSV were included in this observational study. Healthy controls
were asymptomatic adults from whom samples were collected prior to the widespread circulation
of SARS-CoV-2 in the community. Patients with COVID-19 were defined according to WHO
guidance and positive SARS-CoV-2 RT-PCR testing by nasopharyngeal swabs. Patients with
COVID-19 were classified as acute hospitalized (less than 14 days from symptom onset) or
convalescent non-hospitalized (more than 4 weeks since symptom onset and currently
asymptomatic). The severity of inpatient COVID-19 cases was classified based on the adaptation
of the Sixth Revised Trial Version of the Novel Coronavirus Pneumonia Diagnosis and Treatment
Guidance. Moderate cases were defined as respiratory symptoms with radiological findings of
pneumonia. Severe cases were defined as requiring supplemental oxygen. Critical cases were
organ failure necessitating intensive care unit (ICU) care. Patients with RSV and influenza were
defined as acute hospitalized patients with confirmed diagnoses by BioFire® FilmArray® of
nasopharyngeal and oropharyngeal swabs. RSV and Flu patients were classified as moderate if
not requiring oxygen, severe if requiring oxygen and critical if requiring ICU level care. The study
conforms to the principles outlined in the Declaration of Helsinki and received approval by the
appropriate Institutional Review Board (Stanford University and Emory University). All study
procedures were performed after informed consent was obtained.

In the Hong Kong cohort, 36 patients with confirmed COVID-19 disease after RT-PCR
testing on a respiratory sample from the Princess Margaret Hospital, Hong Kong, were invited to
participate in the study after providing informed consent. Samples from 45 healthy controls which
were matched with cases on age and gender were collected from the Hong Kong Red Cross. The
study was approved by the institutional review board of the Hong Kong West Cluster of the
Hospital Authority of Hong Kong (approval number: UW20-169). All study procedures were
performed after informed consent was obtained. Day 1 of clinical onset was defined as the first
day of the appearance of clinical symptoms. The severity of the COVID-19 cases was classified
based on the adaptation of the Sixth Revised Trial Version of the Novel Coronavirus Pneumonia
Diagnosis and Treatment Guidance. The severity of the patients was categorized as follows; Mild
- no sign of pneumonia on imaging, mild clinical symptoms; Moderate - fever, respiratory
symptoms and radiological evidence of pneumonia; Severe - dyspnea, respiratory frequency
>30/min, blood oxygen saturation 93%, partial pressure of arterial oxygen to fraction of inspired

2
oxygen ratio <300, and/or lung infiltrates >50% within 24 to 48 hours; Critical - respiratory failure,
septic shock, and/or multiple organ dysfunction or failure or death. RSV and Flu patients were
classified as moderate if not requiring oxygen, severe if requiring oxygen and critical if requiring
ICU level care.

Phospho-CyTOF analysis of peripheral blood mononuclear cells

Live frozen PBMCs were revived by gentle resuspension in RPMI 1640 medium supplemented
with 10% FBS (complete RPMI). One million live PBMCs were immediately fixed with 1 ml of 2%
paraformaldehyde (Cat #28906, Pierce) for 30 min. After 30 min, the cells were centrifuged at 500
g for 5 min at room temperature. The cells were resuspended in 90% FBS + 10% DMSO (freezing
medium) and frozen at -80°C until staining. Fixed and frozen PBMCs were shipped in dry ice for
further analysis at the Human Immune Monitoring Center at Stanford University. The CyTOF
assay was run in 5 batches of 20 samples each. Each batch was well controlled with equal
distribution of healthy and infected samples in similar age and gender.

Fixed frozen PBMCs were thawed by gentle resuspension in CSM (PBS supplemented
with 2% BSA, 2 mM EDTA, and 0.1% sodium azide), washed twice with CSM and counted. Cells
were permeabilized and barcoded using Cell-ID™ 20-Plex Pd Barcoding Kit (Fluidigm). The
samples were washed with CSM, pooled, and counted. One pooled sample containing a mix of
all barcoded PBMC samples were stained for 30 min with surface antibody cocktail at room
temperature. The sample was then fixed with 4% freshly prepared paraformaldehyde (Alfa Aesar)
for 10 min at room temperature, washed with CSM, permeabilized with 100% methanol (Sigma)
and kept at -80°C overnight. Next day, the cells were washed with CSM, counted and stained with
pre-titrated intracellular antibody cocktail for 30 min at room temperature. Cells were then washed
with CSM, stained with iridium-containing DNA intercalator (Fluidigm), washed with MilliQ water
and acquired on Helios mass cytometer (Fluidigm) in MilliQ water supplemented with 1x EQ four
element calibration beads (Fluidigm).

The FCS files were bead-normalized before data export. The data were processed for
debarcoding in Flowjo software v10 (TreeStar Inc.). Briefly, the bead-normalized file was used to
gate single cells based on DNA content and event length using FlowJo. The single cells were
reimported and debarcoded using Helios software version 7.0.5189. The debarcoded samples
were analyzed using FlowJo or R version 1.2.1335 for downstream tSNE analysis and
visualization.

3
CyTOF data analysis

High-dimensional analysis of phospho-CyTOF data was performed using an R based pipeline

described in (39). Briefly, the raw fcs files were imported into R and the data were transformed to
normalize marker intensities using arcsinh with a cofactor of 5. For visualization, another
transformation was applied that scales the expression of all values between 0 and 1 using
percentiles as the boundary. Cell clustering was performed with 4,000 cells randomly selected
from each sample using FlowSom and ConsensusClusterPlus. The transformed matrix was used
as an input for FlowSom and cells were separated into 20 clusters. To obtain reproducible results
(avoid random start), a seed was set for each clustering. The 20 clusters were manually annotated
based on the lineage marker expression and were merged to produce the final clusters. The
clusters were visualized in two-dimensional space using tSNE. The abundance of cell populations
was determined using Plotabundance function. In parallel, the data are manually gated to identify
25 immune cell subpopulations that were not well-distinguished in tSNE and used for all
quantitation purposes.

Linear modeling of CyTOF data

In order to identify robust distinguishing features of infection status and disease severity, linear
models were constructed for each CyTOF feature via the limma package in R (40), using age,
gender, and batch as covariates. As the batches were site specific, this also accounted for
variability across the two cohorts (Hong Kong and Atlanta). When comparing the features across
disease severity, days post symptom onset was also used as a covariate. Briefly, each feature is
modeled as a linear function of infection status or disease severity plus additional covariates.
Once the model is fit, the estimated coefficients of the comparison of interest are then tested for
significance by computing the associated t-statistic and adjusting for multiple testing.

