SARS-CoV-2 in severe COVID-19 induces a TGF-β-dominated chronic immune response that does not target itself

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1. Version published to 10.1038/s41467-021-22210-3

   Mar 30, 2021
2. 

   ScreenIT

   Mar 1, 2021

   SciScore for 10.1101/2020.09.04.20188169: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IRB: Human Donors: The recruitment of study subjects was conducted in accordance with the Ethics Committee of the Charité (EA 1/144/13 with EA 1/075/19 and EA 2/066/20) and was in compliance with the Declaration of Helsinki.
   Randomization	Analysis was done using averages of 4 fields of view chosen randomly (blind) and then assessed for foci using fluorescent microscopes.
   Blinding	Analysis was done using averages of 4 fields of view chosen randomly (blind) and then assessed for foci using fluorescent microscopes.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Four …

   SciScore for 10.1101/2020.09.04.20188169: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IRB: Human Donors: The recruitment of study subjects was conducted in accordance with the Ethics Committee of the Charité (EA 1/144/13 with EA 1/075/19 and EA 2/066/20) and was in compliance with the Declaration of Helsinki.
   Randomization	Analysis was done using averages of 4 fields of view chosen randomly (blind) and then assessed for foci using fluorescent microscopes.
   Blinding	Analysis was done using averages of 4 fields of view chosen randomly (blind) and then assessed for foci using fluorescent microscopes.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Four healthy adults (3 male and 1 female; average age 47 ± 7 (SEM)) and 13 COVID-19 patients (7 male and 6 female; average age 74 ± 11 (SEM)) with either documented disease history or exposure through natural SARS-CoV-2 infection that were verified by the levels of antigen-reactive antibody IgG titers.	antigen-reactive suggested: None
   Enriched cells were incubated with Fc Blocking Reagent (Milteniy Biotec) following manufacturer’s instructions and subsequently stained up to 5×106 cells per 100µL with the following anti-human antibodies:, CD3 (BW264/56, VioBlue, Miltenyi Biotec, Cat No 130-113-133, 1:400), CD14 (TÜK4, VioBlue, Miltenyi Biotec, Cat No 130-113-152, 1:200), CD16 (REA423, VioBlue, Miltenyi Biotec, Cat No 130-113–958, 1:100), CD27 (MT271, PE, Miltenyi Biotec, Cat No 130-097-926, 1:100) and CD38 (HIT2, APC, BioLegend, Cat No 303510, 1:20).	anti-human antibodies:, CD3 suggested: (RayBiotech Cat# CS-11-0105, RRID:AB_1227994) CD16 suggested: (Miltenyi Biotec Cat# 130-113-958, RRID:AB_2726431) CD27 suggested: None CD38 suggested: None APC suggested: None
   To that end, PBMCs were stained as described but with the following anti-human antibodies: CD19 (LT19, Miltenyi Biotec, Cat No 130-113-728, 1:400), CD3 (BW264/56, VioBlue, Miltenyi Biotec, Cat No 130-113-133, 1:400), CD14 (TÜK4, VioBlue, Miltenyi Biotec, Cat No 130-113-152, 1:200), CD27 (MT271, PE, Miltenyi Biotec, Cat No 130-097-926, 1:100) and CD38 (IB6, PE-Vio770, Miltenyi Biotec, Cat No 130–113-990, 1:200).	anti-human antibodies: CD19 suggested: None CD3 suggested: None
   Stimulated cells were enrichment by MACS for CD137+ and CD154+ cells in two consecutive MS columns (Miltenyi Biotec) and the enriched cells incubated with anti-human Cite-seq antibodies (CD154 Cat No 310849	anti-human Cite-seq suggested: None
   Cells were also stained for 15min with the following anti-human antibodies: CD3 (OKT3, BV785, Biolegend, Cat No 317330, 1:100), CD4 (SK3, PE-Cy5.5, Biolegend, Cat No 35-0047-42, 1:200), CD19 (H1B19, V500, BD Biosciences, Cat No 561121, 1:100), CD14 (TM1, Pacific Orange, DRFZ in-house conjugation, 1:100), CD137 (4B4–1, PE, Miltenyi Biotec, Cat No 130-093-476, 1:25) and CD154 (5C8, biotin, Miltenyi Biotec, Cat No 5190204135, 1:10)	anti-human antibodies: CD3 suggested: None CD19 suggested: (Fluidigm Cat# 3142001, RRID:AB_2651155) CD137 suggested: None CD154 suggested: None biotin , Miltenyi Biotec , Cat No 5190204135 suggested: None
   The phenotype of the SARS-CoV-2-stimulated cells was analyzed by flow cytometry, staining with the following anti-human antibodies: CD3 (OKT3, BV785, Biolegend Cat No 317330, 1:100), CD19 (H1B19, V500, BD Cat No 561121, 1:100), CD14 (TM1, Pacific Orange, in house conjugated, 1:100), CD4 (SK3, PE-Cy5.5, Biolegend Cat No 35-0047-42, 1:200), CD45RA (HI100, BV605, Biolegend Cat No 304133, 1:200), CCR7 (G043H7, A488, Biolegend Cat No 353206, 1:100), CD69 (FN50, PE-CF594, BD Biosciences Cat No 5049599, 1:200), HLA-DR (L243, APC-Cy7, Biolegend Cat No 307617, 1:100), CD154 (24-31, BV421, Biolegend Cat No 310824, 1:100), CD137 (4B4-1, PE, Miltenyi Biotec Cat No 130-093-476, 1:25), IFNγ (4S.B3	CD14 suggested: (BD Biosciences Cat# 340827, RRID:AB_400137) CD4 suggested: None CD45RA suggested: (Bio-Rad Cat# MCA340A488, RRID:AB_321265) CCR7 suggested: (GenWay Biotech Inc. Cat# GWB-EA1DA7, RRID:AB_10269678) A488 suggested: (BioLegend Cat# 310916, RRID:AB_528869) CD69 suggested: None HLA-DR suggested: (BioLegend Cat# 307635, RRID:AB_10897449) APC-Cy7 suggested: (StressMarq Biosciences Cat# SPC-100D-APCCY7, RRID:AB_2822621) BV421 suggested: (BioLegend Cat# 658709, RRID:AB_2563283) IFNγ suggested: None
