Non-productive exposure of PBMCs to SARS-CoV-2 induces cell-intrinsic innate immunity responses

Abstract

Cell-intrinsic responses mounted in vivo in PBMCs during mild and severe COVID-19 differ quantitatively and qualitatively. Whether they are triggered by signals emitted by productively infected cells of the respiratory tract or are, at least partially, resulting from physical interaction with virus particles, remains unclear. Here, we analyzed susceptibility and expression profiles of PBMCs from healthy donors upon ex vivo exposure to SARS-CoV and SARS-CoV-2. In line with the absence of detectable ACE2 receptor expression, human PBMCs were refractory to productive infection. Bulk and single cell RNA-sequencing revealed JAK/STAT-dependent induction of interferon-stimulated genes, but not pro-inflammatory cytokines. This SARS-CoV-2-specific response was most pronounced in monocytes. SARS-CoV-2-RNA-positive monocytes displayed a lower ISG signature as compared to bystander cells of the identical culture. This suggests a preferential invasion of cells with a low ISG base-line profile or delivery of a SARS-CoV-2-specific sensing antagonist upon efficient particle internalization. Together, non-productive physical interaction of PBMCs with SARS-CoV-2-but not SARS-CoV particles stimulates JAK/STAT-dependent, monocyte-accentuated innate immune responses that resemble those detected in vivo in patients with mild COVID-19.

SciScore for 10.1101/2022.02.15.480527: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Ethics	IRB: Withdrawal of blood samples from healthy humans and cell isolation were conducted with approval of the local ethics committee (Ethical review committee of Charité Berlin, votes EA4/166/19 and EA4/167/19).
Sex as a biological variable	not detected.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Cell Line Authentication	Contamination: Cell lines were routinely monitored for absence of mycoplasma and paramyxovirus simian virus 5.

Table 2: Resources

Antibodies
Sentences	Resources
Human ACE2 was detected using a polyclonal goat anti-human ACE2 antibody (1:500, R&D Systems), a horseradish peroxidase (HRP)-labeled donkey anti-goat antibody (1:5000, Dinova) and Super Signal …

SciScore for 10.1101/2022.02.15.480527: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Ethics	IRB: Withdrawal of blood samples from healthy humans and cell isolation were conducted with approval of the local ethics committee (Ethical review committee of Charité Berlin, votes EA4/166/19 and EA4/167/19).
Sex as a biological variable	not detected.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Cell Line Authentication	Contamination: Cell lines were routinely monitored for absence of mycoplasma and paramyxovirus simian virus 5.