In vitro stimulation of peripheral blood leukocytes

Live frozen PBMCs were revived, counted and resuspended at a density of 15 million live cells/ml
in complete RPMI. The cells were rested at 37°C in CO2 incubators for 1 h before stimulation.
After the rest, 100 µl of cell suspension containing 1.5 million cells was added to each well of a
96-well round-bottomed tissue culture plate. Each sample was treated with three conditions, no
stimulation, viral cocktail containing 4 µg/ml R848 and 25 µg/ml polyIC or bacterial cocktail
containing 25 ng/ml LPS, 10 µg/ml Pam3CSK and 0.3 µg/ml Flagellin. All samples contained

4
0.18% v/v DMSO in total volume of 200 µl per well. The samples were incubated at 37°C in CO2
incubators for 1.5 h before addition of 10 µg/ml Brefeldin-A. The cells were incubated for an
additional 3.5 h. The cells were washed with PBS, incubated with PBS containing 2 mM EDTA
for 10 min at 4°C, and stained with Zombie UV fixable viability dye (Biolegend). The cells were
washed with PBS containing 5% FCS, blocked with 5 µl of Human Trustain FcX blocking solution
(BioLegend, Cat #422302) for 5 min before the addition of surface antibody cocktail containing
anti-CD3 (clone SP34-2, BD Biosciences, Cat #562406), anti-CD11c (clone S-HCL-3, BD
Biosciences, Cat #744436), anti-CD123 (clone 7G3, BS Biosciences, Cat #740722), anti-HLA-
DR (clone L243, BioLegend, Cat #307642), anti-CD16 (clone 3G8, BioLegend, Cat #302026),
anti-CD56 (clone HCD56, BioLegend, Cat #318332), anti-CD8a (clone RPA-T8, BD Biosciences,
Cat #612914), anti-CD20 (clone 2H7, BD Biosciences, Cat #612848) and anti-CD14 (clone M5E2,
BD Biosciences, Cat #612902). The cells were stained for 20 min at 4°C in 100 µl volume.
Subsequently, the cells were washed, fixed and permeabilized with cytofix/cytoperm buffer (BD
Biosciences) for 20 minutes. The permeabilized cells were stained with ICS antibodies to IL-1b
(clone H1b-98, BioLegend, Cat #511705), phospho-NF-kB p65 Ser529 (clone B33B4WP, Thermo
Fisher Scientific, Cat #46-9863-42), IFN-a (clone LT27:295, Milteyni Biotec, Cat #130-092-601),
IL-6 (clone MQ2-13A5, BioLegend, Cat #501120), IL-12p40/p70 (clone C8.6, BD Biosciences,
Cat #565023) and Perforin (clone dG9, BioLegend, Cat #308120), TNF-a (clone Mab11,
BioLegend, Cat #502938), MCP-1 (clone 5D3-F7, BioLegend, Cat #502612) and IFN-γ (clone
4S.B3, BD Biosciences, Cat #563563). Cells were then washed twice with perm/wash buffer and
once with staining buffer before analysis using BD Symphony Flow Cytometer. All flow cytometry
data were analyzed using Flowjo software v10 (TreeStar Inc.).

Plasma protein profiling using Olink multiplex panel

All plasma samples were heat-inactivated at 56°C for 15 min to inactivate the virus. A pilot
experiment using 2 plasma samples was performed to determine the effect of heat-inactivation
on cytokine measurements. We tested these samples at two dilutions and the best dilution was
determined for the assay. The inflammation panel used in the current study had minimal effects
on heat-inactivation (Fig. S21). The analytes affected more than 2-fold are highlighted. In the final
assay, all samples were from Emory were quantified using Olink multiplex proximity extension
assay (PEA) inflammation panel (Olink proteomics: www.olink.com) according to the
manufacturer’s instructions and as described before (41). The PEA is a dual-recognition
immunoassay, where two matched antibodies labelled with unique DNA oligonucleotides

5
simultaneously bind to a target protein in solution. This brings the two antibodies into proximity,
allowing their DNA oligonucleotides to hybridize, serving as template for a DNA polymerase-
dependent extension step. This creates a double-stranded DNA “barcode” which is unique for the
specific antigen and quantitatively proportional to the initial concentration of target protein. The
hybridization and extension are immediately followed by PCR amplification and the amplicon is
then finally quantified by microfluidic qPCR using Fluidigm BioMark HD system (Fluidigm
Corporation. South San Francisco, California).

ELISA assays

The Olink data for the three key cytokines, EN-RAGE, OSM and TNFSF14 were validated with
ELISA. EN-RAGE and TNFSF14 were measured using the human EN-RAGE DuoSet ELISA and
Human LIGHT/TNFSF14 Quantikine ELISA kits purchased from R&D Systems, respectively. The
assays were carried out as per the manufacturer’s protocol. Briefly, for EN-RAGE ELISA, 96 well
polystyrene microplates were coated with 100 µl of 1 µg/ml capture antibody overnight at room
temperature. Next morning, the plates were blocked with reagent diluent and plasma diluted 100-
fold was added and incubated for 2 h at room temperature. The plates were washed and
incubated with detection antibody for 2 h, followed by streptavidin-HRP for 20 min. For TNFSF14,
precoated plates were incubated with neat plasma for 2 h as described in the manufacturer’s
protocol. The plates were washed, incubated with detection antibody, Streptavidin-HRP and TMB
substrates as described for EN-RAGE.

OSM was measured using Human Oncostatin-M/OSM ELISA kit from Abcam which uses
Simplestep ELISA technology. Neat plasma samples were added to 96 well plate and incubated
with an antibody cocktail provided, containing tagged capture and detector antibodies. After
incubation for an hour at room temperature, the plates were washed with wash buffer and
developed using TMB substrate. For all three end-point ELISAs, the absorbance was measured
at 450 nm with 570 nm as a background. The concentration of unknown samples were
interpolated from standards using sigmoidal four parameter logistic regression or a linear
regression analysis in Graphpad prism.

CITE-seq single-cell RNA sequencing

Live frozen PBMCs were thawed and 2x washed with RPMI supplemented with 10% FBS and 20
µg/mL DNAse I (Sigma Aldrich). DCs were enriched using the Dynabeads™ DC Enrichment Kit

6
(Invitrogen, 11308D) according to manufacturer’s instructions with 3 – 4 million PBMCs as starting
material. The enriched cells were mixed with total PBMCs at a ratio of 1:2 and mixed cells were
stained with a cocktail of TotalSeq-A antibodies in PBS supplemented with 5% FBS, 2 mM EDTA,
and 5 mg/mL human IgG (Table S2), washed twice with PBS supplemented with 5% FBS, and 2
mM EDTA, and resuspended in PBS supplemented with 1% BSA (Miltenyi), and 0.5 U/µL RNase
Inhibitor (Sigma Aldrich). About 9,000 cells were targeted for each experiment.

Cells were mixed with the reverse transcription mix and subjected to partitioning along
with the Chromium gel-beads using the 10X Chromium system to generate the Gel-Bead in
Emulsions (GEMs) using the 3' V3 chemistry (10X Genomics, Pleasanton, CA). The RT reaction
was conducted in the C1000 touch PCR instrument (BioRad). Barcoded cDNA was extracted
from the GEMs by Post-GEM RT-cleanup and amplified for 12 cycles. Prior to amplification the
cDNA amplification mix was spiked in with ADT additive primer (0.2 µM stock) in order to amplify
the antibody barcodes. Amplified cDNA was subjected to 0.6x SPRI beads cleanup (Beckman,
B23318). Amplified antibody barcodes were recovered from the supernatant and were processed
to generate TotalSeq-A libraries as instructed by the manufacturer (BioLegend, TotalSeq™-A
Antibodies and Cell Hashing with 10x Single Cell 3' Reagent Kit v3 3.1 Protocol). The rest of the
amplified cDNA was subjected to enzymatic fragmentation, end-repair, A tailing, adapter ligation
and 10X specific sample indexing as per manufacturer’s protocol. Libraries were quantified using
Bioanalyzer (Agilent) analysis. Used primers are listed in Table S3.

10x Genomics scRNA-Seq and TotalSeq-A libraries were pooled and sequenced on an
Illumina HiSeq 4000 using the recommended sequencing read lengths of 28 bp (Read 1), 8 bp
(i7 Index Read), and 91 bp (Read 2). Cell Ranger v3.1.0 (10x Genomics) was used to demultiplex
raw sequencing data and quantitate transcript levels against the 10x Genomics GRCh38
reference v3.0.0.