   Flow cytometric analysis of anti-spike protein serum antibody titers: HEK293T cells were transfected with a plasmid expressing wild-type SARS-CoV-2 S protein(Hoffmann et al., 2020).	anti-spike protein serum suggested: None
   Next day, transfected cells were collected and incubated with sera or recombinant anti-SARS-CoV-2 Spike Glycoprotein S1 antibody [CR3022] (ab273073; Abcam) for 30 min, washed twice with PBS/BSA and stained with anti-human IgG-Alexa647 (Southern Biotech)	anti-SARS-CoV-2 Spike Glycoprotein S1 suggested: (Abcam Cat# ab273074, RRID:AB_2847846) CR3022 suggested: (Imported from the IEDB Cat# CR3022, RRID:AB_2848080) anti-human IgG-Alexa647 suggested: None
   ELISA analysis of anti-spike protein, its RBD domain and anti-nucleoprotein serum antibody titers: Antibody titers against various Sars-Cov-2 S protein were measured using COVID-19 Human IgM IgG Assay Kit (Abnova, Cat No ABN-KA5826) with slight modifications: anti-S IgG and IgM were quantified according to manufacturer’s instructions.	ELISA analysis of anti-spike protein , its RBD domain suggested: None anti-nucleoprotein serum antibody suggested: None Human IgM IgG suggested: None anti-S IgG suggested: None
   Quantification of anti-S-RBD and anti-NP Sars-CoV-2 antibody responses has been performed using respective recombinant proteins.	anti-S-RBD suggested: None anti-NP Sars-CoV-2 suggested: None
   Antibodies were detected using anti-human IgG (MP biomedicals), IgM (Sigma-Aldrich), IgA1 (Southern Biotech) and IgA2 (Southern Biotech) coupled with alkaline phosphatase, followed by pNPP substrate.	anti-human IgG suggested: None IgA1 suggested: None IgA2 suggested: None
   Antibody generation: From the VH and VL sequences obtained by scRNA, synthetic GeneBlocks were synthesized (IDTDNA, Leuwen, Belgium) and cloned into expression vectors for human IgG1 and human Igk, essentially as described by Tiller et al. (Tiller et al., 2008), except that we used Gibson assembly for the cloning.	human IgG1 suggested: None
   Fluorescence-coupled antibodies used: IgA2-PE (Clone REA995, Miltenyi, Cat. No.130-117-763, dilution 1:50); CD27-PE (Clone REA499, Miltenyi, Cat. No. 130-114-166, dilution 1:50); CD38-PE (Clone IB6, Miltenyi, Cat. No. 130-113-427, dilution 1:50) and IgA-PE (Clone IS11–8E10, Miltenyi, Cat. No. 130-114-002, dilution 1:50).	IgA2-PE suggested: (SouthernBiotech Cat# 9140-09, RRID:AB_2796664) IgA-PE suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Flow cytometric analysis of anti-spike protein serum antibody titers: HEK293T cells were transfected with a plasmid expressing wild-type SARS-CoV-2 S protein(Hoffmann et al., 2020).	HEK293T suggested: None
   The pre-incubated serum/virus suspensions were then added to VeroE6 cells cultured in 100µL DMEM 5% hiFCS on a 96-well plate, and incubated at 37°C for 1 day before readout (plaques/Fluorescent foci).	VeroE6 suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
   Software and Algorithms
   Sentences	Resources
   Cells were acquired using a LSRFortessa flow cytometer (BD Biosciences) with FACSDiva (BD Biosciences) software and analyzed with FlowJo (Tree Star).	FACSDiva suggested: (BD FACSDiva Software, RRID:SCR_001456) FlowJo suggested: (FlowJo, RRID:SCR_008520)
   Single Cell RNA-library preparation and sequencing: Single cell suspensions were obtained by cell sorting and applied to the 10x Genomics workflow for cell capturing and scRNA gene expression (GEX) and TCR/BCR/CiteSeq library preparation using the Chromium Single Cell 5’ Library & Gel Bead Kit as well as the Single Cell 5’ Feature Barcode Library Kit (10x Genomics).	TCR/BCR/CiteSeq suggested: None
   The cellranger output was further analyzed in R using the Seurat package (version 3.1.1)(Butler et al., 2018).	Seurat suggested: (SEURAT, RRID:SCR_007322)
   Sparse Partial Least Squares Discriminant Analysis (sPLS-DA) was performed using mixOmics R-package by keeping 200 genes in 3 components.	mixOmics suggested: (mixOmics, RRID:SCR_016889)

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   Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

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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.

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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   Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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

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   Results from rtransparent:

   • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
   • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
   • Thank you for including a protocol registration statement.

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   Read the original source
3. 

   Version published to 10.1101/2020.09.04.20188169v2 on medRxiv

   Sep 9, 2020
4. 

   Version published to 10.1101/2020.09.04.20188169v1 on medRxiv

   Sep 8, 2020