Vγ9Vδ2 T cells are potent inhibitors of SARS-CoV-2 replication and exert effector phenotypes in COVID-19 patients

  1. Laetitia Gay
  2. Marie-Sarah Rouviere
  3. Soraya Mezouar
  4. Manon Richaud
  5. Laurent Gorvel
  6. Etienne Foucher
  7. Loui Madakamutil
  8. Bernard La Scola
  9. Amélie Menard
  10. Jérôme Allardet-Servent
  11. Philippe Halfon
  12. Paul Frohna
  13. Carla Cano
  14. Jean-Louis Mege
  15. Daniel Olive

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Abstract

Vγ9Vδ2 T cells play a key role in the innate immune response to viral infections, including SARS-CoV-1 and 2, and are activated through butyrophilin (BTN)-3A. Here, the objectives were to: 1) characterize the effects of SARS-CoV-2 infection on the number, phenotype, and activation of Vγ9Vδ2 T cells in infected patients, and 2) assess the effects of in vitro SARS-CoV-2 infection on the expression of BTN3A and its impact on the activation and response of Vγ9Vδ2 T cells to an anti-BTN3A antibody. Blood Vγ9Vδ2 T cells decreased in clinically mild SARS-CoV-2 infections compared to healthy volunteers (HV). This decrease was maintained up to 28 days and in the recovery period. Terminally differentiated Vγ9Vδ2 T cells tend to be enriched on the day of diagnosis, 28 days after and during the recovery period compared to HV. Furthermore, these cells showed cytotoxic and inflammatory activities as shown by TNFα, IFNγ and CD107a/b increase following anti-BTN3A activation. Moreover, BTN3A upregulation and Vγ9Vδ2 T cell infiltration were observed in a lung biopsy from a fatal SARS-CoV-2 infection, as compared to HV. In vitro , SARS-CoV-2 infection significantly increased BTN3A expression in macrophages and lung cell lines. The activation via BTN3A enhanced the anti-SARS-CoV-2 Vγ9Vδ2 T cells cytotoxicity and IFN-γ and TNFα in SARS-CoV-2 infected patient. Increasing concentrations of anti-BTN3A were accompanied by an inhibition of viral replication. Altogether, these data suggest that Vγ9Vδ2 T cells are important in the immune response against SARS-CoV-2 infection and that activation by an anti-BTN3A antibody may enhance their response.

KEY POINTS

  • SARS-CoV-2 mediates upregulation of the key receptor of Vγ9Vδ2 T cells BTN3A on lung tissues and cell lines as well as monocytes

  • During SARS-CoV-2 infection, Vγ9Vδ2 are differentiated and efficiently degranulate and secrete cytokines upon activation with BTN3A mAb

Article activity feed

  1. ScreenIT

    SciScore for 10.1101/2022.04.15.487518: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: The protocol of the study was approved by an independent national review board (Comité de Protection des Personnes, Ile-de-France XI, ID: 20027-60604) and registered at ClinicaTrials.gov (NCT04816760).
    Consent: All patients provided written informed consent.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    After 30 min incubation, primary antibodies binding were detected with Alexa Fluor-488 anti-mouse (Invitrogen).
    anti-mouse
    suggested: None
    5 μg/ml IgG1 mouse monoclonal antibody 20.1 directed against BTN3A (Imcheck Therapeutics) or 10 μg/ml IgG1 mouse monoclonal antibody BB3 directed against Vδ2-TCR (kind gift of Dr. Pende, Genoa, Italy supplier) were used as primary antibodies.
    BTN3A
    suggested: None
    IgG1
    suggested: None
    Vδ2-TCR
    suggested: None
    A second staining with 10 μg/ml IgG2b mouse monoclonal antibody 9C4 directed against EpCam (Biolegend) allowed the identification of epithelial cells by indirect detection with anti-IgG2b Cyanine-3 labelled secondary Ab (Jackson ImmunoScience, dilution 1:500).
    EpCam
    suggested: None
    anti-IgG2b
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Virus production and cell infection: SARS-CoV-2 strain IHU-MI6 was obtained after VeroE6 cells (ATCC® CRL-1586™) infection in MEM supplemented with 4% FBS as previously described17.
    VeroE6
    suggested: None
    Cytotoxicity assay: Monocytes, MDMs, BEAS-2B and MRC-5 were labeled with 10 μM Cell Proliferation Dye eFluor® 670 (Invitrogen) and then stimulated with virus.
    MRC-5
    suggested: None
    Immunoassays: Monocytes, MDMs, BEAS-2B and MRC-5 were stimulated with virus and co-cultured with Vγ9Vδ2 T cells (effector) at 1:1 E:T ratio in the presence of anti-BTN3A 20.1 Ab (0, 0.1, 1 or 10 μg/ml).
    BEAS-2B
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Virus production and cell infection: SARS-CoV-2 strain IHU-MI6 was obtained after VeroE6 cells (ATCC® CRL-1586™) infection in MEM supplemented with 4% FBS as previously described17.
    SARS-CoV-2
    suggested: None
    Software and Algorithms
    SentencesResources
    The images were acquired on confocal microscope (Zeiss LSM 880 airy scan) using Zen Black software (Zeiss) and Fiji software (ImageJ-win64) used for data treatment.
    Zen Black
    suggested: (Black Zen software, RRID:SCR_018163)
    Fiji
    suggested: (Fiji, RRID:SCR_002285)
    Data were collected on a BD Canto II instrument (BD Biosciences) and analyzed with FlowJo software.
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Statistical Analyses: Statistical analysis was performed using GraphPad Prism (GraphPad Software), for transcriptional analysis using the Student t or nonparametric Mann-Whitney U test and for Spectral Cytometry using non parametric Kruskal-Wallis test, followed by Dunn’s multiple comparisons test or two-way ANOVA, followed by Holm-Sidak’s multiple comparisons test.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    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: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT04816760RecruitingImmune Cells Phenotypes During COVID-19
    NCT04243499RecruitingFirst-in-Human Study of ICT01 in Patients With Advanced Canc…

    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.

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

    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 10.1101/2022.04.15.487518v1 on bioRxiv