CAR Macrophages for SARS-CoV-2 Immunotherapy

  1. Wenyan Fu
  2. Changhai Lei
  3. Zetong Ma
  4. Kewen Qian
  5. Tian Li
  6. Jian Zhao
  7. Shi Hu

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Abstract

Targeted therapeutics for the treatment of coronavirus disease 2019 (COVID-19), especially severe cases, are currently lacking. As macrophages have unique effector functions as a first-line defense against invading pathogens, we genetically armed human macrophages with chimeric antigen receptors (CARs) to reprogram their phagocytic activity against SARS-CoV-2. After investigation of CAR constructs with different intracellular receptor domains, we found that although cytosolic domains from MERTK (CAR MERTK ) did not trigger antigen-specific cellular phagocytosis or killing effects, unlike those from MEGF10, FcRγ and CD3ζ did, these CARs all mediated similar SARS-CoV-2 clearance in vitro . Notably, we showed that CAR MERTK macrophages reduced the virion load without upregulation of proinflammatory cytokine expression. These results suggest that CAR MERTK drives an ‘immunologically silent’ scavenger effect in macrophages and pave the way for further investigation of CARs for the treatment of individuals with COVID-19, particularly those with severe cases at a high risk of hyperinflammation.

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  1. Version published to 10.3389/fimmu.2021.669103
  2. Version published to 10.1101/2020.07.26.222208v2 on bioRxiv
  3. ScreenIT

    SciScore for 10.1101/2020.07.26.222208: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.Randomizationnot detected.Blindingnot detected.Power Analysisnot detected.Sex as a biological variablenot detected.Cell Line AuthenticationThe identities of the cell lines were verified by STR analysis, and the cell lines were confirmed to be mycoplasma free.

    Table 2: Resources

    Antibodies
    SentencesResources
    After coculturing, the cells were harvested and stained with an anti-CD11b APC-Cy7-conjugated antibody (M1/70, BioLegend) and analyzed by FACS using a FACSCalibur flow cytometer (BD Biosciences).
    anti-CD11b
    suggested: None
    The SARS-CoV-2 pseudovirus could not be neutralized with anti-VSV-G antibodies, and no G∗ΔG-VSV was mixed with the SARS-CoV-2 pseudovirus stock.
    anti-VSV-G
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    293 and Vero cells were maintained in DMEM supplemented with 10% fetal bovine serum, and THP-1 cells were maintained in RPMI medium supplemented with 10% fetal bovine serum.
    Vero
    suggested: None
    Lentiviral infection was used to stably express CAR constructs in THP-1 cells.
    THP-1
    suggested: CLS Cat# 300356/p804_THP-1, RRID:CVCL_0006
    FACS-based phagocytosis assay UTD or CAR-expressing THP-1 cells were cocultured with GFP+ 293T cells or GFP+ 293T-S (S+) target cells for 4 h at 37 °C.
    293T
    suggested: RRID:CVCL_LC70
    In vitro cytotoxicity assay 293T and 293T-S cells were used as targets in luciferase-based killing assays including control (UTD) or CAR macrophages.
    293T-S
    suggested: RRID:CVCL_LC70

    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 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.

    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. ScreenIT

    SciScore for 10.1101/2020.07.26.222208: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line AuthenticationAuthentication: The identities of the cell lines were verified by STR analysis, and the cell lines were confirmed to be mycoplasma free.
    Contamination: The identities of the cell lines were verified by STR analysis, and the cell lines were confirmed to be mycoplasma free.

    Table 2: Resources

    Antibodies
    SentencesResources
    After coculturing, the cells were harvested and stained with an anti-CD11b APC-Cy7-conjugated antibody (M1/70, BioLegend) and analyzed by FACS using a FACSCalibur flow cytometer (BD Biosciences).
    anti-CD11b
    suggested: None
    The SARS-CoV-2 pseudovirus could not be neutralized with anti-VSV-G antibodies, and no G*ΔG-VSV was mixed with the SARS-CoV-2 pseudovirus stock.
    anti-VSV-G
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    293 and Vero cells were maintained in DMEM supplemented with 10% fetal bovine serum, and THP-1 cells were maintained in RPMI medium supplemented with 10% fetal bovine serum.
    Vero
    suggested: None
    FACS-based phagocytosis assay: UTD or CAR-expressing THP-1 cells were cocultured with GFP+ 293T cells or GFP+ 293T-S (S+) target cells for 4 h at 37 °C.
    293T
    suggested: RRID:CVCL_LC70)
    In vitro cytotoxicity assay: 293T and 293T-S cells were used as targets in luciferase-based killing assays including control (UTD) or CAR macrophages.
    293T-S
    suggested: RRID:CVCL_LC70)
    Phagocytosis assay: In all cases, SARS-CoV-2 S pseudotyped virions were pelleted (90 min at 14,000 rpm and 4 °C), and after removal of the supernatant, the pellets were resuspended in RPMI medium and incubated with phagocytes (THP-1 cells or CAR macrophages) at 37 °C for 1.5 h.
    THP-1
    suggested: None

    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.

