SARS-CoV-2-reactive T cells in healthy donors and patients with COVID-19

  1. Julian Braun
  2. Lucie Loyal
  3. Marco Frentsch
  4. Daniel Wendisch
  5. Philipp Georg
  6. Florian Kurth
  7. Stefan Hippenstiel
  8. Manuela Dingeldey
  9. Beate Kruse
  10. Florent Fauchere
  11. Emre Baysal
  12. Maike Mangold
  13. Larissa Henze
  14. Roland Lauster
  15. Marcus A. Mall
  16. Kirsten Beyer
  17. Jobst Röhmel
  18. Sebastian Voigt
  19. Jürgen Schmitz
  20. Stefan Miltenyi
  21. Ilja Demuth
  22. Marcel A. Müller
  23. Andreas Hocke
  24. Martin Witzenrath
  25. Norbert Suttorp
  26. Florian Kern
  27. Ulf Reimer
  28. Holger Wenschuh
  29. Christian Drosten
  30. Victor M. Corman
  31. Claudia Giesecke-Thiel
  32. Leif Erik Sander
  33. Andreas Thiel

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Abstract

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  1. ScreenIT

    SciScore for 10.1101/2020.04.17.20061440: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: Study subjects: This study was approved by the Institutional Review board of the Charité (EA2/066/20).
    Consent: After providing written informed consent, 68 healthy donors (Supplementary Table 1) and 18 COVID-19 patients (Table 1) were included in the study.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Flow Cytometry: Stimulation was stopped by incubation in 20 mM EDTA for 5 min and surface staining conducted for 15 min with the following fluorochrome conjugated antibodies titrated to their optimal concentrations: CD38-PE-Vio770 (clone REA671, Miltenyi), CD69-APC-Cy7 (FN50, Biolegend), HLAD-DR-VioGreen (REA805, Miltenyi), CD4-BrilliantViolet605 (RPA-T4, Biolegend), CD8-PerCP (SK1, Biolegend) with 1 mg/ml Beriglobin (CSL Behring) added prior to the staining.
    CD69-APC-Cy7
    suggested: None
    FN50
    suggested: None
    CD4-BrilliantViolet605
    suggested: None
    RPA-T4
    suggested: None
    CD8-PerCP
    suggested: (Miltenyi Biotec Cat# 130-098-057, RRID:AB_2660906)
    Experimental Models: Cell Lines
    SentencesResources
    Recombinant immunofluorescence assays (rIFA) to determine IgG titers against HCoV was done by using VeroB4 cells expressing cloned recombinant coronavirus spike proteins from HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1 as described in Corman et al.39.
    VeroB4
    suggested: CCLV Cat# CCLV-RIE 1146, RRID:CVCL_1912)
    HCoV-NL63
    suggested: RRID:CVCL_RW88)
    Software and Algorithms
    SentencesResources
    Data analysis and statistics: Flow cytometry data were analyzed with FlowJo 9.9.6 (FlowJo LLC)
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Prism 5 (GraphPad Inc.) was used for plotting and statistical analysis.
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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).

    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 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/s41586-020-2598-9
  3. ScreenIT

    SciScore for 10.1101/2020.04.17.20061440: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementMaterials and Methods Study subjects This study was approved by the Institutional Review board of the Charité (EA2/066/20).Randomizationnot detected.Blindingnot detected.Power Analysisnot detected.Sex as a biological variablenot detected.Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    All HD were negative for IgG antibodies specific for S subunit 1 (S1) in contrast to COVID-19 patients (Figure 2f).
    IgG
    suggested: None
          <div style="margin-bottom:8px">
        <div><b>S subunit 1</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Therefore, we additionally tested 18 of the 68 HD for the presence of antibodies specific for the four HCoV (229E, NL63, HKU1, OC43).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>NL63</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>HKU1</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Flow Cytometry Stimulation was stopped by incubation in 20 mM EDTA for 5 min and surface staining conducted for 15 min with the following fluorochrome conjugated antibodies titrated to their optimal concentrations: CD38-PE-Vio770 (clone REA671, Miltenyi), CD69-APC-Cy7 (FN50, Biolegend), HLAD-DR-VioGreen (REA805, Miltenyi), CD4-BrilliantViolet605 (RPA-T4, Biolegend), CD8-PerCP (SK1, Biolegend) with 1 mg/ml Beriglobin (CSL Behring) added prior to the staining.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>CD69-APC-Cy7</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>CD8-PerCP</b></div>
        <div>suggested: (Miltenyi Biotec Cat# 130-113-722, <a href="https://scicrunch.org/resources/Any/search?q=AB_2726263">AB_2726263</a>)</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>SK1</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Experimental Models: Cell Lines</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Recombinant immunofluorescence assays (rIFA) to determine IgG titers against HCoV was done by using VeroB4 cells expressing cloned recombinant coronavirus spike proteins from HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1 as described in Corman et al.39.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>VeroB4</b></div>
        <div>suggested: DSMZ Cat# ACC-33, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_1912">CVCL_1912</a></div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>HCoV-NL63</b></div>
        <div>suggested: <a href="https://scicrunch.org/resources/Any/search?q=CVCL_RW88">CVCL_RW88</a></div>
      </div>
    </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Software and Algorithms</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Stimulation was conducted with 5x106 PBMC in RPMI 1640 medium (Gibco) supplemented with 10% heat inactivated AB serum (Pan Biotech), 100 U/ml penicillin (Biochrom), 0.1 mg/ml streptomycin (Biochrom) and PepMixTM SARS-CoV-2 spike glycoprotein (JPT) peptide pool 1 or 2 in the presence of 1 µg/ml purified anti-CD28 (clone CD28.2, BD Biosciences).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Biochrom</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data analysis and statistics Flow cytometry data were analyzed with FlowJo 9.9.6 (FlowJo LLC).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>FlowJo</b></div>
        <div>suggested: (FlowJo, <a href="https://scicrunch.org/resources/Any/search?q=SCR_008520">SCR_008520</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Prism 5 (GraphPad Inc.) was used for plotting and statistical analysis.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>GraphPad</b></div>
        <div>suggested: (GraphPad Prism, <a href="https://scicrunch.org/resources/Any/search?q=SCR_002798">SCR_002798</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Potent human neutralizing antibodies elicited by SARS-CoV-2 infection. bioRxiv 2020.03.21.990770 (2020). 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. doi:10.1101/2020.03.21.990770 Meng, T. et al.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>bioRxiv</b></div>
        <div>suggested: (bioRxiv, <a href="https://scicrunch.org/resources/Any/search?q=SCR_003933">SCR_003933</a>)</div>
      </div>
    </td></tr></table>

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

    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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

  4. Version published to 10.1101/2020.04.17.20061440v1 on medRxiv