Single-cell RNA seq processing and analysis

Raw count data was filtered to remove cells with a mitochondrial RNA fraction greater than 25%
of total RNA counts per cell. The resultant count matrix was used to create a Seurat (v 3.1.4)
object. Filtered read counts were scaled by a factor of 10,000 and log transformed. The antibody-
derived tag matrix was normalized per feature using center log normalization. The top 2000
variable RNA features were used to perform PCA on the log-transformed counts. Using a scree
plot, we chose the first 25 principle components (PCs) (98.5% of variance explained) to perform

7
further downstream analyses, including clustering and UMAP projections. Clusters were identified
with Seurat SNN graph construction followed by Louvain community detection on the resultant
graph with a resolution of 0.4, yielding 25 clusters. We removed one cluster that was composed
of a majority of dead cells, as judged by the percentage of mitochondrial RNA (>80% before
filtering) and exceptionally low unique features, resulting in 24 final clusters. We further removed
samples from 4 patients with influenza A and RSV infection, as well as 2 convalescent subjects
from the downstream analysis. The R package uwot (v.0.1.7) was used to produce UMAP
projections with the previously selected 25 PCs. All codes used for the analysis are deposited at
https://github.com/scottmk777/COVID.

Statistical analysis

Two-sided Wilcox tests were used in all differentially expressed gene analyses. First, we identified
DEG that distinguished each cluster from the remaining 23 clusters. These differentially
expressed genes were cross-referenced with known immune markers to annotate clusters.
Differentially expressed genes were displayed in heatmaps with a random subsample of up to
500 cells per cluster. Next, we identified DEG in each cluster that distinguished cells derived from
severe and moderate COVID-19 patients from healthy cells within that cluster. DEG were defined
as those with p-value less than 0.05 and a minimum log fold change of 0.25. The DEG from each
cluster were analyzed with overrepresentation analysis using the BTM modules. P-values were
determined by hypergeometric distribution. Significant pathways were those with a p-value <
0.005. For visualization purposes, only the top 10 most significant pathways were selected.
ComplexHeatmap (v.2.1.0) was used to produce all heatmaps.

IFN and inflammatory gene analysis in myeloid cells

To further compare the inflammatory state of myeloid clusters between subjects, we took the
average expression of each patient in the pDC and CDC2 clusters across all genes. We filtered
this gene list to only include the union set of genes that contributed to the significance of BTMs
M150, M127, M165 or M75 in any cluster. Average expression values for each subject were
hierarchically clustered using Euclidean distance.

For the expression analysis of selected interferons and inflammatory cytokines measured
during in-vitro stimulation and Olink plasma analysis, we averaged the gene expression values in
COVID-19 subjects and healthy subjects. We centered and scaled the average expression to

8
enable improved visualization and cross-gene comparisons. Pearson’s correlation coefficients
were calculated between S100A12 and all measured genes. The five most positively and
negatively correlated genes were then plotted for comparison; all adjusted correlation p-values
were smaller than the smallest positive floating-point number precisely calculable in the R
environment (2.22*10^-16) and therefore not comparable.

Flow cytometry for immune cell phenotyping

Live frozen PBMCs were thawed and 2x washed with RPMI supplemented with 10% FBS and 20
µg/mL DNAse I (Sigma Aldrich). Cells were washed with PBS and incubated with Zombie UV
Fixable viability dye (1:1000 in PBS, Biolegend 423108) for 20 minutes at 4°C in the dark. Next,
cells were washed with PBS and blocking buffer (PBS supplemented with 5% FBS, 2 mM EDTA
and 5 mg/mL human IgG). Cells were resuspended in blocking buffer with fluorescently-labeled
antibodies against CD3 (BUV737, BD Bioscience 564308), CD8 (BUV563, BD Bioscience,
612914), CD123 (BUV395, BD 564195), HLA-DR (BV785, Biolegend 307642), CD86 (BV650,
Biolegend 305428), CD14 (BV605, Biolegend 301834), CD1c (BV421, Biolegend 331526), CD56
(PE-CY7, BD 335791), CD370 (PE, Biolegend 353803), CD11c (APC-eFluor780, Life
Technologies 47-0128-42), CD16 (AF700, Biolegend 302026) and CD19 (APC, Biolegend
302212), and incubated for 20 minutes at 4°C in the dark. Cells were washed with PBS and fixed
with Cytofix (BD 554655) for 10 minutes at room temperature in the dark. Cells were washed with
PBS and stored in MACS buffer (PBS supplemented with 5% FBS, 2 mM EDTA) until acquisition
on a BD Symphony Flow Cytometer. All flow cytometry data were analyzed using Flowjo software
v10 (TreeStar Inc.).

Bulk transcriptomics

Frozen PBMCs were thawed and 0.5 – 1 million live cells were solubilized in 1 ml of Trizol. The
samples were frozen at -80°C until RNA isolation. RNA was isolated from each sample using the
miRNeasy Mini kit (Qiagen) and 10 ng of total RNA was used as input for cDNA synthesis using
the Clontech SMART-Seq v4 Ultra Low Input RNA kit (Takara Bio) according to the
manufacturer’s instructions. Amplified cDNA was fragmented and appended with dual-indexed
bar codes using the NexteraXT DNA Library Preparation kit (Illumina). Libraries were validated
by capillary electrophoresis on an Agilent 4200 TapeStation, pooled at equimolar concentrations,
and sequenced on an Illumina NovaSeq6000 at 100SR, yielding 20 million reads per sample.

9
 ENSEMBL IDs were filtered to remove low/non-expressed transcripts (0 reads in >50% of
samples). Gene-level counts were created by averaging counts from all ENSEMBL IDs mapping
to the same gene symbol (IDs mapping to multiple symbols were discarded), using the bioMart
package. For hierarchical clustering of ISGs, genes were z-score normalized across subjects and
then both genes and subjects were grouped by hierarchical clustering via the Ward algorithm,
using Euclidean distance as a distance metric. Visualization was performed using the pheatmap
package in R. For variance analysis of ISGs, principal variance components analysis (PVCA) was
used (42). This approach performs a principal components analysis on the gene expression and
then uses mixed linear modeling to assess the relative contribution to the variance in the first 5
principal components of parameters of interest (age, gender, severity, and days post onset of
symptoms in this case).

Bacterial DNA quantification by PCR

DNA was extracted from 200 µl plasma using QIAamp DNA Mini Kit (QIAGEN, Germantown, MD)
according to manufacturer’s guidelines. DNA was eluted in 30 ul of microbial DNA-free water.
Bacterial DNA quantification was performed by qPCR with SsoAdvanced™ Universal SYBR®
Green Supermix kit on the Bio-Rad CFX96. Universal 16S primers were used as previously
reported (43): EUBF 5’ - TCCTACGGGAGGCAGCAGT - 3’ and EUBR 5’ -
GGACTACCAGGGTATCTAATCCTGTT - 3’. Reactions were composed of 10 µl Supermix (2x),
0.5 ul each primer (10 µM), 4 µl PCR-grade water and 5 µl of DNA template. Reaction conditions
include initial denaturation at 98oC for 3 min followed by 40 cycles of denaturation for 15 s at 90oC,
annealing for 15 s at 60oC and elongation for 60 s at 72oC. The specificity of all qPCR products
was assessed by analysis of a post-PCR dissociation curve performed between 60°C and 95°C.
Each sample was run in triplicate and the mean value was used. The absolute number of copies
of the 16S rRNA gene was determined by comparison with a quantitative standard curve
generated with serial dilution of a microbial DNA standard from Enterococcus faecalis (Sigma
Aldrich). The average quantities of 16S copies per sample were presented as copies/ml plasma.

Quantitation of LPS in plasma

The LPS in plasma was quantified using human embryonic kidney (HEK)-Blue-hTLR4 cells (Cat
#hkb-htlr4) Invivogen, San Diego, CA) according to manufacturer’s guidelines. Briefly, 20 µl of
plasma heat-inactivated at 56ºC for 15 min, was added to 180 µl of HEK-Blue-hTLR4 cell

10
suspension in HEK-Blue detection medium. The cells were incubated at 37oC in 5% CO2 for 9 h
before secreted alkaline phosphatase activity was measured at 620 nm. The absolute quantities
of LPS in plasma were determined by comparison with a quantitative standard curve generated
with serial dilution of standard LPS-B5 (Invivogen, San Diego, CA).

