Outcome of SARS-CoV-2 infection is linked to MAIT cell activation and cytotoxicity

  1. Héloïse Flament
  2. Matthieu Rouland
  3. Lucie Beaudoin
  4. Amine Toubal
  5. Léo Bertrand
  6. Samuel Lebourgeois
  7. Camille Rousseau
  8. Pauline Soulard
  9. Zouriatou Gouda
  10. Lucie Cagninacci
  11. Antoine C. Monteiro
  12. Margarita Hurtado-Nedelec
  13. Sandrine Luce
  14. Karine Bailly
  15. Muriel Andrieu
  16. Benjamin Saintpierre
  17. Franck Letourneur
  18. Youenn Jouan
  19. Mustapha Si-Tahar
  20. Thomas Baranek
  21. Christophe Paget
  22. Christian Boitard
  23. Anaïs Vallet-Pichard
  24. Jean-François Gautier
  25. Nadine Ajzenberg
  26. Benjamin Terrier
  27. Frédéric Pène
  28. Jade Ghosn
  29. Xavier Lescure
  30. Yazdan Yazdanpanah
  31. Benoit Visseaux
  32. Diane Descamps
  33. Jean-François Timsit
  34. Renato C. Monteiro
  35. Agnès Lehuen

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Abstract

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  1. ScreenIT

    SciScore for 10.1101/2020.08.31.20185082: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: The Ethics Committees approved clinical investigations.
    Consent: Informed consent was obtained from each enrolled patient.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Flow cytometry: Surface and intracytoplasmic staining were performed on blood samples or PBMC with the following antibodies: CD3 (OKT3), CD19 (HIB19), CD4 (OKT4), CD8 (SK1), Vα7.2 (3C10), TCR-γδ (B1)
    CD3
    suggested: None
    HIB19
    suggested: None
    CD4
    suggested: None
    CD8
    suggested: None
    TCR-γδ
    suggested: None
    B cells, monocytes, and CD4+ cells were depleted from PBMC of the same healthy donors using Dynabeads™ Untouched™ CD8+ kit (#11348D, Invitrogen, USA) with an in-house antibody mix with the following biotinylated monoclonal anti-human antibodies CD4 (OKT4), CD14 (63D3) and CD19 (HIB19) from Biolegend. 2.105 PBMCs or enriched MAIT cells per well were co-cultured with differentiated macrophages during either 24h or 96 hours.
    anti-human antibodies CD4
    suggested: (BD Biosciences Cat# 557939, RRID:AB_2802162)
    CD14
    suggested: (Dr. Bettina Wagner - Cornell University Cat# CD14 105, RRID:AB_2737335)
    CD19
    suggested: (Miltenyi Biotec Cat# 130-105-171, RRID:AB_2661109)
    Blocking antibodies were used against IL-12p70 (MAB219, R&D systems) at 5μg.mL-1 and IL-18 (D044-3, MBL) at 5μg.mL-1. 1 μg.mL-1 B18R (34-8185-81, eBioscience) was used to block Type I interferons50.
    IL-12p70
    suggested: None
    IL-18
    suggested: (MBL International Cat# D044-3, RRID:AB_592009)
    Experimental Models: Cell Lines
    SentencesResources
    In vitro culture: For virus culture, the Vero E6 kidney epithelial cells line was acquired from the American Type Culture Collection (#CRL-1586, ATCC, USA)
    Vero E6
    suggested: None
    After virus adsorption for 1h at 37°C with plate rocking every 15 min, the viral inoculum was removed and Vero cells were washed with PBS free medium.
    Vero
    suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
    Software and Algorithms
    SentencesResources
    B cells, monocytes, and CD4+ cells were depleted from PBMC of the same healthy donors using Dynabeads™ Untouched™ CD8+ kit (#11348D, Invitrogen, USA) with an in-house antibody mix with the following biotinylated monoclonal anti-human antibodies CD4 (OKT4), CD14 (63D3) and CD19 (HIB19) from Biolegend. 2.105 PBMCs or enriched MAIT cells per well were co-cultured with differentiated macrophages during either 24h or 96 hours.
    Biolegend.
    suggested: (BioLegend, RRID:SCR_001134)
    Statistics and Bioinformatics analysis: All bioinformatics analyses were performed using RStudio (1.2.5) running on R software version 4.0.
    RStudio
    suggested: (RStudio, RRID:SCR_000432)
    Our pipeline combined this workflow with the flowWorkspace R library for FlowJo workspace reading and cell population selection.
    flowWorkspace
    suggested: (flowWorkspace, RRID:SCR_001155)
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Multiparametric matrix correlation plots were produced with the adjusted rcorr function from Hmisc and RcmdrMisc packages to compute matrices of Spearman correlations along with the pairwise p-values corrected for multiple inferences using Holm’s method and visualized with the Corrplot package.
    RcmdrMisc
    suggested: None
    Correlation plots hierarchical clustering were produced with the hclust function included in the Corrplot library.
    hclust
    suggested: (HCLUST, RRID:SCR_009154)
    Principal component analysis (PCA) was processed with FactoMineR library and graphically produced with Factoextra package.
    FactoMineR
    suggested: (FactoMineR, RRID:SCR_014602)
    Factoextra
    suggested: (factoextra, RRID:SCR_016692)
    Heatmaps were plotted using pheatmap library, with data centered to zero and scaled for each parameter.
    pheatmap
    suggested: (pheatmap, RRID:SCR_016418)
    Statistical analyses were performed with GraphPad Prism software version 8.0 and R software version 4.0.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    Results from OddPub: Thank you for sharing your code.

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    In other lung pathologies such as asthma, CD69+-activated NK, ILCs, and MAIT cells are associated with airflow limitation43. We observed a significant correlation between MAIT cells activation and ILC2 frequency and it is already established that ILC2 are important drivers of allergen-induced airway hyperresponsiveness (AHR) after influenza A virus (IAV) infection14. Blood cytokine analysis showed increased amounts of type I interferons and pro-inflammatory cytokines such as IL-6, IL-8 and the immunosuppressive IL-10 cytokines in COVID-19 patients as previously reported28. Presence of elevated levels of IL-1β, IL-6, IL-8, and IL-10 are indicative of cytokine storms that are major potential complications in severe COVID-19 patients. Moreover, IL-15 and IL-18 levels were increased in COVID-19 patients in relation with disease severity and IL-18 strongly correlated with MAIT cell activation in patients with fatal outcome. In these patients, IFNα2 level inversely correlated with increased IL-18. During the early phases of infection in vitro, macrophages produced important amounts of type I interferons which collapsed in later stages, around 4 days post-infection. This is in agreement with the ability of SARS-CoV-2 to suppress type I interferon production through numerous structural and non-structural viral proteins44 and as highlighted in COVID-19 patient plasma analysis of ICU patients. However, long-term in vitro infected macrophages produce increased levels of IL-18 that can a...

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

    IdentifierStatusTitle
    NCT04262921RecruitingFrench COVID Cohort

    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.
    • No protocol registration statement was detected.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. Version published to 10.1038/s41590-021-00870-z
  3. Version published to 10.1101/2020.08.31.20185082v1 on medRxiv