Table 2: Resources

Antibodies
Sentences	Resources
Human ACE2 was detected using a polyclonal goat anti-human ACE2 antibody (1:500, R&D Systems), a horseradish peroxidase (HRP)-labeled donkey anti-goat antibody (1:5000, Dinova) and Super Signal West Femto Chemiluminescence Substrate (Thermo Fisher Scientific).	anti-goat suggested: None
As a loading control, samples were analyzed for ꞵ-Actin expression using a mouse anti-ꞵ-actin antibody (1:5000, Sigma Aldrich) and a HRP-labeled goat anti-mouse antibody (1:10000, Dianova).	anti-ꞵ-actin suggested: None anti-mouse suggested: None
Samples were multiplexed using TotalSeq-A Antibodies purchased from BioLegend (A0256, A0258 and A0259)	TotalSeq-A suggested: None A0256 suggested: (ABclonal Cat# A0256, RRID:AB_2757069) A0259 suggested: (ABclonal Cat# A0259, RRID:AB_2757072)
Flow Cytometry Analysis: PBS-washed cells were PFA-fixed and immunostained for individual surface protein expression using the following antibodies: Anti-CD3-FITC (#561807; BD Biosciences), anti-CD4-APC (#555349; BD Biosciences), anti-CD14-PE (#561707; BD Biosciences), anti-CD19-FITC (#21270193; ImmunoTools), anti-NRP1/CD304-APC-R700 (#566038, BD Biosciences), anti-PD-1/CD279-PE (#21272794; ImmunoTools) and anti-TIM-3/CD366-FITC (#345022; Biolegend).	Anti-CD3-FITC suggested: (Sigma-Aldrich Cat# F0522, RRID:AB_476959) anti-CD4-APC suggested: (Sigma-Aldrich Cat# SAB4700557, RRID:AB_10896237) anti-CD14-PE suggested: (Sigma-Aldrich Cat# SAB4700101, RRID:AB_10897597) anti-CD19-FITC suggested: (Sigma-Aldrich Cat# SAB4700112, RRID:AB_10896899) anti-NRP1/CD304-APC-R700 suggested: None anti-PD-1/CD279-PE suggested: None anti-TIM-3/CD366-FITC suggested: None
To determine ACE2 cell surface expression, cells were immunostained with a goat anti-human ACE2 antibody (#AF933, R&D Systems) followed by immunostaining with a secondary antibody donkey anti-goat Alexa Fluor 488 (#A-11055, Thermo Fisher).	anti-human ACE2 suggested: None
Experimental Models: Cell Lines
Sentences	Resources
Cell Lines and Primary Cells: Vero E6 (ATCC CRL-1586) cells, Calu-3 (ATCC HTB-55) cells and HEK293T (ATCC CRL-3216) cells were cultivated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum, 1% non-essential amino acids (Thermo Fisher Scientific) and 1% sodium pyruvate (Thermo Fisher Scientific) in a 5% CO2 atmosphere at 37°C.	Calu-3 suggested: ATCC Cat# HTB-55, RRID:CVCL_0609)
Virus was grown on Vero E6 cells and concentrated using Vivaspin® 20 concentrators with a size exclusion of 100 kDa (Sartorius Stedim Biotech) in order to remove cytokines of lower molecular weight, including IFNs.	Vero E6 suggested: None
Cells inoculated with culture supernatants from uninfected Vero cells mixed with OptiPro serum-free medium supplemented with 0.5% gelatine and PBS, served as mock-infected controls.	Vero suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
ACE2-positive HEK293T cells were generated by transduction of cells with retroviral vectors generated by transfection of HEK293T cells with MLV gag-pol (Bartosch, Dubuisson, and Cosset 2003), pCX4bsrACE2 (Kamitani et al. 2006) and pVSV-G (Stewart et al. 2003).	HEK293T suggested: None
Recombinant DNA
Sentences	Resources
ACE2-positive HEK293T cells were generated by transduction of cells with retroviral vectors generated by transfection of HEK293T cells with MLV gag-pol (Bartosch, Dubuisson, and Cosset 2003), pCX4bsrACE2 (Kamitani et al. 2006) and pVSV-G (Stewart et al. 2003).	pCX4bsrACE2 suggested: None pVSV-G suggested: RRID:Addgene_138479)
Software and Algorithms
Sentences	Resources
Data analysis was performed using LightCycler Software 4.1 (Roche).	LightCycler suggested: (LightCycler Software, RRID:SCR_012155)
Protein concentration was determined with the Thermo Scientific’s Pierce™	Thermo Scientific’s suggested: None
Quality control of the libraries were performed with the KAPA Library Quantification Kit and Agilent TapeStation.	Agilent TapeStation suggested: (Agilent TapeStation Laptop, RRID:SCR_019547)
(10X Genomics) and further analysed using the Seurat v3.1.4 package (Butler et al. 2018) in R v3.6 (https://www.r-project.org/).	Seurat suggested: (SEURAT, RRID:SCR_007322) https://www.r-project.org/ suggested: (R Project for Statistical Computing, RRID:SCR_001905)
Statistical significance was calculated by performing Student’s t-test using GraphPad Prism.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)

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Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

Results from JetFighter: We did not find any issues relating to colormaps.

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Results from scite Reference Check: We found no unreliable references.

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Non-productive exposure of PBMCs to SARS-CoV-2 induces cell-intrinsic innate immunity responses

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