Hierarchical clustering using CyTOF and cytokine measurements

Features distinguishing between infection status (cell frequencies and functional markers by
CyTOF) and disease severity (cytokine abundancies by Olink) were integrated in order to examine
whether the combination of these features could segregate subjects by both disease state and
severity. Features were z-score normalized across subjects and then both features and subjects
were grouped by hierarchical clustering via the Ward algorithm, using Euclidean distance as a
distance metric. Visualization was performed using the pheatmap package in R.

11
Fig. S1

Mass cytometry analysis of peripheral blood leukocytes.

Kinetics of frequency of plasmablast, CD8 effector T cells and pDCs in Atlanta and Hong Kong cohorts determined by
CyTOF. Each dot is an individual and colors indicate severity of the disease. The blue lines show exptrapolated median
and grey shade defines the confidence limits.
Fig. S2

B cells IgD+ CD27- Effector CD8

Naive
IgD+ CD27+ CM Naive CM Naive
HSC
Memory

HLA-DR
CD34

CD27

CD27
CD20

IgD
IgD- CD27- IgD- CD27+ EM EM
Memory CS
CD45 CD3 CD27 CD38 CD45RA CD45RA
Monocytes Effector CD4
CD8 T
NK T

NK

HLA-DR
HLA-DR

Tregs

CD127
CD14

CD4 T
CD56

CD8
CD16 CD56 CD3 CD4 CD25 CD38

PB/PC
pDC cDC Basophils
CD123

CD123
CD27

Unclassified

CD11c CD38 CD14

Gating strategy to identify 25 different immune cell subpopulations.

Fig. S3

A Plasmablast response
Interv Corticosteroid Antiviral treatment
10.00 ns 10.00 * 10.00 ns Severity

Frequency (% of CD45 cells)

**** **** **** Healthy

Frequency (% of CD45 cells)

Frequency (% of CD45 cells)

**** **** **** Moderate

Severe
1.00 1.00 1.00
ICU

0.10 0.10
0.10

0.01
0.01
0.01

s
lth

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N
B Effector CD8 T cell response
Interv Corticosteroid Antiviral treatment
ns ** *** Severity

Frequency (% of CD8 T cells)

****
Frequency (% of CD8 T cells)

**** **** Healthy

Frequency (% of CD8 T cells)

100.00 100.00 100.00 ****

**** **** Moderate
Severe
ICU

10.00 10.00
10.00

1.00
1.00
1.00
y

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Plasmablast and effector CD8 T cell frequencies in the Hong Kong cohort classified by intervention.
Box plots showing frequencies of plasmablasts (A) or effector CD8 T cells (B) in healthy and infected individuals
treated with IFN-β1, corticosteroid or antivirals.
Fig. S4

A pDC frequency
Interv Corticosteroid Antiviral treatment
ns ns ns

** * **
3.00 3.00
Frequency (% of CD45 cells)

3.00
** ** *
Severity
1.00 1.00 1.00
Healthy
Moderate

0.30 0.30 Severe

0.30
ICU

0.10 0.10
0.10

0.03
0.03
0.03

ls

s
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N
B pS6 (mTOR) in pDCs
Interv Corticosteroid Antiviral treatment
ns ns *
pS6 (FC relative to healthy controls

**** *** ****

2.0 2.0 ****
2.0 ****
****
Severity
1.5 1.5 Healthy
1.5
Moderate

1.0 1.0 Severe

1.0
ICU
0.5
0.5
0.5

0.0
0.0
0.0
y

ls

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C IKB in mDCs
Interv Corticosteroid Antiviral treatment
ve to healthy controls
ve to healthy controls

ns ns ns
ve to healthy controls

* * ****
3 3 3
**** **** ***

Severity
2 Healthy
2 2
Moderate
Severe

1 1 ICU
1

0
0
0
y

ls

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Innate immune responses measured by mass cytometry in the Hong Kong cohort classified by intervention
Box plots showing frequencies of pDCs (A) or signaling molecules pS6 and IKBa in pDCs (B) and mDCs (C),
respectively.
Fig. S5

125
Wilcoxon, p = 0.3 Wilcoxon, p = 0.93 Wilcoxon, p = 0.49 Wilcoxon, p = 0.12 Wilcoxon, p = 0.35

100
Seve
75 hy

50
Sev
ICU
25

0
hy Inf hy Inf hy Inf hy Inf hy Inf

125
Wilco Wilcoxon, p = 0.0051 Wilcoxon, p = 0.21 Wilco Wilco

100

75

50

25

0
hy Inf hy Inf hy Inf hy Inf hy Inf

125
Wilco Wilcoxon, p = 0.015 Wilcoxon, p = 0.00023 Wilco Wilcoxon, p = 0.00092

100

75

50

25

0
hy Inf hy Inf hy Inf hy Inf hy Inf

Flow cytometry analysis of ex vivo stimulated peripheral blood leukocytes.

Box plots showing fraction of CD14+ monocytes (CD3- CD20- CD56- HLA-DR+ CD14+ CD16-/+) in PBMCs of healthy or
infected producing cytokines indicated on the plots. Each dot represents an individual and the colors indicate severity
of clinical disease. In all the box plots, the boxes show median, upper and lower quartiles. The whiskers show 5 – 95
percentiles. Each dot represents an Atlanta cohort donor (n = 14 and 17 for healthy and infected, respectively). The
differences between the groups was measured by Mann-Whitney rank sum test (Wilcoxon, paired = FALSE). The p-
values depicting significance are shown within the box plots.
 Fig. S6

A 15

HGF
TNFSF14 OSM

EN.RAGE
10 IL6
CCL4 X4E.BP1
−log10 P

CSF.1 MCP.2 MCP.3

TNF-a IL.18R1 CCL19 CXCL10
CASP.8 CCL3 CXCL5
IL18 CXCL1
TGF.alpha
5 CXCL11
IL8
FGF.19 MMP.1
CCL20

0
−2 0 2 4
Fold change (Infected/healthy)

B
HGF 4E−BP1 CXCL10 CXCL5
* ** 20 ns ns
15
** ns ns ns
15 ns ns ns ns
15
*** * 15 * *
(log2)(log2)

10
10 10
10
Normalized protein expression NPX

5
5 5 5

0 0 0 0
od y

Se te
re

le U

t
u
SV

od y

Se te
re

le U

t
u
SV

od y

Se te
re

le U

t
u
SV

od y

Se te
re

le U

t
u
SV
en

en

en

en
M alth

M alth

M alth

M alth
Fl

Fl

Fl

Fl
va IC

va IC

va IC

va IC
a

a
ve

ve

ve

ve
R

R
er

er

er

er
sc

sc

sc

sc
e

e
H

H
on

on

on

on
C

FGF19 C
ns 12
ns
Multiplex cytokine
ns analysis in plasma.
(A) A9 volcano
ns plot highlighting differentially abundant molecules in COVID-19 infected individuals compared to
healthy subjects. The horizontal discontinuous line indicates a p value of 0.05 after FDR correction; any molecule
above which is statistically significant. The vertical discontinuous lines represent the order of change in the
6
magnitude. (B) Bar plots showing the other molecules which were atleast 2-fold higher in infected individuals. In all
the box plots, the boxes show median, upper and lower quartiles. The whiskers show 5 – 95 percentiles. Each dot
represents
3 an Atlanta cohort sample (n = 18 healthy, 4 moderate, 18 severe, 12 ICU, 2 convalescent, 8 Flu and 11
RSV). Colors of the dots indicate severity of clinical disease as shown in the legends. The differences between the
groups
0 was measured by Mann-Whitney rank sum test (Wilcoxon, paired = FALSE). The p-values depicting
significance are shown within the plots.
od y

Se te
re

le U

t
u
SV
en
M alth

Fl
va IC
a
ve

R
er

sc
e
H

on
C

Infection_severity
 Fig. S7

Independent validation of Olink data by ELISA

(A) Concentration of EN-RAGE, OSM and TNFSF14 measured by sandwich ELISA kits from R&D Systems
(Human EN-RAGE DuoSet ELISA), Abcam (Human Oncostatin M ELISA) and R&D Systems (Human LIGHT
Quantikine ELISA), respectively. (B) Spearman’s correlation between the protein levels measured by ELISA (X-
axis) and the Olink assay (Y-axis). ELISA values below detection limits were excluded from the correlation analysis.
N = 11 healthy & 29 infected for EN-RAGE, 18 healthy and 37 infected for OSM and TNFSF14 ELISA.
Fig. S8

UMAP representation of PBMCs before QC filtering.