  5. ScreenIT

    SciScore for 10.1101/2020.07.26.222208: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.Randomizationnot detected.Blindingnot detected.Power Analysisnot detected.Sex as a biological variablenot detected.Cell Line AuthenticationThe identities of the cell lines were verified by STR analysis, and the cell lines were confirmed to be mycoplasma free.

    Table 2: Resources

    Antibodies
    SentencesResources
    After coculturing, the cells were harvested and stained with an anti-CD11b APC-Cy7-conjugated antibody (M1/70, BioLegend) and analyzed by FACS using a FACSCalibur flow cytometer (BD Biosciences).
    anti-CD11b
    suggested: None
    The SARS-CoV-2 pseudovirus could not be neutralized with anti-VSV-G antibodies, and no G∗ΔG-VSV was mixed with the SARS-CoV-2 pseudovirus stock.
    anti-VSV-G
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    293 and Vero cells were maintained in DMEM supplemented with 10% fetal bovine serum, and THP-1 cells were maintained in RPMI medium supplemented with 10% fetal bovine serum.
    Vero
    suggested: None
    THP-1
    suggested: None
    FACS-based phagocytosis assay UTD or CAR-expressing THP-1 cells were cocultured with GFP+ 293T cells or GFP+ 293T-S (S+) target cells for 4 h at 37 °C.
    293T
    suggested: RRID:CVCL_LC70
    In vitro cytotoxicity assay 293T and 293T-S cells were used as targets in luciferase-based killing assays including control (UTD) or CAR macrophages.
    293T-S
    suggested: RRID:CVCL_LC70

    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 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.

    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.

  6. ScreenIT

    SciScore for 10.1101/2020.07.26.222208: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.Randomizationnot detected.Blindingnot detected.Power Analysisnot detected.Sex as a biological variablenot detected.Cell Line AuthenticationThe identities of the cell lines were verified by STR analysis, and the cell lines were confirmed to be mycoplasma free.

    Table 2: Resources

    Antibodies
    SentencesResources
    The synthetic receptors were constructed to contain an scFv derived from an antibody recognizing the virus spike protein, CR3022, which has been reported to bind with the receptor-binding domain of the SARS-CoV-2 S glycoprotein with high affinity, and the CD8 transmembrane domain present in the αCD19 CAR for T cells 9.
    CR3022
    suggested: (Imported from the IEDB Cat# CR3022, AB_2848080)
          <div style="margin-bottom:8px">
        <div><b>CD8</b></div>
        <div>suggested: (BioLegend Cat# 391503, <a href="https://scicrunch.org/resources/Any/search?q=AB_2721611">AB_2721611</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After coculturing, the cells were harvested and stained with an anti-CD11b APC-Cy7-conjugated antibody (M1/70, BioLegend) and analyzed by FACS using a FACSCalibur flow cytometer (BD Biosciences).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>anti-CD11b</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The SARS-CoV-2 pseudovirus could not be neutralized with anti-VSV-G antibodies, and no G∗ΔG-VSV was mixed with the SARS-CoV-2 pseudovirus stock.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>anti-VSV-G</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">CAR-mediated macrophage phagocytosis was further confirmed by a luciferase-based killing assay, and our data showed that CARMEGF10, CARγ and CARζ cells eradicated S protein-expressing 293T cells in an antigen-specific manner (Fig. 1D)</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>293T</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All the CAR-expressing macrophages potently inhibited Vero E6 cell infection with the SARS-CoV-2-S pseudotyped virus.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Vero E6</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">293 and Vero cells were maintained in DMEM supplemented with 10% fetal bovine serum, and THP-1 cells were maintained in RPMI medium supplemented with 10% fetal bovine serum.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Vero</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>THP-1</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In vitro cytotoxicity assay 293T and 293T-S cells were used as targets in luciferase-based killing assays including control (UTD) or CAR macrophages.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>293T-S</b></div>
        <div>suggested: <a href="https://scicrunch.org/resources/Any/search?q=CVCL_LC70">CVCL_LC70</a></div>
      </div>
    </td></tr></table>

    Data from additional tools added to each annotation on a weekly basis.

    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.

  7. Version published to 10.1101/2020.07.26.222208v1 on bioRxiv