Immunopathological signatures in multisystem inflammatory syndrome in children and pediatric COVID-19

SciScore for 10.1101/2021.09.24.21263853: (What is this?)

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

Table 1: Rigor

Ethics	IRB: All subjects were recruited following protocols approved by local Institutional Review Boards (IRBs) or from NIH NIAID IRB, as indicated below: Chile: IRB: Comité Ético Científico Facultad de Medicina Clínica Alemana Universidad del Desarrollo, Santiago, Chile Protocol: 2020-41 Title: Infección por Sars CoV-2 y enfermedad Covid-19: caracterización epidemiológica, clínica, virológica e inmunológica (SARS-CoV-2 infection and COVID-19: Epidemiologic, clinical, virologic and immunologic characterization) Pavia, Italy: IRB: Ethics Committee of the Fondazione IRCCS Policlinico San Matteo, Pavia Protocol: 20200037677 Title: Analisi comprensiva della risposta immunitaria innata ed adattativa durante
Sex as a biological variable	not detected.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
SARS-CoV-2 antibody testing: SARS-CoV-2 anti-spike and anti-nucleocapsid antibody testing was performed via luciferase immunoprecipitation systems assay, as previously described [6].	SARS-CoV-2 suggested: None anti-spike suggested: None anti-nucleocapsid suggested: None
PBMC pools were Fc blocked (Human TruStain FcX, BioLegend) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS).	human ‘hashtag’ suggested: None
The fluorescence-labeled antibody cocktail against human CD45 (APC/Cyanine7), CD3 (AF488), CD19 (APC), CCR7 (BV786), CD95 (BV650), IgD (PerCP-Cy5.5) and CD27(PE/Cyanine7; all antibodies obtained from Biolegend) were added at the end of blocking and incubated for 20 minutes at 4°C in the dark.	human CD45 suggested: (BD Biosciences Cat# 563716, RRID:AB_2716864) CD3 suggested: (SouthernBiotech Cat# 8200-30, RRID:AB_2796425) CD19 suggested: (BD Biosciences Cat# 563333, RRID:AB_2738141) CCR7 suggested: (Creative Diagnostics Cat# CPBT-66983GM, RRID:AB_2528776) BV786 suggested: (BD Biosciences Cat# 740991, RRID:AB_2740614) CD95 suggested: (BioLegend Cat# 305642, RRID:AB_2632622) BV650 suggested: None PerCP-Cy5.5 suggested: (BD Biosciences Cat# 560612, RRID:AB_1727457) CD27 suggested: None
Because multiple samples from different timepoints for each donor were collected and could not be demultiplexed by this method alone, ‘hashtag’ antibodies (Biolegend) were used to uniquely label the different time points.	‘hashtag’ suggested: None
The data of several proteins that directly bind with secondary antibodies detected through buffer incubation without any serum were excluded (such as IGHG1, IGHG3 and so on) alongside the controls (such as Rhodamine+IgG64, Anti-human IgG, GST 10ng/ul etc.).	IGHG1 suggested: None IGHG3 suggested: None Anti-human IgG suggested: (LSBio (LifeSpan Cat# LS-C23907-500, RRID:AB_900919)
Multiplex particle-based anticytokine autoantibody screening assay and functional evaluation: Plasma samples were screened for autoantibodies against IFN-α, IFN-β, IFN-ω and IFN-γ in a multiplex particle-based assay [31], in which differentially fluorescent magnetic beads were covalently coupled to recombinant human proteins (2.5 ug/reaction).	IFN-α, IFN-β suggested: None IFN-ω suggested: None
Recombinant DNA
Sentences	Resources
For the comparison of pHC with pCOVID-19, the classification was then repeated after the exclusion of allergic pHC, with similar results.	pCOVID-19 suggested: None
Software and Algorithms
Sentences	Resources
These analyses were completed with IBM SPSS Statistics v.	SPSS suggested: (SPSS, RRID:SCR_002865)
27 and GraphPad Prism version 9 [7].	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)
Trained with Python sklearn library’s RandomForestClassifier object, using parameters: n_estimator=2000, random_state=42 for data set.	Python suggested: (IPython, RRID:SCR_001658)
The statistical analysis of SOMAscan® results was performed using R Studio (R Core Team, 2020) [11], also using a specifically developed webtool for basic data plotting and analysis [12].	SOMAscan® suggested: None
To assess the difference in the overall predictive importance (derived by GENIE3) of each variable with and without IVIG treatment, we summed the interaction strengths associated with each predictor-target pair for a given predictor variable in either treatment condition (steroid treatment applied in both conditions).	GENIE3 suggested: (GENIE3, RRID:SCR_000217)
The resulting values were visualized using the Complexheatmap package in R.	Complexheatmap suggested: (ComplexHeatmap, RRID:SCR_017270)