PBMCs from 12 subjects including healthy donors and patients infected with COVID-19, were enriched for dendritic cells
and profiled for simultaneous transcription and cell-surface proteins using CITE-seq. (A, B) UMAP representation of 63,469
single cells after preprocessing colored by experiment (A) and cluster (B).
Fig. S9

Per-cell QC metrics. After preprocessing, qc metrics were calculated for each cell in each cluster. Shown are the
mitochondrial RNA fraction (top), RNA counts (middle) and unique features (bottom) per cell.
Fig. S10

Top10 distinctive genes per cluster. After QC filtering, we calculated the differentially expressed genes (DEGs) in each
cluster compared to all other cells. Heatmap shows the expression levels of the top 10 DEGs in up to 500 randomly
sampled cells from each cluster.
Fig. S11

CITE-seq antibody expression per cluster. Heatmap showing the abundance of CITE-seq antibodies in up to 500
randomly sampled cells from each cluster after QC filtering. The color indicates the normalized ADT values.
 Freq Freq Freq Freq

0.00
0.25
0.75
1.00
0.00
0.25
0.50
0.75
1.00
0.00
0.25
0.50
0.75
1.00
0.00
0.25
0.50
0.75
1.00
Fig. S12

cells passing
C0−CD4 C0−CD4 C0−CD4 C0−CD4
C1−NK C1−NK C1−NK C1−NK
C2−CD8 C2−CD8 C2−CD8 C2−CD8

0.50 QC filtering
−C MONO_1 C3−C MONO_1 C3−C MONO_1 C3−C MONO_1
−NC MONO C4−NC MONO C4−NC MONO C4−NC MONO
C5−CDC2 C5−CDC2 C5−CDC2 C5−CDC2
6−PLATE_1 C6−PLATE_1 C6−PLATE_1 C6−PLATE_1
C7−B C7−B C7−B C7−B
C9−GRAN C9−GRAN C9−GRAN C9−GRAN
C10−PDC C10−PDC C10−PDC C10−PDC
C MONO_IFN C11−C MONO_IFN C11−C MONO_IFN C11−C MONO_IFN
C12−EOS C12−EOS C12−EOS C12−EOS

Cluster
Cluster
Cluster

C13−PB_1 C13−PB_1 C13−PB_1 C13−PB_1

Participant and time point breakdown by cluster annotation.

4−PLATE_2 C14−PLATE_2 C14−PLATE_2 C14−PLATE_2
C15−PB_2 C15−PB_2 C15−PB_2 C15−PB_2
−C MONO_2 C16−C MONO_2 C16−C MONO_2 C16−C MONO_2
C17−RBC C17−RBC C17−RBC C17−RBC
18−T_IFN C18−T_IFN C18−T_IFN C18−T_IFN
C19−HSC C19−HSC C19−HSC C19−HSC
20−BASO C20−BASO C20−BASO C20−BASO
C21−Cl21 C21−Cl21 C21−Cl21 C21−Cl21
−C MONO_3 C22−C MONO_3 C22−C MONO_3 C22−C MONO_3
C23−T C23−T C23−T C23−T
24−CDC1 C24−CDC1 C24−CDC1 C24−CDC1
Set

Pt_ID

hd5
hd4
hd3
hd2
hd1
set2
set1

cov7
cov6
cov5
cov4
cov3
cov2
cov1

Disease

Severe

Healthy
Healthy

Moderate
COVID−19

Resp.Distress
Shown is the cell fraction by cluster for each experiment (top), subject (middle), disease (bottom) calculated based on all
Fig. S13
innate antiviral response (M150) ● ● ●
●●●●● ● ●●
● ●●
●
● ● ●
● ● ● ●
● ●
●
type I interferon response (M127) ●
●●●●●●●●●●●● ●
● ● ●●●●● ●
●●● ●
●
enriched in activated dendritic cells (II) (M165) ●
● ●
● ●
● ●
● ●
● ●
● ●
●●
● ●
●
antiviral IFN signature (M75) ●
● ●
● ●
● ●
● ●
●●
● ●
●
cell cycle and transcription (M4.0) ●
●
●
●
●
●
●
●
●
● ●
● ● ●

enriched in T cells (I) (M7.0) ●

●
●
●
●
● ●
● ●
● ●
● ●
●
●●●
●

●
● ●
●●● ●
● ●
● ●
●●● ●
●●●●● ●
●
● ●
●
enriched in antigen presentation (II) (M95.0)
● ●
● ●
● ●
● ● ●
● ●
●
● ● ●
● ●

immune activ ● ●
ric cluster (M37.0) ●● ● ●● ●

TLR and inflammatory signaling (M16) ●

● ● ●

●●● ●
Monocyte surface signature (S4) ● ● ● ● ●
● ● ●

regulation of antigen presentation and immune response (M5.0) ● ● ●●●● ● ●●

● ●
● ●●
● ● ● ● ● ● ● ●
●
●
●
●

enriched in antigen presentation (I) (M71) ●

●
● ●
● ●
●●● ● ●
● ●
●●● ●
●
antigen processing and presentation (M200) ●●
●●●●●●●●● ● ●
●
● ●
activated dendritic cells (M67)
● ● ●
● ●
● ●
●
ke receptor signaling (M68) ●
●
● ● ●
● ●
●●●●●●● ●
●
viral sensing & immunity; IRF2 targets network (I) (M111.0) ●
● ●
● ●
● ●
●
viral sensing & immunity; IRF2 targets network (II) (M111.1) ●
● ●
● ●
●
T cell activation and signaling (M5.1)●
● ● ●
●
●
T cell activation (I) (M7.1) ●●
●
● ●
● ●
●

cell adhesion (lymphocyte homing) (M21) ●

●●● ●
●●● Log of 1/P val
T cell differentiation (Th2) (M19) ●
● ●
● 20
Activated (LPS) dendritic cell surface signature (S11) ●
●
●
●
●
● ●
●
10
T cell surface, activation (M36) ●
● ●
●
integrins and cell adhesion (M84) ● ● ●
● ●
●●● 0

