Deciphering the state of immune silence in fatal COVID-19 patients

  1. Pierre Bost
  2. Francesco De Sanctis
  3. Stefania Canè
  4. Stefano Ugel
  5. Katia Donadello
  6. Monica Castellucci
  7. David Eyal
  8. Alessandra Fiore
  9. Cristina Anselmi
  10. Roza Maria Barouni
  11. Rosalinda Trovato
  12. Simone Caligola
  13. Alessia Lamolinara
  14. Manuela Iezzi
  15. Federica Facciotti
  16. Annarita Mazzariol
  17. Davide Gibellini
  18. Pasquale De Nardo
  19. Evelina Tacconelli
  20. Leonardo Gottin
  21. Enrico Polati
  22. Benno Schwikowski
  23. Ido Amit
  24. Vincenzo Bronte

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Abstract

Since the beginning of the SARS-CoV-2 pandemic, COVID-19 appeared as a unique disease with unconventional tissue and systemic immune features. Here we show a COVID-19 immune signature associated with severity by integrating single-cell RNA-seq analysis from blood samples and broncho-alveolar lavage fluids with clinical, immunological and functional ex vivo data. This signature is characterized by lung accumulation of naïve lymphoid cells associated with a systemic expansion and activation of myeloid cells. Myeloid-driven immune suppression is a hallmark of COVID-19 evolution, highlighting arginase-1 expression with immune regulatory features of monocytes. Monocyte-dependent and neutrophil-dependent immune suppression loss is associated with fatal clinical outcome in severe patients. Additionally, our analysis shows a lung CXCR6 + effector memory T cell subset is associated with better prognosis in patients with severe COVID-19. In summary, COVID-19-induced myeloid dysregulation and lymphoid impairment establish a condition of ‘immune silence’ in patients with critical COVID-19.

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  1. Version published to 10.1038/s41467-021-21702-6
  2. ScreenIT

    SciScore for 10.1101/2020.08.10.20170894: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementConsent: Ethics approval statement: All the patients (and/or initially their families) provided written informed consent before sampling and for the use of their clinical and biological data.
    IACUC: This study was approved by the local ethical committee (Prot. n° 17963, and n° 51095, P.I. Vincenzo Bronte); informed consent was obtained from all the participants to the study.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Briefly, peripheral blood was incubated with FcR Blocking reagent (Miltenyi Biotec, Paris, FR) followed by the addition of: antihuman PE-conjugated CD56 (BD Bioscience, San Jose, CA, USA; clone NCAM16.2), FITC-conjugated-CD16 (Biolegend, San Diego, CA, USA; clone 3G8), PerCP-Cy5.5-conjugated CD3 (BD Bioscience, San Jose, CA, USA; clone UCHT1), PE.Cy7-conjugated HLA-DR (eBiosciences, ThermoFisher Scientific, Waltham, MA, USA; clone L243), APC.H7-conjugated CD14 (BD Bioscience, San Jose, CA, USA; clone MφP9), Brilliant Violet 421™-conjugated PD-L1 (BD Bioscience, San Jose, CA, USA; clone MIH1) and aleza fluor 647-conjugated ARG1 (developed in our laboratory [36]) antibodies, Aqua LIVE/DEAD dye (ThermoFisher Scientific, Waltham, MA, USA).
    antihuman PE-conjugated CD56
    suggested: None
    PerCP-Cy5.5-conjugated CD3
    suggested: None
    CD14
    suggested: None
    ARG1
    suggested: None
    From the CD14- fraction the CD66+ low density gradient neutrophils (LDNs) were isolated by the sequential addition of CD66b-FITC antibody (BD Biosciences, NJ, USA) and microbeads anti-FITC (Miltenyi), following manufacturer’s instructions.
    anti-FITC
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    The Spike SARS-CoV2 glycoprotein receptor binding domain (RBD) was expressed in mammalian HEK293 cells at IEO, Milan by Drs.
    HEK293
    suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
    Software and Algorithms
    SentencesResources
    At the end of the culture, cells were stained with anti-CD3-PE/Cy7 (UCHT1, eBioscience, Thermo Fisher Scientific) and CellTrace signal of lymphocytes was analyzed with FlowJo software (Tree Star, Inc. Ashland).
    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:
    However, our study is not flawless and suffers from limitations, including experimental design ones. Firstly, this is not a longitudinal study. Secondly, the absence of BAL samples from mild patients prevents us to generalize and validate the observations made on severe ones. Moreover, the absence of BAL samples from healthy control deprives us of estimating the basal state and composition of the lung immune system. While it is technically possible to collect such samples, it raises important ethical question that need to be considered while designing COVID-19 cohorts. The third limiting factor is the nature of the BAL samples: briefly, a physiologic solution is introduced into the lower respiratory tract and then collected. Such method is able to capture infiltrating immune cells in spite of non-immune cell types (i.e. epithelial cells), as shown by recent autopsy reports [43]. Lung biopsy represents an interesting alternative to BAL but is far riskier, especially for patients suffering from ARDS, such as severe COVID-19 patients, and the amount of available material is limited. One may therefore try to collect samples from deceased patients, however as most patient died after several weeks in ICU, the biological features observed during the autopsy might either be linked to the disease itself or to the extended stay in ICU. In the case of lung biopsy, highly multiplexed imaging techniques such SeqFISH [44] or CODEX [45], but also spatial transcriptomic technologies [46] cou...

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

    IdentifierStatusTitle
    NCT04438629RecruitingEvaluation of Immune Response in COVID-19 Patients

    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.

  3. Version published to 10.1101/2020.08.10.20170894v1 on medRxiv
  4. Version published to 10.21203/rs.3.rs-56689/v1 on Research Square