All sequencing data were then processed with Burrows–Wheeler Aligner (BWA) and the Genome Analysis Toolkit (GATK	BWA suggested: (BWA, RRID:SCR_010910) Genome Analysis Toolkit suggested: None GATK suggested: (GATK, RRID:SCR_001876)
Raw fastq files were trimmed using Trimmomatic v0.39 [15] and mapped to the hg38 human reference genome using BWA-MEM v07.17.	Trimmomatic suggested: (Trimmomatic, RRID:SCR_011848) BWA-MEM suggested: (Sniffles, RRID:SCR_017619)
PCR Duplicates were marked using Samblaster v0.1.2.5 [16] and GATK4 v4.1.9.0 was used to perform BAM recalibration, and HLA*LA [17] was used to call HLA genotypes.	Samblaster suggested: (SAMBLASTER, RRID:SCR_000468) GATK4 suggested: None
The resulting CDR3 sequences were collapsed and filtered to quantify the absolute abundance and frequency of each unique CDR3 region with Adaptive Biotechnologies’ pipeline [19]	Biotechnologies’ suggested: None
10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. d) Bulk RNA sequencing and single cell sample demultiplexing: For each sample, 100,000-500,000 cells were processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD) and standard RNA sequencing libraries were generated using Illumina Truseq library preparation kits.	Genomics suggested: (UTHSCSA Genomics Core, RRID:SCR_012239)
The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol [23].	STAR suggested: (STAR, RRID:SCR_004463)
Lowly expressed genes were removed for each cell type individually using the filterByExpr function from edgeR [26]	edgeR suggested: (edgeR, RRID:SCR_012802)
Differentially expressed genes were identified using the limma voom [27] workflow which models the log of the cpm (counts per million) of each gene.	limma suggested: (LIMMA, RRID:SCR_010943)
Enriched gene sets were identified using the pre-ranked GSEA algorithm implemented in the FGSEA R package [https://www.biorxiv.org/content/10.1101/060012v3].	FGSEA suggested: (fgsea, RRID:SCR_020938)
The pseudobulk gene counts were normalized with the varianceStabilizingTransformation function from DEseq2 (doi:10.1186/s13059-014-0550-8) for the score calculation.	DEseq2 suggested: (DESeq2, RRID:SCR_015687)
For BCR data, V(D)J sequencing contigs from 10x CellRanger output was processed using Immcantation v2.7.0 toolbox (https://immcantation.readthedocs.io/en/latest/index.html).	Immcantation suggested: None
BCR sequence genotype inference and mutation load quantification were performed with reference to the pipeline from Mathew et al. [29] using the TIgGER package [30] and SHazaM package [28].	TIgGER suggested: None SHazaM suggested: None
Autoantibody analysis: Autoantibody analysis was performed using HuProt™ v4.0 human protein microarrays and processed by CDI Laboratories (Baltimore, MD)	HuProt™ suggested: None
Beads were washed again, resuspended in assay buffer, and analyzed on a BioPlex X200 instrument.	BioPlex suggested: (BioPlex, RRID:SCR_016144)
Cells were acquired on a BD LSRFortessa cytometer, gated on CD14+ monocytes, and analyzed with FlowJo software.	FlowJo suggested: (FlowJo, RRID:SCR_008520)

Results from OddPub: Thank you for sharing your code and data.

Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

This study has some limitations. Only a few children with severe pCOVID-19 were included in the study, and therefore the results obtained may not apply to rare cases of life-threatening disease in children. We did not have longitudinal samples available for all MIS-C patients. However, the number of patients included in the study was sufficient to detect early and late signatures of the disease. Finally, while an increasing number of cases of pCOVID-19 due to the delta variant have been recently reported [62], almost all samples were collected prior to the emergence of this variant. Therefore, the impact of the delta variant on innate and adaptive immune responses in children with pCOVID-19 and MIS-C remains to be studied. Despite these limitations, this study has helped identify novel age-, time- and treatment-related immunopathological signatures that characterize MIS-C and pCOVID-19.

Results from TrialIdentifier: We found the following clinical trial numbers in your paper:

Identifier	Status	Title
NCT03394053	Recruiting	The Mechanistic Biology of Primary Immunodeficiency Disorder…
NCT03610802	Recruiting	Send-In Sample Collection to Achieve Genetic and Immunologic…

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

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

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.

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

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Immunopathological signatures in multisystem inflammatory syndrome in children and pediatric COVID-19

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