T cell differentiation via ITK and PKC (M18) ●

●●●
T cell activation (III) (M7.4) ●
●●●
T cell signaling and costimulation (M44) ●
● ●
●
respiratory electron transport chain (mitochondrion) (M238) ●
● ●
●
transcription regulation in cell development (M49) ●
●
●
● % Path is DEG
platelet activation (I) (M32.0) ●
● ●
● ●
● ● 0.3
platelet activation (II) (M32.1) ●
● ●
●
● 0.6
myeloid, dendritic cell activation via NFkB (II) (M43.1) ● ● ●
● ●
●
inflammasome receptors and signaling (M53) ●
● ●
● ●
● ● 0.9

putative targets of PAX3 (M89.1) ●

● ●
●
GF induced, enriched in nuclear receptor subfamily 4 (M94) ●
● ●
●
inflammatory response (M33) ●
● ●
●●●
suppression of MAPK signaling (M56) ●
● ●
●●● ●
●
enriched in antigen presentation (III) (M95.1) ●
● ●
● ●
●
translation initiation factor 3 complex (M245) ●
● ●
● ●
● ●
●
putative targets of PAX3 (M89.0) ●
●●●●●
cytoskeletal remodeling (enriched for SRF targets) (M34) ●
● ●
●
cell movement, Adhesion & Platelet activation (M30) ●
● ●
●
enriched in myeloid cells and monocytes (M81) ●
● ●
●

cell adhesion (M51) ●

● ●
●

chaperonin mediated protein folding (II) (M204.1) ●

● ●
●
chaperonin mediated protein folding (I) (M204.0) ● ●
●
chemokines and inflammatory molecules in myeloid cells (M86.0) ●
● ●
●●● ●
● ●
● ●
● ●
● ●
●
signaling in T cells (I) (M35.0) ●
● ●
●●● ●
● ●
● ●
● ●
● ●
● ●
●
leukocyte differentiation (M160) ●
● ●
● ●
●
ranscription factor network (M20) ●
● ●
● ●
● ●
● ●
● ●
● ●
●
cell cycle and growth arrest (M31) ●
● ●
● ●
● ●
●●●●● ●
●
enriched in NK cells (I) (M7.2) ●
● ●
● ●
●
●
●
●
● ●
●
●
●●●
●
●
enriched in monocytes (II) (M11.0) ●
●
●
●
● ●● ●
● ● ● ●
●
●
●
●
C23-T
C7-B
C20-BAS
Cd24CDC1
C5-CDC2

C11-C MONO_IFN
C21-Cl21
C3- C MONO_1
C16-C MONO_2

C12-EOS
C9-GRAN
C19-HSC
C4-NC MONO

C13-PB_1
C10-PDC
C6-PLATE_1
C17-RBC

C18-T_IFN
C0-CD4
C2-CD8
C1-NK

DEG analysis between COVID-19 infected and healthy subjects. After QC filtering, we calculated for each cluster the
DEGs between cells from COVID-19 infected subjects (n=7) and healthy control subjects (n=5). We then performed
overrepresentation analysis to identify enriched BTM gene sets in the up or downregulated genes. The heatmap shows the
top 10 enriched BTMs that appear in at least 2 cluster. The size of the circles indicates the fraction of BTM genes in the
DEG set, the color indicates the log 1/P value.
Fig. S14
Percent Expressed
C0−CD4 Average Expression C1−NK
2
●

0
● 5
1 ● 10
COVID−19 ● ● ●

● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●

0 COVID−19 ● ● ●

● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●

● 15

−1
● 20

Percent Expressed Average Expression

Healthy ● ● ●

● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●

0
Healthy ● ● ●

● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●

2
● 1 1
● 2
0
● 3
IF L1
IF L2
IF L3
IF G
IF K
IF NE
IF A1

IF NA8
N 2
IF A13

IF NA6
N 5
IF A14
IF A17
N 6
IF A10
IF A7
N 4
N 1
IF W1
B1

IF L1
IF L2
IF L3
IF G
IF K
IF NE
IF A1

IF NA8
N 2
IF A13

IF NA6
IF A15
IF A14
IF A17
N 6
IF A10
IF A7
N 4
N 1
IF W1
B1
IF A

IF A

IF A1

IF NA
IF A2

IF A

IF A

IF NA
IF A2
N

N
N

N
N
N
N

N
N
N
N
N

N
N

N
N
N

N
N
N
IF

IF
−1

C2−CD8 Average Expression C3−C MONO_1 Average Expression

2 2

1 1
COVID−19 ● ● ●

● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●

0
COVID−19 ● ● ●

● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●

−1 −1
Percent Expressed Percent Expressed
Healthy ● ● ●

● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●

0 ●
Healthy ● ● ●

● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●

0.00
● 10 ● 0.25
● 20 ● 0.50
● 30 ● 0.75
IF L1
IF L2
IF L3
IF G
IF K
IF NE
IF A1

IF NA8
N 2
IF A13

IF NA6
N 5
IF A14
IF A17
N 6
IF A10
IF A7
N 4
N 1
IF W1
B1

IF L1
IF L2
IF L3
IF G
IF K
IF NE
IF A1

IF NA8
N 2
IF A13

IF NA6
IF A15
IF A14
IF A17
N 6
IF A10
IF A7
N 4
N 1
IF W1
B1
IF A

IF A

IF A1

IF NA
IF A2

IF A

IF A

IF NA
IF A2
N

N
N

N
N
N
N

N
N
N
N
N

N
N

N
N
N

N
N
N
IF

IF
Average Expression
C4−NC MONO Average Expression C5−CDC2 2
2
1
1
COVID−19 ● ● ●

● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●

0 COVID−19 ● ● ●

● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● 0

−1
−1
Percent Expressed
Percent Expressed
Healthy ● ● ●

● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●

0
Healthy ● ● ●

● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●

●
●

0
1
● 1
● 2
● 2
● 3
● 3
IF L1
IF L2
IF L3
IF G
IF K
IF NE
IF A1

IF NA8
N 2
IF A13

IF NA6
N 5
IF A14
IF A17
N 6
IF A10
IF A7
N 4
N 1
IF W1
B1

IF L1
IF L2
IF L3
IF G
IF K
IF NE
IF A1

IF NA8
N 2
IF A13

IF NA6
IF A15
IF A14
IF A17
N 6
IF A10
IF A7
N 4
N 1
IF W1
B1
●
IF A

IF A

IF A1

IF NA
IF A2

IF A

IF A

IF NA
IF A2
N

N
N

N
4
N
N
N

N
N
N
N
N

N
N

N
N
N

N
N
N
IF

IF

C10−PDC Percent Expressed C11−C MONO_IFN Average Expression

2
●

0.0
● 0.5 1
COVID−19 ● ● ●

● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●
● 1.0 COVID−19 ● ● ●
● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●

0
● 1.5
−1
Average Expression
2 Percent Expressed
Healthy ● ● ●

● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●

1
Healthy ● ● ●

● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●

0
● 1
0 ● 2
● 3
IF L1
IF L2
IF L3
IF G
IF K
IF NE
IF A1

IF NA8
N 2
IF A13

IF NA6
N 5
IF A14
IF A17
N 6
IF A10
IF A7
N 4
N 1
IF W1
B1

IF L1
IF L2
IF L3
IF G
IF K
IF NE
IF A1

IF NA8
N 2
IF A13

IF NA6
IF A15
IF A14
IF A17
N 6
IF A10
IF A7
N 4
N 1
IF W1
B1
IF A

IF A

IF A1

IF NA
IF A2

IF A

IF A

IF NA
IF A2
N

N
N

−1
N
N
N

N
N
N
N
N

N
N

N
N
N

N
N
N
IF

IF

Percent Expressed Average Expression

C16−C MONO_2 ●

0
C18−T_IFN 2
● 5 1
● 10
COVID−19 ● ● ●

● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●

● 15 COVID−19 ● ● ●

● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● 0

● 20 −1
Percent Expressed
Average Expression
Healthy ● ● ●

● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●
2 Healthy ● ● ●

● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●

●
●

0
1
1 ● 2
0 ● 3
IF L1
IF L2
IF L3
IF G
IF K
IF NE
IF A1

IF NA8
N 2
IF A13

IF NA6
N 5
IF A14
IF A17
N 6
IF A10
IF A7
N 4
N 1
IF W1
B1

IF L1
IF L2
IF L3
IF G
IF K
IF NE
IF A1

IF NA8
N 2
IF A13

IF NA6
IF A15
IF A14
IF A17
N 6
IF A10
IF A7
N 4
N 1
IF W1
B1

●
IF A

IF A

IF A1

IF NA
IF A2

IF A

IF A

IF NA
IF A2
N

N
N

4
N
N
N

N
N
N
N
N

N
N

N
N
N

N
N
N
IF

IF

−1

Percent Expressed
C23−T ●

0
● 5
● 10
COVID−19 ● ● ●

● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●

● 15
● 20

Average Expression
Healthy ● ● ●

● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●

0
IF L1
IF L2
IF L3
IF G
IF K
IF NE
IF A1

IF NA8
N 2
IF A13

IF NA6
N 5
IF A14
IF A17
N 6
IF A10
IF A7
N 4
N 1
IF W1
B1
IF A

IF A

IF A1

IF NA
IF A2
N
N
N
N
N

N
N

N
N
N
IF

−1

IFN expression in CITE-seq clusters. Dot plots showing the expression levels of different interferon genes in innate and
adaptive CITE-seq clusters of single cells from COVID-19 infected and healthy subjects. Color indicates the average
expression level in a cluster, size indicates the percent of cells expressing each IFN gene in that cluster.
Fig. S15

A
3 Days_post_symptom_onset
20

2 Severity
Moderate
Severe
ICU

0
Days_post_symptom_onset
Severity

PARP14
DTX3L
STAT2
ADAR
OAS3
BST2
IFITM3
OAS2
PARP9
TDRD7
CNP
PML
IFI35
IRF7
IL15RA
PLSCR1
LGALS9
OGFR
SELL
TRIM25
VCPIP1
ANKFY1
ABTB2
TRIM14
FAM111A
GBP4
GCH1
GBP2
CASP4
TRAFD1
CXCL10
NMI
RTP4
PHF11
TRIM21
GBP3
PSME2
DHX58
RSAD2
IFIT2
UBE2L6
IFI44
EIF2AK2
CMPK2
EPSTI1
LGALS3BP
ISG15
USP18
PARP11
STAT1
DDX60
SAMD9L
DDX58
IFIH1
XAF1
IFIT1
IFIT3
TAP1
PARP12
PNPT1
SP110
PPM1K
ISG20
ZBP1
IRF9
XRN1
HSH2D
RNF213
SMCHD1
ZCCHC2
USP25
MOV10
ZNFX1
CCRL2
NAMPT
EHD4
IL1RN
CCNYL1
HK2
MAX
TMEM140
CLIC4
CD86
PIK3AP1
TIPARP
CD69
NR4A3
TLK2
ATP10A
NLRC5
SLFN5
DCK
GBP5
PYHIN1
PCGF5
AIDA
BBX
P2RY14
PPA1
CD274
RBM43
B C
1.0
Proportion variance explained

0.8

0.6

0.4

0.2

0.0
Age

Severity

Time

Gender

resid

Interferon response genes in bulk RNAseq analysis. (A) Hierarchically clustered heatmap from the bulk RNAseq
dataset, performed using an extended group of subjects (n = 17 healthy and 17 COVID-19 infected subjects), depicting
relative expression levels of interferon-stimulated gene signature described in Mostafavi S, et al. Cell 2016. (B) Correlation
analysis between average ISG expression on the Y-axis measured by bulk RNAseq and plasma IFN-⍺ protein measured
by SIMoA technology. (C) Bar chart representing the proportion of variance explained by the covariates in X-axis.
Fig. S16

cDC cmc
4.0
MFI CD86 (log10)

●●
●●
3.5 ●

●
● ●
●

●
●
●
●
3.0 ● ●
●
● ●

●
●
● ●
●
2.5
y

re

re
lth

lth
at

at
ve

ve
er

er
ea

ea
Se

Se
od

od
H

H
M

Infection_severity
B
Classical Monocytes cytes Myeloid DCs
Perc. Exp. Exp. lvl
Perc. Exp.
CO ● ● ● ● ● ● ●

● Perc. Exp. ● ● ● ● ●

● ●
●
●
●
● ●
● ● ●
● ●

● ●
● ●
Healthy ●
●
● ● ● ●
● ●

● ●
●
● ● ● ● ●

● ● ●

●
● ●
● ● ●
● ●

● ●
C M
L7

F
6
C M
L7

F
6

C M
L7

F
6
IL
IL

TN
TN

IL
TN
S
S

S
C
C

C
O
O

Attenuated inflammatory response in PBMCS. (A) Boxplots showing the log10 MFI of CD86 expression on mDCs and
classical monocytes detected via flow cytometry on samples analyzed in CITE-seq experiment. (B) Dot plots showing the
expression levels of different inflammatory cytokine genes in innate CITE-seq clusters of single cells from COVID-19
infected and healthy subjects. Color indicates the average expression level in a cluster, size indicates the percent of cells
expressing each gene in that cluster.
Fig. S17
C3−C MONO_1 C4−NC MONO C5−CDC2
5
4
3
2
1
0
C10−PDC C11−C MONO_IFN C16−C MONO_2
5 count

HLA−DPA1
4
20
3 15
2 10
1 5
0
C22−C MONO_3 0 2 4 6 0 2 4 6
5
4
3
2
1
0
0 2 4 6
S100A12

C3−C MONO_1 C4−NC MONO C5−CDC2

5
4
3
2
1
0
C10−PDC C11−C MONO_IFN C16−C MONO_2
5
count
HLA−DPB1

4 25
3 20
2 15
10
1
5
0
C22−C MONO_3 0 2 4 60 2 4 6
5
4
3
2
1
0
0 2 4 6
S100A12

C3−C MONO_1 C4−NC MONO C5−CDC2

0
C10−PDC C11−C MONO_IFN C16−C MONO_2
count
25
HLA−DRA

4
20
15
2 10
5
0
C22−C MONO_3 0 2 4 60 2 4 6

0
0 2 4 6
S100A12

C3−C MONO_1 C4−NC MONO C5−CDC2

4
3
2
1
0

C10−PDC C11−C MONO_IFN C16−C MONO_2

4 count
HLA−DQA1

3
2 10

1 5
0

C22−C MONO_3 0 1 2 3 4 5 0 1 2 3 4 5

4
3
2
1
0
0 1 2 3 4 5
S100A12

C3−C MONO_1 C4−NC MONO C5−CDC2

5
4
3
2
1

C10−PDC C11−C MONO_IFN C16−C MONO_2

5 count
HLA−DRB1

4 16
3 12
2 8
1 4

C22−C MONO_3 0 2 4 6 0 2 4 6
5
4
3
2
1

0 2 4 6
S100A12

Correlation between S100A12 and HLA-DR. Dot plots showing the expression levels of S100A12 and selected genes of
the antigen presentation machinery in single cells of monocyte and dendritic cell clusters as detected via CITE-seq.
Fig. S18

RAGE expression in single cells. UMAP representation showing the expression levels of the EN-RAGE receptor RAGE
(AGER) in all cells passing QC filters.
Fig. S19

Systemic release of bacterial products in severe COVID-19 infection

(A) LPS levels measured in plasma correlated to cytokine responses measured by Olink multiplex cytokine assay. (B)
Kinetics of cytokine response in plasma.
Fig. S20

4 Severity
Severity
Healthy
pDCs.pS6 Moderate
2 Severe
ICU
pDCs Convalescent
0 Type
mDCs.Ikba Cell frequency
Cytokine
−2 Functional marker
mDCs.HLA_DR

Effector_CD8_T_cells −4

Plasmablasts

IL6

MCP.3

EN.RAGE

OSM

TNFSF14
Type

Hierarchical clustering of phospho CyTOF and plasma cytokine datasets.

Hierarchical clustering of healthy and COVID-19 infected subjects using immune cell
type frequencies and signaling molecules detected in phospho-CyTOF and circulating
plasma cytokines. The heatmap colors represent row-wise z scores.
Fig. S21

Multiplex cytokine analysis in plasma.

Spearman’s correlation between cytokine responses measured in plasma samples with and without no heat-inactivation.
The changes > 2-fold are highlighted with labels.
Table S1. CyTOF antibody panel

Conjugate Target Clone Source (Cat #)

89Y CD45 HI30 FLUIDIGM (3089003B)
141Pr CD7 CD7-6B7 Biolegend (343102)
142Nd CD19 HIB19 FLUIDIGM (3142001B)
144Nd pPLCg2 (Y759) K86-689.37 FLUIDIGM (3144015A)
145Nd CD4 RPA-T4 FLUIDIGM (3145001B)
146Nd IgD IA6-2 FLUIDIGM (3146005B)
147Sm CD20 2H7 FLUIDIGM (3147001B)
148Nd CD34 581 FLUIDIGM (3148001B)
149Sm CD25 (IL-2R) 2A3 FLUIDIGM (3149010B)
150Nd pStat5 (Y694) 47 FLUIDIGM (3150005A)
151Eu CD123 (IL-3R) 6H6 FLUIDIGM (3151001B)
152Sm pAkt (S473) D9E FLUIDIGM (3152005A)
153Eu pStat1 (Y701) 4a FLUIDIGM (3153005A)
154Sm H3K27ac MABI 0309 ActiveMotiv (39685)
155Gd CD27 L128 FLUIDIGM (3155001B)
156Gd p-p38 (T180/Y182) D3F9 FLUIDIGM (3156002A)
157Gd CD24 ML-5 Biolegend (311102)
158Gd pStat3 (Y705) 4 FLUIDIGM (3158030D)
159Tb CD11c Bu15 FLUIDIGM (3159001B)
160Gd CD14 M5E2 FLUIDIGM (3160001B)
161Dy CD80 2D10.4 FLUIDIGM (3161023B)
163Dy CD56 NCAM16.2 FLUIDIGM (3163007B)
164Dy IkBa L35A5 FLUIDIGM (3164004A)
165Ho pCREB (S133) 87G3 FLUIDIGM (3176005A)
166Er CD16 B73.1 eBioscience (16-0167-82)
167Er CD38 HIT2 FLUIDIGM (3167001B)
168Er CD8a SK1 FLUIDIGM (3168002B)
169Tm CD45RA HI100 FLUIDIGM (3169008B)
170Er CD3 UCHT1 FLUIDIGM (3170001B)
171Yb pERK 1/2 (T202/Y204) D13.14.4E FLUIDIGM (3171010A)
172Yb Ki-67 B56 FLUIDIGM (3172024B)
174Yb HLA-DR L243 FLUIDIGM (3174001B)
175Lu pS6 N7548 FLUIDIGM (3175009A)
176Yb CD127 R3434 Novus Biologicals (DDX0700P)
Table S2. CITE-seq antibody panel

Antigen Clone Tag Dilution Cat # Supplier

CD3 UCHT1 A0034 1:10 300475 Biolegend
CD4 OKT4 A0922 1:10 317451 Biolegend
CD8 RPA-T8 A0080 1:10 301067 Biolegend
CD19 HIB19 A0050 1:10 302259 Biolegend
CD20 2H7 A0100 1:10 302359 Biolegend
CD56 5.1H11 A0047 1:10 362557 Biolegend
CD69 FN50 A0146 1:10 310947 Biolegend
CD28 CD28.2 A0386 1:10 302955 Biolegend
CD95 DX2 A0156 1:10 305649 Biolegend
CD279 (PD-1) RMP1-30 A0004 1:10 109123 Biolegend
CD197 (CCR7) G043H7 A0148 1:10 353247 Biolegend
CD45RO UCHL1 A0087 1:10 304255 Biolegend
CD45RA HI100 A0063 1:10 304157 Biolegend
HLA-DR L243 A0159 1:10 307659 Biolegend
CD14 M5E2 A0081 1:10 301855 Biolegend
CD16 3G8 A0083 1:10 302061 Biolegend
CD11c S-HCL-3 A0053 1:10 371519 Biolegend
CD1c (BDCA1) L161 A0160 1:10 331539 Biolegend
CD370 (CLEC9A) 8F9 A0207 1:10 353807 Biolegend
CD123 6H6 A0064 1:10 306037 Biolegend
CD86 IT2.2 A0006 1:10 305443 Biolegend
CD274 (PD-L1) 29E.2A3 A0007 1:10 329743 Biolegend
CD163 GHI/61 A0358 1:10 333635 Biolegend
CD33 P67.6 A0052 1:10 366629 Biolegend
CD57 QA17A04 A0168 1:10 393319 Biolegend
CD27 O323 A0154 1:10 302847 Biolegend
CD38 HIT2 A0389 1:10 303541 Biolegend
CD25 BC96 A0085 1:10 302643 Biolegend
CD34 581 A0054 1:10 343537 Biolegend
TCRgd B1 A0139 1:10 331229 Biolegend
TCRa7.2 3C10 A0581 1:10 351733 Biolegend
Anti-PE PE001 A0911 1:10 408109 Biolegend
CD127 A019D5 A0390 1:10 351352 Biolegend
CD94 DX22 A0867 1:10 305521 Biolegend
FCER1a AER-37 A0352 1:10 334641 Biolegend
Isotype control, mIgG2a, k MOPC-173 A0091 1:10 400285 Biolegend
Isotype control, mIgG1, k MOPC-21 A0090 1:10 400199 Biolegend
Isotype control, mIgG2b, k MPC-11 A0092 1:10 400373 Biolegend
Isotype control, rIgG2b, k RTK4530 A0095 1:10 400673 Biolegend
Table S3. TotalSeq A primer sequences

ADTadditive primer CCTTGGCACCCGAGAATT*C*C

SI-PCR Primer AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGC*T*C
RPI1 CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCCTTGGCACCCGAGAATTC*C*A
RPI2 CAAGCAGAAGACGGCATACGAGATACATCGGTGACTGGAGTTCCTTGGCACCCGAGAATTC*C*A
RPI3 CAAGCAGAAGACGGCATACGAGATGCCTAAGTGACTGGAGTTCCTTGGCACCCGAGAATTC*C*A
RPI4 CAAGCAGAAGACGGCATACGAGATTGGTCAGTGACTGGAGTTCCTTGGCACCCGAGAATTC*C*A
RPI5 CAAGCAGAAGACGGCATACGAGATCACTGTGTGACTGGAGTTCCTTGGCACCCGAGAATTC*C*A
RPI6 CAAGCAGAAGACGGCATACGAGATATTGGCGTGACTGGAGTTCCTTGGCACCCGAGAATTC*C*A
RPI7 CAAGCAGAAGACGGCATACGAGATGATCTGGTGACTGGAGTTCCTTGGCACCCGAGAATTC*C*A
RPI8 CAAGCAGAAGACGGCATACGAGATTCAAGTGTGACTGGAGTTCCTTGGCACCCGAGAATTC*C*A

Note - * indicates a phosphorothioate bond

Table S4. Raw data of CITE-seq analysis.

Tab 1 shows the expression of top 100 genes in each cell cluster defined by CITE-seq analysis.
The tab 2 shows the differentially expressed genes between healthy (n = 5) and COVID-19 (n = 7)
infected patients. The tab 3 shows statistically significant blood transcriptional modules identified
in each cell cluster. The tab 4 shows correlation between expression of S100A12 and all other
genes measured by CITE-seq analysis.
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